Weak Inhibitors Protect Cholinesterases from Stronger Inhibitors (Dichlorvos): In Vitro Effect of Tiapride

Abstract

Metoclopramide is a benzamide dopamine receptor antagonist and serotonine receptor agonist widely used as an antiemetic and gastric prokinetic drug. In addition, metoclopramide is a weak and reversible inhibitor of cholinesterases. The authors have previously shown that metoclopramide has a cholinesterase protective effect against inhibition by organophosphates (OPs). The putative mode of protective action of metoclopramide is, when administered in excess, competion for the active site of the enzyme with the more potent OP. In the present paper the authors present their results using another benzamide with weak cholinesterase inhibitory properties, tiapride (TIA). The purpose of the study was to quantify in vitro the extent of TIA-conferred protection, using dichlorvos (dichlorovinyl dimethyl phosphate; DDVP) as an inhibitor. DDVP is a moderately toxic (LD₅₀ in rats in the milligram range), non-neuropathic OP. The substance is responsible for a large number of accidental or suicidal exposures. Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood and butyrylcholinesterase (BChE) activities in human plasma were measured photometrically in the presence of different DDVP and TIA concentrations and IC₅₀ was calculated. Determinations were repeated in the presence of increasing TIA concentrations. The IC₅₀ of DDVP increases with the TIA concentration in a linear manner. The protective effect of TIA on cholinesterase could be of practical relevance in the treatment of OP poisoning. The authors conclude that in vivo testing of TIA as an OP protective agent is warranted.

Organophosphorus compounds are serine esterase and protease inhibitors widely used in agriculture as insecticides and acaricides, in industry and technology as softening agents and additives to lubricants, and some of them are declared as chemical warfare agents. Two of them (sarin and VX) were involved in terrorist attacks in Japan (Wiener and Hoffman 2004). The inhibition of esterases (butyrylcholine: EC no. 3.1.1.8) and (acetylcholine: EC no. 3.1.1.7) results from reacting covalently with the active centre serine, i.e., by phosphorylation (Levine 1991).

In 1990 a World Health Organization (WHO) task group noted that there may be 1 million serious unintentional pesticide poisonings each year and, on the basis of a survey of self-reported minor poisoning, estimated that there may be up to 25 million agricultural workers in the developing world suffering an episode of poisoning each year (Jeyaratnam 1990).

Dichlorvos (dichlorovinyl dimethyl phosphate; molecular weight [MW] 221), also known as DDVP, is according to the WHO Toxicity Classification a highly hazardous organophosphorus compound (class Ib). It has a short pH-dependent hydrolisis half life of < 1 day. The IC₅₀ of DDVP for human red blood cell acetylcholinesterase (RBC-AChE) is two orders of magnitude higher than that of paraoxon; for butyrylcholinesterase (BChE) the difference is less pronounced (7:1; data on file).

The effects of poisoning with organophosphorus compounds are well known and have been described extensively (Karalliedde 1999; Namba 1971; Namba et al. 1971; Zoch 1971; Petroianu et al. 1998). Oximes are the only enzyme reactivators clinically available (Johnson et al. 2000). Pralidoxime (PRX) is used as an adjunct to atropine in the treatment of poisoning by most cholinesterase inhibitors. Clinically, whereas atropine relieves muscarinic signs and symptoms, PRX is supposed to shorten the duration of the respiratory muscle paralysis by reactivation of cholinesterases (Johnson et al. 2000). Clinical experience with PRX (and other oximes) is disappointing (van Helden et al. 1996; Peter and Cherian 2000). Recently we have been able to show that metoclopramide confers some degree of protection—both in vitro and in vivo—against inhibition by organophosphates (Petroianu et al. 2003a, 2003b, 2003c). The putative mode of protective action of metoclopramide—when administered in excess—is competition for the enzyme with the more potent organophosphate, so that the enzyme is occupied by the weak inhibitor (benzamide) instead of the potent one (organophosphate) and thus less inhibited.

Tiapride (TIA) is a benzamide derivative structurally related to metoclopramide. TIA blocks dopamine receptors in the brain. It has affinity for dopamine D₂ but lacks affinity for nondopaminergic receptors including, histaminergic H₁, alpha₁- and alpha₂-adrenergic, and serotonergic receptors. TIA has been used successfully in the clinic for a number of years for the treatment of, among others, agitation and aggressiveness in elderly patients (Scatton et al. 2001).

Even at higher doses, TIA does not exceed a D₂-receptor occupancy of 80%, which is in accordance with the finding that TIA rarely causes acute extrapyramidal syndromes. Nevertheless, clinical studies have demonstrated its efficacy in neuroleptic-induced tardive dyskinesia, psychomotor agitation in geriatric patients, and choreatic movement disorders. TIA is well tolerated in daily doses between 300 and 1800 mg (orally, intramuscularly, or intravenously). Adverse events are generally rare and mild (Dose and Lange 2000). Following oral administration of single doses of 200 mg, TIA mean maximum plasma concentrations (C _max) of 1.5 to 1.7 mg/L were measured (Rey et al. 1982; Steele, Faulds, and Sorkin 1993).

TIA is rapidly absorbed when given orally; bioavailability is approximately 70% to 80%. Renal elimination of the unchanged drug accounts for the majority of the administered dose. Hepatic metabolism is minimal (10% to 15%), yielding N-oxide and N-deethylated derivatives. The half-life time of elimination is 229 ± 41 min. The apparent volume of distribution is ≈1.4 L/kg and the average plasma clearance is 270 ml/min. Dose adjustment is necessary in renal impairment. Neither protein binding nor glucuronide, sulphate, or acetyl conjugation is observed (Roos et al. 1986; Norman et al. 1987; Steele, Faulds, and Sorkin 1993).

Similar to metoclopramide, TIA is also a weak inhibitor of cholinesterases (Fontaine and Reuse 1980).

The objectives of this study are

To determine in vitro in human blood the IC₅₀ values of TIA for RBC-AChE and BChE.

To quantify in vitro the protective effect of increasing TIA concentrations on RBC-AChE and BChE against DDVP, as assessed by the IC₅₀ shift.

To quantify in vitro the binding constant K of TIA for RBC-AChE and BChE using DDVP inhibition data (Schild plot).

MATERIAL AND METHODS

RBC-AChE Activity

The RBC-AChE activity was measured in diluted whole blood samples in the presence of the selective butyrylcholinesterase inhibitor ethoproprazine as previously described (Worek et al. 1999). The assay, which is based on Ellman’s method, measures the reduction of dithiobisnitrobenzoic acid (DTNB) to nitrobenzoate (TNB⁻) by thiocholine, the product of acetylthiocholine hydrolysis (Ellman et al. 1961). Freshly drawn venous blood samples were diluted in 0.1 M phosphate buffer (pH 7.4) and incubated with DTNB (10 mM) and ethopropazine (6 mM) for 20 min at 37°C prior to addition of acetylthiocholine. The change in the absorbance of DTNB was measured at 436 nm. The AChE activity was calculated using an absorption coefficient of TNB⁻ at 436 nm (ɛ = 10.6 mM⁻¹ cm¹). The values were normalized to the hemoglobin (Hb) content (determined as cyanmethemoglobin) and expressed as mU/μ mol/Hb (Van Kampen and Zijlstra 1961).

BChE Activity

BChE activity was measured in plasma obtained from heparinized blood after centrifugation (10 min, 500 × g). Plasma was analyzed immediately or kept frozen in 1-ml aliquots until analysis. The assay, which is based on Ellman’s method, measures the reduction of DTNB to nitrobenzoate (TNB⁻) by thiocholine, the product of butyrylthiocholine hydrolysis at 37°C. Diluted plasma samples were incubated with DTNB prior to addition of butyryllthiocholine. The change in the absorbance of DTNB was measured at 436 nm. The BChE activity was calculated using an absorption coefficient of TNB⁻ at 436 nm (ɛ = 10.6 mM⁻¹ cm¹). The values were expressed as μ mol/L/min (Ellman et al. 1961; Worek et al. 1999).

Determination In Vitro in Human Blood of the IC₅₀ Value of TIA for RBC-AChE and BChE

Blood from human volunteers was used (n = 5, 2 males and 3 females). None of the volunteers was on any drugs. Enzyme activities were determined in the absence of and then after addition of TIA and DDVP. DDVP and TIA were added before the incubation period. For the graphical representation and IC₅₀ calculation, the SlideWrite (Advanced Graphics Software, Encinitas, CA, USA) software was used (user defined equation y = a ₀ /[1 + (x/a ₁) exp a ₂]), where a ₁ corresponds to the IC₅₀ value.

Quantification of the Protective Effect of Increasing TIA Concentrations on RBC-AChE and BChE Against DDVP Inhibition, as Assessed by the IC₅₀ Shift In Vitro in Human Blood

IC₅₀ determinations (DDVP for RBC-AChE and BChE) as described above were repeated in the absence of and then in the presence of increasing TIA concentrations. The calculated IC₅₀ values were plotted against the TIA concentrations to obtain an IC₅₀ shift curve. For the graphical representation and calculations, the SlideWrite (Advanced Graphics Software) software was used (equation y = a ₀ + a ₁ x ) where a ₁ represents the slope (tangent; tg α) of the IC₅₀ shift graph. The IC₅₀ shift (tg α) has no units.

Calculation of the Binding Constant K of TIA for RBC-AChE and BChE

The performed measurements (IC₅₀ shift) allow the calculation of the binding constant K of TIA for RBC-AChE and BChE. K (the estimated amount of free substance required to half saturate the maximal binding capacity of RBC-AChE or BChE) is calculated using the Schild plot. The graphical method requires plotting of log (dose ratio −1) versus − log concentration, where the dose ratio is defined as IC₅₀ of DDVP determined in the presence of TIA divided by the IC₅₀ of the DDVP determined in the absence of TIA (Cheng, 2001; Arunlakshana and Schild 1959). For the graphical representation and calculations, the SlideWrite (Advanced Graphics Software) software was used (equation y = a ₀ + a ₁ x ).

RESULTS

Tiapride is a weak inhibitor of RBC-AChE with an IC₅₀ of 259 μ M (95% confidence interval [CI] = 252–264); its ability to inhibit BChE is less pronounced with an IC₅₀ in the low milimolar range (IC₅₀ = 3.5 mM; 95% CI = 3.1–3.8).

TIA’s ability to protect cholinesterases from inhibition by DDVP as assessed by IC₅₀ shift (the slope [tangent] of the IC₅₀ shift graph) is also more pronounced for RBC-AChE, with an absolute numerical value for tg α of 2.67 × 10⁻³ (95% CI = 2.18–3.16 × 10⁻³) or 2.67 nM/μ M. Its ability to protect BChE is less pronounced, with a tg α of 0.89× 10⁻³ (95% CI = 0.78–1.0 × 10⁻³) or 0.89 nM/μ M.

The binding constant K of TIA for RBC-AChE calculated using DDVP IC₅₀ shift data is ≈112 μ M whereas the binding constant for BChE is ≈49 μ M.

Results are summarized below and presented in Figures 1 (IC₅₀), 2 (IC₅₀ shift), and 3 (Shild plots) and in Tables 1A and 1B (IC₅₀ shift data) and 2A and 2B (Shild plot data).

IC50TIA for RBC-AChE	259 μM 95% CI = 252–264
IC50TIA for BChE	3.5 mM 95% CI = 3.1–3.8
DDVP IC50 shift for RBC-AChE	tg α = 2.67 × 10−3 (2.67 nM/μ M) 95% CI = (2.18–3.16) × 10−3
DDVP IC50 shift for BChE	tg α = 0.89 × 10−3 (0.89 nM/μ M) 95% CI = (0.78–1.0) × 10−3
Binding constant K of TIA for RBC-AChE calculated using DDVP IC50 shift data	≈112 μ M
Binding constant K of TIA for BChE calculated using DDVP IC50 shift data	≈49 μ M

DISCUSSION

Present Treatment Options

PRX and related oxime class reactivators are used in the treatment of poisoning by certain cholinesterase inhibitors. Clinically, whereas atropine relieves muscarinic signs and symptoms, PRX is supposed to shorten the duration of the respiratory muscle paralysis by reactivation of cholinesterases. Recently published consensus guidelines for stocking of emergency antidotes in the United States recommend stocking of PRX in each and every hospital at tremendous costs (Dart et al. 2000). However, the clinical experience with PRX is mixed. A recently published review of the topic offers a very balanced view on the use of oxime reactivators. The authors allude to the “disappointment” clinicians have experienced while using oximes and express their view (hope) that higher concentrations might be more effective (by increasing the measurable esterase activity) (Johnson et al. 2000).

Recently the Food and Drug Administration (FDA) approved oral pyridostigmine (3-hydroxymethylpyridinium bromide dimethylcarbamate) for preexposure treatment of some nerve gases; the concept is to block the cholinesterase reversibly using the carbamate, in order to deny access to the active site of the enzyme to the irreversible inhibitor (nerve gas) on subsequent exposure (hence pretreatment). Pretreatment with oral pyridostigmine followed by the conventional atropine plus oxime treatment increased LD₅₀ of soman by at least one order of magnitude as compared to atropine plus oxime. Pyridostigmine pre-treatment is effective only when followed by atropine and oxime; pyridostigmine alone is not effective (Gordon, Leadbeater, and Maidment 1978; Wiener and Hoffman 2004).

Alternative Concepts

Pyridostigmine is a fairly potent cholinesterase inhibitor with an inhibitory constant K in the low nanomolar range. The drug does not penetrate into the central nervous system (CNS) and the maximal dose is limited by peripheral side effects (Marino et al. 1998).

We speculated that a weak inhibitor of cholinesterases applied at high dose might offer similar or superior benefits with less side effects. The concept was previously tested with promissing results using the benzamide metoclopramide both in vitro and in vivo (Petroianu et al. 2003a, 2003b, 2003c). The putative mode of protective action of metoclopramide—when administered in excess—is competition for the enzyme with the more potent organophosphate, so that the enzyme is occupied by the weak inhibitor (benzamide) instead of the potent one (organophosphate) and thus less inhibited. The benzamide D₂ receptor blocker tiapride is structurally related to metoclopramide.

The substance is clinically widely used and well known for its wide margin of safety. Its ability to inhibit cholinesterases, although well known, was considered to be of marginal clinical relevance in the context of its more traditional clinical use (Fontaine and Reuse 1980).

Tiapride Plasma Levels

To assess the possible clinical relevance of any in vitro findings, we performed in Wistar rats both tolerability tests and TIA plasma concentration measurments after intraperitoneal administration. TIA is extremely well tolerated by the animals up to doses of 200 μ mol/rat. (HPLC) determinations of TIA plasma concentration confirmed the t _max of TIA as being in the 120-min range; intraperitoneal administration of 50 and 100 μ mol of TIA/rat resulted in C _max values of ≈4 and 25 μ mol/L, respectively. If higher doses are administered the C _max values of TIA reach triple-digit numbers. These values are of the same order of magnitude as the binding constant K of TIA; it appears therefore that an in vivo effect could be possible.

RBC-AChE

The IC₅₀ value of TIA for the enzyme is in the triple-digit micromolar range (≈252–264 μ M). This value is comparable to previously published values by other groups (Fontaine and Reuse 1980). A marked in vivo inhibitory effect of TIA on RBC-AChE is unlikely. The IC₅₀ value of metoclopramide for the enzyme as published by our group was in the double-digit micromolar range (≈24–42 μ M) (Petroianu et al. 2003a).

BChE

The IC₅₀ value of TIA for the enzyme is much higher than the value for RBC-AChE. The ratio of the two values (IC₅₀ BChE versus IC₅₀ RBC-AChE) using our data is 13. An in vivo inhibition of BChE by high-dose TIA application is unlikely. The IC₅₀ value of metoclopramide for the enzyme as published by our group was in the double-digit micromolar range (≈14–65μ M) (Petroianu et al. 2003c).

IC₅₀ Shift Determinations

The interpretation of IC₅₀ shift determinations is fraught with the same type of problems as the interpretation of IC₅₀ data: the results depend on the experimental conditions. In order to allow comparisons the experimental conditions have to be standardized. Although for both enzymes TIA is capable of increasing the IC₅₀ values thus causing a dose-dependent shift, the a ₁ values, representing the slope (tg α) of the line, indicate that the shift is more pronounced for RBC-AChE (2.67 nM/μ M versus 0.89 nM/μ M). Although no protection data for DDVP using metoclopramide exist, data for for RBC-AChE using two other organophosphates (mipafox and paraoxon) yield a similar picture (1.4 nM/μ M).

Binding Constant K

The slope of the Schild plot (a ₁) for both enzymes is essentially 1, indicative of a competitive mechanism of interaction (DDVP and TIA). For competitive mechanism situations (slope of the Schild plot tg α ≈1), the dissociation equilibrium constant (binding constant) K is equal to the inhibitory constant (Arunlakshana and Schild 1959; Cheng 2001).

The K value of TIA for RBC-AChE is at the high end of the therapeutically achievable range and two orders of magnitude higher than K value of metoclopramide (4.3–6.5 μ M) (Petroianu et al. 2003a). An interaction with RBC-AChE in vivo after very-high-dose TIA administration appears, however, possible.

The K value of TIA for BChE (≈49 μ M), although well within the therapeutically achievable range, is two orders of magnitude higher than K value of metoclopramide (0.5–0.7 μ M) (Petroianu et al. 2003c). An interaction in vivo of TIA with BChE is likely.

CONCLUSION

The calculated values of the inhibitory constant K of TIA are achievable in vivo for BChE and possibly for RBC-AChE. As such, a protective in vivo appears posible. The protective effect of TIA on cholinesterase could be of practical relevance in the treatment of organophosphate poisoning. We conclude that in vivo testing of TIA as an organophosphate protective agent is warranted.