Plasma carboxylesterase

Several drugs, in particular neuropharmacological agents, feature a car-boxylate group esterified to an aminoalkyl moiety. As a rule, such lipophilic compounds are good substrates for hydrolases, and their duration of action is influenced by their rate of hydrolysis (see also Sect. 7.3.4). A simple example is that of procaine (7.56), which is rapidly inactivated by hydrolysis [41] [76a], Various hydrolases catalyze the reaction, in particular plasma cholinesterase and cellular carboxylesterases. As often reported, atropine and scopolamine are rapidly hydrolyzed by plasma carboxylesterases in rabbits (with very large differences between individual animals), but are stable in human plasma [1] [75] [76a] [110]. 

Malathion is an organophosphate cholinesterase inhibitor that is hydrolyzed and inactivated by plasma carboxylesterases much faster in humans than in insects, thereby providing a therapeutic advantage in treating pediculosis (see Chapter 7). Malathion is available as a 0.5% lotion (Ovide) that should be applied to the hair when dry 4-6 hours later, the hair is combed to remove nits and lice. 

Malathion (chemathion, mala-spray) requires conversion to malaoxon (replacement of a sulfur atom with oxygen in vivo, conferring resistance to mammalian species). Malathion can be detoxified by hydrolysis of the carboxyl ester linkage by plasma carboxylesterases, and plasma car-boxylesterase activity dictates species resistance to malathion. The detoxification reaction is much more rapid in mammals and birds than in insects. Malathion has been employed in aerial spraying of relatively populous areas for control of Mediterranean fruit flies and mosquitoes that harbor and transmit viruses harmful to human beings (e.g.. West Nile encephalitis virus). Evidence of acute toxicity from malathion arises only with suicide attempts or deliberate poisoning. 

Li B, Sedlacek M, Manoharan I, Boopathy R, Duysen EG, Masson P, Lockridge O (2005) Butyrylcholinesterase, paraoxonase, and albumin esterase, but no carboxylesterase, are present in human plasma. Biochem Pharmacol 70 1673-1684... 

In humans, erythrocytes contain an esterase that displays genetic polymorphism [86], This esterase has been called esterase D (ES-D), a name without connection to the above-presented A-, B-, and C-classification. Three carboxylesterases named HU1, HU2, and HU3 have been found in human liver microsomes. Other tissues where esterases have been found include brain, plasma, stomach, small intestine, and colon [79]. 

Carboxylesterases (EC 3.1.1.1) can be detected in most mammalian tissues. Besides organs with high carboxylesterase activity such as liver, kidney, and small intestine, esterase activity is present, e.g., in the brain, nasal mucosa, lung, testicle, and saliva. Compared to rat plasma, human plasma contains little carboxylesterase, its esterase activity being essentially due to cholinesterase [61][73][79][89-91],... 

The physiological functions of carboxylesterases are still partly obscure but these enzymes are probably essential, since their genetic codes have been preserved throughout evolution [84] [96], There is some evidence that microsomal carboxylesterases play an important role in lipid metabolism in the endoplasmic reticulum. Indeed, they are able to hydrolyze acylcamitines, pal-mitoyl-CoA, and mono- and diacylglycerols [74a] [77] [97]. It has been speculated that these hydrolytic activities may facilitate the transfer of fatty acids across the endoplasmic reticulum and/or prevent the accumulation of mem-branolytic natural detergents such as carnitine esters and lysophospholipids. Plasma esterases are possibly also involved in fat absorption. In the rat, an increase in dietary fats was associated with a pronounced increase in the activity of ESI. In the mouse, the infusion of lipids into the duodenum decreased ESI levels in both lymph and serum, whereas an increase in ES2 levels was observed. In the lymph, the levels of ES2 paralleled triglyceride concentrations [92] [98],... 

Enol esters are distinct from other esters not because of a particular stability or lability toward hydrolases, but due to their hydrolysis releasing a ghost alcohol (an enol), which may immediately tautomerize to the corresponding aldehyde or ketone. A well-studied example is that of vinyl acetate (CH3-C0-0-CH=CH2), a xenobiotic of great industrial importance that, upon hydrolysis, liberates acetic acid (CH3-CO-OH) and acetaldehyde (CH3-CHO), the stable tautomer of vinyl alcohol [25], The results of two studies are compiled in Table 7.1, and demonstrate that vinyl acetate is a very good substrate of carboxylesterase (EC 3.1.1.1) but not of acetylcholinesterase (EC 3.1.1.7) or cholinesterase (EC 3.1.1.8). The presence of carboxylesterase in rat plasma but not in human plasma explains the difference between these two preparations, although the different experimental conditions in the two studies make further interpretation difficult. 

For example, the hydrolysis of phenyl acetate (7.15) by carboxylesterase isozymes was investigated over a broad pH range, allowing many insights into their catalytic mechanisms [30] (see Chapt. 3). This substrate was also used together with various inhibitors to characterize esterases in human and rat tissues [31], Thus, the approximate values of Km (in pM) and Vmax (in pmol min 1 (g tissue)-1) in human tissues were 300 and 60 in liver micro-somes, 200 and 40 in liver cytosol, and 1500 and 250 in plasma, respective-... 

A variety of hydrolases catalyze the hydrolysis of acetylsalicylic acid. In humans, high activities have been seen with membrane-bound and cytosolic carboxylesterases (EC 3.1.1.1), plasma cholinesterase (EC 3.1.1.8), and red blood cell arylesterases (EC 3.1.1.2), whereas nonenzymatic hydrolysis appears to contribute to a small percentage of the total salicylic acid formed [76a] [82], A solution of serum albumin also displayed weak hydrolytic activity toward the drug, but, under the conditions of the study, binding to serum albumin decreased chemical hydrolysis at 37° and pH 7.4 from tm 12 1 h when unbound to 27 3 h for the fully bound drug [83], In contrast, binding to serum albumin increased by >50% the rate of carboxylesterase-catalyzed hydrolysis, as seen in buffers containing the hydrolase with or without albumin. It has been postulated that either bound acetylsalicylic acid is more susceptible to enzyme hydrolysis, or the protein directly activates the enzyme. 

In human blood, only 6-acetylmorphine was formed from heroin, with no 3-acetylmorphine or morphine being detected. Four kinetically distinct enzyme activities were seen, namely one in plasma, one in the cytosol of red blood cells, and two in red blood cell membranes [92], In human plasma at 37°, hydrolysis to 6-acetylmorphine occurs with a tm value of some minutes, the enzyme responsible being cholinesterase [90] [93]. These and other results tend to indicate that the formation of morphine from 6-acetylmorphine is due to tissue carboxylesterases, in particular cerebral enzymes [94],... 

Natural (-)-cocaine (7.57, Fig. 7.8), which has the (2/ ,3S)-configuration, is a relatively poor substrate for hepatic carboxylesterases and plasma cholinesterase (EC 3.1.1.8), and also a potent competitive inhibitor of the latter enzyme [116][121], In contrast, the unnatural enantiomer, (+)-(2S,3/ )-cocaine, is a good substrate for carboxylesterases and cholinesterase. Because hydrolysis is a route of detoxification for cocaine and its stereoisomers, such metabolic differences have a major import on their monooxygenase-catalyzed toxification, a reaction of particular effectiveness for (-)-cocaine. 

The L-dopa esters were also examined for their enzymatic hydrolysis in human plasma and/or by purified pig liver carboxylesterase (EC 3.ELI Table 8.1). In human plasma under the conditions of study, hydrolysis again followed pseudo-first-order kinetics. In all but two cases examined, enzymatic hydrolysis was slightly faster than chemical hydrolysis. For the methyl ester, rates of chemical and enzymatic hydrolysis were comparable, whereas the /erf-butyl ester was not hydrolyzed in plasma and was protected from chemical hydrolysis presumably by becoming bound to plasma proteins. 

In the case of carboxylesterase-catalyzed hydrolysis (Table 8.1), the Michaelis constant consistently indicated relatively low affinity for the enzyme, with a variation between substrates of one order of magnitude. Even less variation was seen in the maximal velocity of the reaction. It is interesting to note that, for the four compounds where comparisons are possible, a direct relationship exists between the rate of hydrolysis in plasma and the Vmax of carboxylesterase hydrolysis, suggesting comparable catalytic mechanisms. 

The best correlation equations obtained for the Km values in the presence of carboxylesterase (CE) or human plasma (HP) are given below as Eqns. 8.3 and 8.4, respectively. The statistical quality of the equations can be assessed by r2, the squared correlation coefficient, and q2, the cross-validated correlation coefficient (a measure of the predictive power of the equation, which is considered as acceptable when q2> 0.4). Both equations are statistically sound and have acceptable predictive power. 

Two examples of aryl esters are given in Table 8.5, namely the 4-chloro-phenyl and 4-nitrophenyl esters of nicotinic acid (8.33). Under physiological conditions of pH and temperature, these two compounds were clearly much more susceptible to chemical hydrolysis than the alkyl and arylalkyl esters in Table 8.5. Their affinity for carboxylesterase and human plasma hydrolases, as assessed by the Michaelis constant Km, was generally higher, while nothing can be said regarding Vmax values. 

A number of prodrugs in clinical use are esters of fatty acids. For example, haloperidol decanoate is of interest in slow-release preparations. This compound was hydrolyzed by such hydrolases as purified carboxylesterase but was reported to be stable in human blood or plasma and in a variety of rat tissue homogenates [107], The source of this apparent lack of reactivity was competitive binding to blood and tissue proteins. In other words, protein binding sequesters this very lipophilic prodrug and prevents enzymatic hydrolysis, thereby slowing its activation and prolonging its in vivo effects. 

In contrast to the A-monosubstituted carbamates, the A,A-disubstituted analogues (8.124 and 8.125, R = R R"NCO R = Me or Et R" = Me, Et, i-Pr, etc.) proved very stable at pH 7.4 in both buffer and plasma, with less than 5% degradation in 4 d. In fact, these compounds were potent inhibitors of plasma cholinesterase (EC 3.1.1.8), with K values ranging from 600 to 3 nM. Although these carbamates were stable in plasma, they underwent rapid bioactivation in liver, as demonstrated with mouse and rat liver microsomes. For example, the A,A-dimethylcarbamate (8.124, R = Me2NCO) was bioactivated in rat liver microsomes with t1/2 of ca. 30 min. Two routes of bioactivation were postulated, namely direct carboxylesterase-catalyzed hydrolysis, and cytochrome P450 mediated A-dealkylation to a more labile A-monosubstituted carbamate. 

Most of the hemiesters 8.136 underwent no or little enzymatic degradation in human plasma, in agreement with the known inertness of hemiesters toward cholinesterase (see Chapt. 7). In contrast, very rapid hydrolysis was usually seen in pig and rat liver preparations, indicating the involvement of carboxylesterases. The only inert compound was the 3,3-dimethylglutarate hemiester of paracetamol (8.136, X = C(CH3)2CH2, Fig. 8.12). Data on the hydrolysis of such prodrugs by human hepatic enzymes will be welcome. 

Esters, amides, hydrazides, and carbamates can all be metabolized by hydrolysis. The enzymes, which catalyze these hydrolytic reactions, carboxylesterases and amidases, are usually found in the cytosol, but microsomal esterases and amidases have been described and some are also found in the plasma. The various enzymes have different substrate specificities, but carboxylesterases have amidase activity and amidases have esterase activity. The two apparently different activities may therefore be part of the same overall activity. 

Capecitabine is a fluoropyrimidine carbamate prodrug that has nearly 70-80% oral bioavailability. It undergoes extensive metabolism in the liver by the enzyme carboxylesterase to an intermediate, 5 -deoxy-5-fluorocytidine. This in turn is converted to 5 -deoxy-5-fluorouridine by the enzyme cytidine deaminase. The 5 -deoxy-5-fluorouridine metabolite is then hydrolyzed by thymidine phosphorylase to fluorouracil in the tumor. (The expression of thymidine phosphorylase is significantly higher in a broad range of solid tumors than in corresponding normal tissue.) Peak plasma levels are achieved in about 1.5 hours, and peak fluorouracil levels are reached at 2 hours after oral administration. 

Tissue esterases have been divided into two classes the A-type esterases, which are insensitive, and the B-type esterases, which are sensitive to inhibition by organo-phosphorus esters. The A esterases include the arylesterases, whereas the B esterases include cholinesterases of plasma, acetylcholinesterases of erythrocytes and nervous tissue, carboxylesterases, lipases, and so on. The nonspecific arylesterases that hydrolyze short-chain aromatic esters are activated by Ca2+ ions and are responsible for the hydrolysis of certain organophosphate triesters such as paraoxon (Figure 10.10B). 

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