the pharmacokinetics, metabolism, and tissue residues of b ......shared by phenethanolamine...

173

1Presented at a symposium titled “Pharmacology, Toxicology,and the Illegal Use of b-Adrenergic Agonists,” July 1996, at theASAS 88th Annu. Mtg., Rapid City, SD.

2Mention of trademark or proprietary product does not constitutea guarantee or warranty of the product by the USDA and does notimply its approval to the exclusion of other products that may alsobe suitable.

Received October 21, 1996.Accepted April 21, 1997.

The Pharmacokinetics, Metabolism, and Tissue Residuesof b-Adrenergic Agonists in Livestock1,2

D. J. Smith

USDA, ARS Biosciences Research Laboratory, Fargo, ND 58105

ABSTRACT: Since the early 1980s the usefulnessof dietary b-agonists to improve the efficiency of feedutilization and(or) to enhance carcass leanness inlivestock species has been well documented. Less welldocumented are the pharmacokinetic properties, bi-otransformation pathways, and tissue residue profilesof b-agonists used to enhance leanness in experimen-tally or illegally treated animals. Pharmacokineticdata for clenbuterol, cimaterol, fenoterol, L-644,969,ractopamine, salbutamol, and terbutaline have beenpublished but biotransformation and tissue residuestudies for these compounds in livestock species aresparse. In general, b-agonists having halogenatedaromatic ring systems are metabolized by oxidativeand conjugative pathways and have long plasma half-lives, whereas b-agonists having hydroxylated aro-

matic rings are metabolized solely by conjugation andhave relatively short plasma half-lives. b-Agonistshaving high oral bioavailabilities, long plasma half-lives, and relatively slow rates of elimination havehigh oral potencies in humans. Residues of suchillegally used compounds in edible tissues of livestockrepresent a genuine risk to consumers. Conversely, b-agonists having low oral bioavailabilities, shortplasma half-lives, and rapid rates of elimination havelow oral potencies in humans. Residues of suchcompounds in edible tissues of properly treatedanimals would not likely represent a credible risk toconsumers of such products. The reviewed dataindicate that the development of a safe and effective b-agonist for use in livestock is possible.

Key Words: Beta-Adrenergic Agonists, Residues, Pharmacokinetics, Food Safety

1998 American Society of Animal Science. All rights reserved. J. Anim. Sci. 1998. 76:173–194

Introduction

Phenethanolamine b-adrenergic agonists ( b-agonists) conform to the general structure shown inFigure 1. For a b-agonist to have biological activity, itmust have a substituted six-membered aromatic ring,hydroxyl group bonded to the b-carbon in the Rconfiguration, positively charged nitrogen in theethylamine side chain, and bulky substituent (R ofFigure 1) on the aliphatic nitrogen to confer specificityfor the b-receptor (Carlstrom et al., 1973; Weiner,1980). These elements are common to allphenethanolamine b-agonists and, with the exceptionof the bulky group on the aliphatic nitrogen, are alsocommon to the natural adrenergic neurotransmittersepinephrine and norepinephrine.

In 1933, Easson and Stedman proposed that b-adrenergic receptors ( b-receptors) bind b-agonists atthree points on the molecule: the b-hydroxyl group, thealiphatic nitrogen, and the aromatic ring. Subsequentstudies indicate that omissions of, or substitutions to,any of these regions may have pronounced effects onreceptor binding and agonistic activity (reviewed byRuffolo, 1991). Site-directed mutagenesis of b-adrenergic receptors has validated the hypothesis ofEasson and Stedman (1933) by showing that specificamino acids within the b-adrenergic receptor areresponsible for interacting with the charged aliphaticamine, substituents of the aromatic ring, and b-hydroxyl groups of b-adrenergic agonists (Wallis,1993; Hieble et al., 1995).

The physiological activity of a b-agonist depends onits inherent activity at the receptor and on itsabsorption, rates of metabolism and elimination, anddistribution to target tissues. The chemical charac-teristics governing a b-agonist’s activity at its receptormay also influence its absorption, distribution,metabolism, and elimination. Thus, in determiningthe mechanisms of b-adrenergic action, the phar-macokinetic properties of individual molecules shouldbe considered. The purpose of this review is toconsider the pharmacokinetic properties, elimination

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Figure 1. The general structure of phenethanolamineb-adrenergic agonists and common substitution patternsof aromatic rings present on b-agonists. For b-adrenergicagonists, the R group adjacent to the aliphatic nitrogenis always bulky, commonly a t-butyl group, isopropylgroup, alkylphenyl or alkylphenol. The aromatic ringlabels m- and p- indicate the meta- and para- positionsrelative to the phenthanolamine b-carbon.

characteristics, and residues of b-agonists in livestock.Because the metabolism and pharmacokinetic charac-teristics of b-agonists are largely unknown inlivestock, the available literature from rodent, dog,rabbit, and human studies will also be reviewed.

Chemical Properties of Phenethanolamineb-Adrenergic Agonists

It is a mistake to overgeneralize the physiologicalfunctions of a class of compounds. For example, eventhough testosterone, estrogen, and aldosterone aresteroid hormones and have some chemical propertiesin common, their physiological functions are different.Likewise, glycine and lysine are amino acids and areincorporated into the primary structures of proteins,but they serve different roles within the secondary,tertiary, and quarternary structures of specific pro-teins. Similarly, phenethanolamine adrenergic agentsshare common structural qualities, but not allphenethanolamines are b-agonists. Some are b-an-tagonists, some activate a-receptors, and some arespecific for subclasses within each of the a- and b-receptor subfamilies. Even within the b-adrenergicagonist subfamily, the chemical and pharmacokinetic

characteristics of a specific agonist may differ con-siderably from another b-agonist. The following dis-cussion will focus on the general chemical propertiesshared by phenethanolamine b-adrenergic agonists,namely the aromatic group, b-hydroxyl group, andaliphatic nitrogen. Many of the properties listed belowwill also be properties of some a-adrenergic agonistsand antagonists, and some b-antagonists. To be sure,the exact chemical nature of a given compounddepends on its specific chemical composition.Nevertheless, because the ultimate biological activityof a given molecule is a function of the chemistry ofthe molecule itself, it is worthwhile to review themajor chemical characteristics of b-agonist molecules.

Aromatic Rings. Aromatic rings attached to the b-carbon (Figure 1) are essential for biological activityof b-agonists. They are generally substituted withhydroxyl groups, halogens, amines, hydroxymethylgroups, cyano groups, or various combinations of theabove (Baker and Kiernan, 1983; Kruger et al., 1984;Anderson et al., 1987). The chemical substitutionspresent on the aromatic ring greatly influence thelongevity of the b-agonist within mammalian or aviantissues and the compound’s efficacy at the receptor.

The pattern of aromatic substitution is importantfor biological activity because a serine hydroxyl withinthe b-receptor probably hydrogen-bonds to substi-tuents at the 3- ( meta- ) or 4- ( para- ) carbons (Figure1; Wallis, 1993; Hieble et al., 1995). For compoundswith aromatic hydroxyl groups, the hydroxyl groupmust be unhindered to maintain activity. For exam-ple, bambuterol (a bis-dimethylformyl ester pro-drugof terbutaline) must be hydrolyzed to terbutaline byesterase to be activated (Morgan, 1990) andglucuronidation of the p-hydroxyl group of salbutamol(Figure 2) eliminated salbutamol’s pharmacologicalactivity (Martin et al., 1971). Conversely, hydroxyla-tion of the p- position of C-78 (Figure 2) resulted in ametabolite with greater activity than the non-hydrox-ylated parent compound (Yamamoto et al., 1977).

The pattern of aromatic substitution is also a majordeterminant in the route of metabolism of b-agonists.For example, catechols such as isoproterenol (Figure2) and dobutamine (Figure 3; Murphy et al., 1976)are rapidly deactivated by methylation of the3-hydroxyl group of the aromatic ring by catechola-mine O-methyl transferases ( COMT; Kopin, 1985),severely limiting their effectiveness after oral ad-ministration. Other patterns of aromatic hydroxyla-tion have been developed that retain activity at the b-receptor, but they are resistant to deactivation byCOMT. Resorcinols (terbutaline, fenoterol; Figure 2),saligenins (salmeterol, salbutamol), and simplephenols (ractopamine, ritodrine; Figure 2) are b-agonists that are not substrates for COMT. However,resorcinol, saligenin, and phenolic b-agonists (Figure1) are rapidly deactivated by enzymes in the liver andintestine. Because of this, aromatic substitutions forwhich conjugative biotransformation enzymes have

RESIDUES AND METABOLISM OF b-AGONISTS 175

Figure 2. Structures of b-adrenergic agonists.

Figure 3. Structures of b-adrenergic agonists.

low activity were generated. Nearly all of thesearomatic groups contain halogen atoms substitutingfor hydroxyl groups. Clenbuterol, mabuterol, andperhaps cimaterol (Figure 2) are examples of suchsubstances. The halogen atoms do not inhibit bindingto the receptor, but they do prevent the rapidmetabolic deactivation that occurs with ring hydrox-ylated b-agonists. Mabuterol and clenbuterol weredesigned specifically to resist rapid metabolic degrada-tion by enzymes active toward aromatic hydroxylgroups (Morgan, 1990).

Halogen substitution increases the lipophilicity ofthe aromatic portion of a b-agonist relative to ahydroxylated agonist. For example, dichloroaniline(the aromatic substituent present on clenbuterol) hasa log P value (octanol/water partition coefficient) 2.1units higher than salicyl alcohol (saligenol; thearomatic substituent present on salbutamol andsalmeterol) (Wallis, 1993). Perhaps as a conse-quence, it has been claimed that clenbuterol is morelipophilic than other b-adrenergic agonists (Nazzal,1985; Witkamp and van Miert, 1992) and it has beeninferred that clenbuterol readily partitions into adi-pose tissue (Witkamp, 1996).

Alkyl Amine. Claims that clenbuterol is lipophilicmight be true if dichloroaniline were not associated

with the phenethanolamine portion of clenbuterol.Because aliphatic amines present on b-agonists havean alkaline pKa they exist in the protonated (ionized)form in blood and other tissues at physiological pH(7.4). For example, the aliphatic amine pKa forfenoterol is 8.5 or 10 (Moffat et al., 1986);isoproterenol, 10.1 (Tariq and Al-Badr, 1985); rac-topamine 9.4 (Turberg et al., 1995); salbutamol, 9.3(Lund, 1994); and terbutaline 10.1 (Ahuja andAshman, 1990). If the b-agonist were not ionized atthe receptor, it would not have biological activity(Carlstrom et al., 1973; Hieble et al., 1995). Even if ab-agonist is administered in the free base form(unprotonated nitrogen), the pKa of the aliphaticnitrogen atom dictates that the vast majority of

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molecules will become protonated in the stomach ifadministered in the feed, or in the blood if given i.v.(Takacs-Novac et al., 1995). Thus, the dissociationconstant of the aliphatic nitrogen common to all b-agonists dictates that the molecule will not partitioninto adipose tissue unless other regions on the samemolecule have sufficient lipophilicity to interact withfat.

Consistent with the claim that b-agonists are notgenerally lipophilic is the fact that the commerciallyavailable salts of phenethanolamine b-agonists arevirtually insoluble in non-polar solvents such asmethylene chloride, ethyl acetate, and ether. Thesame compounds are readily soluble in methanol orwater (Turberg et al., 1995; and see individual drugentries for b-agonists in Moffat, 1986; Reynolds et al.,1989; and Lund, 1994).

Free bases of b-agonists are lipophilic, which hasperhaps led to the widespread, erroneous conclusionthat b-agonists will typically accumulate in adiposetissue. The lipophilic properties of the free base of b-agonists have been used during their analyses intissues and feeds. Salts of the analyte of interestpresent in the aqueous matrix are converted to thefree base by adjustment of the matrix to pH 10, orgreater, with alkali, and the b-agonist is extractedfrom the aqueous solution into a solvent such as ethylacetate, ether, or methylene chloride (Wilson et al.,1994; Turberg et al., 1995).

Salmeterol (Figure 3) is a notable exception to theabove discussion because it is a b-agonist that is trulylipid-soluble in spite of the fact that it has a chargedaliphatic nitrogen. The phenethanolamine portion ofsalmeterol is identical to salbutamol, but its N-alkylgroup consists of a long N-arylalkyloxyalkyl sidechain. Whereas salbutamol is approximately 11 A intotal length, salmeterol is 25 A, with the side chainmaking up about 17 A of the total length of themolecule (Johnson, 1995). Because of its lipid solubil-ity, salmeterol’s side chain has a great affinity for cellmembranes, whereas the side chain of salbutamol isnot long enough for the molecule as a whole to havelipid-soluble characteristics. As a result, salbutamolremains in the extracellular compartment of tissues,whereas salmeterol has a great affinity for cellmembranes. In fact, salmeterol is greater than 10,000times more lipophilic than salbutamol (Johnson,1995). The long N-arylalkyloxyalkyl side chain ofsalmeterol essentially “anchors” the molecule intocellular membranes, allowing the chargedphenethanolamine portion of the molecule to interactwith the b-receptor at the surface of the cell. It isbeyond the scope of this review to detail the ex-perimental evidence for the mechanism of action ofsalmeterol, but several recent reviews (Jack, 1991;Wallis, 1993; Johnson, 1995) provide excellent in-sights into the development of this unique b-agonist.

β-Hydroxyl Group. The b-carbon of phenethanola-mine b-agonists is chiral, resulting in the presence of

levorotatory and dextrorotatory stereoisomers in com-mercial preparations. At the receptor level, a properlyoriented hydroxyl group at the b-carbon is essentialfor biological activity (Ruffolo, 1991). For direct-acting b-adrenergic agonists, biological activity isassociated exclusively with the levorotatorystereoisomer (Ruffolo, 1991). This has been shown tobe true for clenbuterol (Martin et al., 1985; Waldeckand Widmark, 1985), fenoterol (Kaiser et al., 1978),isoproterenol (Mersmann and McNeel, 1992), sal-butamol (Mazzoni et al., 1994), ractopamine (Thomp-son et al., 1980; Yen et al., 1989; Ricke et al., 1996),terbutaline (Jeppson et al., 1984), and others (Shawet al., 1981; Yen et al., 1983; Johnson, 1995). Thecompound L-644,969, a b-agonist that was developedfor use as a leanness-enhancing agent, is the stereoiso-merically pure R,R isomer (Zang and Grieve, 1995).Whereas “inactive” stereoisomers of some compoundsmay not be devoid of biological activity (Ariens, 1984;Crossley, 1992), distomers (inactive or weakly activeisomers) of b-agonists are generally orders of magni-tude less potent than the levorotatory isomers, espe-cially for direct-acting b-agonists.

The importance of the benzylic hydroxyl group indetermining receptor specificity is exemplified by acomparison of the structural isomers dobutamine andractopamine (Figure 3). The aryl group attached tothe b-carbon of dobutamine is a catechol, whereas thisaryl group is a simple phenol for ractopamine;ractopamine, however, has a hydroxyl group presentat the b-carbon that is absent on dobutamine. Bothmolecules have identical phenylalkyl groups bonded tothe ethanolamine. In terms of their physiologicalactivities, ractopamine is a full agonist at the b2receptor, a partial agonist at the b1 receptor, and hasno agonistic activity at a-adrenergic receptors (Col-bert et al., 1991). Dobutamine, on the other hand, isan a- and b-adrenergic agonist in which the levorota-tory stereoisomer has a-adrenergic activity and thelevorotatory and dextrorotatory stereoisomers are b-adrenergic agonists (Ruffolo et al., 1981). The lack ofthe b-hydroxyl group in dobutamine allows themolecule to interact and act on the a-adrenergicreceptor (Ruffolo, 1991).

Pharmacokinetics of b-Adrenergic Agonists

Absorption and Elimination Studies. Peak plasmalevels of b-adrenergic agonists generally occur within1 to 3 h after oral administration in humans(reviewed by Morgan, 1990), and a similar pattern isobserved after oral administration to farm animals. Incalves (Meyer and Rinke, 1991), plasma clenbuterolpeaked at about .5 ng/mL 2 to 7 h after initialtreatment with 5 mg/kg BW; after 21 d of this dosage,plasma clenbuterol peaked at 1.1 ng/mL at 4 h afterdosing. The doubling of the peak plasma concentrationwas caused by accumulation of clenbuterol over time.


In lactating cows dosed chronically with oral clen-buterol (5 mg/kg BW twice daily), plasma clenbuterolreached a plateau (5 to 5.5 ng/mL) after 5 to 7 d(Stoffel and Meyer, 1993) and at 2 ng/mL plasmaafter 1 to 3 d of treatment at a similar dosage in adifferent study (von Ahnen, 1994). Calves receivingdaily clenbuterol doses (10 mg/kg BW) had maximalplasma concentrations after 10 d of treatment, andaccumulation in plasma over time was clearly evident(Sauer et al., 1995).

Multi-day studies performed in cattle with sal-butamol have shown plasma levels of the parent drugpeaked at roughly the same concentrations as clen-buterol, even though the dose of salbutamol wasseveral times greater than the corresponding clen-buterol dose. For example, peak salbutamol concentra-tions in plasma were 4.8 and 4.0 ng/mL 3 to 4 h afteran oral dose of 78 mg/kg BW (Pou et al., 1992) on d 1and d 10 of dosing, respectively. In lactating dairycattle that received oral salbutamol (twice daily; 50mg/kg BW), plasma salbutamol levels peaked atapproximately 6 ng/mL (von Ahnen, 1994). In con-trast to clenbuterol, there was no evidence for theaccumulation of salbutamol residues in plasma follow-ing repeated dosing (Pou et al., 1992; von Ahnen,1994).

The appearance of terbutaline in blood after oraladministration to livestock species has been reportedonly for dairy cattle (von Ahnen, 1994) and chickens(Malucelli et al., 1994). In dairy cows dosed twicedaily with 50 mg/kg BW, plasma terbutaline concentra-tions ranged from below the limit of quantification (.5ng/mL) to about 4 ng/mL during the course of a6-d treatment period. Plasma concentrations werevariable within and among animals. In broiler chick-ens fed terbutaline (10 ppm) for 14 consecutive days,plasma terbutaline at slaughter (0 withdrawalperiod) averaged 42.8 ng/mL. Plasma concentrationsof clenbuterol and salbutamol were 1.2 and 25.0 ng/mL, respectively, when chickens were slaughteredwith a zero withdrawal period after 14 d of dietaryexposure to either 1 ppm clenbuterol or 10 ppmsalbutamol (Malucelli et al., 1994).

Although the presence of a b-agonist in the plasmais credible evidence of absorption, it is not a goodmeasure for the extent of drug absorption. Totalabsorption can be estimated by measuring the urinaryexcretion of radioactivity following oral administrationof a radiolabeled b-agonist. Total absorption can beunderestimated, however, if metabolites are excretedin bile, or secreted in saliva or milk, after the parentdrug is absorbed. Biliary excretion can be estimatedby measuring the fecal excretion of radioactivity afterparenteral administration of a radiolabeled drug or bybile collection using bile duct-cannulated animals.Both approaches have been used in the assessment ofb-agonist absorption.

The apparent absorption of b-agonists, as assessedusing radiolabeled test compound, is shown in Table 1.

Clenbuterol (Smith and Paulson, 1997) and ractopa-mine (Dalidowicz et al., 1992; Smith et al., 1993) arethe only b-agonists for which absorption studies inlivestock have been published. Ractopamine wasextensively and rapidly absorbed in turkeys (Smith etal., 1993) and swine (Dalidowicz et al., 1992).Clenbuterol absorption by bovine calves was rapid;radioactivity in the blood averaged 160 ppb (clen-buterol equivalents) within 1 h of an oral dose (3 mg/kg BW; Smith and Paulson, 1997). By 48 h afterdosing, less than 50% of the total dose administeredhad been excreted in the urine and less than 2% of thedose was excreted in the feces. The balance of theradioactivity remained in the carcass and intestinaltract. The absorption of clenbuterol was extensive;recovery of the radiolabel in the urine and nongas-trointestinal components of the carcass accounted foronly 76% of the total dose administered.

The use of radiolabeled b-agonists to determineabsorption has been more prevalent for those drugsthat have been registered for use in humans than forthose used in livestock. Like absorption in livestock,absorption of most b-agonists was rapid and extensivein humans and lab animals (Table 1). For mostcompounds, excretion of radioactivity following oral ori.v. administration of a radiolabeled b-agonist wasnearing completion by 48 h (Table 1). Although thistrend is probably true for livestock species as well,there may be some exceptions. For example, bovinecalves orally dosed with 3 mg/kg BW [14C]clenbuterolHCl excreted less than one-half of the administeredradiolabel via the urine within 48 h after dosing. Incontrast, dogs given an oral dose of 2.5 mg/kg BW of[14C]clenbuterol HCl had excreted 72% of the ad-ministered radioactivity in urine 48 h after dosing(Zimmer, 1976b). Rabbits excreted 92% of the radi-oactivity present in a 2.5 mg/kg BW oral dose of[14C]clenbuterol HCl in the urine within a72-h period; 83% of the administered radioactivity wasexcreted during the first 24 h (Zimmer, 1976b).

Biliary excretion of b-agonists, or their metabolites,seems to be species-specific for any given compound.For example, biliary elimination of ractopamine is ofmajor importance in rats and turkeys (35 to 60% ofadministered radioactivity; Smith and Paulson, 1994;Smith and Paulson, unpublished observations), but itseems to be of less importance in swine, in which 88%of an oral dose of [14C]ractopamine HCl was elimi-nated in urine (Dalidowicz et al., 1992). Biliaryexcretion of terbutaline is substantial in rats (30% ofan intra-arterial dose was excreted within 3 h ofadministration; Eriksson et al., 1975), but is of lesserimportance in dogs (about 2% of an administereddose; Nilsson et al., 1973a) and humans (2% of anadministered dose; Nilsson et al 1973b). The biliaryelimination of salmeterol is important in dogs and rats(42 to 72% of administered radioactivity; Manchee etal., 1993).

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Table 1. Excretion of b-adrenergic agonists in various animals following different routes of administration

aHours after dosing that observation was made.bMatrix in which radioactivity was excreted.cApparent absorption; expressed as a percentage of the administered dose.dFor entries in which the matrix was bile or feces, absorption was not determined directly. The values are provided so that the contribution

of biliary excretion after absorption can be estimated.eUrinary and carcass radioactivity (not including radioactivity associated with the gastrointestinal tract) combined.fForty-five percent of the dose was excreted in urine within 24 h.

PercentageCompound and animal Dose Route Timea, h Excretab absorptionc,d Reference

ClenbuterolBovine 3 mg/kg Oral 48 Urine 76e Smith and Paulson, 1997Dogs 2.5 mg/kg Oral 72 Urine 74 Zimmer, 1976bHumans 20 mg Oral 96 Urine 80 Zimmer, 1976aRabbit 2.5 mg/kg Oral 96 Urine 92 Zimmer, 1976bRat 2 mg/kg i.v. 72 Feces 19 Kopitar and Zimmer, 1976Rat 2 mg/kg Oral 72 Urine 67 Kopitar and Zimmer, 1976

FenoterolDog 50 mg/kg Oral 70 Urine 43 Rominger and Pollmann, 1972Dog 1−2 mg/kg i.v. 46 Feces 12 Rominger and Pollmann, 1972Humans 90 mg/kg Oral 48 Urine 39 Rominger and Pollmann, 1972Mouse 1 mg/kg Oral 72 Urine 45 Kojima et al., 1980bMouse 1 mg/kg i.v. 72 Feces 16 Kojima et al., 1980bRat 1 mg/kg Oral 72 Urine 16 Kojima et al., 1980a

MabuterolHuman 41 mg Oral 192 f Urine 80 Guentert et al., 1984Rat 1 mg/kg Oral 48 Urine 62 Yuge et al., 1984Rat 1 mg/kg i.v. 48 Feces 29 Yuge et al., 1984

RactopamineRat 7.7 mg/kg Oral 24 Urine 29 Smith and Paulson, 1994Rat 7.7 mg/kg Oral 24 Bile 59 Smith and Paulson, 1994Swine 40 mg Oral 168 Urine 88 Dalidowicz et al., 1992Turkey 6.7 mg/kg Oral 48 Urine 52 Smith et al., 1993Turkey 4.4 mg/kg Oral 24 Bile 37 Smith and Paulson, unpublished

SalbutamolDog 12.5 mg/kg Oral 24 Urine 72 Martin et al., 1971Human 10 mg Oral 24 Urine 90 Martin et al., 1971Human 4−8 mg Oral 72 Urine 76 Evans et al., 1973Rabbit 50 mg/kg Oral 48 Urine 63 Martin et al., 1971Rat 25 mg/kg Oral 48 Urine 56 Martin et al., 1971Rat 25 mg/kg i.v. 5 Bile 20 Martin et al., 1971

SalmeterolDog .2 mg/kg Oral 48 Urine 13 Manchee et al., 1993Dog .2 mg/kg Oral 8 Bile 42 Manchee et al., 1993Human 10 mg/kg Oral 72 Urine 23 Manchee et al., 1993Rat 2 mg/kg Oral 48 Urine 10 Manchee et al., 1993Rat 2 mg/kg i.v. 48 Feces 72 Manchee et al., 1993

TerbutalineDog 1 mg/kg Oral 96 Urine 75 Nilsson et al., 1973aDog .5 mg/kg i.v. 6 Bile 2 Nilsson et al., 1973aHuman 51−62 mg/kg Oral 72 Urine 40 Nilsson et al., 1972Human 3 mg/kg i.v. 72 Feces 3 Nilsson et al., 1972Rat 5 mg/kg Oral 72 Urine 44 Conway et al., 1973Rat .1 mg/kg i.v. 12 Bile 33 Nilsson et al., 1973a

Salbutamol and clenbuterol are secreted into themilk of dairy cows (Stoffel and Meyer, 1993; vonAhnen, 1994) and terbutaline was detected in milkfrom women (Boreus et al., 1982; Lonnerholm andLindstrom, 1982). Terbutaline was also inconsistentlyfound in bovine milk (von Ahnen, 1994). Concentra-tions of clenbuterol in bovine milk averaged 12.5 ng/mL (range 5.5 to 22.5 ng/mL) in a study by Stoffeland Meyer (1993) (the clenbuterol dose was 10 mg/kgBW) and ranged from 3 to 9 ng/mL milk from cows

dosed twice daily with 5 mg/kg BW (von Ahnen,1994).The parent drug was concentrated into milk, relativeto plasma levels, in cows and women. It is unknownwhether clenbuterol, salbutamol, or terbutalinemetabolites are also excreted into milk.

The site(s) of absorption for most b-agonistsis(are) generally unknown. Mabuterol was not ab-sorbed from the stomach but was extensively absorbedalong the entire small intestine of rats (Yuge et al.,1984). Terbutaline was stereoselectively absorbed


Table 2. Plasma half-lives of b-agonists in various animals

aDistribution half-life after i.v. infusion.bAccording to the cited authors this value is based on limited data.cPhase of elimination ( a, b, g) not indicated.dCalculated after i.v. infusion.eCalculated after oral administration.fPlasma half-life after i.v. administration of either R- or S-salbutamol.gPlasma half-life of total radioactivity (parent + metabolites).

T1/2, h

Compound and animal a b g Reference

CimaterolCattle .04a .91 Byrem et al., 1992

ClenbuterolCattle 18 55 Stoffel and Meyer, 1993Cattle 19 57 120b Sauer et al., 1995Horse 10c Kallings et al., 1991Human .9 33.9 Zimmer, 1976aHuman 30 Yamamoto et al., 1985Rabbit 9 Yamamoto et al., 1985Ratd 3.6 26.6 Kopitar and Zimmer, 1976Rate 1.9 25.4 Kopitar and Zimmer, 1976Rat 30 Yamamoto et al., 1985

FenoterolGuinea pig .6 Kords, 1975Mouse 2c Kojima et al., 1980bRat .04 .72 Koster et al., 1985bWomen .1 .9 Warnke et al., 1992Women .2 4.9 von Mandach et al., 1995

MabuterolHuman 20−30 Guentert et al., 1984

RitodrineHuman 1.3−2 Gandar et al., 1980

SalbutamolHuman 3.9 Morgan et al., 1986Human 2−3f Boulton and Fawcett, 1996Rabbit .75e Perreault et al., 1992Rabbit .68d Perreault et al., 1992Rabbit 1.5g Martin et al., 1971

TerbutalineHuman 15−18 Nyberg, 1984Human 14 Borgstrom et al., 1989aHuman 11 Bredberg et al., 1992

from the duodenum in humans (Borgstrom et al.,1990), and the levorotatory ( −) isomer was absorbedat almost twice the rate of the inactive dextrorotatorystereoisomer. The pH of the intestinal tract mayinfluence the site of absorption. The low pH of thestomach in nonruminants or the abomasum inruminants favors the formation of a cation at thephenethanolamine nitrogen, whereas the more neutralnature of the duodenum, jejunum, and ileum wouldreduce the extent of ionization and increase passiveabsorption across the intestinal mucosa. The author isunaware of any published report demonstrating thesite of absorption of b-agonists in ruminants.

Table 2 shows the plasma half-lives of several b-agonists in humans and other animals. The generalpattern evident from these studies is that halogenatedb-agonists have longer plasma half-lives than the b-agonists bearing hydroxyl groups on their aromaticrings. The terminal plasma half-lives of clenbuteroland mabuterol are greater than 20 h in all species

with the exception of rabbits. In contrast, the terminalhalf-lives of fenoterol, salbutamol, and ritodrine inhumans varies from less than 1 h to about 5 h.Terbutaline has an intermediate terminal half-life of11 to 14 h in humans. With the exception of twoclenbuterol studies and one cimaterol study, theplasma half-lives of other b-agonists in livestockspecies either have not been determined or are notpublished.

The half-life of clenbuterol in cattle, estimated fromthe urinary excretion of parent clenbuterol, rangedfrom 18 h for the initial distribution phase to 65 h forthe terminal half-life (Meyer and Rinke, 1991).Hooijerink et al. (1991) estimated the half-life ofclenbuterol in cattle to be approximately 36 h. Dataare not available for the urinary half life of other b-agonists in livestock species.

Although most b-agonists are well absorbed, theyare not equally available to target tissues afterabsorption. The systemic availability, absolute availa-

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bility, or bioavailability of a drug is the fraction of anadministered agent that reaches systemic circulationin its unchanged form (Gibaldi, 1991). Unless ametabolite is the biologically active form of a drug, theintact drug molecule must reach target tissues inorder for the drug to have an effect.

Bioavailability is often measured by determiningthe ratio of the area under the curve ( AUC)generated from a graph of plasma drug concentrationvs time after i.v. administration with the AUC derivedafter an alternative route of drug administration.Common alternate routes of administration are oral,i.p., s.c., i.m., or topical application. When the AUCafter oral administration is equal to the AUC after i.v.administration, the drug is said to be 100% bioavaila-ble. When the ratio of the AUC after oral and i.v.administration is less than 1, then the drug is lessthan 100% bioavailable (Peng, 1990; Hinchcliff et al.,1991).

Alternatively, bioavailability may also be estimatedby evaluating the urinary excretion of the parent drugafter the administration of a radioisotopically labeledcompound. In general, the greater the fraction ofparent compound excreted, relative to the totalamount of radioactivity excreted, the greater is thebioavailability of the drug. Similarly, bioavailabilityat any point in time can be assessed by measuring thefraction of the plasma radioactivity that is composedof parent drug.

A survey of the literature clearly reveals that thereare few published data on the systemic availability ofb-agonists in livestock species. Smith et al. (1993)showed that only 8% of an oral dose of ractopaminewas excreted in the urine unchanged by turkeys.Additionally, Dalidowicz et al. (1992) indicated thatswine excreted approximately 4 to 16% of the parentcompound in the urine following a single oral dose ofractopamine. After repeated dosing, the amount ofparent ractopamine in the urine increased to 36 to85% of the total urinary radioactivity. In rats given asingle oral dose of [14C]ractopamine, only 2% of theurinary radioactivity was parent ractopamine (Smithand Paulson, 1994). In contrast, 40% of the urinaryradioactivity excreted by cattle after a single oralclenbuterol dose was parent compound (Smith andPaulson, unpublished data).

Studies employing nonradioactive b-agonists incattle have shown that 18% of an i.v. cimaterol dosewas eliminated in the urine 8 h after administration(Byrem et al., 1992) and 40 to 70% of an oralsalbutamol dose (1 mg/kg BW) was excreted intourine as parent (Montrade et al., 1995). The latterstudy suggests that salbutamol has a relatively highoral bioavailability in cattle. Salbutamol has low oralbioavailability in other species (see below), suggest-ing that there may be species differences in the firstpass metabolism of salbutamol in cattle compared toother species.

Table 3 summarizes the percentage of parent b-agonist in plasma and (or) urine of humans, labora-tory animals, and livestock dosed with various b-adrenergic agonists. Few data are published forlivestock. The table is arranged by the pattern ofaromatic substitution on the agonist. In general, thedata indicate that following oral administration, thequantity of unchanged drug in plasma or urine is lessthan 50% of the total radioactivity present. Forcatechols, resorcinols, phenols, and saligenins, levelsof unchanged drug account for 25% or less of theurinary radioactivity or dose, with the exception ofterbutaline in dogs and salbutamol in cattle.Halogenated phenethanolamines have greater percen-tages of unchanged drug in plasma or urine after oraladministration. In contrast, the proportion of parentdrug present in urine of animals after i.v. administra-tion is greater than after oral administration regard-less of the compound. These data suggest thatintestine and liver play an important role in thebiotransformation of parent drug after oral adminis-tration.

No estimates of b-agonist bioavailability in farmanimals have been derived from pharmacokineticstudies using the ratio of the plasma AUC after oraland i.v. dosing. Bioavailability estimates of b-agonistsin humans, calculated from pharmacokinetic studies,were summarized by Morgan (1990) and have beenreviewed by Witkamp (1996).

Pathways of b-AdrenergicAgonist Biotransformation

Several factors influence whether a given b-agonistis readily bioavailable after oral administration. Asdiscussed above, absorption is not generally a limitingfactor for most b-adrenergic agonists. High rates ofbiotransformation followed by rapid elimination inurine or bile severely limit the bioavailability of someagonists, even to the detriment of their clinicaleffectiveness in humans (Morgan, 1990) and perhapsother species (Mersmann, 1995).

The metabolic pathways of biotransformation forthe phenolic, resorcinolic, catecholic, and saligenic b-agonists are exclusively through conjugation withsulfate or glucuronic acid (Table 4). The addition ofglucuronic acid, or sulfate, or both (Smith et al.,1995), facilitates rapid excretion in the urine and(or)bile (Sipes and Gandolfi, 1986). Conjugation of thearomatic hydroxyl groups is the only confirmed site ofconjugative metabolism when a b-agonist carries anaromatic hydroxyl group. For compounds bearing anaromatic hydroxyl group, no conjugates of the benzylichydroxyl ( b-hydroxyl) have been reported, suggestingthat the aromatic ring hydroxyl is a much bettersubstrate for the UDP-glucuronosyl transferases thanthe benzylic hydroxyl. A glucuronide conjugated to theb-hydroxyl group of clenbuterol was reported by


Table 3. Estimate of drug bioavailability as assessed by the presence of parent drug in urineor plasma of livestock, rodents, or humans after treatment with b-adrenergic agonists

aPercentage of radioactivity in plasma comprised of parent drug. Generally measured at peak concentrations (Cmax) of radioactivity in theplasma or serum.

bPercentage of radioactivity in urine comprised of parent drug.ci.d. = intraduodenal dose.dPlasma radioactivity from one patient, dose of .2 mg/kg.eCalculated from Table 3 of Conolly et al., 1972.fUrine collected during 1st d of a 4-d dosing regimen.gUrine collected during the 4th d of a 4-d dosing regimen.hParent expressed as a percentage of the total dose.

Compound and animal Dose Route Plasmaa Urineb Reference

Catechol

IsoproterenolDog 1 mg/kg i.d.c 10 8−24 Conolly et al., 1972Dog .27−.64 mg/kg i.v. 75 60−80 Conolly et al., 1972Human .04−.2 mg/kg Oral 0d 14.1e Conolly et al., 1972Human .06−.44 mg/kg i.v. 80 60 Conolly et al., 1972

Halogen

CimaterolCattle 47−65 mg/kg i.v. 18.3 Byrem et al., 1992

ClenbuterolCalf 3 mg/kg Oral ∼40 Smith and Paulson, unpublishedDog 2.5 mg/kg Oral 32.8 41.1 Zimmer, 1976bHuman 20 mg total Oral 75 66.4 Zimmer, 1976aRabbit 2.5 mg/kg Oral 39.4 67.1 Zimmer, 1976bRat 2 mg/kg Oral 45.2 69.7 Kopitar and Zimmer, 1976

MabuterolHuman 40−42 mg Oral 46 30 Guentert et al., 1984

Phenol

RactopamineSwine 30 mg/kg feed Oral 4−16f Dalidowicz et al., 1992Swine 30 mg/kg feed Oral 36−85g Dalidowicz et al., 1992Rat 9.9 mg/kg Oral 1.9 Smith and Paulson, 1994Rat 9.0 mg/kg i.p. 22.6 Smith and Paulson, 1994Turkey 6.7 mg/kg Oral 8 Smith et al., 1993

RitodrineHuman 84−445 mg i.v. 24 Kuhnert et al., 1986

Resorcinol

TerbutalineDog 1 mg/kg Oral 50 72 Nilsson et al., 1973aDog .5 mg/kg i.v. 91 Nilsson et al., 1973aHuman 51−58 mg/kg Oral 15 6h Nilsson et al., 1972Human 51−58 mg/kg i.v. > 50 60h Nilsson et al., 1972Rat 1 mg/kg Oral 3 8 Nilsson et al., 1973aRat 1 mg/kg i.v. 48 Nilsson et al., 1973aRat 5 mg/kg Oral 3h Conway et al., 1973Rat 5 mg/kg i.p. 19h Conway et al., 1973

Saligenol

SalbutamolCattle 1 mg/kg Oral 50−70 Montrade et al., 1995Human 4−8 mg Oral 20 <30 Evans et al., 1973Human .02−.2 mg i.v. >50 >>50 Evans et al., 1973Human 4 mg Oral 31.8h Conway et al., 1973Human i.v. 64.2h Morgan et al., 1986

Schmid et al. (1990), suggesting that in the absenceof an aromatic hydroxyl group, the b-hydroxyl groupmay serve as a substrate for the UDP-glucuronosyltransferases.

The exact site of conjugation for many b-agonistshas not been unambiguously resolved becausemethods such as mass spectrometry and enzyme or

acid hydrolysis (techniques often used to identify orcharacterize drug conjugates) do not generally permitthe exact site of conjugation to be determined. Unlesscare is taken to validate the activities of enzymes usedto hydrolyze drug conjugates in urine, quantitativeerrors can be introduced quite easily into studies inwhich the objective is to quantify the total amount of

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Table 4. Biotransformation pathways of b-adrenergic agonists in various animals

aConjugates of parent compound only.bAn X within the appropriate column indicates that the parent drug, an oxidative metabolite(s), or a conjugative metabolite was present

in excreta.cThe presence of a metabolite in the urine was suggested by authors, but no identification was made. The metabolite was unlikely to be a

conjugate because it appeared as a peak in the gas chromatographic analysis of cimaterol.dNI = not investigated.eIdentification of urinary oxidative and conjugative metabolites is in progress.fA ractopamine sulfate, glucuronide-diconjugate was isolated and identified.gA ractopamine di-glucuronide was tentatively identified.

Conjugatesa

Compound and animal Matrix Parentb Oxidative Glucuronide Sulfate Other Reference

CimaterolCattle Urine X ≈c NId NI Byrem et al., 1992

ClenbuterolCattlee Urine X X X NI NI Smith and Paulson,

unpublishedDog Urine X X X X Schmid et al., 1990Human Urine X X NI NI NI Zimmer, 1976aRabbit Urine X X NI NI NI Zimmer, 1976bRat Urine X X NI NI NI Kopitar and Zimmer,

1976FenoterolMice Urine X NI X X NI Kojima et al., 1980bHuman Plasma X NI X X NI Hildebrandt et al., 1994Rat Urine X None X None None Kojima et al., 1980aRat Bile None None X None None Kojima et al., 1980a

RactopamineRat Urine X None X None Smith and Paulson, 1994Rat Bile None None X X Xf Smith et al., 1995Swine Urine X None X None Xg Dalidowicz et al., 1992Turkey Urine X None X None Smith et al., 1993Turkey Bile None None X None None Smith and Paulson, 1993

RitodrineHuman Urine NI NI X X NI Brashear et al., 1990

TerbutalineDog Urine X NI None X NI Nilsson et al., 1973aHuman Urine X NI None X NI Nilsson et al., 1972;

Tegner et al., 1984Rat Urine X NI X None NI Nilsson et al., 1973a

drug excreted into urine. Before using enzymes toidentify metabolites, enzyme activity should be vali-dated with glucuronide or sulfate standards. For ring-hydroxylated b-agonists, it is possible to synthesizeauthentic glucuronide standards (Smith et al., 1993).

The oxidative metabolism of b-agonists bearingaromatic hydroxyl groups, other than salmeterol(Manchee et al., 1993), has not been confirmed in anyspecies. Oxidation of salbutamol by cattle has beensuggested by Montrade et al. (1995), but definitiveevidence for such a metabolite has not been presented.The use of radiolabeled salbutamol would help confirmthe formation of such a metabolite. Salmeterol ismetabolized through aliphatic oxidation, conjugation,and through o-dealkylation (Manchee et al., 1993).

Clenbuterol is reportedly metabolized by oxidativeand conjugative pathways in dogs (Figure 4; Schmidet al., 1990). With regard to the conjugative pathwaysthat were reported for parent clenbuterol, glucuroni-dation of the b-hydroxyl group and the aliphaticnitrogen, sulfation of the aromatic amine, and ethyla-

tion of the benzylic hydroxyl (formation of a clen-buterol b-ethyl ether) were said to occur in dogs(Schmid et al., 1990).

These conjugation pathways seem to be unique toclenbuterol. Glucuronidation of the aliphatic nitrogenand benzylic hydroxyl groups have not been reportedfor other b-adrenergic agonists, nor has ethylation ofthe b-hydroxyl group. Ethylation of the b-hydroxylgroup is a novel pathway for drug metabolism and theauthor is not aware of other drugs that are conjugatedin such a manner. A mechanism for the activation ofeither the benzylic hydroxyl group or of ethanol beforeconjugation is unknown. The formation of a clen-buterol ethyl ether during sample work-up is not anunreasonable explanation for the appearance of such ametabolite if the samples were ever exposed to acidicethanol. We (D. J. Smith and V. J. Feil, unpublishedobservations) demonstrated the formation of such anartifact by proton nuclear magnetic resonance spec-troscopy and mass spectroscopy when ractopamine isexposed to acidic methanol and a postulated mechan-


Figure 4. The pathways of oxidative metabolism ofmabuterol and clenbuterol in dogs and rats. Adaptedfrom Schmid et al. (1990) and Horiba et al. (1984).

ism for such a reaction has been published by Sill etal. (1987). By analogy, a similar “metabolite” could beformed from clenbuterol. If the clenbuterol b-ethylether is a biologically formed metabolite of clen-buterol, then the mechanism and site of its formationdeserves further study because of its unusual charac-ter.

The oxidative pathways through which clenbuterolis metabolized, as reported by Schmid (1990), aresimilar to those reported for mabuterol in rats (Figure4; Horiba et al., 1984), specifically, oxidation of the t-butyl group by the introduction of a hydroxyl group,oxidative deamination of the carbon-nitrogen bond atthe phenethanolamine a-carbon to yield a diol (formabuterol) and ultimately mandelic acid analogs. Themandelic acid analogs are then oxidized to theglyoxcylic acid and decarboxlyated to the benzoic acidanalogs and excreted, or conjugated with glycine to

yield the corresponding hippuric acid analogs. Schmid(1990) also reported the formation of 1,6,-dichloro-4-hydroxy analine from clenbuterol. Horiba et al.(1984) did not provide data to support the isolation ofglucuronic acid or sulfate conjugates of mabuterolmetabolites, but their isolation techniques likelywould hydrolyze sulfate and some glucuronic acidconjugates.

These studies are in excellent agreement with dataof Yamamoto et al. (1977) indicating that thechlorinated b-agonist C-78 [o-chloro-a- ( tert-but-laminomethyl)-benzyl alcohol hydrochloride; Figure 2]was metabolized to some extent to mandelic andbenzoic acid analogs. The main biotransfromationpathway for C-78 was hydroxylation of the aromaticring at the para- position. This metabolite had greaterpharmacological activity than the parent drug.

The metabolism of halogenated b-agonists inlivestock has not been published. Boenisch and Quirke(1992) suggested that the same pattern of clenbuterolmetabolism that is present in dogs is present in “foodproducing animals”, but they did not specify species,doses, or other experimental variables. They also didnot address whether the novel metabolites present indog urine were also present in the excreta of livestock.

In livestock, the involvement of liver and intestinein the biotransformation of b-adrenergic agonists isbasically supported by circumstantial evidence. Theextensive biliary elimination of ractopamine con-jugates in turkeys and rats suggests that liver plays amajor role in the formation of those conjugates (Smithand Paulson, 1993; Smith et al., 1995). In laboratoryspecies, biliary elimination of salbutamol conjugateshas been attributed to their formation in the liver(Martin et al., 1971).

Studies with laboratory species have establishedthe involvement of liver and intestine in the pre-systemic removal of b-agonists such as fenoterol andsalbutamol from systemic circulation. Koster et al.(1986) demonstrated that fenoterol was rapidlyglucuronidated in microsomes and cells from liver andintestine. The maximal rate of glucuronidation wasgreater for enterocytes than for hepatocytes but wassimilar for hepatic and intestinal microsomes. The Kmfor glucuronidation in enterocytes, however, was muchgreater than for hepatocytes, suggesting that theintrinsic clearance of fenoterol would be greater inliver. Ritodrine, salbutamol, and terbutaline were alsoshown to be glucuronidated by intestinal tissues atrates that could limit bioavailability (Koster et al.,1985a). These data have been corroborated by in vivostudies showing that the pre-systemic intestinalextraction ratio for fenoterol was .93 and for liver was.67. Thus, for some b-agonists, the intestine has agreater capacity to glucuronidate than does liver(Koster et al., 1985b), and liver has a large capacityfor glucuronidation.

Perreault et al. (1993) provided direct evidence forthe involvement of liver and intestine in thepresystemic elimination of salbutamol in rabbits by

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demonstrating that its extraction ratio by the intes-tine and liver was .92 and .71, respectively. Theestimated bioavailability of salbutamol was only .013and .15 after intra-duodenal and intra-portal adminis-tration, respectively. These results are also consistentwith those of Kurosawa et al. (1993), who noted thatonly 8% of an intraduodenally administered dose ofsalbutamol was present in systemic circulation inrabbits, even though over 93% of the dose wasabsorbed.

Hepatic UDP-glucuronosyl transferases from rabbitliver microsomes, immobilized onto Sepharose beads,have been used to synthesize milligram quantities ofractopamine glucuronides (Smith et al., 1993), andsimilar preparations from swine liver have been usedto synthesize sub-gram quantities of ractopamineglucuronides. During these synthetic efforts, thecapacity of the liver from a variety of species toproduce the enzymes required to metabolize ractopa-mine has been demonstrated. These findings suggestthat a number of other b-agonist-glucuronide con-jugates could also be synthesized in high yields usingthis technique.

Because b-agonists exist as mixtures ofstereoisomers they are susceptible to stereoselective orstereospecific metabolism. Stereospecific metabolismoccurs when only one stereoisomer of a racemate ismetabolized through a given pathway; stereoselectivemetabolism occurs when the pathways of metabolismfor individual stereoisomers are similar, but the ratesof metabolism differ. Stereospecific and stereoselectivemetabolism of b-agonists have not been extensivelystudied in any species, but evidence for each has beendemonstrated.

Smith et al. (1993) demonstrated the stereospecificglucuronidation of ractopamine HCl in turkeys. Allfour stereoisomers of ractopamine were glucuroni-dated at the N-methylpropyl phenol (the right-handring of ractopamine as shown in Figure 3), but onlythe RR and RS stereoisomers were glucuronidated atthe ethanolamine phenol of ractopamine (the leftphenol as shown in Figure 3). Thus, glucuronidationof the ethanolamine phenol was stereospecific for theRR and RS stereoisomers of the RR,SS and RS,SRracemates of ractopamine.

Stereoselective biotransformation is more commonthan stereospecific biotransformation. Examples ofstereoselective biotransformation of b-adrenergicagonists exist for terbutaline, fenoterol, and sal-butamol. Stereoselective biotransformation of clen-buterol (and other b-agonists) likely occurs but hasnot been investigated. The ( −)R stereoisomer ofsalbutamol has been demonstrated to be metabolizedto its sulfate conjugate (the major metabolite ofsalbutamol in humans) more rapidly than the inactive(+)S isomer by cytosolic fractions of liver, intestine,platelets (Walle et al., 1993), and by lung and bypulmonary epithelial cell cytosols (Eaton et al., 1996).

The intrinsic clearance ( Vmax ÷ Km) was 9.8 to 13.3times faster for the pharmacologically active ( −)Risomer than for the inactive (+)S isomer in thesetissues. Results of such studies suggest that the active( −)R isomer would be cleared more rapidly byhumans than the inactive (+)S isomer and it wasconfirmed by Boulton and Fawcett (1996). Theydemonstrated that the ratio of the ( −)R/(+)Sstereoisomers of salbutamol in humans dropped fromunity immediately after an i.v. infusion to .66 after 8h. The relative decrease in the ( −)R isomer wasbecause clearance was greater, and the terminal half-life of the ( −)R isomer was shorter than for the (+)Sisomer. When salbutamol was given orally, the ( −)R/(+)S ratio of the stereoisomers in plasma was .3 overthe duration of the 8-h study. As a result of the morerapid clearance of the ( −)R isomer, its oral bioavaila-bility was .3, compared with the oral bioavailability of.7 for the inactive (+)S isomer.

For terbutaline, both pharmacokinetic data andmetabolism data indicate that the inactivestereoisomer is metabolized and cleared more rapidlythan the active isomer. In vitro studies of Walle andWalle (1989) have demonstrated that the ratio ofsulfation of (+ ) terbutaline to ( −) terbutaline by ratliver cytosol was about 7.3. Because sulfation is amajor pathway of terbutaline elimination in humans(Nilsson et al., 1972), the ( −) stereoisomer wouldlikely be cleared less rapidly than the (+ ) isomer. Thishas been observed in humans following a single i.v. ororal dose or after repeated oral administration (Borg-strom et al., 1989a,b).

The stereoselective metabolism of other b-agonistshas not been extensively studied and not many dataexist. Especially important would be the determina-tion of the stereoselective or stereospecific metabolismof drugs such as clenbuterol and salbutamol, whichhave been used illegally in livestock. Residues of suchdrugs could be more potent than predicted if theresidue remaining in an edible tissue werepredominantly the active stereoisomer. Conversely, ifthe stereochemical composition of a drug residueincluded mainly inactive stereoisomers, then theresidue would likely represent less risk to consumersof such products.

Residues of b-Adrenergic Agonists in Livestock

Little is known about the total residues (parentplus metabolites) remaining in edible tissues ofanimals that have been treated with b-agonists. Onlythree studies have been published in which the totalradioactive residues present in the edible tissues oflivestock species have been quantified (Dalidowicz etal., 1992; Smith et al., 1993; Smith and Paulson,1997). Of these studies, only Dalidowicz et al., (1992)reported tissue residues after repeated doses of thetest drug, conditions that would simulate animal


exposure during efficacy experiments or under illicitconditions.

Data from studies in which radiolabeled b-agonistswere used clearly show that ractopamine HCl andclenbuterol HCl are not lipophilic. Total radioactiveresidues in swine adipose tissue were 20 ppb after a0-d withdrawal period from a diet containing 30 ppmractopamine, but after a 48-h withdrawal periodradioactive residues were not detectable (detectionlimit of 10 ppb; Dalidowicz et al., 1992). In contrast,total residues in liver and kidney were still detectable48 h after withdrawal of the compound. Total radioac-tive residues in adipose tissue of cattle given an oralbolus dose of 3 mg/kg BW [14C]clenbuterol HCl were1.1 ppm 48 h after dosing with higher concentrationspresent in liver and kidney (Smith and Paulson,1997). Total residues in liver of [14C]ractopamine-treated swine were 19 times greater than the adiposetissue residues at a zero withdrawal period(Dalidowicz et al., 1992) and total radioactiveresidues in bovine calf liver after [14C]clenbuteroltreatment were over four times those present inadipose tissue (Smith and Paulson, 1997). Similarly,radioactivity did not accumulate in adipose tissue of[14C]clenbuterol-, [3H]fenoterol-, or [3H]terbutaline-treated rats, mice, or rabbits (Kopitar and Zimmer,1976; Kojima et al., 1980a,b; Hsu et al., 1994). Asdescribed above, the chemistry of the aliphatic nitro-gen of phenethanolamine b-agonists suggests thatpartitioning into adipose tissue is not likely underphysiological conditions.

Radioactive residue levels were generally greatestin excretory tissues of [14C]ractopamine HCl-treatedanimals, with liver and kidney having the highestlevels of residues in both swine (Dalidowicz et al.,1992) and turkeys (Smith et al., 1993). In swine,kidney residues were greater than liver residues atzero withdrawal, suggesting the importance of thekidney in the excretion of ractopamine metabolites(over 88% of an oral dose of ractopamine was excretedin the urine of swine; Dalidowicz et al., 1992). Inturkeys, a species in which biliary excretion ofmetabolites is quantitatively as important as theurinary excretion of metabolites (Smith and Paulson,unpublished observations), liver residues were threetimes greater than kidney residues (Smith et al.,1993). After a 24-h withdrawal period, only 45 and11% of the total residues present after a 0-d with-drawal period remained in liver and kidney, respec-tively, illustrating the rapid decline of residues inthose tissues (Dalidowicz et al., 1992).

Residue depletion studies with clenbuterol arecurrently being conducted at the USDA BiosciencesResearch Lab in Fargo but have not been completed,so comments on the depletion of radioactive residueswill be restricted to a single dose study with bovinecalves and to rodent data. Total radioactive residueswere greatest in lungs, followed by liver and kidney ofcalves 48 h after a single oral dose (3 mg/kg BW) of

[14C]clenbuterol (Smith and Paulson, 1997). Concen-trations of total residues in skeletal muscle were aboutone-fifth that in liver and were roughly equivalent tothe total radioactive residues present in adiposetissue. Kopitar and Zimmer (1976) determined thattotal residues were greatest in lung and livers of ratstreated orally or i.v. with clenbuterol (Kopitar andZimmer, 1976), and muscle residues were consistentlyless than 5% of the total residue present in liver.

Depletion of total radioactive residues after clen-buterol administration has been studied in rats butnot in other animals. After i.v. administration totalresidues in liver dropped rapidly from 9% of the totaldose (2 mg/kg BW) at 1 h after administration to .8%of the administered dose after 24 h. Total liverresidues remained at .5% of the dose 48 and 72 h afterdosing, suggesting that a portion of the total residuemeasured at early time periods could be bound.Kidney residues also dropped rapidly during the first12 h after i.v. infusion of [14C]clenbuterol but re-mained constant from 24 to 72 h after dosing. Afteroral administration, kidney residues depleted to un-detectable levels at 48 h, but liver residues remainedconstant from 48 to 72 h.

Table 5 shows the relationship between totalradioactive residues in liver and kidney with theresidues of parent b-agonist for ractopamine HCl andclenbuterol HCl for swine and calves, respectively.Note that the doses administered in each study weredifferent in terms of amount and duration. Smith andPaulson (1997) administered a single oral dose of 3mg/kg BW [14C]clenbuterol HCl, whereas Dalidowiczet al. (1992) administered dietary [14C]ractopamineHCl (20 mg/kg feed) over a 7-d period. In the latterstudy, neither feed intake nor the size of the hog wasstated. Assuming that a 60-kg hog was fed and usingthe National Research Council (NRC, 1988) estimatefor feed intake (3.1 kg feed/d), then the daily dose of[14C]ractopamine HCl was 1.03 mg/kg BW. If the hogswere 80 kg, then the dose was approximately .8 mg/kgBW. Table 5 reveals two obvious differences betweenclenbuterol and ractopamine. First, total radioactiveresidues 48 h after a single clenbuterol administrationwere about 50 times greater in liver and kidney thantotal residues remaining in liver and kidney after a7-d exposure period to roughly one-third of theractopamine HCl dose. Comparing the residue dataafter 48 h of withdrawal for both compounds, the totalresidues in livers of clenbuterol-treated calves were 72times greater than the total residues in ractopamine-treated swine, and 123 times greater in kidney.

The second difference is the percentage of parentcompound remaining in each tissue. For clenbuterol,parent compound represented 44 and 63% of the totalradioactive residues in liver and kidney, respectively,after a 48-h withdrawal period. The amount of parentractopamine remaining in livers and kidneys of swineafter the same withdrawal period, however, was 5.5and 16.7%, respectively.

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Table 5. The relationship between the total radioactive residues present in tissuesof swine or calves treated with [14C]clenbuterol HCl or ractopamine HCl

aData taken from Dalidowicz et al., 1992; animals were provided 20 mg/kg ractopamine HCl in the feedfor a 7-d period and were slaughtered at the indicated withdrawal times.

bData taken from Smith and Paulson, 1997; [14C]clenbuterol HCl was administered as a single oraldose of 3 mg/kg BW; two calves were slaughtered after a 48-h withdrawal period.

Liver residues, ppm Kidney residues, ppm

Compound and withdrawal, h Total Parent % Parent Total Parent % Parent

Swine

Ractopaminea

24 .106 .015 14.1 .116 .032 27.548 .073 .004 5.5 .048 .008 16.772 .056 .002 3.6 .036 .003 8.3

Calves

Clenbuterolb

48 5.04 2.23 44.2 5.90 3.71 62.9

A summary of data derived from clenbuterol residuestudies in cattle is shown in Table 6 and for sheep,chickens, and trout is shown in Table 7. Directcomparison of the data is difficult because differentdosages, feeding periods, and withdrawal times wereused within and across species. In many studies thedosage was calculated as milligrams/kilogram BW,and in others the dosage was expressed as milligrams/kilogram diet (and animals were either limit-fed orwere given ad libitum access to the feed). To makecomparisons even more difficult, some reports havenot provided either feed intake data or the size of theanimals fed, which disallows the expression of thedosage on a milligram/kilogram BW basis.

Nevertheless, several conclusions can be made fromthe data. The accumulation of clenbuterol in liver andkidney is dosage- (Elliot et al., 1993a) and time-dependent (Elliot et al., 1993b). In cattle, theaccumulation of clenbuterol in liver reached a maxi-mum after 15 d of treatment (Elliot et al., 1993a).Parent clenbuterol depleted fairly rapidly from liverand kidney during the first 48 h after withdrawal(Malucelli et al., 1994) but depleted more slowly after48 h. The initial half-life of liver and kidney clen-buterol residues in calves was estimated to be 41 and31 h, respectively; the slower portion of the elimina-tion curve was characterized by tissue half-lives forliver and kidney of 170 and 153 h, respectively (Saueret al., 1995). A biphasic pattern of clenbuterolelimination is also characteristic for plasma and urine(Stoffel and Meyer, 1993; Sauer et al., 1995),although residues deplete more rapidly from plasmaand urine than from tissues. In urine, the initialelimination half-life of clenbuterol was 10 to 28 h,followed by a slower elimination phase characterizedby a half-life of 60 to 78 h (Meyer and Rinke, 1991;Sauer et al., 1995).

Liver residues of clenbuterol remained in the partsper billion range between 16 and 39 d after thetermination of treatment (16 mg/kg BW) and in theparts per trillion range as long as 56 d after

withdrawal (Elliot et al., 1993c). These data aregenerally representative of the depletion of clenbuterolin other species with the exception of rainbow trout. Introut, clenbuterol residues remained as high as 24 ppbin livers for 30 d after a withdrawal period wasinitiated (Brambilla et al., 1994). The relatively highdose of 5 ppm of dietary clenbuterol administered inthis study undoubtedly contributed to the high tissueresidues and prolonged time required for depletionfrom tissues. Sheep fed 3.1 ppm dietary clenbuterol for14 d had liver clenbuterol residues of 3.6 ppmfollowing a 15-d withdrawal period. These residueswere 35-fold less than the hepatic clenbuterol of trout,which suggests that trout may eliminate the drugmore slowly than other species.

Tissue clenbuterol concentrations decrease mostslowly from the eye and perhaps hair or feathers(Malucelli et al., 1994). If clenbuterol accumulatesappreciably in any tissue, then it does so in pigmentedocular tissues or in pigmented hair rather than inadipose tissue. Clenbuterol residues accumulated inthe retina and choroid of the eye rather than inunpigmented tissues of the eye (Sauer et al., 1995;Smith and Paulson, 1997). Melanin is the componentwithin the eye that is thought to be responsible forbinding clenbuterol (Howells et al., 1994; Sauer andAnderson, 1994) and other b-agonists such as sal-butamol and salmeterol (Howells et al., 1994). Ocularresidues of clenbuterol in albino rats, which lackpigmentation, were comparable to ocular residues ofclenbuterol in control animals, whereas strains of ratswith pigmented eyes showed clear evidence of ocularclenbuterol accumulation (Dursch et al., 1995). Asimilar observation was made with regard to clen-buterol binding in hair; white hair bound lessclenbuterol than brown hair, which bound less thanblack hair in guinea pigs (Polettini et al., 1995) andcattle (Dursch et al., 1995; Smith and Paulson, 1997).The mechanism of binding has been proposed to bethrough a combination of ionic and hydrophobic forces,


Table 6. Comparison of residues of clenbuterol in tissues of clenbuterol-treated cattle

aNA = not available.bClenbuterol residues present in the retina or retina/choroid of the eye.cClenbuterol residues present in the whole eye homogenate.dValues represent a mean of the values reported for retinal clenbuterol residues as measured by enzyme immunoassay and gas-

chromatography-mass spectroscopy.eTotal radioactive residues, reported as clenbuterol HCl equivalents.

Tissue concentration, ppb

Dose and duration n Withdrawal, d Liver Kidney Eye Reference

16 mg·kg−1·d−1 Elliot et al., 1993c30 d 2 0 85.9 NAa 1,671b

2 16 3.0 NA 181b

2 39 .7 NA 116b

2 56 .4 NA 89b

16mg·kg−1·d−1 Elliot et al., 1993b30 d 2 0 61.7 NA 1,675b

2 15 1.9 NA 181b

5 mg·kg−1·d−1 Meyer and Rinke, 199121 d 3 0 39.0 32.7 118c

2 3.5 1.6 1.2 57.5c

2 14 .6 <.1 15.1c

16 mg·kg−1·d−1 Elliot et al., 1993a1 d 2 0 29.2 NA 329bd

7 d 2 0 57.8 NA 1,692bd

15 d 2 0 194.7 NA 2,661bd

30 d 2 0 73.8 NA 1,686bd

2 15 2.4 NA 165bd

2 35 .7 NA 73bd

2 56 .4 NA 72bd

2 84 <.2 NA 59bd

2 112 <.2 NA 24bd

2 140 <.2 NA 17bd

20 mg·kg−1·d−1 Biolatti et al., 199442 d 6 0 24.0 15.6 90b

3 mg/kg Smith and Paulson, 19971 d 2 2 2,200 3,700 84,500e

but a clear understanding of the mechanisms involvedhas not been developed.

A combination of mechanisms probably exists thatgoverns the binding of b-agonists to melanin. Not allb-agonists have the same propensity to bind to oculartissues in vivo or in vitro, even when their structuresare similar. For example, Manchee et al. (1993)demonstrated that salmeterol binds to ocular tissuesin pigmented rats after in vivo exposure. Their datacorroborated in vitro data that show that salmeterolhas a propensity to bind to melanin (Sauer andAnderson, 1994). However, this was not true forsalbutamol, which is structurally identical tosalmeterol in the phenethanolamine portion of themolecule. Salbutamol bound poorly to melanin duringin vitro studies (Howells et al., 1994; Sauer andAnderson, 1994) and also bound poorly to oculartissues during in vivo studies (relative to clenbuterol;Polettini et al., 1995). Salmeterol is more hydrophobicthan salbutamol (Johnson, 1995), but its hydropho-bicity in relationship to clenbuterol is unknown.During in vitro studies, clenbuterol and salmeterolhad roughly equal abilities to bind to melanin.

Whatever the mechanism of binding, clenbuterol isdepleted very slowly from ocular tissues and clen-buterol residues have been detected readily in wholeeye homogenates of cattle 15 (Meyer and Rinke,1991) or 42 d after termination of clenbuteroltreatment (Elliot et al., 1995). When retinal extractswere evaluated after a 56-d withdrawal period,clenbuterol concentrations were 55 to 123 ppb. Theselevels were comparable to clenbuterol residues presentin liver after a 0-d withdrawal period (Elliot et al.,1993c). Because of the high concentrations of residueremaining in ocular tissues or in hair after longwithdrawal periods, these tissues have been suggestedas matrices for detecting illegal clenbuterol use. In theUnited States, where clenbuterol has not been ap-proved for use in livestock, hair or eyes would be anexcellent matrix; however, in Europe, where clen-buterol is registered for therapeutic use, these tissuesmight have limited usefulness (Elliott et al., 1995).

Table 8 summarizes the published studies thathave investigated residues of b-agonists other thanclenbuterol in livestock species. In chickens receiving10 ppm dietary salbutamol or terbutaline, salbutamol

SMITH188

Table 7. Residues of clenbuterol in chickens, sheep, and trout

aIntake of feed containing clenbuterol was restricted to .5 kg/d.bWhole eye homogenate.cNA = not available.



Clenbuterol-treated chickens

1 ppm in diet Malucelli et al., 199414 d 5 0 66.6 24.3 89.9b

5 1 22.2 2.6 NAc

5 2 13.1 2.0 NA5 3 7.1 1.2 NA5 7 10.8 1.5 NA5 14 2.3 .2 6.6b

5 43 <.1 <.1 2.6b

Clenbuterol-treated sheep

.13 ppm in dieta (1.6 mg/kg BW) Elliot et al., 1993a14 d 3 0 20.2 16.4 NA

3 5 2.1 1.2 NA3 10 .7 .1 NA3 15 .6 .1 NA

1.3 ppm in diet (16 mg/kg BW) Elliot et al., 1993a14 d 3 0 162.0 83.9 NA

3 5 24.6 2.8 NA3 10 4.9 .4 NA3 15 3.2 .4 NA

3.1 ppm in diet (39.2 mg/kg BW) Elliot et al., 1993a14 d 3 0 302.4 267.2 NA

3 5 51.4 4.8 NA3 10 14.8 .5 NA3 15 3.6 1.0 NA

Clenbuterol-treated trout

5 ppm in diet Brambilla et al., 199415 d 5 0 440 NA NA21 d 5 0 320 NA NA

5 6 290 NA NA5 9 205 NA NA5 15 128 NA NA5 21 78 NA NA5 30 24 NA NA

residues were approximately twice those of terbutalineafter a 0-d withdrawal period for liver and kidney, butafter a 1-d withdrawal period residues of each drugfell to similar levels (Malucelli et al., 1994). In thesame study, residues of clenbuterol in liver and kidneywere consistently lower than residues of terbutaline orsalbutamol, but the dietary level of clenbuterol wasone-tenth that of either salbutamol or terbutaline. Asa proportion of the administered dose, salbutamol andterbutaline residues were lower than clenbuterolresidues. Likewise, when one considers the 2- to20-fold greater dose, residues of parent ractopamine inliver and kidneys of swine (Dalidowicz et al., 1992)were proportionally lower than the residues of clen-buterol, terbutaline, or salbutamol in chickens(Malucelli et al., 1994).

One study has investigated the residues of sal-butamol in calves (Montrade et al., 1995), butradiolabeled salbutamol was not used, so total

residues are not known. Two calves were treated dailywith an oral dose of 1 mg/kg BW salbutamol for sevenconsecutive days. One animal was slaughtered with nowithdrawal period and the other animal was slaugh-tered after a 7-d withdrawal period. Liver residues ofsalbutamol were almost 4 ppm at zero withdrawal andhad fallen to .110 ppm after a 7-d withdrawal period.

Discussion

Clenbuterol residues present in tissues of illegallytreated calves have caused acute poisonings in con-sumers of the tainted liver (Martınez-Navarro, 1990;Pulce et al., 1991; Salleras et al., 1995). Conse-quently, b-adrenergic agonists as a class have comeunder intense scrutiny as potential toxins in the foodsupply, and legislation in Europe has banned their useas leanness-enhancing agents for the foreseeable


Table 8. Residues of salbutamol and terbutaline in liver, kidney, and eye

aTwo calves were used in the cited study; a calf slaughtered after a 7-d withdrawal period was reported to have liver residuesapproximately 35 times higher than a calf slaughtered after no withdrawal period. Likewise, kidney residues reported for the calf slaughteredafter the 7-d withdrawal period were 13 times greater than the residues reported for the calf slaughtered after no withdrawal period. Personalcommuncation with Dr. Montrade has confirmed that the questionable data reported by Montrade et al. (1995) were transposed into thewrong columns. Thus, the liver containing the lower concentration of residues was from the calf slaughtered with the longer withdrawalperiod.

bNA = not available.



Salbutamol-treated chickens

10 ppm in diet Malucelli et al., 199414 d 5 0 334 110 85

5 1 118 16 NAb

5 2 47 10 NA5 3 19 5 NA5 7 44 9 NA5 14 4 <1 195 43 <1 <1 4

Salbutamol-treated cattlea

1 mg/kg BW Montrade et al., 19957 d 1 0 3,920 130 NA

1 7 110 10 NA

Terbutaline-treated chickens

10 ppm in diet Malucelli et al., 199414 d 5 0 165 55 22

5 1 98 18 NA5 2 48 5 NA5 3 37 4 NA5 7 18 4 NA5 14 7 <2 <25 43 <2 <2 <2

Ractopamine-treated swine

20 ppm in diet Dalidowicz et al., 19924 d 6 1 15 32 NA

6 2 4 8 NA6 3 2 3 NA

future. Resistance to the use of phenethanolamineleanness-enhancing agents in livestock is widespread(Witkamp and van Miert, 1992; Witkamp, 1996),even though it has been recognized that the Europeanban on anabolic agents has “provoked new safetyproblems rather than solving existing problems”(Meyer, 1991). The European ban on b-adrenergicagonists has not eliminated their illegal use in Europe(Kuiper et al., 1996), and abuse of b-agonists has alsobeen documented in the United States (Mitchell,1996).

The propensity for clenbuterol toxicosis to occur inindividuals who have eaten contaminated animalproducts is related to its oral potency. Table 9 showsthe doses of b-agonists required for clinical efficacy inhumans after oral administration. A dose as low as 10mg (total mass, not 10 mg/kg BW) of clenbuterol is aneffective orally administered bronchodilating agent,but for salbutamol or terbutaline to be orally effective2,000 to 5,000 mg (total mass) must be administered.For ritodrine, an oral dose of 10,000 mg, preceded by12 h of intravenous ritodrine infusion, is required for

clinical effectiveness. For continued therapy, clen-buterol need only be administered twice daily,whereas other b-agonists should be administered 3 to12 times daily. There are obviously large differences inoral potencies among b-agonists.

Just as the dose of clenbuterol required for clinicaleffectiveness differs from terbutaline and ritodrine byseveral orders of magnitude, the highest dose ofclenbuterol that would have no observable effect( NOEL) in humans may also differ from terbutalineand ritodrine by the same orders of magnitude. Thishas important implications for the development of b-agonists for use in meat-producing animals. The oralNOEL of clenbuterol in humans is 2.5 mg (total mass;Boenisch and Quirke, 1992), but there are nopublished NOEL available for other b-agonists. Gener-ally, NOEL for products developed for animal produc-tion purposes are not determined in humans, butclenbuterol is registered for human use in somecountries. However, because of the difference in theoral potencies among these b-agonists, it is likely thatthe oral NOEL for ritodrine, fenoterol, terbutaline,

SMITH190

Table 9. Therapeutic doses of b-adrenergic agonists used in humans for bronchodilation or tocolytic purposes

aSingle administration; note most b-agonists require multiple administrations during a 24-h period.bThe dose on a body weight basis was calculated assuming an average adult body weight of 60 kg.cNOEL = highest dose with no observable effect.dNA = not available.eOral therapy with ritodrine is usually preceded by intravenous ritodrine infusion (15−35 mg/min) for a duration of 12 h.

Oral dosea

Compound mg mg/kg BWb Doses/day NOELc Reference

Clenbuterol 10 to 20 .16 to .33 2 2.5 mg/d Prather et al., 1995Fenoterol 2,500 to 5,000 42 to 83 3 NAd Heel et al., 1978Ritodrine 10,000e 167 12 NA PDR, 1995bSalbutamol 2,000 to 4,000 33 to 67 3 to 4 NA Price and Clissold, 1989Terbutaline 5,000 83 3 NA PDR, 1995a

and salbutamol, are orders of magnitude greater thanthe NOEL for clenbuterol.

The rationale for determining the NOEL for a givenb-agonist in humans is that the NOEL is used byregulatory agencies to calculate acceptable daily in-takes ( ADI) of drug residues. The ADI (NOELdivided by an appropriate safety factor) representsthe amount of total residue that a model adult human(60 kg) could safely consume each day throughout alifetime (Guyer and Miller, 1994). The safe tissueconcentration for chemical residues is calculated fromthe ADI using a food factor published by the FDA(FDA, 1994). Thus, the allowable amount of a drugresidue in any edible tissue is ultimately dependent onthe NOEL of the compound.

The NOEL for clenbuterol in humans is 2.5 mg(Boenisch and Quirke, 1992). This means that aperson could be expected to ingest 2.5 mg of clenbuterolper day (.04 mg·kg−1·d−1 for a 60-kg human) with noobservable effects. The ADI is calculated with a safetyfactor to account for human variability; as describedby Boenisch and Quirke (1992), a safety factor of 10(rather than 100) was applied to clenbuterol becausethe NOEL was determined in humans, rather than inlaboratory animals. Thus, the ADI for clenbuterol is250 ng/d (total mass; Boenisch and Quirke, 1992; 4.1ng·kg−1·d−1 for a 60-kg human).

No NOEL have been published for b-agonistsdeveloped for use in animal production (cimaterol;L646,969; ractopamine; Anderson et al., 1990). As aresult, a direct comparison of ADI among different b-agonists is difficult, if not impossible. Data have beenpublished on the cardiovascular effects of butopaminein humans (Thompson et al., 1980). Butopamine isthe RR stereoisomer of ractopamine (see Reynolds etal., 1989 monograph on butopamine; Yen et al., 1989;U. S. Pharmacopoeia, 1997). Ractopamine is a mix-ture of RR, RS, SR, and SS stereoisomers (U. S.Pharmacopoeia, 1997). It could be argued that aNOEL for butopamine in humans could be constructedfrom the data of Thompson et al. (1980) because theirstudy was essentially a titration of butopamine doseon cardiovascular variables. Because the biological

activity of ractopamine likely resides with the RRstereoisomer (Yen et al., 1989; Ricke et al., 1996), aNOEL for ractopamine in humans can also beestimated from this data. It should be emphasizedthat the NOEL calculated for ractopamine in thismanner is for discussion purposes only and in no wayrepresents a NOEL established by a regulatoryagency.

Butopamine administered to humans by i.v infusionat .04 mg/(kg·min) for 1 h (144 mg total mass for a60-kg person) had no effects on heart rate or othercardiovascular variables. This dose of butopaminecorresponds to 576 mg of ractopamine, because rac-topamine is composed of RR, RS, SR, and SSstereoisomers. If one accepts 576 mg of ractopamine asan estimated i.v. NOEL in humans (9.6 mg·kg−1·d−1

for a 60-kg human), then one could calculate the ADIfor ractopamine and compare it with clenbuterolbecause its ADI is known. In the calculation ofclenbuterol’s ADI, a safety factor of 10 was usedbecause the data were generated in humans (Boenischand Quirke, 1992). Dividing the estimated NOEL ofractopamine (576 mg) by the same safety factor leadsto an ADI of 57.6 mg/d for ractopamine.

The estimated ADI of i.v.-administered ractopamineis 230 times greater than the ADI calculated from oralexposure to clenbuterol. Because ractopamine is apara- substituted phenolic phenethanolamine b-agonist (the phenol rings of ractopamine are identicalto ritodrine; see Figure 3), its oral bioavailability inhumans is likely very poor. Because effective oraldoses of b-agonists are generally 5 to 10 times greaterthan the effective parenteral doses (Morgan, 1990),the oral NOEL for ractopamine would likely be 5 to 10times greater than the NOEL estimated from dataobtained after i.v. infusion. Thus, the oral potency ofractopamine in humans is likely three orders ofmagnitude less than the oral potency of clenbuterol.

Obviously, the argument stated above is based onseveral assumptions, namely, that the RRstereoisomer of ractopamine is its active isomer, that aNOEL for ractopamine can be estimated from thestudy of Thompson et al. (1980) of butopamine inhumans, and that the bioavailability of ractopamine is


poor in humans, but each assumption is based on solidprinciple. Clinical data of b-agonists approved forhuman therapeutic purposes indicate the potencies oforally administered b-agonists may differ by severalorders of magnitude, so it would not be unrealistic forthe potency of clenbuterol and ractopamine, or other b-agonists, to differ by such a wide margin. This beingthe case, it is likely that the NOEL for b-agonists oflow oral potency are correspondingly greater than forb-agonists of great oral potency. Likewise, the safetissue concentrations, or maximum residue limits, ofb-adrenergic agonists with low oral potency should begreater than for high potency b-agonists such asclenbuterol.

If these assumptions are true, and the wealth ofdata available on the use of b-agonists in humanssuggests that they are, then it is likely that effectiveb-adrenergic agonists can be developed for use inanimal production, and that residues of such com-pounds in edible tissues would present negligible riskto consumers. Indeed, the available data suggest thatthe risk of human poisoning from the consumption ofresidues present in edible tissues of animals properlytreated with approved, low oral potency b-agonistswould be orders of magnitude less than risks as-sociated with the consumption of tissues from animalstreated illegally with high potency b-adrenergicagonists.

Implications

The reviewed data indicate that the metabolismand the disposition of b-adrenergic agonists in mam-mals and avians are dependent on the chemicalproperties of the particular b-agonist. As such, the oralpotencies of b-agonists may differ by as much as threeorders of magnitude. Clenbuterol has received muchattention in recent years because it has been usedillicitly in animal production, resulting in clenbuterol-contaminated tissues that have produced acute toxic-ity in consumers. Not all b-adrenergic agents are asorally potent as clenbuterol, however, and it is likelythat safe and effective b-agonists could be developedfor use in animal production.

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