Substrate Report for: 6-monoacetylmorphine

As the most popularly abused one of opioids, heroin is actually a prodrug. In the body, heroin is hydrolyzed/activated to 6-monoacetylmorphine (6-MAM) first and then to morphine to produce its toxic and physiological effects. It has been known that heroin hydrolysis to 6-MAM and morphine is accelerated by cholinesterases, including acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE). However, there has been controversy over the specific catalytic activities and functional significance of the cholinesterases, which requires for the more careful kinetic characterization under the same experimental conditions. Here we report the kinetic characterization of AChE, BChE, and a therapeutically promising cocaine hydrolase (CocH1) for heroin and 6-MAM hydrolyses under the same experimental conditions. It has been demonstrated that AChE and BChE have similar kcat values (2100 and 1840 min(-1), respectively) against heroin, but with a large difference in KM (2170 and 120muM, respectively). Both AChE and BChE can catalyze 6-MAM hydrolysis to morphine, with relatively lower catalytic efficiency compared to the heroin hydrolysis. CocH1 can also catalyze hydrolysis of heroin (kcat=2150 min(-1) and KM=245muM) and 6-MAM (kcat=0.223 min(-1) and KM=292muM), with relatively larger KM values and lower catalytic efficiency compared to BChE. Notably, the KM values of CocH1 against both heroin and 6-MAM are all much larger than previously reported maximum serum heroin and 6-MAM concentrations observed in heroin users, implying that the heroin use along with cocaine will not drastically affect the catalytic activity of CocH1 against cocaine in the CocH1-based enzyme therapy for cocaine abuse.

Heroin is de-acetylated in the body to morphine in two steps. The intermediate 6-acetylmorphine (6-AM) is formed rapidly and is considered important for the pharmacological effect of heroin. In urine drug testing, an atypical pattern of morphine and 6-AM is known to occur in low frequency. The aim of this study was to investigate this atypical pattern in more detail and to identify responsible substances for a possible inhibition of the conversion from 6-AM to morphine. Urine samples were selected from a routine flow of samples sent for drug testing. Out of 695 samples containing morphine and 6-acetylmorphine, 11.5% had the atypical pattern of a 6-AM to morphine ratio above 0.26 as derived from a bimodal frequency distribution. An in vitro study of the conversion of 6-acetylmorphine to morphine in human liver homogenates demonstrated that a number of known carboxylesterase inhibitors were able to inhibit the reaction mimicking the situation in vivo. Compound 3 (3,6-Dimethoxy-4-acetoxy-5-[2-(N-methylacetamido)ethyl]phenanthrene) a substance formed from thebaine during the production of heroin was found to be a strong inhibitor. Liquid chromatography-mass spectrometry was used to identify possible inhibitors present in vivo. This part of the investigation demonstrated that several components may contribute to the effect. It is concluded that inhibition of liver carboxylesterase activity is a possible mechanism causing the atypical pattern and that one candidate compound is the result of the heroin production process. An inhibition of 6-AM metabolism is likely to increase the pharmacological effect of heroin and may be related to a higher risk of lethal toxicity.

As the most active metabolite of heroin, 6-monoacetylmorphine (6-MAM) can penetrate into the brain for the rapid onset of heroin effects. The primary enzymes responsible for the metabolism of 6-MAM to the less potent morphine in humans are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The detailed reaction pathways for AChE- and BChE-catalyzed hydrolysis of 6-MAM to morphine have been explored, for the first time, in the present study by performing first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the two enzymatic reaction processes follow similar catalytic reaction mechanisms, and the whole catalytic reaction pathway for each enzyme consists of four reaction steps. According to the calculated results, the second reaction step associated with the transition state TS2(a)/TS2(b) should be rate-determining for the AChE/BChE-catalyzed hydrolysis, and the free energy barrier calculated for the AChE-catalyzed hydrolysis (18.3 kcal mol(-1)) is 2.5 kcal mol(-1) lower than that for the BChE-catalyzed hydrolysis (20.8 kcal mol(-1)). The free energy barriers calculated for the AChE- and BChE-catalyzed reactions are in good agreement with the experimentally derived activation free energies (17.5 and 20.7 kcal mol(-1) for the AChE- and BChE-catalyzed reactions, respectively). Further structural analysis reveals that the aromatic residues Phe295 and Phe297 in the acyl pocket of AChE (corresponding to Leu286 and Val288 in BChE) contribute to the lower energy of TS2(a) relative to TS2(b). The obtained structural and mechanistic insights could be valuable for use in future rational design of a novel therapeutic treatment of heroin abuse.

As the most popularly abused one of opioids, heroin is actually a prodrug. In the body, heroin is hydrolyzed/activated to 6-monoacetylmorphine (6-MAM) first and then to morphine to produce its toxic and physiological effects. It has been known that heroin hydrolysis to 6-MAM and morphine is accelerated by cholinesterases, including acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE). However, there has been controversy over the specific catalytic activities and functional significance of the cholinesterases, which requires for the more careful kinetic characterization under the same experimental conditions. Here we report the kinetic characterization of AChE, BChE, and a therapeutically promising cocaine hydrolase (CocH1) for heroin and 6-MAM hydrolyses under the same experimental conditions. It has been demonstrated that AChE and BChE have similar kcat values (2100 and 1840 min(-1), respectively) against heroin, but with a large difference in KM (2170 and 120muM, respectively). Both AChE and BChE can catalyze 6-MAM hydrolysis to morphine, with relatively lower catalytic efficiency compared to the heroin hydrolysis. CocH1 can also catalyze hydrolysis of heroin (kcat=2150 min(-1) and KM=245muM) and 6-MAM (kcat=0.223 min(-1) and KM=292muM), with relatively larger KM values and lower catalytic efficiency compared to BChE. Notably, the KM values of CocH1 against both heroin and 6-MAM are all much larger than previously reported maximum serum heroin and 6-MAM concentrations observed in heroin users, implying that the heroin use along with cocaine will not drastically affect the catalytic activity of CocH1 against cocaine in the CocH1-based enzyme therapy for cocaine abuse.

Heroin is de-acetylated in the body to morphine in two steps. The intermediate 6-acetylmorphine (6-AM) is formed rapidly and is considered important for the pharmacological effect of heroin. In urine drug testing, an atypical pattern of morphine and 6-AM is known to occur in low frequency. The aim of this study was to investigate this atypical pattern in more detail and to identify responsible substances for a possible inhibition of the conversion from 6-AM to morphine. Urine samples were selected from a routine flow of samples sent for drug testing. Out of 695 samples containing morphine and 6-acetylmorphine, 11.5% had the atypical pattern of a 6-AM to morphine ratio above 0.26 as derived from a bimodal frequency distribution. An in vitro study of the conversion of 6-acetylmorphine to morphine in human liver homogenates demonstrated that a number of known carboxylesterase inhibitors were able to inhibit the reaction mimicking the situation in vivo. Compound 3 (3,6-Dimethoxy-4-acetoxy-5-[2-(N-methylacetamido)ethyl]phenanthrene) a substance formed from thebaine during the production of heroin was found to be a strong inhibitor. Liquid chromatography-mass spectrometry was used to identify possible inhibitors present in vivo. This part of the investigation demonstrated that several components may contribute to the effect. It is concluded that inhibition of liver carboxylesterase activity is a possible mechanism causing the atypical pattern and that one candidate compound is the result of the heroin production process. An inhibition of 6-AM metabolism is likely to increase the pharmacological effect of heroin and may be related to a higher risk of lethal toxicity.

As the most active metabolite of heroin, 6-monoacetylmorphine (6-MAM) can penetrate into the brain for the rapid onset of heroin effects. The primary enzymes responsible for the metabolism of 6-MAM to the less potent morphine in humans are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The detailed reaction pathways for AChE- and BChE-catalyzed hydrolysis of 6-MAM to morphine have been explored, for the first time, in the present study by performing first-principles quantum mechanical/molecular mechanical free energy calculations. It has been demonstrated that the two enzymatic reaction processes follow similar catalytic reaction mechanisms, and the whole catalytic reaction pathway for each enzyme consists of four reaction steps. According to the calculated results, the second reaction step associated with the transition state TS2(a)/TS2(b) should be rate-determining for the AChE/BChE-catalyzed hydrolysis, and the free energy barrier calculated for the AChE-catalyzed hydrolysis (18.3 kcal mol(-1)) is 2.5 kcal mol(-1) lower than that for the BChE-catalyzed hydrolysis (20.8 kcal mol(-1)). The free energy barriers calculated for the AChE- and BChE-catalyzed reactions are in good agreement with the experimentally derived activation free energies (17.5 and 20.7 kcal mol(-1) for the AChE- and BChE-catalyzed reactions, respectively). Further structural analysis reveals that the aromatic residues Phe295 and Phe297 in the acyl pocket of AChE (corresponding to Leu286 and Val288 in BChE) contribute to the lower energy of TS2(a) relative to TS2(b). The obtained structural and mechanistic insights could be valuable for use in future rational design of a novel therapeutic treatment of heroin abuse.

Diacetylmorphine deacetylates rapidly to 6-monoacetylmorphine and then to morphine. The immunomodulatory effects of diacetylmorphine are under investigation by several groups utilising various methods including in vitro cell culture; however, diacetylmorphine stability under these conditions is unknown. The aim of this study was to quantify diacetylmorphine degradation under cell culture conditions and to determine the mechanism by which this occurs. Diacetylmorphine degradation in a mouse splenocyte mitogenesis assay was investigated. Morphine and 6-monoacetylmorphine were quantified using HPLC with UV detection. After 6 h, approximately 73% of diacetylmorphine had been hydrolysed in the presence of cells. The half-life of diacetylmorphine was 1.4 h in cell media alone and 1.2-2.2 h in incubations containing cells, while the half-life of 6-monoacetylmorphine was 3.1 h in cell media alone and 0.99-1.2 h in incubations containing cells. 6-Monoacetylmorphine and morphine formation were found to be dependent on incubation time and diacetylmorphine concentration, and were not dependent on esterase activity, mitogen concentration, presence of erythrocytes and cell media evaporation. Only morphine formation was dependent on lymphocyte concentration. 6-Monoacetylmorphine formation was independent of cells and appeared to be due to the conditions of the cell culture (pH and temperature), while morphine formation was dependent to a greater extent on cells, but independent of esterase activity. The study highlights the limitations of conclusions made in previous studies which have not recognised diacetylmorphine instability.

1. In human blood, heroin is rapidly hydrolysed by sequential deacylation of two ester bonds to yield first 6-monoacetylmorphine (6-MAM), then morphine. 2. Serum butyrylcholinesterase (BCHE) hydrolyses heroin to 6-MAM with a catalytic efficiency of 4.5/min per mumol/L, but does not proceed to produce morphine. 3. In vitro, human erythrocyte acetylcholinesterase (AChE) hydrolyses heroin to 6-MAM, with a catalytic efficiency of 0.5/min per mumol/L under first-order kinetics. Moreover, erythrocyte AChE, but not BCHE is capable of further hydrolysing 6-MAM to morphine, albeit at a considerably slower rate. 4. Both hydrolysis steps by erythrocyte AChE were totally blocked by the selective AChE inhibitor BW284c51 but were not blocked by the BCHE-specific inhibitor, iso-OMPA (tetraisopropylpyrophosphoramide). 5. The brain synaptic form of AChE, which differs from the erythrocyte enzyme in its C-terminus, was incapable of hydrolysing heroin. 6. Heroin suppressed substrate hydrolysis by antibody-immobilized erythrocyte but not by brain AChE. 7. These findings reveal a new metabolic role for erythrocyte AChE, the biological function of which is as yet unexplained, and demonstrate distinct biochemical properties for the two AChE variants, which were previously considered catalytically indistinguishable.

Concomitant i.v. use of cocaine and heroin ("speedballing") is prevalent among drug-abusing populations. Heroin is rapidly metabolized by sequential deacetylation of two separate ester bonds to yield 6-monoacetylmorphine and morphine. Hydrolysis of heroin to 6-monoacetylmorphine is catalyzed by pseudocholinesterase. The pathway for hydrolysis of 6-monoacetylmorphine to morphine in vivo has yet to be established. Pseudocholinesterase and two human liver carboxylesterases [human liver carboxylesterase form 1 (hCE-1) and human liver carboxylesterase form 2 (hCE-2)] catalyze the rapid hydrolysis of ester linkages in cocaine. This investigation examined the relative catalytic efficiencies of hCE-1, hCE-2 and pseudocholinesterase for heroin metabolism and compared them with cocaine hydrolysis. Enzymatic formation of 6-monoacetylmorphine and morphine was determined by reverse-phase high-performance liquid chromatography. All three enzymes rapidly catalyzed hydrolysis of heroin to 6-monoacetylmorphine (hCE-1 kcat = 439 min-1, hCE-2 kcat = 2186 min-1 and pseudocholinesterase kcat = 13 min-1). The catalytic efficiency, under first-order conditions, for hCE-2-catalyzed formation of 6-monoacetylmorphine (314 min-1 mM-1) was much greater than that for either hCE-1 or pseudocholinesterase (69 and 4 min-1 mM-1, respectively). Similarly, the catalytic efficiency for hydrolysis of 6-monoacetylmorphine to morphine by hCE-2 (22 min-1 mM-1) was substantially greater than that for hCE-1 (0.024 min-1 mM-1). Cocaine competitively inhibited hCE-1-, hCE-2- and pseudocholinesterase-catalyzed hydrolysis of heroin to 6-monoacetylmorphine (Ki = 530, 460 and 130 microM, respectively) and 6-monoacetylmorphine hydrolysis to morphine (Ki = 710, 220 and 830 microM, respectively). These data demonstrate that metabolism of cocaine and heroin in humans is mediated by common metabolic pathways. The role of hepatic hCE-2 is particularly important for the hydrolysis of heroin to 6-monoacetylmorphine and of 6-monoacetylmorphine to morphine.

Selective inhibition of peripheral esterases by tri-ortho-tolyl phosphate in the mouse resulted in an increase in the analgetic activity of heroin, without affecting the activity of morphine. In vitro inhibition of esterases by paraoxon reduced the affinity of heroin for the opiate receptor, while that of morphine was unaffected. These results suggest that both central and peripheral esterases are involved in the metabolism of heroin and that interference with critical esterases can alter its pharmacologic and toxicologic effects.

The pharmacokinetics of intravenously administered heroin and its derived metabolites, 6-O-monoacetylmorphine, morphine, and the glucuronidated conjugates of morphine, were studied in dogs at doses of 0.1-0.5 mg/kg. The spontaneous hydrolysis of the sampled biological fluids was inhibited by tetraethyl pyrophosphate so that the heroin concentration at the times of sampling could be analyzed for the first time. Heroin is concomitantly rapidly metabolized and distributed among body tissues. Metabolic clearance of 2916 +/- 321 ml/min are largely extrahepatic and are sixfold greater than hepatic blood flow. Nevertheless, the terminal half-life of 60-90 min resembles that of morphine and is maintained by the rate-determining return of distributed heroin from esterase-free tissues. Normal renal clearances of 43 +/- 6 ml/min result in 1.6 +/- 0.2% of the dose being renally excreted unchanged. The large overall volume of distribution, 344 +/- 60 liters, is indicative of heroin's wide distribution and lipophilicity, which rapidly equilibrates heroin in the plasma with the cerebrospinal fluid. Heroin is concomitantly metabolized almost equally to 6-O-monoacetylmorphine and morphine. The monoacetylmorphine is metabolized concomitantly to morphine and glucuronide conjugates in a 4:3 ratio and exercises its own activity. Its time course is close to that of heroin, although the total clearance (2200 ml/min) and overall volumes of distribution (90-170 liters) were less. The integrated model of transformations and eliminations was constructed with concomitant metabolism of the heroin metabolite, 6-O-monoacetylmorphine, to morphine and glucuronide conjugates. The assumption that the glucuronide conjugates partition into the bile and systemic circulation in the same ratio as does the conjugate of the derived morphine metabolite gave pharmacokinetic parameters consistent with the morphine pharmacokinetics studied previously and provided excellent fits of the plasma level-time curves of all of the derived metabolites of heroin.