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Cocaine-related deaths have increased since the early 1990s in Europe, including the UK. Being multi-factorial, they are difficult to define, detect and record. The European Monitoring Centre for Drugs and Drug Addiction commissioned research to: describe trends reported to Special Mortality Registries and General Mortality Registers; provide demographic and drug-use characteristic information of cases; and establish how deaths are identified and classified. A questionnaire was developed and piloted amongst all European Monitoring Centre for Drugs and Drug Addiction Focal Point experts/Special Mortality Registries: 19 (63%) responded; nine countries provided aggregated data. UK General Mortality Registers use cause of death and toxicology to identify cocaine-related deaths. Categorisation is based on International Classification of Diseases codes. Special Mortality Registries use toxicology, autopsy, evidence and cause of death. The cocaine metabolites commonly screened for are: benzoylecgonine, ecgonine methyl ester, cocaethylene and ecgonine. The 2000s saw a generally accelerating upward trend in cases, followed by a decline in 2009. The UK recorded 2700–2900 deaths during 1998–2012. UK Special Mortality Registry data (2005–2009) indicate: 25–44 year-olds account for 74% of deaths; mean age=34 (range 15–81) years; 84% male. Cocaine overdoses account for two-thirds of cases; cocaine alone being mentioned/implicated in 23% in the UK. Opioids are involved in most (58%) cocaine overdose cases.

This study is part of a larger project designed to investigate the prevalence of psychoactive drug (PAD) use among Brazilian drivers. In this paper we describe the development and validation of an analytical method to analyze 32 prescription and illicit PADs (amphetamines, benzodiazepines, cocaine, cannabis, opioids, ketamine and m-CPP) and metabolites in oral fluid samples collected with a Quantisal™ device. Samples were extracted with ethyl acetate:hexane and analyzed by LC-MS/MS. Instrumental LOD ranged from 0.26 to 0.65 ng/mL. Mean procedural recoveries at 1.3 ng/mL (LLOQ) ranged from 50% to 120% for 24 compounds. Recoveries were concentration independent, with the exception of femproporex, heroin and ecgonine methyl-ester (EME) for which the recovery decreased significantly at higher levels (13 and 52 ng/mL). RSD was20% for all compounds at all spiking levels. Ion suppression due to the matrix was20% for most compounds, and higher than 60% for EME and diethylpropion. Analysis was performed against a in-matrix standard curve. About 10% of the 2235 oral fluid samples collected from drivers on Brazilian Federal highways were positive (≥LOD) for at least one analyte investigated. Alone or in combination with other drugs, cocaine/metabolites were the analytes most detected in the samples (129; 5.8%), followed by amphetamines/metabolite (69; 3.1%), benzodiazepines (28; 1.2%), cannabinoids (23; 1.1%) and opioids (8; 0.4%). Detection of at least two PADs from different classes accounted for 9.3% of the 236 positive samples. Cocaine was found at higher levels in the samples (up to 1165 ng/mL). Preventive measures aimed at reducing the use of PADs by drivers in Brazil will certainly contribute to decrease the country's highway death rates.

Opiates, cocaine, and metabolites were quantified by liquid chromatography-mass spectrometry (LC-MS) in 284 urine specimens, collected thrice weekly, to monitor possible drug relapse in 15 pregnant heroin-dependent women. Opiates were detected in 149 urine specimens (52%) with limits of quantification (LOQ) of 10-50 microg/L. Morphine, morphine-3-glucuronide, and/or morphine-6-glucuronide were positive in 121 specimens; 6-acetylmorphine, a biomarker of heroin ingestion, was quantifiable in only 7. No heroin, 6-acetylcodeine, papaverine, or noscapine were detected. One hundred and sixty-five urine specimens (58%) from all 15 participants were positive for one or more cocaine analytes (LOQ 10-100 microg/L). Ecgonine methylester (EME) and/or benzoylecgonine were the major cocaine biomarkers in 142. Anhydroecgonine methylester, a biomarker of smoked cocaine, was positive in six; cocaethylene and/or ecgonine ethylester, biomarkers of cocaine and ethanol co-ingestion, were found in 25. At the current Substance Abuse Mental Health Services Administration cutoffs for total morphine (2000 microg/L), codeine (2000 microg/L), 6-acetylmorphine (10 microg/L), and benzoylecgonine (100 microg/L), 16 opiate- and 29 cocaine-positive specimens were identified. Considering 100 microg/L EME as an additional urinary cocaine biomarker would identify 51 more positive cocaine specimens. Of interest is the differential pattern of opiate and cocaine biomarkers observed after LC-MS as compared to gas chromatography-mass spectrometry analysis.

Understanding cocaine and metabolites urinary excretion following smoking is important for interpretation of urine test results in judicial, workplace and treatment settings. In National Institute on Drug Abuse approved studies on a secure research unit, six subjects smoked placebo, 10, 20, and 40 mg cocaine with a precise dose delivery device and six different subjects smoked 42 mg cocaine in a glass pipe. Urine specimens (n = 700) were collected for up to seven days and analyzed for cocaine (COC), benzoylecgonine (BE), ecgonine methylester (EME), m-hydroxybenzoylecgonine (mOHBE), p-hydroxybenzoylecgonine (pOHBE), norbenzoylecgonine (NBE), and ecgonine (EC) by gas chromatography–mass spectrometry. Results (mean ± SE) for the 40-mg precise delivery doses are as follows: COCBEEMEmOHBEpOHBENBEECCutoff(ng/mL)10201025252550Cmax(ng/mL)4085±23039196±15694638±1548222±69540±227614±347852±211Tmax(h)2.2±0.36.6±0.95.6±1.47.8±0.54.4±1.56.0±2.09.3 ±1.3Lastpositive(h)5510616455553280 Mean Cmax for all analytes linearly increased with increasing dose. Tmax was not dose-dependent. All metabolites were detected in some subjects within 2 h. EC concentrations were significantly higher after smoked cocaine in a precise delivery coil compared to a glass “crack” pipe.

Objectives Cocaine intoxication leads to over 500,000 emergency department visits annually in the United States and ethanol cointoxication occurs in 34% of those cases. Cardiotoxicity is an ominous complication of cocaine and cocaethylene overdose for which no specific antidote exists. Because infusion of lipid emulsion (Intralipid) can treat lipophilic local anesthetic toxicity and cocaine is an amphipathic local anesthetic, the authors tested whether lipid emulsion could attenuate cocaine cardiotoxicity in vivo. The effects of lipid emulsion were compared with the metabolically inert sulfobutylether-β-cyclodextrin (SBE-β-CD; Captisol) in an isolated heart model of cocaine and cocaethylene toxicity to determine if capture alone could exert similar benefit as lipid emulsion, which exhibits multimodal effects. The authors then tested if cocaine and cocaethylene, like bupivacaine, inhibit lipid-based metabolism in isolated cardiac mitochondria. Methods For whole animal experiments, Sprague-Dawley rats were anesthetized, instrumented, and pretreated with lipid emulsion followed by a continuous infusion of cocaine to assess time of onset of cocaine toxicity. For ex vivo experiments, rat hearts were placed onto a nonrecirculating Langendorff system perfused with Krebs-Henseleit solution. Heart rate, left ventricle maximum developed pressure (LVdevP), left ventricle diastolic pressure, maximum rate of contraction (+dP/dtmax), maximum rate of relaxation (–dP/dtmax), rate-pressure product (RPP = heart rate × LVdevP), and line pressure were monitored continuously during the experiment. A dose response to cocaine (10, 30, 50, and 100 μmol/L) and cocaethylene (10, 30, and 50 μmol/L) was generated in the absence or presence of either 0.25% lipid emulsion or SBE-β-CD. Substrate-specific rates of oxygen consumption were measured in interfibrillar cardiac mitochondria in the presence of cocaine, cocaethylene, ecgonine, and benzoylecgonine. Results Treatment with lipid emulsion delayed onset of hypotension (140 seconds vs. 279 seconds; p = 0.008) and asystole (369 seconds vs. 607 seconds; p = 0.02) in whole animals. Cocaine and cocaethylene induced dose-dependent decreases in RPP, +dP/dtmax, and –dP/dtmaxabs (p

This case discusses the pharmacodynamic interaction between cocaine and propranolol, resulting in hypertension and exaggerated cocaine toxicity.

Hepatocellular damage is thought to occur as a result of cytochrome P450-mediated oxidation of cocaine to norcocaine (NC), a precursor of the hepatotoxic nitrosonium ion. However, this damage occurs only in male mice, with females exhibiting minimal biochemical and histological signs of hepatocellular stress. The objective of this study was to determine the plasma time course and tissue disposition of cocaine and its metabolites to further investigate the role that metabolism may play in the gender difference observed. Male and female CF-1 mice were orally administered 20 mg/kg cocaine hydrochloride once daily for 7 days. Blood samples were withdrawn at various time points post-injection and analyzed for cocaine and its metabolites benzoylecgonine (BE), norcocaine, ecgonine methyl ester (EME), and ecgonine (E). In addition, tissue concentrations of cocaine and its metabolites were determined in liver, heart, brain, and kidney tissue. The results demonstrated that the plasma elimination half-life of cocaine is nearly three times longer in males versus females. Non-hepatotoxic hydrolysis metabolites BE, EME, and E were higher in female tissues while norcocaine was detected in tissues of male animals only. This study revealed that differences in cocaine pharmacokinetics and the resultant differences in the biodisposition of cocaine and its metabolites in tissues contribute to the mechanism of gender difference seen in cocaine hepatotoxicity.

Erythroxylum coca var. coca Lam. And Erythroxylum novogranatense var. novogranatense (Morris) Hieron (E.n. var. novogranatense) were grown in a controlled environment for 52 weeks to monitor the content of hygrine, tropinone, tropacocaine, methyl ecgonine, cocaine and cis- and trans-cinnamoylcocaine in seeds, plant parts, and organs during juvenile growth. Embryos and endosperms of var. coca contained cocaine while only embryos of var. novogranatense contained cocaine. Tropacocaine was present in hypocotyls, stems and roots of var. novogranatense, but not in var. coca. Trans-cinnamoylcocaine was the most abundant alkaloid in cotyledons (12 weeks after seeding) and leaves (24 weeks after seeding) in both var. coca and var. novogranatense. After 52 weeks of seeding growth, cocaine was the preeminent alkaloid in leaves of both taxons with a cocaine content of 0.66 percent in var. coca and 1.04 percent in var. novogranatense.

A series of 2beta-isopropyl ester analogs of cocaine (7-11) was synthesised and evaluated in an in vitro dopamine transporter (DAT) binding assays. Ecgonine HCl (5) was obtained from (-)-cocaine (1) by hydrolysis using 1 N HCl. Acid catalysed esterification of 5 using 2-propanol and HCl gas afforded 2beta-isopropyl ecgonine (6). Compounds 7-9 were obtained via esterification of the 3beta-hydroxyl group of 6 using the appropriate acid chloride. Compound 10 was obtained via selective hydrolysis and re-esterification of 7 using 2-propanol and HCl gas. Compound 11 was obtained by reduction of 9 using H(2)/Pd-C. Compounds 7, 10 and 11 showed high binding affinity to the DAT (as indicated from the inhibition of the binding of [(3)H]WIN 35,428 (3)) with IC(50) values (mean +/- S.E.M.) 208.5 +/- 9.5, 47.43 +/- 1.79 and 11.25 +/- 3.37 nM, respectively). Compound 7 is comparatively as active as cocaine, 10 is ca. fivefold more active than cocaine and 11 is ca. 20-fold more active than cocaine and even twice more active than the radioligand 3. Compound 11, like its methyl ester analog (2' aminococaine), exhibited the highest affinity to the DAT. These results, along with previous results, emphasise the importance of a hydrogen-bond donor group at the 2'-position of cocaine and its isopropyl ester analogs to enhance binding affinity to the DAT.

We have compared the pharmacokinetics of bolus dose cocaine administration with that of its three most important metabolites; norcocaine, ecgonine methylester, and benzoylecgonine and assessed whether kinetics are dose dependent at two equimolar doses equivalent to cocaine hydrochloride 2.5 and 5 mg kg respectively. Forty-nine male Sprague-Dawley rats were randomly divided into 8 groups to receive iv either high (14.7 umol kg ) (HI) or low (7.3 umol kg ) (LO) bolus doses of cocaine or one of its metabolites. Arterial blood samples for cocaine and metabolite analysis were taken repetitively over the next 3 h. Equimolar bolus doses of these congeners showed biexponential plasma concentration decay curves which were fitted to a two compartment model and subjected to noncompartmental analysis. The plasma concentration time profiles were significantly different for the HI and LO doses administered for each congener. The elimination half-lives of cocaine and norcocaine were similar (28–33 min), that for ecgonine methylester (60–71 min) was approximately twice this and for benzoylecgonine was 40–44 min. Cocaine clearance (155–158 ml/kg/min) was found to be in the range found in other rat studies. Ecgonine methylester clearance and benzoylecgonine clearance were found to be one quarter and one eighth of this value respectively. The pharmacokinetic profile of these congeners was not dose dependent when the two doses administered were compared.

In humans, the plasma enzyme butyrylcholinesterase, BChE (EC 3.1.1.8), mediates the in vivo plasma hydrolysis of cocaine to the pharmacologically inactive metabolite ecgonine methyl ester, EME. This enzyme has been purified from human plasma to investigate the potential as a treatment for cocaine intoxication. Cocaine (2.1 μg mL−1) was incubated in plasma with a BChE concentration in the normal range (3.02 μg mL−1) and in plasma with enhanced BChE concentrations of 9.14, 20.8 and 37.8 μg mL−1, respectively for time periods up to 120 min. Cocaine and the hydrolytic products, ecgonine methyl ester and ecgonine, were quantified simultaneously by gas chromatography-mass spectrometry (GC-MS). The enhancement of plasma BChE concentration resulted in a dramatic increase in the rate of hydrolysis of cocaine. There was a stoichimetric conversion of cocaine to the inactive hydrolysis product, ecgonine methyl ester. Accordingly, the half-life of cocaine in plasma decreased significantly with enhanced BChE concentration. At plasma BChE concentrations of 3.02, 9.14, 20.8 and 37.8 μg mL−1, half-life values of 116, 35.8, 21.4 and 9.0 min, respectively were observed. The marked reduction in cocaine half-life provides evidence supporting the potential therapeutic use of BChE for the treatment of cocaine intoxication. © 1998 John Wiley & Sons, Ltd.

The most common complications of cocaine ingestion are on the cardiovascular and central nervous systems and produce chest pain and generalized seizures. In humans, decreased levels of butyrylcholinesterase (BChE) (EC 3.1.1.8) have been associated with sustained effects of cocaine and life-threatening complications. Administration of purified human BChE has previously been demonstrated to protect against cocaine-associated cardiovascular toxicity in rats. A shift in the metabolism of cocaine as well as enhanced metabolism may be the underlying mechanism of the enzyme. Therefore, levels of the parent drug and four metabolites were determined in rat plasma after i.p. administration of a lethal cocaine dose, followed by i.v. administration of BChE. Plasma and brain concentrations of cocaine were lowered by 80% after BChE administration. Furthermore, the metabolic profile of cocaine in the plasma was altered. The concentration of ecgonine methylester was doubled although the concentration of ecgonine, a secondary metabolite of cocaine, was reduced. The level of benzoylecgonine was reduced by one-half while norcocaine was absent. Cocaine-associated effects upon the central nervous system were also shown to be reduced by administration of BChE to conscious rats. Furthermore, our studies in the cat have also shown that purified BChE shifts the metabolic profile of cocaine (1 mg/kg) to the pharmacologically inactive products ecgonine methylester and ecgonine. Pretreatment with BChE (0.27, 1.0, and 10.0 mg/kg) ameliorated the hypertensive effects of cocaine (1 mg/kg) by reducing the duration and the extent of BP elevation by 66%. Administration of the enzyme, 1 min after cessation of cocaine infusion, resulted in an immediate attenuation in the cocaine-induced broadening of the QRS complex. These results suggest that BChE could be an effective and rapid therapy for the treatment of life-threatening cocaine-induced cardiovascular effects in human while clearing the total body burden of cocaine.

Rodent animal models have been widely used for studying neurologic and toxicological events associated with cocaine abuse. It is known that the mouse is more susceptible to cocaine-induced hepatotoxicity (CIH) than the rat. However, the causes behind this species-dependent sensitivity to cocaine have not been elucidated. In this study, cocaine metabolism in the mouse and rat was characterized through LC-MS-based metabolomic analysis of urine samples and were further compared through calculating the relative abundance of individual cocaine metabolites. The results showed that the levels of benzoylecgonine, a major cocaine metabolite from ester hydrolysis, were comparable in the urine from the mice and rats treated with the same dose of cocaine. However, the levels of the cocaine metabolites from oxidative metabolism, such as N-hydroxybenzoylnorecgonine and hydroxybenzoylecgonine, differed dramatically between the two species, indicating species-dependent cocaine metabolism. Subsequent structural analysis through accurate mass analysis and LC-MS/MS fragmentation revealed that N-oxidation reactions, including N-demethylation and N-hydroxylation, are preferred metabolic routes in the mouse, while extensive aryl hydroxylation reactions occur in the rat. Through stable isotope tracing and in vitro enzyme reactions, a mouse-specific α-glucoside of N-hydroxybenzoylnorecgonine and a group of aryl hydroxy glucuronides high in the rat were identified and structurally elucidated. The differences in the in vivo oxidative metabolism of cocaine between the two rodent species were confirmed by the in vitro microsomal incubations. Chemical inhibition of P450 enzymes further revealed that different P450-mediated oxidative reactions in the ecgonine and benzoic acid moieties of cocaine contribute to the species-dependent biotransformation of cocaine.

The predominant cocaine metabolites were tested for central nervous system effects by intracerebroventricular (ICV) administration in rats. We found two types of responses: cocaine, norcocaine (NC), benzoylecgonine (BE), and benzoylnorecgonine (Nor BE) produced stimulatory effects, whereas ecgonine methyl ester (EME) and ecgonine (EC) resulted in no specific effect or sedation. A novel metabolite interaction was revealed when rats were pretreated with EME, which inhibited both analgesia and seizures by subsequently administered cocaine. Pretreatment with EC inhibited both cocaine and BE seizures and seizure-associated death. Direct injection of EME into the nucleus accumbens significantly suppressed systemic cocaine potentiation of intracranial electrical self-stimulation of the ventral tegmental area, whereas corticotropin releasing hormone injected ICV selectively potentiated BE-induced seizures and death. These results confirm multiple, metabolite-mediated activities in the central nervous system. Pharmacological interactions of the metabolites with each other and/or with neurohormones may help explain some of the pathophysiological effects seen in human chronic cocaine abuse.

The stability of nine illicit drugs and metabolites in influent wastewater at relevant conditions (20 °C and at pH 7.5) was evaluated during 26 h (in 1 h intervals) to assess whether significant in-sewer processes of degradation or formation occur. This assessment is important for the sewage epidemiology approach, which uses concentrations of illicit drugs and metabolites in influent wastewater to back-calculate amounts of these substances used by a community or population. The results of this study showed that amphetamine, methamphetamine, ecstasy and EDDP (metabolite of methadone) are stable in wastewater for 12 h and longer. For benzoylecgonine and methadone, a modest formation in the course of time was observed (0.2% per h and 0.4% per h, respectively), while for cocaine (40% degradation after 12 h), ecgonine methylester (20% degradation after 12 h), and 6-monoacetylmorphine (20% degradation after 12 h), a clear decrease in concentrations in time was seen. These findings suggest that for compounds with a significant stability issue, it is important to take their behavior into account when performing back-calculations in sewage epidemiology. However, this study also highlights the need of future research regarding transformation pathways of the individual compounds.

We investigated whether alcohol pretreatment would affect the disposition and metabolic pattern of intravenously (i.v.) administered cocaine in pigs. Six pigs (Group A) received alcohol (1 g/kg/day) and six pigs (control; Group D) received an equal volume of isocaloric dextrose 44% in water for 10 days via an intragastric tube. On day 11, arterial samples were taken for five hours following an intravenous administration of cocaine hydrochloride (4 mg/kg). Plasma concentrations of cocaine and its major metabolites were analyzed by HPLC method. Significant decrease in plasma half-life (10 +/- 1.2 vs. 18.7 +/- 1.4 min), and significant increases in apparent volume of distribution (73 +/- 6 vs. 51 +/- 31) and clearance (5.37 +/- 0.6 vs. 1.82 +/- 0.1 l/min) were seen in alcohol pretreated pigs as compared with control pigs (P0.05). Significant increases in plasma concentrations of benzoylecgonine (P0.05), and insignificant differences in ecgonine methyl ester and norcocaine levels were seen between the two groups. Neither ecgonine nor cocaethylene was detected in the blood samples. Our data show that alcohol administration for ten days accelerated the elimination of i.v. administered cocaine and altered its metabolic pattern in pigs.

Urinary excretion of ecgonine (EC) was compared to that of cocaine, benzoylecgonine, ecgonine methyl ester and minor metabolites, meta-hydroxybenzoylecgonine, para-hydroxybenzoylecgonine, and norbenzoylecgonine, following controlled administration of oral, intravenous, intranasal, and smoked cocaine. Urine EC concentrations peaked later than all other analytes and had longer detection times than the other minor metabolites. With a 50 ng/mL cutoff concentration and following low doses of 10 to 45 mg cocaine by multiple routes, detection times extended up to 98 h. Maximum concentrations (Cmax) were 6-14 mole % of those for benzoylecgonine, Cmax increased with dose, time to maximum concentration (Tmax) was independent of dose, and route of administration did not have a significant impact on Cmax or Tmax for metabolites. EC is an analyte to consider for identifying cocaine use due to its stability in urine and long detection times.

When cocaine is smoked, a pyrolytic product, methyl ecgonidine (anhydroecgonine methyl ester), is also consumed with the cocaine. The amount of methyl ecgonidine formed depends on the pyrolytic conditions and composition of the illicit cocaine. This procedure describes detection of cocaine and 10 metabolites--cocaethylene, nor-cocaine, nor-cocaethylene, methyl ecgonine, ethyl ecgonine, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine and ecgonine--in blood and urine. In addition, the detection of pyrolytic methyl ecgonidine and three metabolites--ecgonidine (anhydroecgonine), ethyl ecgonidine (anhydroecgonine ethyl ester) and nor-ecgonidine (nor-anhydroecgonine)--are included. The newly described metabolites, ethyl ecgonidine and nor-ecgonidine, were synthesized and characterized by gas chromatography-mass spectrometry (GC-MS). All 15 compounds were extracted from 3 mL of blood or urine by solid-phase extraction and identified by a GC-MS method. The overall recoveries were 49% for methyl ecgonine, 35% for ethyl ecgonine, 29% for ecgonine and more than 83% for all other drugs. The limits of detection were between 0.5 and 4.0 ng/mL except for ecgonine, which was 16 ng/mL. Linearity for each analyte was established and in all cases correlation coefficients were 0.9985-1.0000. The procedure was applied to examine the concentration profiles of analytes of interest in post-mortem (PM) blood and urine, and in urine collected from living individuals (LV). These specimens previously were shown to be positive for the cocaine metabolite, benzoylecgonine. Ecgonidine, the major metabolite of methyl ecgonidine, was present in 77% of PM and 88% of the LV specimens, indicating smoking as the major route of cocaine administration. The new pyrolytic metabolites, ethyl ecgonidine and nor-ecgonidine, were present in smaller amounts. The urine concentrations of nor-ecgonidine were 0-163 ng/mL in LV and 0-75 ng/mL in PM specimens. Ethyl ecgonidine was found only in PM urine at concentrations 0-39 ng/mL. Ethanol-related cocaine metabolites, ethyl ecgonine or cocaethylene, were present in 69% of PM and 53% of cocaine-positive LV specimens, implying alcohol consumption with cocaine use. The four major metabolites of cocaine--benzoylecgonine, ecgonine, nor-benzoylecgonine and methyl ecgonine--constituted approximately 88 and 97% of all metabolites in PM and LV specimens, respectively. The concentrations of nor-cocaine and nor-cocaethylene were consistently the lowest of all cocaine metabolites. At benzoylecgonine concentrations below 100 ng/mL, ecgonine was present at the highest concentrations. In 20 urine specimens, benzoylecgonine and ecgonine median concentrations (range) were 54 (0-47) and 418 ng/mL (95-684), respectively. Therefore, detection of ecgonine is advantageous when benzoylecgonine concentrations are below 100 ng/mL.

Butyrylcholinesterase is a major cocaine-metabolizing enzyme in humans and other primates, catalyzing hydrolysis to ecgonine methylester. Increasing butyrylcholinesterase activity may be a treatment for cocaine addiction. We evaluated the effect of 30-min pretreatment with horse-derived butyrylcholinesterase (5-15,000 U i.v.) or with the selective butyrylcholinesterase inhibitor cymserine (10 mg/kg i.v.) on the metabolism of cocaine (17 mg/kg i.p.) in anesthetized rats. Venous blood samples were collected for two hours after cocaine administration and later assayed for cocaine and metabolites by gas chromatography/mass spectroscopy. Whole brains were collected after the last blood sample and similarly assayed. Butyrylcholinesterase significantly increased plasma and brain ecgonine methylester levels and decreased cocaine plasma half-life from 26.2 min (saline) to 16.4 min (15,000 U). Butyrylcholinesterase had no significant effect on plasma or brain cocaine or benzoylecgonine levels. Cymserine had no effect on any variable. These findings suggest that butyrylcholinesterase treatment may have benefits in enhancing cocaine metabolism and in increasing levels of ecgonine methylester, which may have a protective action against cocaine.

Human monoclonal antibodies (mAbs) designed for immunotherapy have a high potential for avoiding the complications that may result from human immune system responses to the introduction of nonhuman mAbs into patients. This study presents a characterization of cocaine/antibody interactions that determine the binding properties of the novel human sequence mAb 2E2 using three-dimensional quantitative structure-activity relationship (3D-QSAR) methodology. We have experimentally determined the binding affinities of mAb 2E2 for cocaine and 38 cocaine analogues. The K(d) of mAb 2E2 for cocaine was 4 nM, indicating a high affinity. Also, mAb 2E2 displayed good cocaine specificity, as reflected in its 10-, 1500-, and 25000-fold lower binding affinities for the three physiologically relevant cocaine metabolites benzoylecgonine, ecgonine methyl ester, and ecgonine, respectively. 3D-QSAR models of cocaine binding were developed by comparative molecular similarity index analysis (CoMSIA). A model of high statistical quality was generated showing that cocaine binds to mAb 2E2 in a sterically restricted binding site that leaves the methyl group attached to the ring nitrogen of cocaine solvent-exposed. The methyl ester group of cocaine appears to engage in attractive van der Waals interactions with mAb 2E2, whereas the phenyl group contributes to the binding primarily via hydrophobic interactions. The model further indicated that an increase in partial positive charge near the nitrogen proton and methyl ester carbonyl group enhances binding affinity and that the ester oxygen likely forms an intermolecular hydrogen bond with mAb 2E2. Overall, the cocaine binding properties of mAb 2E2 support its clinical potential for development as a treatment of cocaine overdose and addiction.

Background: Concurrent use of cocaine and alcohol results in formation of a cocaine homolog and metabolite—cocaethylene. Methods: To characterize cocaethylene pharmacology, ten paid volunteer subjects were given deuterium-labeled (d 5 ) cocaine (0.3, 0.6, and 1.2 mg/kg and cocaine placebo) by a 15-min constant rate intravenous injection 1 h after a single oral dose of ethanol (1 g/kg) or ethanol and cocaine placebo using a double-blind, crossover design. Six of the same volunteers subsequently received a 1.2 mg/kg dose of cocaine alone. A small (7.5 mg) nonpharmacologically active dose of deuterium-labeled cocaethylene-d 3 was concurrently administered with the cocaine to enable calculation of absolute cocaethylene formation and clearance. Plasma and urine cocaine, cocaethylene, and benzoylecgonine concentrations, physiologic and subjective effects were measured. Results: When co-administered with ethanol, 17±6% (mean±S.D.) of the cocaine was converted to cocaethylene. Cocaethylene peak plasma concentrations and AUC increased proportionally to the cocaine dose. Ethanol ingestion prior to cocaine administration decreased urine benzoylecgonine levels by 48% and increased urinary cocaethylene and ecgonine ethyl ester levels. Subjects liked and experienced more total intoxication after the combination of cocaine and ethanol than after either drug alone. Conclusions: In the presence of ethanol, the altered biotransformation of cocaine resulted in 17% of an intravenous cocaine dose being converted to cocaethylene and relatively lower urinary concentrations of benzoylecgonine.

Recent reports have shown that anti-cocaine catalytic monoclonal antibody 15A10 reduces the toxic effect of cocaine by increasing its breakdown to systemically inert products ecgonine methylester and benzoic acid. This study reports the microencapsulation of antibody 15A10 using biodegradable poly (lactic-glycolic) acid (PLGA) by double emulsion technique. Formulation parameters such as protein loading, polymer molecular weight and the presence of zinc carbonate were studied for their effects on in-vitro release of antibody from microspheres. The initial burst release was decreased by the reduction of the protein (as % of total ingredients) in the formulation. Although changing the polymer molecular weight did not cause a reduction in initial burst release, it was effective in improving the release rate. The inclusion of zinc carbonate in microsphere preparation resulted in increase in initial burst release. An in-vivo study in mice revealed the presence of antibody in blood up to ten days following subcutaneous injections. These data demonstrate a potential for a sustained-release formulation of monoclonal antibody 15A10 for treatment of cocaine addiction.

Cocaine metabolism has been studied previously with respect to the formation of predominant hydrolytic or hepatotoxic metabolites via oxidative pathways. In the present study, cocaine and eight of its metabolites (norcocaine, ecgonine methyl ester, benzoylecgonine, benzoylnorecgonine, 3-hydroxy-benzoylecgonine, cocaethylene, norcocaethylene, and ecgonine ethyl ester) were incubated with microsomes from rat liver, kidney, lung, and brain. Qualitative analysis of the metabolites produced was performed using solid phase extraction (SPE), trimethylsilylation, and GC/MS. It was found that the metabolites with a free carboxylic group (e.g., benzoylecgonine) were not further oxidized by microsomal enzymes and their presence in urine or blood may therefore be due to hydrolysis of the respective alkylated entities. Although microsomes from all organs exhibited oxidative metabolism, significant differences were noted. Kidney microsomes produced essentially the same results as liver, but aryl hydroxylated metabolites were not found in incubations with lung and brain microsomes. N-Hydroxy-norcocaine was found only in traces with brain microsomes. It appears that cocaine is converted to N-hydroxy-norcocaine (which is the precursor of toxic metabolites) not only in the liver but also in other organs of rat. This might be relevant in the development of lung toxicity observed in smokers of cocaine ("crack").

Tobacco smoking and cocaine use often co-occurs and the frequency of smoking has been positively correlated with the likelihood of cocaine use. In addition, nicotine pretreatment has been shown to increase the rate of cocaine self-administration in rats and to enhance cue-induced cocaine craving in humans. The present study was conducted to investigate whether nicotine pretreatment via a transdermal patch alters the behavioral, physiological, and pharmacokinetic effects of an acute dose of cocaine in nondependent human volunteers. Seven male tobacco smokers who used cocaine occasionally provided informed consent and participated in this placebo-controlled, four-visit study. Following pretreatment with a transdermal nicotine patch (placebo, 14 mg), subjects were challenged with an acute dose of intranasal cocaine (placebo, 0.9 mg/kg). Nicotine pretreatment attenuated cocaine-induced increases in reports of "high" and "stimulated" and increased the latency to detect cocaine effects and cocaine-induced euphoria. Nicotine did not alter cocaine's effects on heart rate, skin temperature, and blood pressure or plasma cocaine, benzoylecgonine (BE), or ecgonine methylester (EME) concentrations. Our findings indicate that nicotine pretreatment alters some of the positive subjective effects of cocaine in humans without affecting cocaine's effects on physiologic responses or pharmacokinetic profiles.

The in vitro stability of cocaine (COC) was monitored in fresh whole blood and plasma stabilized with potassium fluoride (0.25%) for as long as 15 days. The samples were stored at 4 degreesC, 20 degreesC and 40 degreesC. Additionally, fresh plasma samples containing either benzoylecgonine (BZE), ecgonine methyl ester (EME) or ecgonine (ECG) were stored at 4 degreesC and 20 degreesC. Data were established using subsequent solid-phase extraction procedures and high-performance liquid chromatography coupled to atmospheric pressure ionization mass spectrometry for isolation and quantitation of COC, BZE, EME, and ECG. COC, BZE, and EME concentrations decreased with increasing storage temperature and time after an apparent first-order reaction kinetic. Only ECG appeared to be stable at storage temperatures as high as 20 degreesC for the entire observation period. At 40 degreesC, the amount of ECG produced from hydrolysis of COC still totalled 80% of the initial COC concentration. Hydrolysis of COC to EME occurred more rapidly in plasma than in blood. The dynamic degradation profiles obtained were dependent on the storage temperature. The conversion of COC to BZE, EME, and ECG appeared to be stoichiometric at all time intervals at storage temperatures of 4 degreesC and 20 degreesC. The presence of any hydrolysis product of COC in blood or plasma constitutes confirmatory evidence of COC incorporation, and determination of ECG seems most promising even in samples stored under unfavorable conditions.

Rationale: The importance of genetic factors in the development of alcoholism has been demonstrated repeatedly. However, the impact of a family history of alcoholism on the development of other drug use has been less thoroughly studied. Objective: The present study was conducted to investigate whether individuals with a positive family history of alcoholism (FHP) differ from individuals without a history (FHN) in their pharmacokinetic profile, subjective and physiological response to an acute intranasal dose of cocaine (0.9 mg/kg). Methods: Nine FHP and nine FHN male occasional cocaine users provided informed consent and participated in this double-blind, placebo-controlled, two-visit study. Responses to cocaine were assessed via a joystick device, the Addiction Research Center Inventory, visual analog scales and heart rate. Plasma concentrations of cocaine and its metabolites, benzoylecgonine and ecgonine methylester also were measured. Results: There were no significant differences between FHP and FHN subjects in subjective reports of intoxication, physiologic responses or plasma cocaine and benzoylecgonine concentrations following cocaine administration. Plasma levels of the cocaine metabolite ecgonine methylester were significantly higher in FHP subjects from 50 to 120 min post-cocaine administration compared to FHN subjects. Conclusions: Our findings indicate that family history of alcoholism does not appear to influence the behavioral and physiological responses to acute cocaine administration, but that some aspects of cocaine metabolism may be different between the two groups.

The aim of this study was to determine the effect of chronic cocaine infusion on urine cocaine, ecgonine methylester and benzoylecgonine concentrations to establish if they varied with dose and duration of cocaine administration. Male rats were continuously infused with cocaine at either 6 or 18 mg kg−1 daily for 13 days. Three urine samples taken over the course of the infusion period showed that cocaine, ecgonine methylester and benzoylecgonine concentrations varied with the dose administered and the duration of administration. Cocaine, ecgonine methylester and benzoylecgonine concentrations were 2–3 times greater in the high-dose group than the low-dose group at each sampling time point. These decreased, respectively, from 7.0 ± 1.1, 26.7 ± 4.5 and 29.5 ± 5.4 μg mL−1 to 2.5 ± 0.5, 10.5 ± 1.8 and 11.8 ± 1.5 μg mL−1 in the high-dose group and from 1.0 ± 0.2, 7.8 ± 1.5 and 6.3 ± 0.1 μg mL−1 to 0.5 ± 0.1, 4.0 ± 0.6 and 3.1 ± 0.4 μg mL−1 in the low-dose group (P < 0.05) over the infusion period. We also studied the pharmacokinetic and metabolic profile of an intravenous bolus dose of 2.5 mg kg−1 cocaine hydrochloride after a similar cocaine infusion in rats. Cocaine pharmacokinetics and the profile of ecgonine methylester, benzoylecgonine and norcocaine were no different from rats chronically infused with saline for the same period. Altered cocaine metabolism could not explain the effect of the duration of cocaine infusion on altered metabolite concentrations in urine. Ecgonine methylester/benzoylecgonine urine concentration ratios did not alter with duration of infusion (1.2 ± 0.2 and 1.1 ± 0.2 in the high-dose group at the first and last time point) and were not affected by the dose of cocaine (1.3 ± 0.6 and 1.2 ± 0.1 at corresponding times in the low-dose group (P > 0.05)). We conclude that chronic cocaine infusion does not alter cocaine metabolism. This was not reflected by absolute cocaine metabolite urine concentrations, which varied with time, but was represented by urine ecgonine methyl ester/benzoylecgonine concentration ratios.

Hot plate testing of rats was performed to determine the optimal analgesic doses of intracerebroventricularly (ICV) administered cocaine, significant cocaine metabolites, and selected structurally similar molecules. Optimal, (subseizure) analgesic doses for cocaine and selected cocaine analogues were (in microM): cocaine, 0.37; cocaethylene, 0.09; benzoylecgonine, 0.35; norcocaine, 0.43; and ecgonine, 2.1. Ecgonine methyl ester was not analgesic at < or = 3.7 microM. These results, in conjunction with findings on other structurally similar molecules suggest that, of the molecules tested, (a) a hydrophobic group at the C-3 attached carbon is critical for analgesia; (b) a hydrophobic C-2 ester group can enhance analgesic activity (e.g., cocaethylene); (c) the N-methyl position is minimally important for analgesia; and (d) isomeric configurational changes can influence analgesia.

The hydrolytic metabolism of cocaine into benzoylecgonine, ecgonine methyl ester, and ecgonine was studied in the human hepatoma cell line Hep-G2 and in the nontumorigenic fetal hepatic cell line WRL-68. Also, the toxicological response of these cells to cocaine was compared to previously published results obtained with perfused liver cells and in vivo systems. Our experiments indicated that Hep-G2 appear to have similar metabolic and toxicological patterns to in vivo and perfused cell systems. The WRL-68 tissue culture system was found to be less similar. These results suggest Hep-G2 cells can be utilized to study cocaine metabolism and toxicology, and possibly in studies involving other xenobiotic compounds.

Cocaine, a naturally occurring alkaloid, is known to affect the immune system. The present authors have demonstrated that peritoneal macrophages, isolated from mice injected with cocaine, have an increased capacity for the production of reactive oxygen intermediates (ROI) and a decreased capacity for the production of reactive nitrogen intermediates (RNI). The present studies were done to determine the effects of certain cocaine metabolites on the induction of ROI and RNI by peritoneal macrophages. C57BL/6 mice were injected i.p. with either saline or 5 mg/kg of one of the following: cocaine, norcocaine, benzoylecgonine, ecgonine methyl ester HCl or ecgonine HCl. The ROI were measured using a chemiluminescence assay and the RNI were measured as nitrite secretion following exposure of isolated M phi to interferon gamma and LPS. Isolated peritoneal M phi from mice injected i.p. with cocaine, norcocaine and benzoylecgonine exhibited an increase in the production of ROI and a concomitant decrease in the production of RNI. However, injections of either ecgonine methylester HCl or ecgonine HCl had no effect on the induction of either ROI or RNI by murine peritoneal M phi. The cocaine metabolites, norcocaine and benzoylecgonine, have been reported to cause hepatic and/or cerebral toxicity. The present study also demonstrated that injection of these metabolites in vivo, also altered M phi functions which were measured in vitro.

We studied the incorporation rates of cocaine and its major metabolites, benzoylecgonine (BE) and ecgonine methylester (EME), into hair from blood. It was demonstrated in our previous report (Nakahara et al. 1992a) that the incorporation rate of cocaine from blood into hair was much higher than those of BE and EME following administration of cocaine. In this study, the incorporation rates of these drugs into rat hair were investigated following independent administration of BE and EME at 10 mg/kg per day for 5 days, and co-administration of cocaine, BE-d3 and EME-d3 at 3, 0.5 and 0.5 mg/kg per day for 10 days. When BE and EME were administered to rats independently, levels of both these drugs were hardly detectable or quite low in hair, though their AUCs in plasma were very high. On co-administration of cocaine, BE-d3 and EME-d3, the deuterium labeled metabolites in hair were negligibly lower in comparison with the unlabeled ones and in particular the concentration of BE in rat hair was significantly higher than that of BE-d3 although the AUC of BE-d3 was higher than that of BE. It was concluded that the incorporation rates of BE and EME into hair were very low in comparison with that of cocaine and most of the BE detected in hair would be a hydrolytic product derived from cocaine in hair matrix after incorporation.

To the Editor: We have previously reported a method for the analysis of ecgonine together with other polar cocaine metabolites in postmortem blood (1). The findings in that manuscript and our subsequent experience (2,3) have shown that ecgonine is consistently present in postmortem blood specimens; its origins, however, are usually open to debate and discussion (4-6). Application of the method in carefully preserved whole blood specimens from cocaine addicts reporting to an emergency room have also shown ecgonine to be consistently present (3). This suggests that at least in high doses, often chronic cocaine administration, ecgonine will be present during life also. Our intent in the work that led to our original publication was to investigate the utility of ecgonine as a potentially longerlived marker than benzoylecgonine of past cocaine use. All our findings to date however have indicated that the detection windows for benzoylecgonine and ecgonine are approximately the same, at least at our limit of detection (10 ng/mL) (1-3). Other workers have documented the instability of both cocaine and its monoester metabolites, benzoylecgonine and ecgonine methyl ester, during in vitro storage of blood (4,5). It is likely that at least some of the loss of these primary metabolites is via a secondary de-esterification to ecgonine. Consequently, the true value of an assay for ecgonine may be its ability to detect what is likely the final in vitro degradation product following cocaine use. Ecgonine would thus be the analyte of choice in cases in which a specimen has been collected without an enzyme-inhibiting preservative or at temperatures known to be inadequate for the preservation of cocaine or its primary monoester metabolites (4,5). We report a case in which a defendant in a homicide case relied in part on his recent cocaine use as a component of his defense. The specimen was collected in a tube containing EDTA, a chelating agent with no significant enzyme-inhibiting properties, and was stored for approximately 18 months, some of which time included at least some period of room-temperature storage. During that time it was subjected to an analysis for a variety of drugs including cocaine, benzoylecgonine, cocaethylene, and ecgonine methyl ester, all of which proved negative. Following this lengthy storage, the specimen was submitted to us for analysis for the presence of ecgonine, according to the method described in our earlier work (1,2). The testing disclosed the presecnce of ecgoinine in the blood at a concentration of 557 ng/mL. The specimen was negative for other cocaine-related compounds and metabolites including cocaine, benzoylecgonine, ecgonine methyl ester, ecgonine ethyl ester, cocaethylene, and norcocaine. The assay used was a selected ion-monitoring assay; however, the presence of ecgonine was confirmed by full scan mass spectrometry of its derivative. To our satisfaction, this demonstrated that the subject, sometime prior to the collection of the blood specimen, had in fact ingested cocaine, which may have been corroborative of his defense. We did not attempt to estimate the dose, the time since ingestion, or the likely effects of the ingestion on the subject proximate to the time of the specimen collection. The complete absence of other cocaine metabolites suggests that the ecognine was at least partly an artifact of the inadequate preservation of the specimen. Based on this experience, we strongly suggest that ecognine analysis may be an appropriate test to demonstrate cocaine use when the provenance of the specimen is unknown, when preservation has been inadequate, or when other cocaine metabolites have been shown to be undetectable.

To determine whether changes in pituitary responsiveness might account for the lack of corticotropin (ACTH) stimulation following 6 consecutive days of continuous cocaine administration (5 or 25 mg/kg/day, via osmotic minipumps), the hormonal response of vehicle- or cocaine-pretreated male rats was compared. Intravenous injections of synthetic corticotropin-releasing factor (CRF) (0.2, 1, or 5 μg/Kg) elicited dose-dependent increases in ACTH secretion irrespective of whether rats had been previously exposed to cocaine or not. Similarly, in both vehicle- and cocaine-pretreated rats ACTH response to acute injections of the drug was identical, indicating that pituitary corticotrophs remained responsive following continuous administration of cocaine. To determine and compare plasma concentrations of cocaine and its metabolites after continuous or acute administration of the drug, pharmacokinetics analysis of concentration vs. time was ascertained. Circulating concentrations of cocaine from rats continuously exposed to the drug were relatively low throughout the 6 days of exposure. In contrast, intravenous injections of cocaine produced peak concentrations of the drug that were significantly higher than those measured during continuous cocaine infusion. Such peak concentrations in cocaine correlated with marked increases in plasma ACTH levels. Plasma concentrations of the metabolites benzoylecgonine and methyl ester ecgonine followed a pharmacokinetic clearance similar to that of the parent compound, with low concentrations detected during continuous exposure whereas high concentrations were observed following intravenous injections of the drug. Our results suggest two nonmutually exclusive conclusions. First, there may be a critical threshold of cocaine plasma concentrations (as indicated by our results as being over 800 ng/ml) that are necessary for activation of the hypothalamic-pituitary-adrenal axis to occur. Second, stimulation of ACTH secretion may only occur during rapid systemic increases in drug concentrations.

Cocaine is a local anesthetic containing a tertiary amine structure derived from the amino alcohol base, ecgonine. Ecgonine is methylated and esterified with benzoic acid to form cocaine or benzoylmethylecgonine (1). Cocaine was first isolated in 1857 and its medical uses have included application as a topical anesthetic in nasal surgery. The drug appears to be metabolized in humans and other mammals by means of plasma and tissue esterases (2). The major urinary metabolites appear to be the water soluble substances, benzoylecgonine and ecgoninemethy-lester (3). Cocaine can also be N-demethylated in the body and the resulting metabolite, norcocaine, has been reported to have good biological activity in a variety of systems (4,5,6).

The effects of acid (NH4Cl, 60 mg/kg) or alkaline (NaHCO3, 400 mg/kg) pretreatment on tissue disposition and urinary excretion of (3H)-cocaine and its metabolites were assessed in rats. Animals were decapitated 4 hr after 1.9 microCi in 40 mg/kg cocaine (Cl-) in experiments on the biological disposition and the plasma, brain, and liver were assayed. In experiments on urinary excretion, 10 microCi were given and urine was assayed after 18, 24, and 48 hr. Pretreatment with NH4Cl produced a decrease of rates of cocaine + norcocaine in the brain as well as of the polar metabolites (benzoylecgonine, ecgonine benzoylnorecgonine). Forty-eight hours after injection of (3H)-cocaine, NH4Cl produced an increase of the rates of cocaine and its metabolites in the urine. Pretreatment with NaHCO3 causes a decrease of total radioactivity (cocaine + metabolites) in the plasma and in the liver, as well as a decrease of the levels of cocaine + norcocaine in the liver, as well as an increase of the rate of polar metabolites that are excreted in the urine. These data suggest that the decrease of pH after acid treatment produces ionization of basic molecules; these do not penetrate as well in the tissues for which they have no affinity. As a consequence, the urinary excretion is more important. It is possible that alkaline treatment accelerates the excretion of cocaine and its metabolites because it increases diuresis.

1. 1. The activity of some cocaine metabolites (norcocaine, benzoylnorecgonine and ecgonine) and of a stereoisomer of cocaine, pseudococaine, was examined on the high affinity uptake of dopamine into isolated striatal synaptosomes. 2. 2. High affinity uptake of dopamine (defined as uptake at 0.5 μM dopamine concentration) into synaptosomal fractions isolated by gradient centrifugation from rat striatum was inhibited competitively by norcocaine and pseudococaine, as well as by cocaine, with KI 50S of 8 x 10 −5 M, 5 x 10 −4 M and 7 x 10 −6 M, respectively. 3. 3. Benzoylnorecgonine and ecgonine methylester inhibited dopamine uptake about 50% at 5 x 10 −3 or higher. Benzoylecgonine and ecgonine showed no inhibition at these concentrations but the benzoyl-derivatives inhibited dopamine uptake slightly at concentrations of 10 −7 or lower.

1. The metabolism of [3H]norcocaine, N-hydroxy[3H]norcocaine and cocaine-N-oxide has been investigated in rats after i.v. injection. 2. The biological t 1/2 of norcocaine (dose 2 mg/kg i.v.) in plasma, liver and brain were 0.4, 1.6, 0.5 h, respectively and the compound was not detectable in the central nervous system 6 h after injection. The % dose of norcocaine excreted unchanged in urine and faeces in 96 h were 0.7 and 1.0, respectively. Benzoylnorecgonine, norecgonine, norecgonine methyl ester and an unidentified compound were excreted in urine. 3. The biological t 1/2 of N-hydroxynorcocaine (5 mg/kg i.v.) in brain and plasma were 0.3, 1.6 h respectively and only 1.3 and 1.6% of dose were excreted unchanged in urine and faeces in 96 h. N-Hydroxybenzoylnorecgonine and N-hydroxynorecgonine methyl ester were the major urinary metabolites. N-hydroxynorcocaine was not metabolized to norcocaine in vitro by liver microsomes. Doses of greater than 7.5 mg/kg i.v. resulted in death of rats by cardiorespiratory arrest. 4. Cocaine-N-oxide (50 mg/kg i.v.) yielded ecgonine-N-oxide methyl ester as its major metabolite; other minor metabolites were cocaine (0.5%), norcocaine (1%), benzoylecgonine, ecgonine, ecgonine-N-oxide, along with minor amounts of unmetabolized compound. Lethality of cocaine-N-oxide (100 mg/kg i.v.) was possibly due to metabolism to norcocaine and cocaine.

Structure-activity relationships were determined for cocaine congeners in counteracting the depolarization induced by the action of veratridine on voltage-dependent sodium channels of synaptoneurosomes from mouse brain cortex, and their potency was compared to those determined previously on Na+ uptake and batrachotoxinin binding. Cocaine, norcocaine, (+)-pseudococaine, (-)-pseudococaine, (+)-neopseudococaine, benzoyltropine, benzoylpseudotropine, ecgonine methylester, atropine, WIN-35,428, WIN-35,140, WIN-35,065-3, WIN-35,004, and procaine were tested for their potency in inhibiting depolarization as measured by the distribution of the lypophilic cation [3H]triphenylmethylphosphonium across the membrane. All of the tested compounds inhibited the veratridine-induced depolarization in a competitive manner. The structure-activity relationships were similar to those for inhibition of 22Na+ uptake in mouse brain homogenates, and the potency of these local anesthetics in inhibiting veratridine-induced uptake of [3H]triphenylmethylphosphonium correlated well with their potency in inhibiting [3H]batrachotoxinin A 20-α-benzoate binding in mouse brain synaptosomes.

The present study investigated the actions of cocaine and some of its metabolites and analogs upon the synaptosomal (P2) synthesis and release of dopamine appearing from [14C]phenylalanine. Also examined was the influence of reserpine upon these actions of cocaine. The P2 preparations from the rat caudate nucleus were incubated with the drugs for [14C]phenylalanine and, after filtration, the particulates and medium fractions were analyzed for [14C]dopamine and [14C]phenylalanine. Labelled dopamine in the medium was taken as a measure of its release by any addition of drug. Cocaine, norcocaine and the synthetic cocaine analogs WIN 35428 and WIN 35-065(2) each stimulated both the total (medium plus particulates) formation and the release of dopamine, while benzoylecgonine and ecgonine were without effect. Reserpine inhibited the synthesis and enhanced the release of dopamine. An addition of reserpine blocked the stimulating effect of cocaine, norcocaine, WIN 35428 and WIN 35-065(2) on synthesis and furthermore, these drugs had, in the same experiments, inhibitory effects on the synthesis, additive to the reserpine-induced inhibition. Benzoylecgonine and ecgonine were only weakly inhibitory in the presence of reserpine. Stimulation, rather than additive inhibition, by cocaine was observed in the presence of exogenous dopamine, and amphetamine increased the synthesis in the presence of either reserpine or added dopamine. Pretreatment of rats with reserpine also blocked the stimulation of synthesis by cocaine and WIN 35428. The uptake of labelled substrate was not affected by addition of drug, or by the pretreatment.

Analyzing the fators of “Cocaineinactivation” by rabbit's liver the following results were obtained. (1) By the biological estimation of cocaine it was demonstrated that the liver extract of rabbits could inactivate cocaine and its important mechanism was suggested to be destruction. (2) Its destructive products, methyl alcohol, bezoic acid and ecgonine, were chemically demonstrated. (3) The quantitative micro-determination of benzoic acid was testified to be albe to measure benzoic acid derived from cocaine by rabbitt's liver extract. (4) Using this method, it could be proved that the production of benzoic acid was enzymatically performed. (5) The destructive ability was compared among the tissues of rabbits and the livers of several animals, and the liver of rabbits showed the most active destruction.

The effect of ecgonine, tropine, tropinone and some of their derivatives, tropane, N-methylpyrrolidine, and N-methylpiperidine on impulse summation in the CNS, the conditioned avoidance reflex, antagonism with hexobarbital, synergism with cocaine, and also their toxicity (LD50) were investigated experimentally. Benzoylecgonine, the methyl ester of ecgonine, ecgonine itself, tropine, pseudotropine, carbomethoxytropinone, tropinone, tropane, N-methylpyrrolidine, and N-methylpiperidine were shown to have a definite stimulating effect on the activity of the CNS and, in this respect, are similar to cocaine. It is concluded that compounds whose molecules contain the structure of tropane or one of its fragments have a central stimulant effect and that the stimulant action of cocaine on the CNS may be associated with the tropane moiety of its molecule.

Monkeys were chronically treated with cocaine, 1 mg/kg subcutaneously twice daily for the first week and four times daily for the subsequent 21 weeks, followed by the same dose of [3H] cocaine injected intravenously. The t12 values of cocaine in the plasma, cerebrospinal fluid, liver, kidney and heart of acutely and chronically treated monkeys were 1.32, 0.85; 0.91, 0.75; 1.60, 1.29; 0.90, 0.91; and 1.02, 0.91 h, respectively. In areas of the central nervous system (CNS) the values for the acute and chronic group ranged between 0.75 – 0.90 and 0.62 – 0.84 h, respectively. With the exception of the temporal cortex, cerebellum and caudate nucleus, the t12 were not significantly different in the two groups. Norcocaine, benzoylnorecgonine, benzoylecgonine, and minor amounts of ecgonine were the metabolites of cocaine in the brain of monkeys. Significant amounts of total radioactivity due to benoylnorecgonine and benzoylecgonine persisted in the CNS of chronically treated monkeys. Norcocaine constituted approximately 3, 21, 8, and 6% of the values of cocaine in the CNS areas in the acutely treated monkeys, and 14, 13, 14, and 16% in the chronically treated ones, 0.25, 0.5, 1 and 2 h after cocaine injection. A biphasic pattern of peak levels in the plasma and bile of chronically treated monkeys indicated enterohepatic circulation of cocaine. Brain to plasma and CSF to plasma ratios of cocaine were higher in the chronic than in the acute group. The amounts of cocaine excreted in urine and feces as a percentage of dose were 0.23 – 5.1 and 0.1 – 0.23 in the acute group, 0.54 – 7.0 and 0.1 – 0.14 in the chronic group. Major excretion of radioactivity occurred in urine within 24 h and the mean values of the total radioactivity in urine and feces (96 h) in the acute and chronic groups were 60.4 and 63.2% of the dose, respectively. Norcocaine, benzoylnorecgonine, benzoylecgonine, ecgonine methyl ester, ecgonine, and unidentified compounds were the urinary metabolites of cocaine in the two groups. The percentage of benzoylecgonine was higher, that of benzoylnorecgonine lower, in the chronic than in the acute group and the values of other metabolites were not markedly different in the two groups. Data support the postulate that chronic treatment of monkeys with cocaine does not produce dispositional tolerance.

1. Beagle dogs were chronically treated with cocaine, 5 mg/kg subcutaneously twice daily for 6 weeks, followed by same dose of [3H]cocaine given intravenously. 2. The t1/2 values of cocaine in plasma, liver, spleen and heart, in acutely and chronically treated dogs, were: 1-2, 1-1; 2-2, 1-8; 1-8, 1-3; 2-0, 1-2 h, respectively. In both groups, cocaine disappeared from all areas of the central nervous system 12-24 h after injection but significant amounts of radioactivity due to benzoylnorecgonine and benzoylecgonine persisted in the CNS even 1 week after administration of cocaine. Brain-to-plasma ratios of cocaine were lower in chronically-treated than in acutely-treated dogs 2 and 4 h after injection. 3. Norcocaine, benzoylnorecgonine, benzoylecgonine and ecgonine were metabolites of cocaine in dog brain in both groups. Norcocaine and benzoylnorecgonine were present in higher amounts in brains of chronically treated dogs. Rate of disappearance of norcocaine was similar to cocaine in both groups. 4. The amounts of cocaine excreted in urine and faeces as percentage of dose were 0-9-5-0, 1-1-6 in the acute and 2-2-3-3 and 0-2-0-3 in the chronically treated dogs. Major excretion of radiactivity occurred in urine within 24 h in both groups. Total radioactivity (65% of dose) in urine plus faeces was similar in both groups. 5. Norcocaine, benzoylnorecgonine, benzoylecgonine, ecgonine, norecgonine, ecgonine methyl ester and unidentified compounds were urinary metabolites of cocaine in both groups. Benzoylnorecgonine and ecgonine were excreted in higher amounts and benzoylecgonine and norecgonine in lower amounts in the acute than in the chronically treated dogs. 6. The possible role of persistence of benzoylnorecgonine and benzoylecgonine (which possessed potent stimulant activity intracisternally) in the CNS is discussed.

To assess the role that action on sodium channels may play in the physiological effects of cocaine and to obtain information on the structure-activity relationships of this action, cocaine, norcocaine, N-allynorcocaine, (+)-pseudococaine, (-)-pseudococaine, (+/-)-allococaine, (+/-)-allopseudococaine, ecgonine, ecgonine methyl ester, O-benzoylecgonine and atropine were tested for their effects on sodium channels. The method employed was a sodium channel specific equilibrium 22Na+ uptake assay with rat brain membrane homogenates. All of the compounds listed with the exception of the ecgonines were found to be single affinity competitive inhibitors of veratridine activation of sodium channels. Ecgonine showed no inhibition at concentrations as high as 10(-3) M while ecgonine methyl ester and O-benzoylecgonine showed inhibition only at very high concentrations. The order of inhibition potencies was found to (+)-pseudococaine congruent to norcocaine congruent to N-allynorcocaine greater than cocaine greater than (-)-pseudococaine congruent to (+/-)-allopseudococaine greater than (+/-)-allococaine greater than atropine greater than O-benzoylecgonine greater than ecgonine methyl ester greater than ecgonine. This ordering of potencies is in good agreement with published reports of the local anesthetic potencies of these agents.

Antisera which react with the major metabolites of cocaine have been prepared in rabbits and a hemagglutination-inhibition (HI) test which detects these metabolites in urine or serum in concentrations of 1 ng/ml is described. A comparison of this test with alternate detection procedures shows excellent agreement at comparable sensitivity levels. HI tests on simian and human biological specimens suggest that the use of cocaine remains detectable for at least 3 days after administration of a minimal pharmacologically active dose. Combination of physical separation of drug metabolites with immunoassay procedures indicates that benzoylecgonine and ecgonine are the immunoreactive cocaine metabolites found in human urine. While it was possible to apply the HI test at maximal sensitivity to human sera and to murine or simian urine specimens, interference was encountered with some human urine specimens. Preliminary data suggest that by reducing the sensitivity of the test to a threshold of 200 ng/ml this interference can be overcome.

A simple and economical radioimmunoassay for benzoylecgonine in blood or urine is described. Haemolysis, decomposition, common anticoagulants and sodium fluoride do not affect the results. A commercially available antiserum is used at a dilution of 1:600. The tracer is radioiodinated p-hydroxybenzoylecgonine. It is prepared by reacting p-acetoxybenzoic anhydride with methyl ecgonine followed by mild hydrolysis and radioiodination by the Iodo-gen method. The product is purified on a disposable silica cartridge and has a specific activity of about 30 TBq mmol−1. Polyethylene glycol is used to separate the bound and free fractions in the assay. The range of the dose-response curve is 0–400 ng ml−1 benzoylecgonine. The assay is largely specific for benzoylecgonine. Cocaine, ecgonine, methylecgonine and cinnamoyl cocaine have slight or negligible cross-reactivities. The inter-assay coefficient of variation is 7·5% and the recovery of benzoylecgonine from ‘spiked’ blood is 103%. The ‘cut-off’ is 20 ng ml−1 benzoylecgonine for both blood and urine. Radioimmunoassay and high-performance liquid chromatography results agree well.

The hydrolysis of cocaine and its N-demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.