Cocaine Detoxification by Human Plasma Butyrylcholinesterase

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

145, 363–371 (1997)

TO978187

Cocaine Detoxification by Human Plasma Butyrylcholinesterase1 Thomas J. Lynch,*,2 Carol E. Mattes,*,† Anu Singh,† Roy M. Bradley,* Roscoe O. Brady,‡ and Kenneth L. Dretchen† *Pharmavene, Inc., Gaithersburg, Maryland 20850; †Department of Pharmacology, Georgetown University Medical Center, Washington, DC 20007; and ‡6026 Valerian Lane, Rockville, Maryland 20852 Received July 18, 1996; accepted April 17, 1997

Cocaine Detoxification by Human Plasma Butyrylcholinesterase. Lynch, T. J., Mattes, C. E., Singh, A., Bradley, R. M., Brady, R. O., and Dretchen, K. L. (1997). Toxicol. Appl. Pharmacol. 145, 363–371. The ability of human plasma butyrylcholinesterase (BChE) to detoxify cocaine in vivo was evaluated. Intravenous administration of BChE, at doses sufficient to increase the plasma levels of the enzyme as much as 800-fold, produced no adverse effects on the cardiovascular, autonomic, or central nervous systems of rats. Most of the enzyme could be recovered in the plasma immediately after administration and remained active with a b-t12 of 21.6 { 2.4 hr. Pretreatment of chloralose–urethane anesthetized rats with BChE, 0.1–7.8 mg/kg, decreased the hypertensive and arrhythmogenic effects produced by cocaine and increased the lethal dose of cocaine by three- to fourfold. Treatment of conscious rats with 1 and 10 mg/kg BChE decreased the incidence of seizures and deaths produced by a prior dose of cocaine (80 mg/kg, ip). These results suggest that BChE would provide a safe and highly efficacious treatment for cocaine intoxication. q 1997 Academic Press

Cocaine abuse, a leading cause of death among urbandwelling young adults, continues to be a significant social and medical problem (Marzuk et al., 1995). The most severe adverse reactions to cocaine include cardiac arrhythmias, myocardial infarction, stroke, seizures, and sudden death. No current therapies are generally effective against all the toxic effects of cocaine, and the physician is limited to treating individual symptoms (Shrank, 1992; Merigan et al., 1994; Hollander, 1995). Moreover, some effects of cocaine can be exacerbated by concurrent treatment of other symptoms such as myocardial infarction, seizures, and cardiovascular abnormalities (Nelson and Hoffman, 1995). For instance, b-adrenergic blockade by drugs such as propranolol may lead to hypertensive crisis from the unopposed a-adren1

This work was supported, in part, by Grant R43-DA07007 from the National Institute on Drug Abuse. A preliminary report of some of these data was presented at the 1992 FASEB meeting (Dretchen, K. L., Singh, A., Bradley, R. M., and Lynch, T. J. (1992). FASEB J. 6, A1282). 2 Present address: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, 1401 Rockville Pike, HFM-340, Rockville, MD 20852.

ergic activity of cocaine. A more satisfactory clinical approach might be to reduce the toxicity of cocaine by accelerating its metabolic inactivation. The biological activities of cocaine, as well as certain muscle relaxants (e.g., succinylcholine) and local anesthetics, are terminated principally through hydrolysis by plasma butyrylcholinesterase (BChE; EC 3.1.1.8), also referred to as pseudocholinesterase (Vitti and Boni, 1985; Whittaker, 1986; Ambre, 1987; Jatlow, 1988). Lower than normal levels of plasma BChE have been reported in patients experiencing adverse reactions to cocaine (Devenyi, 1989; Hoffman et al., 1992a; Om et al., 1993). In one study, emergency room patients with lifethreatening reactions to cocaine had a mean plasma BChE value lower than those with non-life-threatening symptoms (Hoffman et al., 1992a). In one study, emergency room patients with life-threatening reactions to cocaine had a mean plasma BChE value lower than those with non-life-threatening symptoms (Hoffman et al., 1992a). In the most extreme cases, patients homozygous for a ‘‘silent’’ variant of BChE express no detectable BChE activity (Liddell et al., 1962). Patients with low levels of BChE or defective variants of BChE experience unusually prolonged responses to succinylcholine because of the reduced rate at which the drug is metabolized (Bauld et al., 1974; Whittaker, 1986). Similarly, plasma from homozygotes for the ‘‘atypical’’ variant of BChE has little or no ability to hydrolyze cocaine in vitro, and the hydrolysis of cocaine in plasma from heterozygotes (wild type/atypical) proceeds at about half the normal rate (Jatlow et al., 1979; Stewart et al., 1979). Therefore, it has been suggested that individuals with defective variants of BChE may also be at higher risk of life-threatening reactions to cocaine (Jatlow et al., 1979; Stewart et al., 1979; Devenyi, 1989). It seems reasonable that a given dose of cocaine might produce higher peak levels and a significantly longer halflife if the metabolism of the drug was impaired by either low levels or atypical forms of BChE. This could lead to a more intense and long-lasting response to the drug, which could exacerbate the toxicity of cocaine in these individuals. Moreover, larger and/or repeated doses of cocaine would saturate the available BChE, even in normal individuals,

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0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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leading to a typical overdose of the drug. In both cases, increasing the levels of plasma BChE should increase the rate at which cocaine is inactivated in vivo and may therefore be of value in treating patients experiencing adverse reactions to cocaine. Here we describe an efficient method for the large-scale purification of BChE from human plasma. The enzyme produced by this method is virtually homogeneous and has the characteristics of wild-type BChE purified by conventional procedures. When administered intraveneously to rats, the enzyme remains active in the plasma for prolonged periods and has no discernable adverse effects on the cardiovascular, autonomic, or central nervous systems. Exogenous human plasma BChE is able to counter certain toxic effects of cocaine on the cardiovascular and central nervous systems of rats. Of particular importance is the ability of BChE to rescue an animal from a prior lethal dose of cocaine. These results suggest the potential therapeutic value of BChE in the treatment of cocaine intoxication. METHODS Materials. Human plasma Fraction IV-4 (Cohn et al., 1946) was obtained through the Plasma Derivatives Program of the Jerome H. Holland Laboratory, American Red Cross (Rockville, MD), and was stored at 0707C until used. Fraction IV-4 contains approximately 90% of the BChE but only 6–7% of the protein present in whole plasma (Surgenor and Ellis, 1954). DEAE-Sepharose Fast Flow was from Pharmacia (Uppsala, Sweden); enzyme substrates and inhibitors were obtained from Sigma (St. Louis, MO). (0)-Cocaine (98–100% purity) was obtained from the National Institute on Drug Abuse (Bethesda, MD). All other chemicals were of reagent grade. Procainamide (Sigma) was coupled to CH-Sepharose (or to ECH-Sepharose, 6-amino hexanoic acid-Sepharose, Pharmacia) in the presence of 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; Pierce, Rockford, IL). The swollen agarose was resuspended at room temperature in 3 volumes of 17 mM procainamide (fourfold molar excess over the carboxyl groups of the agarose), and solid EDC was added to the stirred slurry to a final concentration of 170 mM. The pH was maintained between 5.0 and 5.5 for the first 4 hr of the reaction, which was continued overnight. Coupling was monitored by the absorbance of the unbound procainamide, assuming an extinction coefficient at 278 nm of 13,600 M01rcm01. The final density of coupled ligand was 11–20 mmolrml01 of packed agarose. BChE assays. Except where indicated, BChE was assayed using 50 mM benzoylcholine as substrate, at 257C in 67 mM phosphate, pH 7.4, 0.2 mg/ml bovine serum albumin, by monitoring absorbance at 240 nm (Kalow and Lindsey, 1955). The concentration of BChE in the assays was adjusted to 6–50 ng/ml to ensure linearity with respect to time and enzyme concentration. In vivo activities of BChE were determined in plasma clarified from whole blood samples at 1500g for 5 min. BChE was also assayed under similar conditions with 4 mM acetylthiocholine, 4 mM butyrylthiocholine, or 4 mM propionylthiocholine as substrates, all in the presence of 254 mM 5,5*-dithio-bis-(2-nitrobenzoic acid), monitoring absorbance at 410 nm (Ellman et al., 1961; Deitz et al., 1973). The inhibition of BChE (with benzoylcholine as substrate) was determined after incubating the enzyme for 20 min at 257C with 0.5 to 5 mM dibucaine, 10 to 100 mM NaF, or 10 to 100 mM 1,5-bis-(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284C51). The rates of inactivation of BChE by 1 to 10 mM physostigmine or 0.5 to 5 mM tetraisopropylpyrophosphoramide (iso-OMPA) were determined under the same assay conditions, immediately after adding the inhibitor.

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The protein concentration of purified BChE was measured by its absorbance at 280 nm, assuming an extinction coefficient of 1.8 mlrmg01rcm01 (Lockridge et al., 1987). This value was confirmed by quantitative amino acid analysis (data not shown). The protein concentrations of other samples were determined by the method of Bradford (1976) or by a modified Lowry assay (Stoscheck, 1990). Electrophoresis was performed in the presence of SDS (Laemmli, 1970) or under native conditions (Davis, 1964). Native gels were stained either with Coomassie blue or with a cytochemical reaction for esterase activity utilizing naphthol butyrate and Fast blue RR tetrazolium (Allen and Hunter, 1960; McComb et al., 1965). Purification of BChE. The entire procedure was carried out at 4–87C. The precipitated proteins in 2 kg of Fraction IV-4 were resuspended in 5 volumes of cold deionized water and extracted overnight with constant stirring. The suspension was titrated to pH 4.4 with 1 M citric acid and clarified by centrifugation at 13,000g for 1 hr. A more convenient clarification method was also used. After extracting Fraction IV-4 for 4–6 hr, the suspension was titrated to pH 4.4 and then allowed to settle overnight. The suspension could then be clarified at 2500g for 30 min. The resulting supernatant was diluted with 1 volume of cold deionized water and applied directly to a 3.5-liter DEAE-Sepharose column that had been equilibrated with 25 mM sodium acetate, pH 4.4 (buffer A). The column was washed with 6–9 liters of buffer A and eluted with a 10-liter linear gradient of 0–200 mM NaCl in buffer A. Fractions containing BChE activity were pooled, titrated to pH 6.5 with 0.5 M Na2HPO4 , and loaded on a 200-ml procainamide–agarose column equilibrated with 25 mM phosphate, 1 mM EDTA, pH 7.4 (buffer B). The column was washed with 2– 2.5 liters of 250 mM NaCl in buffer B, after which BChE was eluted either by a 1-liter gradient to 1 M NaCl in buffer B or, more conveniently, by step elution with 1 M NaCl. The pooled enzyme was dialyzed overnight against 20–40 volumes of 25 mM phosphate buffer, pH 7.4 (buffer C), or, more conveniently, diafiltered against 5 volumes of buffer C (50-kDa MWCO membrane; Millipore, Bedford, MA). After buffer exchange, the pool was applied to a 300-ml DEAE-Sepharose column equilibrated with buffer C and eluted with a 2-liter gradient of 0–200 mM choline chloride in buffer C. The BChE pool was loaded directly on a second procainamide affinity column equilibrated with buffer B, with a bed volume of 20–50 ml. The column was washed with 12 bed volumes of 250 mM NaCl and eluted with 1 M NaCl, all in buffer B. The pooled BChE was concentrated to Ç5 mg/ml in a stirred pressure cell fitted with a 50-kDa MWCO membrane (Filtron, Northborough, MA) and dialyzed into 20 mM phosphate, 154 mM NaCl, 0.5 mM EDTA, pH 7.4. The concentrated enzyme was filter-sterilized through a 0.22-mm membrane (Millipore, Millex-GV) and stored at 47C. Under these conditions purified BChE retained more than 90% of its original activity after 18 months. Animal experiments. All experiments were performed on male Sprague–Dawley rats weighing 250–350 g, in accordance with the requirements and recommendations in the Guide for the Care and the Use of Laboratory Animals (U.S. Public Health Service, NIH Publication No. 8523, 1985) and in the Guiding Principles in the Use of Animals in Toxicology (Society of Toxicology). For neurological testing on a rotorod and inclined plane, rats were injected with 7.8 mg/kg BChE via the tail vein. The neurological tests were performed 1 hr, 1 day, 2 days, and 3 days after injection of the enzyme. Cardiovascular and pharmacokinetic experiments were performed on rats anesthetized by intraperitoneal administration of 60 mg/kg a-chloralose and 800 mg/kg urethane. Catheters were placed in the femoral artery (to monitor blood pressure) and femoral vein (to deliver the enzyme and/or cocaine as appropriate) and lead II ECG was recorded. BChE in concentrations ranging from 0.1 to 22.5 mg/kg was infused while monitoring blood pressure, heart rate, and EKG profile. Blood samples were drawn before and immediately after infusion of enzyme, and at appropriate intervals thereafter to determine the recovery of the enzyme. Blood pressure and heart rate were also monitored in BChE-treated rats immediately following neurological tests 1 hr, 1 day, 2 days, or 3 days after administering the enzyme. In all cases, similar

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TABLE 1 Enzymatic Properties of Purified BChE Specific activitiesa Substrate Acetylthiocholine Butyrylthiocholine Propionylthiocholine Benzoylcholine

a b

Inhibitionb Rate

Inhibitor

{ { { {

Dibucaine Fluoride Physostigmine iso-OMPA BW284C51

498 861 998 195

12.9 24.4 20.9 7.3

Inhibition or rate constant Ki Ki ki ki Ki

Å Å Å Å Å

1.4 4.6 4.5 1.6 4.3

1 1 1 1 1

1006 M 1005 M 106 M01rmin01 104 M01rmin01 1005 M

Activities in mmolrmin01rmg01 { SE. Assayed in the presence of 50 mM benzoylcholine, 0.2 mg/ml bovine serum albumin, pH 7.4, 257C.

measurements made on animals not treated with BChE served as controls. Finally, the cardiovascular responses to phenylephrine (0.5–50 mg/kg) and acetylcholine (0.02 mg/kg–1.0 mg/kg) were assessed in untreated controls and in animals pretreated with 7.8 mg/kg of BChE. The same system was used to evaluate the ability of BChE to counter the effects of cocaine on cardiovascular function: hypertension at low doses of cocaine, ventricular arrhythmia, and death at higher doses of cocaine. Increasing doses of cocaine (1–25 mg/kg) were infused at equal intervals via the femoral vein into untreated controls and into rats previously treated with 0.1–7.8 mg/kg BChE. Cardiovascular function was monitored throughout the treatment period, and the effects of each dose of cocaine were allowed to completely dissipate before a subsequent dose of the drug was administered. The ability of BChE to reverse the effects of a prior dose of cocaine was determined in conscious rats, each of which had been implanted with a jugular catheter exiting through the back of the neck through which the enzyme was delivered. The rats received intraperitoneal injections of 80 mg/ kg cocaine, approximately the LD50. Control animals received no further treatment; experimental animals received 1–10 mg/kg BChE (iv) 3 min after the injection of cocaine, when the response to cocaine was at its peak in most animals. The half-life of BChE in the plasma of four rats implanted with jugular catheters was determined over the course of 48 hr following treatment with 7.8 mg/kg BChE delivered through the catheters. Whole blood (0.5 ml) was withdrawn through the catheters at defined intervals (see Fig. 2) and equal volumes of saline were administered as volume replacements with each sample taken. Plasma was prepared for analysis of BChE activity and total protein concentrations. During this period of time, the plasma protein concentrations varied as much as 33%, presumably as a result of repeated sampling and the consequent hemodilution. To correct for this variation, BChE levels were expressed as specific activities with respect to total plasma protein.

RESULTS

Biochemical Properties of BChE BChE was produced from Fraction IV-4 with a final purity exceeding 90% and an overall yield of 30–40%. Typically, 2 kg of Fraction IV-4 is derived from 200 liters of plasma, from which 150–200 mg of BChE was isolated. The enzymatic activities of purified BChE with various substrates are given in Table 1. As expected, the activities are maximal with butyrylthiocholine and propionylthiocholine, which dis-

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tinguishes BChE from acetylcholinesterase. The specific activity of the enzyme with benzoylcholine as substrate, 195 mmolrmin01rmg01, is a criterion for the purity of the enzyme and compares favorably with the maximum previously reported value of 200 mmolrmin01rmg01 (Lockridge et al., 1987). The sensitivities of the purified enzyme to various inhibitors are also shown in Table 1. Strong inhibition by isoOMPA and relatively weak inhibition by BW284C51 also distinguish BChE from acetylcholinesterase (Whittaker, 1986). Furthermore, the sensitivities of the purified enzyme to the inhibitors, dibucaine, fluoride, and RO 02-0683 are those of the normal phenotype (Harris and Whittaker, 1961; Kalow and Genest, 1957; Deitz et al., 1973; Evans and Wardell, 1984). Hence, atypical variants of BChE, if present, are below the limits of detection even though pooled plasma is the ultimate source of the enzyme. These preparations of BChE have also been characterized by electrophoresis under native conditions and in the presence of SDS (Fig. 1). On nondenaturing gels, a single band of Mr Ç390,000 is seen after staining with Coomassie blue, which agrees well with the mass of the native enzyme determined hydrodynamically (Muensch et al., 1976). This band also reacts in a cytochemical reaction for esterases, so the BChE activities can be assigned to the major protein species present in these preparations. On reducing SDS gels, purified BChE runs as a Mr Ç90,000 band (major component, equivalent to the monomeric subunit) and a minor band of Mr Ç180,000, which has previously been reported to be a covalently linked dimer which survives reduction with b-mercaptoethanol (Lockridge et al., 1979; Lockridge and La Du, 1982). In Vivo Activity of BChE Anesthetized rats were serially injected (iv) with increasing amounts of 5 mg/ml BChE, and plasma samples taken after each injection were assayed for BChE activity. Doses of BChE of 4.5 to 22.5 mg/kg and of 0.7 to 3 mg/kg produced

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FIG. 1. Purified BChE. Lane 1: On nondenaturing gels, purified BChE migrates as a single protein, visualized by Coomassie blue staining. Lane 2: On an identical gel, the same band seen in lane 2 stains intensely in a cytochemical reaction for esterase activity. Lane 3: After reduction with bmercaptoethanol, purified BChE migrates on SDS gels as an Mr Ç90,000 monomer and a minor band approximately twice this size, previously reported to be a dimer coupled by bonds refractory to b-mercaptoethanol. Positions of standards (Mr 1 1003) are indicated to either side.

linear increases in plasma BChE levels. After single iv doses of 0.1–7.8 mg/kg BChE, plasma BChE increased 7-fold to nearly 800-fold over basal levels, and the average recoveries of the injected enzyme varied from 57.8 to 129.4%. The plasma t12 of 7.8 mg/kg BChE was determined in four animals over 48 hr. The clearance of BChE from the plasma followed biphasic kinetics, with an a-t12 of 3.4 { 0.05 hr and a b-t12 of 21.6 { 2.4 hr (Fig. 2). No effect on the neurological function of rats treated with 7.8 mg/kg BChE was apparent from the rotorod and inclined plane tests at any time from 1 hr to 3 days after treatment with the enzyme. Neither were blood pressure, heart rate, or lead II ECG affected by BChE in doses ranging from 0.1 to 23 mg/kg iv. In normal animals the mean blood pressure was 81.2 { 5.5 mm Hg and the heart rate was 405 { 12 BPM. No significant changes were observed in either of these values or in the EKG patterns immediately after infusion of the enzyme or during the subsequent 3 days. The effect of the enzyme on autonomic function was assessed by comparing the cardiovascular responses to acetylcholine and phenylephrine in control and BChE-treated animals. Increasing plasma BChE levels would be expected to enhance the turnover of acetylcholine, which is a substrate for the enzyme. Control responses to acetylcholine (0.5–1 mg/kg) were first obtained for each animal, after which 7.8 mg/kg BChE was administered iv. The response to higher doses of acetylcholine (1–1000 mg/ml) was then redetermined. Treatment with the enzyme decreased the normal, hypotensive response to acetylcholine about 120-fold (Fig. 3A). For example, in enzyme-treated animals, there was no detectable response to 1 mg/kg acetylcholine, and only a 30%

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decrease in blood pressure after 1000 mg/kg acetylcholine (a dose that would be fatal in control animals). Three days after treatment with BChE, the response to acetylcholine was still shifted about 3-fold to the right. In contrast, treatment with 7.8 mg/kg BChE had no significant effect on the hypertensive response to phenylephrine (Fig. 3B). The reflex bradycardia produced by all doses of phenylephrine was similar in both groups of animals. For example, 5 mg/kg of phenylephrine produced a heart rate of 318 { 19 BPM in control animals and a rate of 320 { 39 BPM in rats immediately after treatment with 7.8 mg/kg BChE. The lack of any effect of BChE on the reflex bradycardia produced by phenylephrine indicates that cholinergic synapses are inaccessible to the enzyme. This result and the normal neurological performance of enzyme-treated animals indicate that even large increases in plasma BChE levels do not influence cholinergic function. Protection against Cocaine Toxicity In untreated anesthetized animals, low doses (õ2 mg/ kg, iv) of cocaine produced transient hypertension whereas higher doses (2 mg/kg, iv) produced ventricular arrhythmia and death within 30 sec (Fig. 4 and Table 2). Prior treatment of the animals with BChE (0.1–7.8 mg/kg) provided significant protection against the effects of cocaine at all doses. For instance, the hypertension caused by 1 mg/kg cocaine was reduced by more than twofold by treatment with 7.8 mg/kg and only modest changes in blood pressure were seen at normally lethal doses of cocaine (Fig. 4). In a separate series of experiments, 4 mg/kg cocaine, iv, elicited transient hypotension (average decrease of 18.4 { 11.0 mm Hg) and mild arrhythmias in BChE-treated animals (7.8 mg/kg, n Å 5), but all survived. Finally, treatment with as little as 0.1 mg/kg BChE increased the lethal dose of cocaine by threefold (Table 2). Conscious rats were also used to test the ability of BChE to reverse the toxic effects of cocaine. At a dose of cocaine of 80 mg/kg, delivered intraperitoneally, nearly half of the animals not receiving subsequent treatment with BChE developed convulsions and died (Table 3). On average, convulsions developed 5–6 min after treatment with cocaine, and death occurred in about 9 min. Prior to these events, the animals became increasingly hyperactive (up to the point at which seizures commenced), which was the first symptom of the potentially fatal treatment with cocaine. In two other groups of animals treated with the same dose of cocaine, 1 or 10 mg/kg BChE was delivered through jugular catheters after hyperactivity was well established but prior to the onset of convulsions (i.e., 2–3 min after the ip injection of cocaine). Of those animals treated with 10 mg/kg BChE, only 1 developed convulsions and all survived (Table 3). Treatment with 1 mg/kg BChE reduced the incidence of convulsions to 3 of 12 animals and only 1 animal died.

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FIG. 2. Elimination half-life of BChE from plasma. Residual BChE activity was determined at the time points indicated after iv injection of 7.8 mg/ kg BChE into each of four rats. Enzyme activity (measured with benzoylcholine as substrate) was normalized by plasma protein concentration to correct for changes in plasma protein concentration due to repeated sampling. Observed specific activities (mmolrmin01rmg01 of plasma protein, solid symbols) were fitted to a one-compartment (solid line) or two-compartment model (dashed line). The results from a single animal are represented.

DISCUSSION

The results of this study indicate that human plasma BChE could provide an effective treatment for cocaine intoxication. Three elements crucial to the development of a practical therapeutic have been demonstrated here: (i) a method for efficiently producing large quantities of the enzyme; (ii) the apparent safety of BChE itself, even when administered at extremely high doses; and (iii) the efficacy of exogenous BChE in reversing the toxic effects of cocaine on the cardiovascular and central nervous systems. The method for purifying BChE from Cohn Fraction IV4 described here was adopted from other methods utilizing whole plasma as the starting material (Lockridge and La Du, 1978; Lockridge et al., 1987). Because Fraction IV-4 is enriched in BChE and produced in large quantities by commercial human plasma fractionators, it offers a practical source for large-scale production of the enzyme. It is notable that the starting material is derived from large numbers of plasma donors, who presumably represent various phenotypes of BChE. Nevertheless, the purified enzyme exhibits the enzymatic properties of wild-type BChE (Table 1), and its ability to hydrolyze BChE has previously been demonstrated (Mattes et al., 1996a). A recombinant form of human BChE might be considered an alternative to the plasma-derived enzyme (Prody et al., 1987; Arpagaus et al., 1990). However, despite the expres-

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sion of small quantities of the recombinant enzyme in experimental systems (Soreq et al., 1989; Neville et al., 1992; Vellom et al., 1993), the commercial production of recombinant BChE may not be feasible at this time since the enzyme is a large, heavily glycosylated protein of complex tertiary and quaternary structure (Lockridge et al., 1987; Lockridge, 1988; Chatonnet and Lockridge, 1989; see also, Sussman et al., 1991; Cygler et al., 1993). A third approach is suggested by the recent development of catalytic antibodies capable of hydrolyzing cocaine (Landry et al., 1993). However, the catalytic efficiency of these antibodies, 50- to 500-fold lower than that of human BChE (Mattes et al., 1996), will need to be increased before they offer a practical alternative to plasma-derived BChE. Of great importance is the apparent safety of exogenous BChE. Even large doses of the enzyme produced no adverse effects on cardiovascular function or behavior. These results predict that BChE could be safely used to treat patients, even when such treatment might increase plasma BChE levels severalfold. This prediction is supported by two observations. First, cruder preparations of BChE than those used in this study have been used clinically to reverse the prolonged apnea caused by the treatment of BChE-deficient patients with succinylcholine (Goedde et al., 1967; Scholler et al., 1977) or with mivacurium (Naguib et al., 1995; Ostergaard et al., 1995). In these studies, no adverse effects of the enzyme treatment were reported. Second, rare individuals

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FIG. 4. Effect of BChE on cocaine-induced hypertension. In animals not treated with BChE, 1 mg/kg cocaine produced a 56% increase in blood pressure. In animals pretreated with 7.8 mg/kg BChE, the same dose of cocaine produced only a slight increase in blood pressure, not significantly different from normal values. Higher doses of cocaine (1.5 and 2.5 mg/kg) produced only modest increases in blood pressure in the enzyme-treated animals. (Average of doses ranged from 1.8 to 3.0 mg/kg cocaine. One animal experienced a transient period of arrythmia and a decrease in blood pressure; the data from this animal were excluded from the average.) Note that 2 mg/kg cocaine is lethal in animals not treated with BChE (see Table 2). The enzyme treatment alone had no effect on blood pressure. For all groups, n Å 5. *Significantly different from control, p õ 0.5 (Student t test).

FIG. 3. Effect of BChE on responses to acetylcholine and phenylephrine. (A) Immediately following treatment with 7.8 mg/kg BChE, the dose of acetylcholine needed to produce an equivalent decrease in blood pressure was about 100-fold greater than that in untreated controls. Three days after treatment with 7.8 mg/kg BChE, the decrease in blood pressure produced by increasing doses of acetylcholine was similar to that in untreated controls. (B) Treatment of animals with 7.8 mg/kg BChE had no significant effect on the increase in blood pressure evoked by increasing doses of phenylephrine. The same result was obtained 3 days after enzyme treatment (data not shown). For all groups, n Å 4.

have been identified who express elevated levels of plasma BChE (Yoshida and Motulsky, 1969; Delbruck and Henkel, 1979; Krause et al., 1988). This condition produces no known symptoms in the effected individuals other than a resistance to drugs such as succinylcholine. Therefore, it is likely that the clinical use of BChE would have few, if any, adverse side effects. It should be noted that while the recovery and in vivo stability of the enzyme was excellent (b-t12 of 21.6 hr), the biphasic clearance of BChE from the plasma was not expected given the large size of the protein. In contrast to the results obtained here, Raveh et al. (1993) reported a t12 of

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46 hr for human BChE in the rat, fit to a single compartment model. The reason for the differences between the two studies is unknown, but both suggest the prolonged stability of BChE in vivo. In comparison, the half-life of plasma BChE in humans is approximately 11 days (Ostergaard et al., 1988). The administration of human BChE significantly reduced the toxic effects of cocaine on the cardiovascular and central nervous systems of rats. This result complements the observation that the inhibition of BChE by iso-OMPA increases the susceptibility of mice to cocaine (Hoffman et al., 1992b).

TABLE 2 Lethal Dose of Cocainea Pretreatment with BChE (mg/kg) 0 0.1 1.0 7.8

(n (n (n (n

Å Å Å Å

4) 2) 3) 4)

Dose of cocaine (mg/kg) 1.96 { 0.61 6.00 8.57 { 0.72* 8.72 { 2.32*

a Intravenous dose of cocaine ({SE) needed to produce cardiovascular collapse in chloralose–urethane anesthetized rats. * Significantly different from controls, p õ 0.05 (Student t test).

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TABLE 3 Cocaine-Induced Convulsions/Deaths a Dose of BCheb (mg/kg)

Convulsions/total

Deaths/total

0 1.0 10

5/12 3/12 1/14*

5/12 1/12* 0/14*

a

80 mg/kg cocaine delivered ip to all animals. Indicated dose of BChE delivered iv, 3 min after treatment with cocaine. * Significantly different from controls, p õ 0.05; (x2 test). b

However, others have obtained the opposite result: inhibition of endogenous BChE by iso-OMPA in rats and pigs has been reported to decrease the toxic effects of cocaine on the cardiovascular, respiratory, and central nervous systems (Kambam et al., 1992, 1993). It may be that the differences in the results of cholinesterase inhibition reflect differences in the metabolism of cocaine in the various species. The underlying reason for this apparent contradiction need not be resolved here, since the direct test of the efficacy of exogenous BChE in this setting is the administration of exogenous BChE, not the inhibition of the endogenous enzyme. It is assumed that the effectiveness of BChE in countering the toxicity of cocaine is due at least in part to the ability of BChE to hydrolyze cocaine to inactive metabolites (Misra et al., 1975; Stewart et al., 1977; Ambre et al., 1982; Matsubara et al., 1984). Evidence supporting this assumption is presented in Mattes et al. (1997). It is also possible that other consequences of BChE’s catalytic activity contribute to its effectiveness. For instance, the two major pathways by which pharmacologically active metabolites of cocaine are produced in significant quantities are oxidation and hydrolysis. The major active product of oxidation, norcocaine (the N-demethylated derivative), is also hydrolyzed by BChE with even greater efficiency than is cocaine itself (Inaba et al., 1978; Stewart et al., 1979; Gatley, 1991). Therefore, the hydrolysis of norcocaine may also contribute to the protective effect of BChE in vivo. The two major hydrolytic products of cocaine are benzoylecgonine and ecgonine methylester, which together with unmetabolized cocaine comprise ú90% of an excreted dose of the drug (Ambre et al., 1988; Jatlow, 1988). Benzoylecgonine, which is not produced by BChE, is far more potent than cocaine in inducing seizures (when injected intracranially) and vascular spasm (Misra et al., 1975; Madden and Powers, 1990; Konkol et al., 1992), leading to the suggestion that some of cocaine’s toxicity may be due to benzoylecgonine (Konkol et al., 1992; Brogan et al., 1992). In contrast, the hydrolysis of cocaine by BChE produces only ecgonine methylester (and benzoic acid), which lacks the pharmacological activity of cocaine (Misra et al., 1975; Stewart et al., 1977; Ambre et al., 1982; Matsubara et al., 1984). It is

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therefore possible that increasing the levels of plasma BChE, as in this study, would shift the metabolism of cocaine toward ecgonine methylester and away from the toxic metabolite, benzoylecgonine. Experimental support for this idea is reported in Mattes et al. (1997). The experimental models in this study were chosen to evaluate the efficacy of BChE in reversing cocaine’s effects on the cardiovascular and central nervous systems. It was anticipated that BChE would influence the effects of cocaine on the heart and vasculature. However, it was unclear whether BChE, which should be unable to cross the blood– brain barrier, would affect the activity of cocaine on the CNS. In fact, BChE proved to be highly efficacious in both cases. This observation suggests that the peripheral activity of BChE is somehow able to reduce the concentration of cocaine (and/or its active metabolites) in the CNS, possibly through a mass action redistribution of the drug as the level of cocaine is reduced in the plasma. The reduction of cocaine levels in the brain by iv administration of BChE is described in Mattes et al. (1997). In principle, BChE should be effective in treating the effects of cocaine that arise in either of two circumstances. The first is the conventional overdose of cocaine, which saturates even a normal individual’s enzymatic capacity to inactivate the drug. In this case, the administration of supranormal levels of BChE should overcome the saturation of the endogenous enzyme and speed recovery. This idea is supported by clinical evidence that adverse reactions to cocaine are correlated with low plasma BChE levels (Devenyi, 1989; Hoffman et al., 1992a; Om et al., 1993). The second instance would be the individual carrying at least one allele for the atypical variant (or other defective form) of BChE. In vitro studies have clearly established that the atypical form of BChE is virtually incapable of hydrolyzing cocaine (Stewart et al., 1979; Jatlow et al., 1979). Although no relevant clinical study has been undertaken, the biochemical data dictate that such individuals are deficient in their ability to metabolize cocaine and should, therefore, be at significantly greater risk of serious, adverse consequences of cocaine abuse. In these individuals, treatment with BChE should be of great, if not critical, benefit. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Mark Sale of the Department of Clinical Pharmacology at Georgetown University Medical Center for calculating the plasma half-life of BChE. The authors thank Dr. William N. Drohan, the Jerome H. Holland Laboratory, American Red Cross, for the generous gift of human plasma Fraction IV-4 and for his many constructive suggestions during the course of this study.

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