Ecgonine Methyl Ester Research Articles

Ecgonine methyl ester protects against cocaine lethality in mice.

Plasma cholinesterase (PChE) metabolizes cocaine to ecgonine methyl ester (EME). Limited data demonstrate that EME is a mild vasodilator. Exogenous PChE protects against cocaine-induced seizures and lethality. It is unclear whether this protective effect results from enhanced degradation of cocaine, the loss of active metabolites (benzoylecgonine, norcocaine), or the production of a beneficial metabolite (EME). This study was designed to further investigate the pharmacologic effects of EME. All experiments used female ICR Swiss albino mice weighing 20-30 grams. Mice were acclimated to 12 h alternating light-dark cycles and given food and water ad libitum. Using a randomized, blinded protocol, 80 mice were then pretreated with either IP EME (50 mg/kg) in a 0.9% sodium chloride solution or an equal volume of 0.9% sodium chloride solution as control. Five minutes later, all animals received 126 mg/kg of cocaine IP and were observed for seizures and death. Fatality was compared using a Fisher's exact test, and the time to seizures and death were compared using a Mann-Whitney U statistic. Pretreatment with EME increased survival following cocaine (9/40 vs. 2/40, for EME vs. control, respectively, p<0.05). The median times to seizure and death for both groups were 2.0 vs. 1.5 min (p>0.05), and 4.5 vs. 4.6 min (p>0.05) (EME vs. control for seizures and death, respectively). In this animal model, EME is protective against cocaine lethality. This effect is consistent with the previously described vasodilatory effects of EME. Further studies are indicated to determine whether the increase in EME produced by exogenous PChE administration contributes to the benefits that occur when PChE is given to cocaine-poisoned animals.

Three-dimensional quantitative structure-activity relationship modeling of cocaine binding by a novel human monoclonal antibody.

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.

Urine Testing for Cocaine Abuse: Metabolic and Excretion Patterns following Different Routes of Administration and Methods for Detection of False-Negative Results

Although cocaine is typically the second-most identified drug of abuse in drug-testing programs, there is surprisingly little quantitative information on excretion patterns following different routes of administration. This report details the urinary excretion and terminal elimination kinetics for cocaine and eight metabolites [benzoylecgonine (BZE), ecgonine methylester (EME), norcocaine (NCOC), benzoylnorecgonine (BNE), m-hydroxy-BZE (m-HO-BZE), p-hydroxy-BZE (p-HO-BZE), m-hydroxy-COC (m-HO-COC), and p-hydroxy-COC (p-HO-COC)]. Six healthy males were administered approximately equipotent doses of cocaine by the intravenous (IV), smoking (SM), and inhalation (IN) routes of administration. Urine specimens were collected for a minimum of three days after drug administration, screened by immunoassay (EMIT and TDX, 300 ng/mL), and analyzed by GC-MS. Mean Cmax values were generally as follows: BZE > EME > COC > BNE approximately p-HO-BZE > m-HO-BZE > m-HO-COC > NCOC > p-HO-COC. Elimination half-lives for cocaine and metabolites were generally shorter following s.m., intermediate after i.v., and longest following i.n. administration. m-HO-BZE demonstrated the longest half-life (mean range 7.0-8.9 h), and cocaine displayed the shortest (2.4-4.0 h). Mean detection times were extended progressively by lowering cutoff concentrations. The maximum increases were approximately 55% at 50 ng/mL for the TDx assay (e.g., the detection time for the last consecutive positive changed from 32.8 h to 50.6 h for i.v. cocaine) and up to 39% for GC-MS at a cutoff concentration of 40 ng/mL (e.g., the detection time for the last consecutive positive changed from 34.8 h to 48.1 h for i.v. cocaine). Sensitivity, specificity, and predictive values for EMIT and TDx were comparable at the 300-ng/mL cutoff concentration; but at lower cutoff concentrations, predictive values of positive results for TDx were diminished indicating a higher risk of false-positive results, that is, positive results that failed to meet administrative cutoff criteria. Detection of positive results was significantly enhanced through the use of the "Zero Threshold Criteria Method", a method developed by the authors to differentiate false-negatives from true-negatives. The method was based on establishing mean immunoassay response (MIR) baselines and variance (SD) in assays of drug-free specimens. Arbitrary thresholds (MIR + 0.5 SD, MIR + 1 SD, MIR + 2 SD) were utilized to evaluate all negative specimens. Apparent true positives were identified by the presence of BZE at or above 40% GC-MS cutoff concentrations. With these criteria, up to 111 false-negative specimens were confirmed as true-positive specimens; this was in addition to the 208 true positives detected at recommended cutoff concentrations. This represents a 50% increase in positive detection rates through the use of this methodology. Such methodology is recommended for further evaluation by drug-testing programs for enhancement of positive detection rates and as an alternative to creatinine testing for dealing with dilute specimens that test negative by initial tests, but contain quantifiable concentrations of drugs of abuse.

Quantitation of cocaine and its major metabolites in human saliva using gas chromatography-positive chemical ionization-mass spectrometry (GC-PCI-MS).

An analytical method for the simultaneous determination of cocaine (COC) and its major metabolites, ecgonine methyl ester (EME) and benzoylecgonine (BEG), in saliva was developed. The method involves liquid-liquid extraction in Toxitubes A, derivatization with 99:1 (v/v) N,O-bis-trimethylsilyltrifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) and gas chromatography-mass spectrometry (GC-MS) determination. The detector response thus obtained is linear over the range 25-1000 ng/mL, with a reproducibility better than 93% and a recovery close to 100% for the three analytes. The limits of detection achieved were 0.9 ng/mL for EME, 2.2 ng/mL for cocaine, and 0.2 ng/mL for BEG, and the limits of quantitation were 3.0 ng/mL for EME, 7.4 ng/mL for cocaine, and 0.8 ng/mL for BEG. The proposed method was applied to 48 saliva samples from cocaine users, 96% of which were positive for the drug and/or its metabolites. Saliva is thus a suitable biological fluid for determining cocaine, EME, and BEG by GC-PCI-MS.

The effect of liquid chromatography eluents and additives on the positive ion responses of cocaine, benzoylecgonine, and ecgonine methyl ester using electrospray ionization

We investigated the effect of common chromatography eluents and additives on the positive ion responses of ecgonine methyl ester (EME), benzoylecgonine (BZE), and cocaine (COC) using electrospray ionization (ESI). Primarily [M+H] + ions were observed, although decomposition of EME and COC to ecgonidine methyl ester gave a sizable peak at m/ z 182.0. The results showed that the sensitivity for the test analytes was greatest in a mobile-phase consisting of a 1:1 mixture of 60% acetonitrile/40% acetone:100 mM ammonium acetate. There was no evidence of a correlation between sensitivity of [M+H] + ions and solution pH. Adducts derived from addition of ammonium salts and ammonium hydroxide, along with cluster ions were not observed, although cationization did occur for BZE (<1.0–23%). Signal intensities for COC (p K a=8.61) obtained under acidic conditions (pH=2.55–2.80) and basic conditions (pH=9.19–10.02) did not vary, suggesting that mechanisms other than in-solution ionization maybe key in formation of ions by the electrospray process.

Studies on metabolic pathways of cocaine and its metabolites using microsome preparations from rat organs.

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").

Cocaethylene metabolism and interaction with cocaine and ethanol: role of carboxylesterases.

Carboxylesterases are important in the metabolism of cocaine, catalyzing the hydrolysis of cocaine to its two major metabolites, benzoylecgonine and ecgonine methyl ester. In the presence of ethanol, some cocaine undergoes transesterification with ethanol instead of hydrolysis with water producing the active metabolite, cocaethylene. The metabolic fate of cocaethylene is unknown, but given its structural similarity to cocaine, it was hypothesized that cocaethylene would also be metabolized by carboxylesterases and its elimination decreased in the presence of ethanol, as is cocaine's. Dogs were given cocaine alone, cocaethylene alone, cocaine and ethanol, cocaethylene and ethanol, and cocaine and cocaethylene on separate study days and sequential blood samples drawn. Plasma concentrations of cocaine, benzoylecgonine, and cocaethylene were determined by high-performance liquid chromatography. The pharmacokinetic dispositions of cocaine and cocaethylene were similar with clearance values of 0.91 +/- 0.22 and 0.79 +/- 0.16 l/min, and volumes of distribution of 2.6 +/- 0.82 and 2.7 +/- 0.47 l/kg, respectively. Both cocaine and cocaethylene clearances were decreased about 20% when given with ethanol. Following administration of cocaethylene alone, benzoylecgonine achieved similar plasma concentrations as those attained following cocaine alone, which indicates that benzoylecgonine is a major metabolite of cocaethylene. Carboxylesterases play an important role in the elimination of both cocaine and cocaethylene.

Detection of Cocaine Analytes and Opiates in Nails from Postmortem Cases

Postmortem fingernail and toenail specimens were utilized for the detection of cocaine analytes and opiates. Nail clippings were obtained from suspected overdoses and/or drug users. Nail clippings were washed with methanol and then solubilized in 0.1M potassium phosphate (pH 5.0). Analytes were isolated from the solubilized nails and methanolic washes by solid-phase or liquid-liquid extraction techniques. Quantitation was performed by gas chromatography-mass spectrometry. Clippings from each hand and foot were analyzed separately for comparison purposes. Concentrations of all the analytes were significantly higher in fingernails than in toenails. Cocaine analytes were detected in the nails of 15 cases with the following concentration ranges (ng/mg): cocaine (n = 15) 1.2-414.1; benzoylecgonine (n = 15) 1.4-170.3; ecgonine methyl ester (n = 14) 0.19-27.0; norcocaine (n = 12) 0.11-32.7; cocaethylene (n = 2) 0.08-2.93. Opiates were detected in 17 cases with the following concentration ranges (ng/mg): morphine (n = 15) 0.05-407.9; 6-acetylmorphine (n = 15) 0.04-504.0; codeine (n = 9) 0.06-8.84; hydromorphone (n = 4) 0.02-0.45. Oxycodone and hydrocodone were each detected in one case. The extent of drug incorporation due to external contamination was evaluated by comparing the differences in the amount of drugs detected in fingernails and toenails, along with the corresponding washes of those nails. These results demonstrate the usefulness of nails as postmortem specimens in detecting drugs of abuse.

Analysis of reaction products of cocaine and hydrogen peroxide by high-performance liquid chromatography/mass spectrometry.

The change of chemical structure of cocaine in the presence of hydrogen peroxide, a main component of hair dye and decolorant treatments, was studied. High-performance liquid chromatography/mass spectrometry (LC/MS) was used for the separation and identification of cocaine derivatives. After a mixture of cocaine and hydrogen peroxide solutions was incubated at 39 degrees C (this temperature is commonly used when the hair is treated with hair dye or decolorant) for 24 h, six reaction products were detected by LC/MS. Two of them were ecgonine methyl ester and benzoylecgonine, which are metabolites of cocaine. The other reaction products were assumed to be ortho-, meta- and para-hydroxycocaines and dihydroxycocaine, in each of which the benzene ring was hydroxylated by the reaction. These five reaction products (except for dihydroxycocaine) were found immediately after mixing cocaine and hydrogen peroxide. Therefore, the above reaction products might be present in the hair of cocaine users that had treated their hair with hair dye or decolorant.

Drugs of abuse monitoring in blood for control of driving under the influence of drugs.

Driving under the influence of drugs is an issue of growing concern in the industrialized countries as a risk and a cause for road accidents. In forensic toxicology, the increasing number of samples for determination of drugs in blood is mainly due to zero-tolerance laws in several countries and well-trained police officers who can better recognize drivers under the influence of drugs of abuse. This review describes procedures for detection of the following drugs of abuse in whole blood, plasma, and serum: amphetamine, methamphetamine, 3,4-methylenedioxy methamphetamine (MDMA), N-ethyl-3, 4-methylenedioxyamphetamine (MDEA), 3,4-methylenedioxyamphetamine (MDA), cannabinoids (delta-9-tetrahydrocannabinol [THC], 11-hydroxy-delta-9-THC, 11-nor-9-carboxy-delta-9-THC), cocaine, benzoylecgonine, ecgonine methyl ester, cocaethylene, the opiates (heroin, 6-monoacetylmorphine, morphine, or codeine), and methadone as well as gamma-hydroxybutyric acid (GHB), lysergic acid diethylamide (LSD), phencyclidine (PCP), and psilocybin/psilocin. For many of the analytes, sensitive immunologic methods for screening are available. Gas chromatography-mass spectrometry (GC-MS) is still the state-of-the-art method for confirmatory analysis or for screening and confirmation in one step. Liquid chromatography-mass spectrometry (LC-MS) procedures for such purposes are also included in this review. Basic data about the biosample assayed, internal standard, workup, GC or LC column and mobile phase, detection mode, reference data, and validation data of each procedure are summarized in two tables.

Cocaine permeability and metabolism in colonic T-84 epithelial cell line

We studied the uptake, transport and metabolism of cocaine in human intestine using the colonic T-84 monolayers as a model. T-84 cells were grown in DMEM/Ham's F-12 medium containing 6 % newborn calf serum at 37 ° C on 1.0 μm collagen inserts. The cells develop into a polarized monolayer with the apical surface facing the upper chamber and the basolateral surface facing the lower. The monolayers develop a transepithelial resistance of ≥ 600 Ω cm 2 in 7 days. Varying concentrations of cocaine HCl was added in a serum free medium to the luminal side only, and after 30 min and 60 min samples from the luminal and serosal aspect were removed for analysis. Cocaine and its metabolites were measured by G C/MS. Cocaine transport across T-84 monolayers increased linearly with increasing concentration of cocaine, with no significant difference between 30 min and 60 min of exposure. Transepithelial resistance did not change even at 800 ng of cocaine suggesting no effect on monolayer viability. The metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) but not norcocaine were detected in both luminal and serosal side. The concentrations of BE and EME in the luminal side were significantly higher than in the serosal. Combined recovery of cocaine, BE and EME from luminal and serosal sides were 52 – 80 % of total added cocaine. While fresh medium did not metabolize cocaine, media previously exposed to the monolayer (cell-free medium) caused a significant breakdown into BE and EME, suggesting that esterases may be released into the medium. These results indicate transfer of cocaine across this model of intestinal epithelial cell line is by simple diffusion and is concentration dependent. These studies imply that cocaine in swallowed amniotic fluid can be absorbed by the fetal gastrointestinal tract.

Effects of cocaine and cocaine metabolites on cardiovascular function in squirrel monkeys

The effects of cocaine and the cocaine metabolites norcocaine, ecgonine methyl ester, benzoylecgonine and cocaethylene were evaluated in conscious squirrel monkeys for their effects on blood pressure and heart rate. Norcocaine, ecgonine methyl ester and benzoylecgonine are produced in vivo following cocaine use. Cocaethylene is produced in vivo following concurrent cocaine and alcohol use. Increases in both blood pressure and heart rate were observed following cocaine doses of 0.3–3.0 mg/kg. Ecgonine methyl ester and benzoylecgonine had no effect on either parameter up to doses of 10.0 mg/kg. Norcocaine increased blood pressure, but was less potent than cocaine. Norcocaine did not affect heart rate at doses up to 3.0 mg/kg. In contrast to the other metabolites, cocaethylene increased blood pressure and heart rate similarly to cocaine. These results suggest that ecgonine methyl ester and benzoylecgonine are devoid of cardiovascular effects at doses comparable to cocaine and would not be expected to contribute to cocaine's overall cardiovascular effects. Norcocaine's effect on blood pressure might contribute to the cardiovascular effects of cocaine, but this metabolite is produced only at low levels in vivo. The one metabolite that might be expected to contribute to cocaine's overall cardiovascular effect is cocaethylene, although the degree of this contribution is not clear.

COMPARISON OF TWO CHROMATOGRAPHIC METHODS FOR THE DETERMINATION OF COCAINE AND ITS METABOLITES IN BLOOD AND URINE

After a solid-phase extraction, two chromatographic procedures (GC/FID and HPLC/UV detector) for the determination of cocaine and its metabolites (benzoylecgonine and ecgonine methyl ester) in human blood and urine have been developed. Linear calibration curves were obtained in urine over the concentration ranges of 0.16–4 μg/mL and 0.2–4 μg/mL, by GC and HPLC, respectively; and, in blood, between 0.1 and 2 μg/mL, by both methods. The recoveries ranged from 66% to 83% for GC, and from 61% to 95% for HPLC. The limit of detection was found to be below 76 ng/mL for all the analytes. Precision and accuracy were studied with acceptable results. The methods have been applied simultaneously to quantitate the compounds in real samples; concentrations of cocaine and benzoylecgonine determined by GC/FID correlated well with those determined by HPLC/UV in most cases.

Analysis of cocaine, benzoylecgonine, ecogonine methyl ester, and ecgonine by high-pressure liquid chromatography-API mass spectrometry and application to a short-term degradation study of cocaine in plasma.

A method for the determination of cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and ecgonine (ECG) in plasma by liquid chromatography-mass spectrometry (LC-MS-MS) was developed. The analytes were isolated from human plasma by subsequent solid-phase extraction and were separated on a Zorbax Eclipse XDB-C8 narrow-bore column using an ammonium acetate buffer/acetonitrile/methanol gradient. A Turbolonspray source was used for ionization. The analytes were characterized by their particular molecular ion and several fragments. Multiple reaction monitoring (MRM) was used for isolation and quantitation. The assay was rapid, highly sensitive, and reliable. The method was applied to monitor the in vitro degradation of cocaine in plasma. Fresh unpreserved and preserved (0.25% KF) plasma samples were spiked with 1,000 ng cocaine/mL. Aliquots of both series were stored at 4 degrees C and 20 degrees C and were analyzed at selected storage times of up to 15 days. In all samples, degradation of cocaine that was dependent on storage time and temperature and on sample preservation could be observed. The formation of BE did not occur to a significant extent (< 12%, referred to the initial concentration of COC), and its concentration was slightly higher in preserved compared with unpreserved plasma at both storage temperatures chosen. EME was formed in considerably higher amounts compared to BE. As already observed for COC, its formation and degradation were dependent on storage time, temperature, and preservation. EME is suggested to be the major source of ECG, which was detectable in all samples after 1-2 days of storage. Although the degradation of COC was shown to be highly dynamic in nature, the sum of all hydrolysis products of COC accounted for the initial COC concentration at any particular time of storage. Therefore, production of hitherto unknown degradation products of COC seems unlikely. Moreover, the common transformation product of BE and EME appeared to be stable, and ECG is suggested as a promising postcollection artifact.

Detection of Cocaine and Its Metabolites in Breast Milk

A method was developed for measuring cocaine and its metabolites, benzoylecgonine, ecgonine methyl ester, norcocaine, ecgonine ethyl ester, cocaethylene, and m-hydroxybenzoylecgonine, in breast milk by gas chromatography/mass spectrometry. Limits of detection for this method ranged from 2.5 to 10 ng/mL, and limits of quantitation ranged from 5 to 50 ng/mL. For each of the compounds measured by this method, linear response was demonstrated to 750 ng/mL. Breast milk was collected from 11 mothers who admitted to drug use during pregnancy and ten drug-free volunteers serving as control subjects. Cocaine was detected in six of the specimens obtained from drug-exposed subjects, and in none of the drug-free control subjects. In breast milk specimens where cocaine and one or more of its metabolites were detected, the concentration of parent compound was greater than any of the metabolites. The highest cocaine concentration found was over 12 microg/mL. Breast-fed infants of cocaine abusing mothers may be exposed to significant amounts of drug orally.

2′-Substituted analogs of cocaine: synthesis and dopamine transporter binding potencies

A series of 2'-substituted cocaine analogs (4-8) was prepared and evaluated in an in vitro dopamine transporter (DAT) binding assay. Compounds 4-7 were prepared by esterifying the 3 beta-hydroxyl group of ecgonine methyl ester (3) using the appropriate acid chloride in the presence of Et3N and benzene. Compound 3 was obtained from cocaine (1) by hydrolysis using 1N HCl to afford ecgonine.HCl which was subjected to acid catalyzed esterification using methanol saturated with HCl gas. Compound 8 was obtained by hydrogenation of 7 using H2/Pd-C. The IC50 values were calculated from displacement experiment of the radioligand [3H]WIN-35,428 (2). 2'-Aminococaine (8) showed high binding affinity to the DAT (14- and 1.3-fold more active than cocaine and the radioligand 2, respectively). These results, along with previous results, emphasize the importance of a hydrogen-bond donor group at the 2'-position of cocaine to enhance binding affinity to the DAT.

Importance of vacutainer selection in forensic toxicological analysis of drugs of abuse.

The enzymatic degradation of cocaine in blood samples, even during transport to a forensic laboratory, is a common problem in toxicological analysis. This can be avoided by the use of blood-sampling devices such as gray-top Vacutainers containing the cholinesterase inhibitor sodium fluoride. In the present study, which included 147 authentic cases, blood samples were collected into two different tubes, one containing fluoride/oxalate and one without stabilizing agents. In all cases, both samples were analyzed for drugs of abuse using Abbott FPIA immunoassays after precipitation and gas chromatography-mass spectrometry (GC-MS) for quantitative analysis. The cannabinoid immunoassay showed markedly lower values in the fluoride-containing samples; this was investigated further and could be explained by hemolysis of these samples. In addition, the concentrations of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) were lower in these samples. A stability study with the THCCOOH acyl glucuronide showed that it is unstable in unpreserved serum, which could explain our observation. GC-MS quantitative data for amphetamine and derivatives, opiates, delta9-tetrahydrocannabinol, and 11-hydroxy-delta9-tetrahydrocannabinol were essentially identical; however, they also differed substantially for cocaine, cocaethylene, ecgonine methylester, and benzoylecgonine. Unexpectedly, the concentrations of benzoylecgonine in unpreserved serum were almost half as high as in the fluoride-containing samples.

A catalytic antibody against cocaine attenuates cocaine's cardiovascular effects in mice: a dose and time course analysis

The murine monoclonal antibody 15A10 (mAb 15A10), elicited by a transition-state analog for cocaine hydrolysis, has previously been shown to metabolize cocaine in vitro and in vivo. The present experiments were designed to evaluate further the in vivo effectiveness of mAb 15A10 in blocking cardiovascular effects of acute cocaine administration. Balb/c mice were implanted with a femoral artery catheter utilized for mean arterial pressure (MAP) monitoring, and administered intravenous (i.v.) pretreatments of either mAb 15A10 (10, 32, 100 and 300 mg/kg) or vehicle prior to cocaine injection (100 mg/kg, i.p.). A time course analysis for mAb 15A10's effect was also conducted, for which either vehicle or 100 mg/kg mAb 15A10 was infused 1, 3, 10 and 30 days prior to cocaine treatment. During the cardiovascular recording sessions, mice were awake and freely moving within a limited area. Increases in MAP (∼25 mm Hg) following cocaine injection were dose-dependently attenuated by mAb 15A10. The antibody-attenuated cocaine-induced increases in MAP at 1- and 3-day pretreatment times, and reduced mortality at some of the time points studied. With 100 mg/kg antibody, plasma cocaine levels were significantly decreased early in the recording session, whereas levels of ecgonine methyl ester increased significantly. Although 10-fold greater quantities of antibody are required to observe significant effects in mouse, compared to our previous studies in rats, the present mouse model provides a convenient paradigm for investigating catalytic and non-catalytic antibodies.

The disposition of benzoylecgonine in maternal and fetal rats

We tested our hypothesis that pregnancy alters the pharmacokinetic profile of benzoylecgonine, and that this metabolite accumulates in the fetus longer than in the mother. Chronically catheterized near-term pregnant and nonpregnant female Sprague–Dawley rats received an intravenous infusion of benzoylecgonine over a period of 30 min. Adult or fetal blood and tissue samples were obtained either at the end of the infusion or 6 h postinfusion for analysis of benzoylecgonine and other cocaine metabolite concentrations via gas chromatography/mass spectrometry (GC/MS). Pregnancy altered benzoylecgonine pharmacokinetics. At the end of the infusion, benzoylecgonine concentration in the fetal plasma was markedly lower than in the maternal plasma with a fetal/maternal ratio of 0.14±0.01. A significantly lower concentration of benzoylecgonine was found in both maternal and fetal brain at 0 h postinfusion, with tissue/plasma concentration ratios of 0.04 and 0.24, respectively, suggesting that benzoylecgonine does not readily penetrate into the brain. At 6 h, the fetal concentration of benzoylecgonine was significantly higher than in the corresponding maternal blood and tissues. Ecgonine methyl ester, a metabolite of benzoylecgonine was found in the maternal liver, but not in the fetus. In addition, the amniotic fluid concentration of benzoylecgonine became significantly higher in the 6-h postinfusion samples as compared to the end of infusion value, suggesting that repeated intrauterine exposure to cocaine may cause an accumulation of benzoylecgonine in the fetus.

Nicotine alters some of cocaine's subjective effects in the absence of physiological or pharmacokinetic changes

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.