Studies on hydrolytic and oxidative metabolic pathways of anhydroecgonine methyl ester (methylecgonidine) using microsomal preparations from rat organs

During smoking of cocaine-base (crack), anhydroecgonine methyl ester (AEME, methylecgonidine) is formed in large amounts as a pyrolysis product of cocaine and is absorbed in the lungs. The metabolism of AEME was studied in the present investigation using microsome preparations from rat liver, lung, kidney, and brain. Potential metabolites of AEME were synthesized and used as substrate to complement the experiments. Analysis of the incubation mixtures was performed using gas chromatography-mass spectrometry and nanoelectrospray multiple-stage mass spectrometry. Screening for metabolites was focused on postulated oxidative pathways, chemical and enzymatic hydrolysis, and ethanol dependent transesterification as known from cocaine metabolism. Enzymatic hydrolysis of AEME to anhydroecgonine (AE), which was inhibited by sodium fluoride, was found in all microsomal preparations. Liver microsomes exhibited the highest activity, brain microsomes the lowest. Anhydronorecgonine methyl ester (ANEME) and anhydroecgonine methyl ester N-oxide were identified as AEME metabolites of liver and lung microsomes only. In the presence of ethanol AEME was metabolized to anhydroecgonine ethyl ester and anhydronorecgonine ethyl ester. Further metabolism of AE or ANEME was not observed. No N-hydroxy-anhydronorecgonine derivatives were found which could represent precursors of cytotoxic metabolites as known to be formed from cocaine.     

A pyrolysis product, anhydroecgonine methyl ester (methylecgonidine), is in the urine of cocaine smokers

A method using combined gas chromatography/mass spectrometry (GC/MS) for determination of the cocaine pyrolysis product anhydroecgonine methyl ester (AEME) in urine is described. Using this method, we found that human subjects who smoked cocaine under laboratory conditions excreted substantial amounts of AEME in their urine. Little, if any, AEME was excreted in the urine when the same subjects were administered cocaine by intravenous and intranasal routes. AEME may be a useful marker for cocaine (crack) smoking. The pharmacology of AEME is unknown. The possibility that AEME may play a role in the effects associated with cocaine smoking needs to be examined.     

Neurotoxicity of anhydroecgonine methyl ester, a crack cocaine pyrolysis product

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaine-AEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaine-AEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.     

Stability studies of some glycolamide ester prodrugs of niflumic acid in aqueous buffers and human plasma by HPLC with UV detection

Glycolamide esters (compounds 1-17) of 2-(3-trifluoromethyl-phenylamino)nicotinic acid (niflumic acid, CAS 4394-00-7) have been synthesized and evaluated as possible prodrugs. In-vitro hydrolysis studies were conducted at selected pH values (1.2, 3.5, 4.8, 7.4 and 7.8) and in human plasma at 37 +/- 0.5 degree C using HPLC with UV detection. The aqueous (pH 7.4 and 7.8) and enzymatic rates of hydrolysis were substantially affected by the nature of promoieties in this series. The compounds showed good chemical stability in the buffers of low pH values (1.2, 3.5 and 4.8) and appreciable hydrolysis under alkaline conditions and in human plasma. They exhibited long hydrolytic half-lives of 7-46 h in aqueous buffer solutions (pH 7.4 and 7.8) and 14-21 min in human plasma, respectively. It was observed that N,N-disubstituted and cyclic glycolamide derivatives showed 2 fold more hydrolysis in the alkaline pH than monosubstituted derivatives, whereas the piperidino and thiomorpholino derivatives did not undergo chemical hydrolysis. The compounds contain two possible sites for hydrolysis with an increased hydrolytic susceptibility at the terminal aliphatic carbonyl site in aqueous buffers and human plasma solutions. They were found to be cleaved at two hydrolytic carbonyls, namely the nicotinyl (2-5 % in enzymatic hydrolysis) and the aliphatic site (7-55 % and 70-85 % in buffer and plasma hydrolysis, respectively) as revealed by HPLC analysis. The glycolamide ester prodrugs of niflumic acid underwent chemical and enzymatic hydrolysis to release mainly the metabolite 2-(3-trifluoromethyl-phenylamino) nicotinic acid carboxymethyl ester (III) and not the parent drug 2-(3-trifluoromethyl-phenylamino)nicotinic acid. The structure of the metabolite was confirmed by liquid chromatography-mass spectroscopy (LCMS).     

A comprehensive study of the stability of cocaine and its metabolites

The stability of cocaine (COC) in blood bank blood, postmortem human whole blood, and buffers was evaluated with consideration for the presence of the degradation products, benzoylecgonine (BE) and ecgonine methyl ester (EME). At COC concentrations commonly seen, the rate of COC hydrolysis was independent of concentration. COC was stable in blood for at least 150 days if the blood was adjusted to pH 5 and preserved with 2% NaF or organophosphates and maintained at 4 degrees C or lower. Without preservation, most COC hydrolyzed to EME. The addition of a pseudocholinesterase (PChE) inhibitor without a reduction of pH caused COC to hydrolyze to BE. COC also hydrolyzed to BE in phosphate buffer. The rate of COC hydrolysis in all studies increased with increasing pH and temperature. COC was more stable in unpreserved postmortem blood than blood bank blood due to the lower pH of the former. The incubation of COC in enzyme solutions provided further evidence of the generally accepted hypothesis that COC is hydrolyzed to EME by PChE and to BE by chemical hydrolysis. In unpreserved blood, BE was more stable than EME at room temperature. There was little loss of BE or EME at refrigerated temperature over a period of 35 days and no evidence that EME or BE could be hydrolyzed enzymatically.     

Profiling cocaine residues and pyrolytic products in wastewater by mixed‐mode liquid chromatography–tandem mass spectrometry

This work provides a new analytical method for the determination of cocaine, its metabolites benzoylecgonine and cocaethylene, the pyrolytic products anhydroecgonine and anhydroecgonine methyl ester, and the pharmaceutical levamisole in wastewater. Samples were solid‐phase extracted and extracts analyzed by liquid chromatography–tandem mass spectrometry using, for the first time in the illicit drug field, a stationary phase that combines reversed‐phase and weak cation‐exchange functionalities. The overall method performance was satisfactory, with limits of detection below 1 ng/L, relative standard deviations below 21%, and percentages of recovery between 93% and 121%. Analysis of 24‐hour composite raw wastewater samples collected in Santiago de Compostela (Spain) and Brasilia (Brazil) highlighted benzoylecgonine as the compound showing the highest population‐normalized mass loads (300–1000 mg/day/1000 inhabitants). In Brasilia, cocaine and levamisole loads underwent an upsurge on Sunday, indicating a high consumption, and likely a direct disposal, of cocaine powder on this day. Conversely, the pyrolytic product resulting from the smoke of crack, anhydroecgonine methyl ester, and its metabolite anhydroecgonine were relatively stable over the four days, agreeing with a non‐recreational‐associated use of crack.     

Kinetics of phosphoramide mustard hydrolysis in aqueous solution

Hydrolysis of phosphoramide mustard was investigated using HPLC, 31P NMR, and GC-MS with specific deuterium labels. The hydrolysis of phosphoramide mustard in sodium phosphate buffers was found to follow apparent first-order kinetics. The rate of hydrolysis was temperature and pH dependent, being slower under acidic conditions. The hydrolysis was not catalyzed by hydroxyl ion, and its pH dependence appeared to be the result of a change in the mechanism of hydrolysis at different pH values. At a pH value approximately above the pKa of the phosphoramide mustard nitrogen, the major hydrolytic pathway of phosphoramide mustard was via the formation of the aziridinium ion, followed by nucleophilic attack. At pH values below its pKa, cleavage of the P-N bond predominated. At pH 7.4, the formation of an aziridinium ion was followed by a rapid hydrolysis to yield the monohydroxy and, subsequently, the dihydroxy products. The hydrolysis at this pH was adequately described by consecutive first-order kinetics. Seven species in the hydrolytic mixture have been identified as intact phosphoramide mustard, N-(2-chloroethyl)-N-(2-hydroxyethyl)phosphorodiamidic acid, N,N-bis-(2-hydroxyethyl)phosphorodiamidic acid, phosphoramidic acid, phosphoric acid, N,N-bis-(2-chloroethyl)amine, and N-(2-chloroethyl)-N-(2-hydroxyethyl)amine by GC-MS with the aid of deuterium labels. Phosphoramide mustard was found to be stabilized by chloride ion. The stabilization was linearly related to the chloride ion concentration, and the mechanism was found to be via the formation of phosphoramide mustard from the aziridinium and chloride ions. Phosphoramide mustard was significantly more stable in human plasma and in 5% human serum albumin as compared to aqueous buffers, an observation that may be important in vivo.     

Metabolism and pharmacokinetics of vinyl acetate

The hydrolysis of vinyl acetate (formation of acetic acid) has been studied in vitro with rat liver and lung microsomes, rat and human plasma and purified esterases (such as acetylcholine esterase, butyrylcholine esterase, carboxyl esterase). Characterization of the kinetic parameters revealed that rat liver microsomes and purified carboxyl esterase (from porcine liver) displayed the highest activity.In order to establish the rate of metabolism of vinyl acetate in vivo, rats were exposed in closed desiccator jar chambers, and gas uptake kinetics were studied. The decay of vinyl acetate was dose-dependent, indicating possible saturation of metabolic pathway(s). The maximal clearance (at lower concentrations) of vinyl acetate from the system (30 000 ml/h per kg body weight) was similar to the maximal ventilation rate in this species. This indicated that under conditions when metabolic enzymes are not saturated the metabolic rate is mainly determined by pulmonary uptake.The exposure of rats to vinyl acetate resulted in a transient exhalation of significant amounts of acetaldehyde into the closed exposure system. This indicates the presence of this metabolic intermediate of vinyl acetate in the organism in vivo.     

The influence of plasma butyrylcholinesterase concentration on the in vitro hydrolysis of cocaine in human plasma

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.     

Hydrolysis of suxamethonium by different types of plasma

1. A method, based on the use of the isolated frog rectus abdominis preparation, is described for studying quantitatively the hydrolysis of suxamethonium at low concentrations.

2. Rates of hydrolysis of butyrylcholine, 10 mM, benzoylcholine, 0·05 mM, and suxamethonium, 0·025 mM, by plasma from different species and human plasma with genetic variants of cholinesterase were measured.

3. The rates of hydrolysis of suxamethonium by different types of plasma vary widely. Human plasma with usual cholinesterase and monkey plasma hydrolyse suxamethonium more speedily than do the plasma of cats, dogs and rats, and human plasma with either atypical or fluoride resistant cholinesterase. This is only to a small extent attributable to differences in enzyme concentration and not explained by the presence of an inhibitor.

4. The K_m values for butyrylcholine are very similar for different types of plasma but the K_I values for suxamethonium in the system plasma-butyrylcholine-suxamethonium vary greatly.

5. These results and observations on the inhibition by decamethonium of the hydrolysis of butyrylcholine are consistent with the interpretation that the rates of hydrolysis of suxamethonium, 0·025 mM, obtained with different types of plasma vary because they are a function of two variables, the affinity of the ester for cholinesterase and the stability of the monosuccinyl derivative of the cholinesterase. It seems that human plasma with atypical cholinesterase hydrolyses suxamethonium much slower than does human plasma with usual cholinesterase mainly or solely because of differences in affinity of the ester for the two enzymes. On the other hand, cat plasma appears to hydrolyse suxamethonium much slower than does human plasma with usual cholinesterase mainly because the monosuccinyl derivative of cholinesterase in cat plasma is much more stable than that in human plasma. The reverse might apply for monkey plasma.

6. Inhibition by dibucaine or sodium fluoride of the hydrolysis of benzoylcholine is not a general guide to rates of hydrolysis of suxamethonium by different types of plasma.

     

Unanticipated acyloxymethylation of sumatriptan indole nitrogen atom and its implications in prodrug design

Sumatriptan is a potent and selective 5-HT(1B) and 5-HT(1D )agonist used in the symptomatic treatment of migraine; it shows poor oral bioavailability ascribed, in part, to its low lipophilicity. In an attempt to develop acyloxymethyl prodrugs of sumatriptan suitable for oral administration, we carried out the reaction of sumatriptan with chloromethyl esters. To our surprise, acyloxymethylation occurred preferentially at the indole nitrogen rather than at sulfonamide nitrogen, reflecting a difference either in product stability or in the nucleophilicities of the indole and sulfonamide anions. The hydrolysis of the corresponding N(1)-acyloxymethyl derivatives was studied in aqueous buffers and in human plasma, by HPLC. N(1)-Acyloxymethyl derivatives of sumatriptan are rapidly hydrolysed to the chemically stable N(1)-hydroxymethylsumatriptan at pH 1-13. Slow formation of the parent drug was observed only at high pH values. Hydrolysis of sumatriptan derivatives is slower in human plasma than in phosphate buffer and also generates N(1)-hydroxymethylsumatriptan rather than the parent drug. These results indicate that N(1)-acyloxymethyl derivatives of sumatriptan cannot be considered as true prodrugs of sumatriptan.     

Selective induction of xenobiotic metabolizing esterases/amidases of liver by methaqualone consumption

The present investigation reports the influence of po and ip methaqualone administration on the hydrolytic metabolism of acetylsalicylic acid, procaine, p-nitrophenylacetate, acetanilid, and butyrylcholine in the liver, kidney, and brain of male rats. Oral administration of methaqualone (60 mg/kg/day) to rats for 20 days caused 41.0, 46.5, and 55.0% stimulation of acetylsalicyclic acid esterase I, acetylsalicyclic acid esterase II, and acetanilid N-deacetylase, respectively, in the liver. Under such conditions, the activities of other esterases remained unaffected. The responses of tissue esterases to ip methaqualone treatment (40 mg/kg/day for 6 days) were similar to those observed after po methaqualone administration. Since a single po dose of methaqualone failed to produce any alteration in the rate of metabolism of acetylsalicylic acid, procaine, p-nitrophenylacetate, acetanilid, and butyrylcholine within 20 hr, it may be interpreted that the stimulation of acetylsalicylic acid and acetanilid metabolism is possibly due to selective enhanced de novo synthesis of the enzymes/isozymes necessary for the hydrolysis of the two drugs. The ability of the kidney and brain to metabolize the esters/amides was not modified by po or ip methaqualone pretreatment suggesting the possibility of noninducible forms of renal and neuronal esterases/amidases.     

Hydrolysis of suxamethonium by plasma from subjects responding to the drug with prolonged apnoea,and by plasma from their relatives

Summary The hydrolysis of benzoylcholine, 0.05 mM, butyrylcholine, 10 mM, and suxamethonium, 0.025 mM, by the plasma of 10 subjects who developed prolonged apnoea following suxamethonium administered during anaesthesia, were measured. Plasmas from all available relatives in four families were similarly studied. The plasma of 3 subjects contained only atypical cholinesterase and hydrolysed suxamethonium, relative to butyrylcholine, at a rate of only 1.6% of that seen with usual human cholinesterase. One subject appeared to have only atypical cholinesterase on the basis of dibucaine and fluoride numbers. Suxamethonium hydrolysis, however, was sixteen times greater than that for the 3 homozygous atypical subjects. Family studies and inhibition of butrylcholine hydrolysis by decamethonium established that this subject was heterozygous with about 20% of the total cholinesterase in the usual form. Two other subjects were also heterozygous for usual and either atypical or fluoride resistant genes. One of them hydrolysed suxamethonium at 25% of the usual rate, but the other had a normal rate of suxamethonium hydrolysis. Four subjects had no detectable anomaly of plasma cholinesterase, and hydrolysed suxamethonium normally. The apparent affinity of suxamethonium for usual and atypical cholinesterase was also determined and the significance of measurements of the hydrolysis of suxamethonium in relation to prolonged apnoea produced by the drug is discussed.     

酮洛芬异丙酯体外水解动力学研究

目的：研究酮洛芬异丙酯（KPI）在不同pH的磷酸盐缓冲溶液，大鼠血浆和大鼠肝匀浆中的水解动力学。方法：采用经典恒温法进行试验，通过HPLC法测定KPI的浓度变化。结果：KPI在水溶液中的水解为伪一级动力学反应，同时受H^＋和OH^-催化，碱催化速率常数均大于酸催化常数，表明KPI在水溶液中受OH^-催化更为显著．其在水溶液中的最稳定的pH（pHm）在7．4左右。KPI在血浆和肝匀浆中的水解属酶催化反应，速率较快，且在血浆中的水解速度快于在肝匀浆中的水解，其20％大鼠血浆中的水解半衰期为0．9min，20％肝匀浆则为7．1min。结论：KPI作为酮洛芬的前体药物其主要水解部位为血浆。     

Kinetics of hydrolysis of meclofenoxate hydrochloride in human plasma

The kinetics of hydrolysis of meclofenoxate hydrochloride in human plasma have been compared with those of clofibrate. The hydrolysis rate in fractionated plasma was determined in the presence and absence of a plasma esterase inhibitor, tetraethyl pyrophosphate. The kinetic data indicated that clofibrate decomposed only by esterase-induced hydrolysis, which was inhibited by binding of clofibrate to plasma proteins. In contrast to clofibrate, meclofenoxate decomposed rapidly in human plasma via spontaneous hydrolysis as well as esterase-induced hydrolysis. The spontaneous hydrolysis appeared to be inhibited by some components present in the esterase fraction isolated from plasma, while no significant inhibition of the hydrolysis by protein binding was observed.     

On the prodrug potential of novel aldose reductase inhibitors with diphenylmethyleneaminooxycarboxylic acid structure.

Diphenylmethyleneaminooxycarboxylic acids were found to represent novel type inhibitors of the enzyme aldose reductase. Ester derivatives of the most active compound (3c) (IC(50)=33 microM) were prepared as potential prodrugs and the rate of degradation was studied by treatment with buffers, plasma, and various hydrolytic enzymes. Whereas all compounds were not hydrolysed at physiological pH, incubation in the presence of enzyme led to hydrolysis. The rate of enzymatic degradation, however, depended on the nature of the ester function. Whereas the isopropyl ester (4) turned out to be the most stable compound, the ethyl ester (2c) could be cleaved in the presence of esterase and lipase, respectively. The benzylic and aromatic esters were found to be hydrolysed rapidly in the presence of lipase (benzyl ester, 7), or in plasma, by cholinesterase and esterase (phenyl ester, 6), respectively.     

Stability of cucurbitacin E in human plasma: chemical hydrolysis and role of plasma esterases

It was shown previously that the anticancerous cytotoxic oxygenated triterpenes, cucurbitacin E (Cuc E) and its deacetylated form, cucurbitacin I (Cuc I), interacted differently with human serum albumin. In this study, the biochemical stability of Cuc E was investigated in vitro by reverse‐phase high performance liquid chromatography. The hydrolysis rate in acidic and alkaline solutions, and in enzymatic conditions in human plasma and in purified plasma esterase solutions of butyrylcholinesterase and albumin, was compared with that measured in phosphate buffer saline (pH 7.4). Cuc E hydrolysis was detected in all the in vitro tests, but the extent of hydrolysis varied according to the different enzymatic and non‐enzymatic conditions. A remarkable rapid hydrolysis of Cuc E was detected in acidic and alkaline solutions. A significant rate of hydrolysis of Cuc E was monitored in human plasma and was associated with the detection of Cuc I. The stability of Cuc E was greatly enhanced in the presence of albumin. However, purified butyrylcholinesterase had no effect on Cuc E stability. Among specific inhibitors of plasma esterases, only EDTA increased Cuc E stability, suggesting that paraoxonase is the human plasma esterase involved in the hydrolysis of Cuc E. Copyright © 2009 John Wiley & Sons, Ltd.     

Isosorbide-based aspirin prodrugs: II. Hydrolysis kinetics of isosorbide diaspirinate

Aspirin prodrugs have been intensively investigated in an effort to produce compounds with lower gastric toxicity, greater stability or enhanced percutaneous absorption, relative to aspirin. This report describes the hydrolysis kinetics and aspirin release characteristics of isosorbide diaspirinate (ISDA), the aspirin diester of isosorbide. ISDA underwent rapid hydrolysis when incubated in phosphate buffered human plasma solutions (pH 7.4) at 37 degrees C, producing appreciable quantities of aspirin. In 30% human plasma solution the half-life was 1.1 min and 61% aspirin was liberated relative to the initial ester concentration. The hydrolysis kinetics of ISDA were monitored in aqueous solution at 37 degrees C over the pH range 1.03-9.4. The aqueous hydrolysis followed pseudo-first-order kinetics over several half-lives at all pH values, resulting in a U-shaped pH rate profile. Salicylate esters and salicylic acid were formed during these processes. The hydrolysis characteristics of ISDA were also investigated in pH 7.4 phosphate buffered solutions containing alpha-chymotrypsin [EC 3.1.1.1] (t(1/2)=200.9 min), carboxyl esterase [EC 3.1.1.1] (t(1/2)=31.5 min), human serum albumin (t(1/2)=603 min), purified human serum butyrylcholinesterase [EC 3.1.1.8] (80 micro g/ml; t(1/2)=9.4 min; 55% aspirin), purified horse serum butyrylcholinesterase (100 micro g/ml; t(1/2)=1.85 min;11% aspirin) and in 10% human plasma solution in the presence of physostigmine (3 micro M). The results indicate that a specific enzyme present in human plasma, probably human butyrylcholinesterase, catalyses aspirin release from isosorbide diaspirinate.     

Ketorolac-dextran conjugates: synthesis, in vitro and in vivo evaluation

Ketorolac is a non-steroidal anti-inflammatory drug. Dextran conjugates of ketorolac (KD) were synthesized and characterized to improve ketorolac aqueous solubility and reduce gastrointestinal side effects. An N-acylimidazole derivative of ketorolac (KAI) was condensed with a model carrier polymer, dextran of different molecular masses (40000, 60000, 110000 and 200000). IR spectral data confirmed formation of ester bonding. Ketorolac contents were evaluated by UV-spectrophotometric analysis. The molecular mass was determined by measuring viscosity using the Mark-Howink-Sakurada equation. In vitro hydrolysis studies were performed in aqueous buffers (pH 1.2, 7.4, 9) and in 80% (V/V) human plasma (pH 7.4). At pH 9, a higher rate of ketorolac release from KD was observed as compared to aqueous buffer of pH 7.4 and 80% human plasma (pH 7.4), following first-order kinetics. In vivo biological screening in mice and rats indicated that conjugates retained analgesic and anti-inflammatory activities with significantly reduced ulcerogenicity compared to the parent drug.     

Serotonergic responsiveness in human cocaine users

BACKGROUND: Animal experiments show that repeated cocaine injections induce changes in brain serotonin (5-hydroxytryptamine, or 5-HT) function which can be detected by altered neuroendocrine responsiveness to serotonergic drug challenge. Studies of human cocaine users given a serotonergic challenge have produced inconsistent results. METHODS: Hormone responses evoked by the 5-HT releaser D,L-fenfluramine (FEN) were examined in eight human cocaine users who resided on a closed research ward. FEN (60 mg oral) was given after a 7-day cocaine-free period and 3 days after a 5-day period of daily double-blind administration of intranasal cocaine (96 mg) and active placebo (4 mg cocaine). Plasma cortisol and prolactin levels were measured after FEN challenges, and after cocaine and placebo administration. RESULTS: Cocaine significantly elevated plasma cortisol levels to a similar degree on the first and fifth days of administration, but did not alter prolactin levels on either day. The first FEN challenge significantly increased plasma prolactin and cortisol, whereas the second challenge increased only prolactin. CONCLUSIONS: Intranasal cocaine increases plasma cortisol without affecting prolactin, with no evidence for tolerance. The reduction in FEN-induced cortisol secretion after cocaine exposure suggests that deficits in 5-HT transmission during early cocaine abstinence might contribute to the maintenance of drug dependence.