The Use of Toxicokinetics D BruceCampbell · (toxicokinetics) in order that conclusions based only...

Molecular NeurobiologyCopyright © 1995 Humana Press Inc.All rights of any nature whatsoever reserved.ISSN0893-7648/95/11 (1-3)'_ 193-216/$8.80

The Use of Toxicokinetics

for the Safety Assessment of Drugs Acting in the Brain

D. BruceCampbell

Servier Research and Development, Fulmer Hall, Windmill Road, Fulmer, Slough, SL3 6HH, UK

Abstract

Pharmacological and toxicological studies undertaken on drugs that affect the brain arefrequently performed in disparate species under various experimental conditions, at doses oftengreatly in excess of those expected to be administered to humans, and the findings areextrapolated implicitly or explicitly with scant regard to differences in the biodisposition of thedrugs. Such considerations are necessary since:

1. Species;2. Strain;3. Gender;4. Route;5. Dose;6. Frequency and time of administration;7. Temperature;8. Coadministration of drugs; and9. Surgical manipulation

are but some of the factors that have been shown to influence the kinetics and metabolism of

drugs. This article, using MDMA and other phenylethylamines as examples, provides evidencefor the need to measure the exposure of the drugs and their active metabolites in blood and brain(toxicokinetics) in order that conclusions based only on dynamic, biochemical, or histologicalevidence are more pertinent. Further, the combined use of toxicokinetic-dynamic modeling canlead to a better appreciation of the mechanisms involved and a more useful approach to thecalculation of safety margins.

Index Entries: Pharmacokinetics; pharmacodynamics; metabolism; allometry; phenyl-ethylamines, MDMA; mathematical modeling.

Molecular Neurobiology 193 Volume 11, 1995

194 Campbell

Introduction What Is Toxicokinetics?

The choice of dosage, frequenc_ and experi- Toxicokinetics has been defined as "the gen-mental design for neurotoxicological investiga- eration of pharmacokinetic data either as antions of drugs of abuse in animals appears to integral component in the conduct of nonclini-

have evolved by experimental trial and error, cai toxicity studies or in specially designedOnce a dose and regimen are found, then they supportive studies, in order to assess systemicare used by others to replicate the study design exposure" (ICH2,1993). In practice, samples ofand hopefully the results. Little consideration body fluids or tissues are taken to measure thewould seem to be made regarding the rel- drug or active metabolite level, often at theevance of the chosen dose or the route of same time as other biochemical measures, to

administration, either for the interpretation of assess the rate and extent of exposure duringthe study or for the possible extrapolation all pivotal toxicological studies. Repeat sam-to humans. Often, in the last paragraph of the piing may in some cases be hazardous to the

conclusion, authors attempt to give their conduct of the study, and satellite groups heldresults some clinical perspective by a simple under similar conditions can be used to obtain

comparison of the doses used in animals to sufficient samples. Sometimes it is difficult toachieve their effect and those used in humans, take multiple samples from single animals toUnfortunately, animals have different kinetic measure certain kinetic parameters, such as theand metabolic profiles from those found in area under the plasma curve, and techniques,humans, and the extent of exposure of the such as composite sampling, may be employed.active compounds may not simply be related In this case, instead of taking four to sixto dosage. For example, the amphetamines samples from each animal (serial sampling) orgenerally have a half-life of approx 2 h, but are killing an animal at each time-point (destruc-generally administered sc every 12 h for 4 d, tive sampling), two to three samples are takenwhereas in humans, these drugs of abuse have from each animal, but at different times to

a half-life of approx 10 h and are probably cover the whole profile. This reduces the stresstaken by mouth once or twice a week (Cook et on the serial animals and the total number of

al., 1992). In the past, such sophistication was destructive animals (Campbell and Jochemsen,hampered by the lack of suitable analytical 1994). Another method of overcoming thetechniques, but not so today, where relatively problem is to use a microdialysis probe tosimple methods can measure drugs like sample plasma or tissue effluents, but sampleMDMA and its metabolites with blood samples size is small and analytical methods need to beas low as 200 I_L (Michel et al., 1993). particularly sensitive.

This brief article attempts to explain why theextrapolation of results from animals to humans

based on dosage alone without concomitant Why Is Toxicokinetics Necessary?measurement of blood or tissue levels can lead

to the misinterpretation of data, particularly The extrapolation of animal data to humanswhen the metabolites are probably more active has relied on the use of the highest dose in ani-than the parent drug. Recently, the importance mals that produces a no effect (NOEL) or theof this concept has led to the drafting, and in lowest dose (LOEL) that produces a measur-principle acceptance, by regulatory agencies able toxic effect, if this can be defined. Forand industrialists alike of toxicokinetic guide- many neurotoxins, this is understandable sincelines on the need for measurement of drug lev- there may be no "normal" plasma levels inels in all toxicological or safety studies in humans, and it would be difficult to undertakeanimals so that more meaningful extrapolation studies to examine this. The same is true, toto humans can be made (ICH2, 1993). some extent, for the drugs of abuse, but often

Molecular Neurobiology Volume 11, 1995

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Use of Toxicokinetics 195

some data do exist that could provide a basis Table 1for extrapolation. There are two basic reasons Possible Effects of High Dosage and Specieswhy simple dosage comparisons can be incor- Differences on Kinetics and Brain Concentrationsrect. First, there are frequently large differences Kinetics Species High dose Brain levels

in the rates and routes of elimination between Absorption +' Saturation $species, which, as will be explained, is often (act/pass),dependent on the relative body weight of the Distributionspecies. Second, the higher doses and the Protein +++ Saturation troutes of administration that are used in toxi- bindingcological studies can dramatically alter the kin- Tissue uptake ++' Saturation 1TM$'etics, leading to nonlinear drug exposure. In (act)addition, the experimental procedure, coadmin- Eliminationistration of another drug, anesthetic, housing Renal +_ Saturation tconditions, age of the animal, gender, naivety Metabolism +++ Saturation tof animals, and so on, can also influence the $ (MetabY

biodisposition of any test compound let alone ,? increase, $ reduce, + small, ++ large, +++ very largethe effect, species differences can be expected.

bact = active transport, pass = passive diffusion.This brief article examines the kinetic rlnsufficientinformation available.

processes of absorption, distribution, and q.¥take into the brain is saturated.elimination during toxicological studies with 'Uptake into other tissues is reduced, allowing greater

particular emphasis on species and dosage dif- uptake into the brain.tActive or toxic metabolite levels will be reduced if

ferences (Table 1) explaining how they may metabolism of parent drug is saturated.influence their interpretation.

oral dosing, but without changing the norfen-Absorption fluramine concentrations owing to marked, but

disproportionate first-pass hepatic metabolismThere are relatively few differences in the (Zaczek et al., 1990; Gordon, 1991 unpublished).

relative rates and extent of absorption between Little is known regarding route-specific absorp-species, and often animal models are used with lion or metabolism for other phenylethylamines.some success to compare new formulations(Clarke and Smith, 1984). Similarly, over a

small range of doses, unless the drug is actively Distributionabsorbed, this process is linear. At very high

toxicological doses, a reduction in total absorp- Plasma Protein Bindingtion can occur, particularly if the compoundhas a poor dissolution or has a local effect, It is generally accepted that it is the free drugsometimes limiting the total amount of drug that is active, and Smith (1993) has shown thatthat can be put into the systemic circulation by there can be more than a 20-fold differencethis route. In neurotoxicological studies, many in the unbound concentrations for certain drugsdrugs are administered ip or sc, and, although in animals compared to humans, and that thesethis may overcome problems of dose-related differences cannot easily be predicted. It wouldreductions in absorption, by-passing the initial appear from those drugs studied that the freehepatic metabolism can lead to large differ- active levels of drug are generally lower inences in the relative proportion of drug to humans compared to mouse, rat, or dog. Thismetabolite seen in oral dosing. Thus, for fen- can become very important if brain levels arefluramine, the parent drug levels are increased not measured, particularly for those com-fivefold after sc dosing in the rat compared to pounds with high plasma protein binding


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196 Campbell

where only small changes in binding, for exam- '° 1

pie from 98 to 96%, can increase free levels by , t _ -. "' '"_'_""-200% and thus the potential for brain uptake. _"_'"'_'_'In addition, the amount of free drug can _-' '-.-...

increase with dosage owing to the saturation i _"' ''" ' -of binding sites, but this will depend on to _ ,o -.. ,._

which plasma protein the drug is bound. Pro-, ,] ' _'"_ __ ' _.=tein binding is relatively low for the amphet- · ' -..

amines (<50%) (Campbell, 1978), and because '°1 c,_. _ _..._this is specific to albumin, which has high 0 _.0, 0.... 0 ,o0amounts present in plasma, more than 500 ,-, ..,,,, ,_,,_g/mL of a drug with a mol wt of 200 wouldbe needed before saturation occurs, assuming Fig. 1. Relationship between the brain plasmaone binding site. For drugs that bind to the ratio of d-fenfluramine (O), d-noffenfluramine (B),lower circulating levels of ch-glycoprotein, the and body weight for different species.drug levels at which saturation may occur will

be correspondingly lower (_20 lig/mL). Simi- in brain than the parent drug, which is unex-lac considerations hold for drugs that bind to pected since the partition coefficient, and,lipoprotein, but less information on these inter- therefore, lipid-solubility, is lower for theactions is available. Unfortunately, these con- metabolite, but without a significant difference

siderations are rarely taken into account when in the plasma protein binding (Campbell,comparing exposure of drugs and can poten- 1978). This would suggest a specific transporttially lead to big discrepancies in the calcula- or binding for the metabolite. Similar speciestion of safety margins, differences in brain uptake have been reported

for fluoxetine and norfluoxetine, using theTissue Uptake more physiological technique of MRS in

For drugs that act as central neurotoxicants, humans after therapeutic dosing (Karson et al.,it has been assumed that the uptake and distri- 1992; Renshaw et al., 1992), and brain excision

for rats (Caccia et al., 1992) and for at least onebution into the brain are similar between spe-cies. This concept has rarely been challenged, other drug, alapride, a novel nootropic agentsince it is not common practice to measure (Lapka, 1991). These species differences,brain levels in animals, and because of the therefore, may be more common than previ-

extreme difficulty in extrapolating the results ously thought.to humans. Recent results with fenfluramine There are a number of possible reasons for

these observations, including species differ-and norfenfluramine using postmortem tissueafter overdosage (30--40 mg/kg) suggest that ences in:the uptake into the human brain (Fleisher and 1. Brain fat and protein composition;Campbell, 1969; Holmes and Gordon, 1989) is 2. Specific binding sites;as much as seven times lower than that found 3. Cerebral blood flow, which could be

in other species (Caccia et al., 1982; Zaczek et expected to be greater for small animalsal., 1990; Caccia, personal communication), in relation to brain size;and that the brain plasma ratio seems to be 4. Specific and active carrier-mediated uptakeinto the brain, which has been previouslyrelated to the body weight of the animal, reported for amphetamine (Pardridge andalthough some variation is seen in primates Connor, 1973);(Fig. 1). Interestingly, in all these species stud- 5. An active removal of the drugs from theied and particularly in monkeys, there is a ten- brain;dency for norfenfluramine levels to be higher 6. Differences in plasma protein binding; and


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7. Sample dilution effects, since the human volume of distribution (Boxembaum, 1982),brain is 3-10 times larger, relative to body according to the formula:

weight, compared to other animals (Boxem- Y = al4Pbaum and Fertig, 1984). log y = log a + b. log W

Indeed, Dedrick (1986) has suggested that

"the large primates are poor surrogates for the where y is the kinetic parameter, W the bodyhuman" and that these interspecies differences weight, b the slope of the line, and a a constantin brain levels may explain the lower toxicity related to each drug. The slopes of these regres-seen in humans compared to other animals for sions have been found to be very similar for aanticancer agents, such as carmustine, when large number of drugs, with b for clearanceadministered by intracarotid infusion, approx 0.7, the half-life 0.25, and the volume of

distribution 1.0 (Campbell, 1994a). Thus, if thekinetics is known in one or more species, the

Metabolism value in humans, in principle, can be calcu-lated. For example, the half-life in the rat for

Species Differences amphetamine and methylamphetamine is 2 h,and using the above formula, the value inThere are a number of examples of the quali- humans can be expected to be 8 h, which is

tative differences in the metabolism of drugs similar to that actually found (Campbell, 1978;between species, including a virtual absence of Cook et al., 1992). Although this approach mayo-demethylation or acetylation in the dog, or be applicable to a number of drugs with highglucuronide formation in the cat, or aromati- clearance or those renally eliminated, for oth-zation in New World but not Old World mon- ers, the prediction to humans is unreliablekeys, but perhaps it is less well known that (Boxembaum, 1982), particularly for those withthere are quantitative differences that can be a low metabolic clearance (Campbell, 1994c).

predicted from the weight of the animal. Most The human lives longer than can be expectedphysiological functions appear to be related to from allometric scaling considerations corn-the size of the organ or tissues concerned, pared to all other mammals, and the use of awhich in turn is proportional to the body life-span correction factor has been shown toweight. Thus, for the small rodents, pro- provide better estimates, particularly usingcesses, such as breathing, heart and metabolic data from rats and Old World Monkeys

rate, blood flow, and renal and hepatic clear-/ (Campbell, 1994c). From these allometric con-ances, all occur at a relatively faster rate than siderations, it follows that parent drug expo-that found in larger animals and humans. Since sure will often be lower in animals (approx 12longevity is also proportional to weight, the times for rats and 3.5 times for cynomolgussmaller animal will live for a shorter time, but monkeys) compared with humans when thethe total number of physiologicals event will same dosage is administered in terms of mg/kgbe approximately the same for all mammals and therefore, toxicity should be correspond-(Mordenti, 1986). This means that for most ingly lower. This is often not the case. Thedrugs, the total body clearance will be con- opposite can be true perhaps owing to the moresiderably faster in the rat compared to humans, extensive conversion to active and sometimesproducing a much shorter half-life and reduced reactive and toxic metabolites in the liver orexposure for the same dosage expressed in brain of experimental animals.terms of mg/kg. This size proportionality is

known as allometric scaling, and a linear rela- Metabolic Pathwaystionship can be found for a large number of

drugs when the log of the body weight is plot- Many of the drugs of abuse are relativelyted against the log of the clearance, half-life, or lipid-soluble, little is excreted unchanged in


198 Campbell

the urine, and they will undergo extensive clearance owing in part to their reaction withmetabolism in the liver. However, because they tissues. For example, MDMA in the rat is metabo-are weak bases with pic a values of approx 9, lized by at least seven different reactions:

the absolute amount of renal excretion and 1. Ring hydroxylation;therefore metabolism will depend on the uri- 2. Demethylenation of the methylenedioxynary pH with acidic urine increasing the renal bridge;involvement up to fivefold. This may have 3. With subsequent o-methylation of the hydroxyimplications on the rate and, to a lesser extent, group; orroutes of metabolism when different animals 4. Oxidation to quinoline;

with different diets are studied under varying 5. Demethylation of the alkyl side chain;external conditions. Little has been done to 6. Cyclization of the trihydroxy derivatives to

the corresponding indoles;study these effects, but they may partially 7. Oxidative deamination of the nitrogen toexplain some of the interspecies differences polar inactive metabolites; and finallyreported on amphetamine metabolism (Dring 8. Glucuronidation and sulfation of hydroxylet al., 1970; Caldwell et al., 1972). Although for groups, the mechanism and intermediatesmany drugs, metabolic degradation is a deac- and, indeed, the importance of whichtivation process to more polar compounds that remain unknown (Fig. 2) (Lira and Foltz,can be more easily excreted, it is becoming 1988, 1991b; Hiramatsu et al., 1991; Patel etclearer that for others, particularly those with al., 1991;Lin et al., 1992)long-term toXicities, such as carcinogenicity, Table 2 attempts to summarize the possiblemetabolism can produce reactive intermedi- involvement of each of the putative metabo-ates, which may be present in the body for lites so far identified. In brief it would al_pearrelatively short periods, making measurement that all com_.pounds with an intact methylene-and identification difficult (Mansuy, 1989). i_y br!dge h-_'_e little ' l_toyac effect__aasAn example of this is the conversion of the measured by--_olon_d 5-_IT-depletion, evenneurotoxin 1-methyl-4-phenyl-1, 2, 3, 6 though they readily enter the brain aftegr,p.g.r-tetrahydropyridine (METE), to the free radi- ipl_eral in'ection (Lim and Fol 988_. Conver- 1cai 1-methyl-_phenylpyridine (MI>P+), which gion from MDMA ko'the ye MDg. was

is cleared more slowly in primate brains con't- not thought to be important, since pretreat- /pared to the rodents (Johannessen et al., 1985) ment with the cytochrome P450 inhibitor, pip- [and could explain the greater sensitivity in eronyl butoxide, had no effect on the acute(monkeys (Boyce et al., 1984). Similarly; it has depletion of 5-HT ($chmidt and Taylor, 19_.}been suggested (Molliver et al., 1986; Paris and but without actual--measurements on 'the _or-Cunningham, 1991) that long-lasting deple- mation of MDA in the brain and organ speci-tion of 5-HT when 3-4 methylenedioxy ficity of this inhibitor (Murray and Reidy,methylamphetamine (MDMA), and 34 meth- 1990), these results must be interpreted withylenedioxy amphetamine (MI>A)are adminis- caution. The more polar catechols and o-tered systemically, but not when injected methylated derivatives formed by hepaticintraventricularly, is caused by the formation demethylenation by the cytochrome 2Eof toxic metabolites. Formation of chemically lsozymes (Tucker et al., 1994) and rinreactive metabolites had been previously hydroxylation by cytochrome 2B4 and 2Dl\hypothesized for parachloroamphetamine (Kumagai et al., 1992b,c) may not be able to /(PCA) (Ames et al., 1977; Sherman et al., 1975) cross the blood-brain barrier, but are found inand reactive intermediates found in brain cerebral tissue, since metabolism of the parent

microsomal preparation (Miller et al., 1986), drug also occurs in the brain (Lim and Foltz,but the search for such compounds is generally 1988; Lin et al., 1992). However they do not

hampered by their relatively low levels com- appear to have any 5-HT-depleting effectspared with other metabolites and their rapid when injected intraventricularly. Similarly, [3-



(8)c,_,pt_,,t co_,hm_, administered, metabolism may be sufficient tom_,_by_ _y_=7.b_,,_,_. : _ _h_,t_, produce toxic levels of metabolites in small,

/_o J_CH2--CFi--N" f ,,CH=; but sensitive areas. However, until definitive(_0,_,--.(I II I I _'H measurement is made in brain tissue of those

fornmtiM _ L _ _ _1 · 'o/'_,/_ _'.. c,3 compounds known to be toxic, together with;_ . m c,,_-,,, some form of kinetic-dynamic analysis, the rel-(3)O-mmbylmkm (I) _ toim_le

byd.,x_,.-,o., evance of the active metabolite hypothesis willremain unresolved.

Fig. 2. Proposed metabolic routes of MDMA. In addition to the suggestion that these com-pounds in themselves may be metabolized to

hydroxylation of the side chain-producing ct- reactive metabolites, there is also evidence sug-methyl norepinephrine does not change 5-HT gesting that they may release such large quan-levels, but production of this metabolite, if it tities of catecholamines, particularly from theoccurs, could contribute to the hemodynamic peripheral circulation, that the catabolism inchanges seen in acute toxicity, such as the brain is saturated, leading to toxic path-hypotension and bradycardia (Yeh and Hsu, ways forming known neurotoxins, such as 6-OH1991), or even the mood-elevating effects. In DA, from dopamine after methylamphetaminecontrast, the ring trihydroxy compounds have (MA) (Seiden and Vosmer, 1984; Stone et al.,a greater effect on dopamine after icv injection 1988) or 5,7-DHT from serotonin after PCA(Elayan et al., 1992; Johnson et al., 1992), and (Berger et al., 1992), but again definitive stud-these metabolites could be responsible for the ies are awaited.long-term depletion in this catecholamine These detailed studies on one drug highlightobserved after MDMA administration. How- the difficulties in the interpretation of toxico-ever, although they can be formed in the liver, logical studies without the benefit of drugbecause of their polarity, it is unlikely that they and/or metabolite levels to assess exposure.would cross the blood-brain barrier, and there Even this approach may be invalidated ifis no evidence to suggest so far that they are highly reactive toxic metabolites and free radi-actually metabolized in the brain. Further work cals are formed, and are only present in bloodis necessary to see if they are present in the or tissues for a few seconds, with little possi-brain after large doses, although this is inher- bility of them being measured. Once formed,ently difficult because of the low levels, they can damage the lipid membranes of nervedifficulties of analysis, and poor stability, terminals or inactivate key synthetic enzymes,Similarly, the quinone formed by reduction of such as tryptophan hydroxylase (Stone et al.,the dihydroxy compounds forms glu- 1989). Indeed, the smaller animals with faster

tathione adducts, which can be blocked by rates of metabolism may actually be more sus-oxygen-free radical inactivators, such as super- ceptible to toxicity because of these reactions.oxide dismutase, highly suggestive of a reac- Much of the studies described above have usedtive intermediate. However, although this has the rat, and although some metabolic studiesbeen shown to occur in liver microsomes from have been undertaken in mice and humans

DMA, information of the same metabolic acti- (Lim and Foltz, 1989, 1991a), little is known

vation in the brain is lacking. Certa' l_e about the metabolism in other species. Somebrain does have the 9bilit_Z _ metabolize drugs authors have suggested that the reduced sensi-_sln_ CY D_yme, and this may have tivity of MDMA seen in the mice comparedevolved as a protecti_-mechanism against with other species could be a consequence ofplant alkaloids ingested by early humans different rates or routes of metabolism (Ston e(Bdt'to and Wedlund, 1992). Although cerebral et al., 1987; Lim and Foltz, 1991a), and that pri-metabolic activitymay be <1% compared to the mates are the most suitable species to compareliver, when high doses of these compounds are with humans since it is suggested that they


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MolecularNeurobiology 201 Volume11, 1995

202 Campbell

may have a more similar metabolism to humans Table 3(Ricaurte, 1989). Unfortunately, the only sub- Species Differencesstituted amphetamine that has been systemati- in the Metabolic Ratio---cally studied, fenfluramine, has shown marked The Relative Amountdifferences in the rates of metabolism between of Norfenfluramine (N-Fen)

animals and humans (Caccia et al., 1982; to Fenflttramine (Fen)inthe BodyFollowing Oral Administration

Marchant et al., 1992), in particular, the conver-sion to the more active norfenfluramine. Thus, Plasma AUC ratio

for most animals, with the exception of the Species NFen/Fenmouse, the relative exposure of norfenflu- Mouse 0.3ramine to fenfluramine expressed as the areas Humans 0.7under the plasma concentration time curve Rat 2.8(Table3) is 3- to more than 30-foldgreater than Pig 3.0in humans, with the primates being the least Dog 5.2similar to humans. Work in our laboratories Guinea pig 9.5*

Squirrel 11.0has also shown that only fenfluramine and Cynomolgus 16.0norfenfluramine can be found in the brain of Rhesus 27.0rodents, but an additional as yet unidentified Baboon 37.0

metabolite observed in plasma may be present Caccia et al. (1982)and unpublished.in the squirrel monkey's brain (Fig. 3). '(Fuller et al. [1988]ip administration).

An additional complexity to take intoaccount is that all the inferences described

above have relied on the depletion of 5-HT as across the range of doses used. In practice, thisthe index of toxicity, and recent work in mice is often the exception rather than the rulehas shown that toxicity, as measured by owing to a saturation of many kinetic pro-increased levels of GFAP, argyrophilia, and cesses, particularly those that are actively orirreversible reductions in tryptophan hydroxy- enzymatically linked (Table 1). Thus, satura-lase, occurs at high doses of MDMA and MDA tion of hepatic metabolism or renal eliminationwithout necessarily large depletions of 5-HT, can lead to relatively large increases in bloodwhereas fenfluramine at similar doses produc- levels, whereas on the contrary, saturation oflng a reduction in $-HT shows no toxicity, as absorption would lead to reduced exposure.measured by these indices (Miller and Many processes of nonlinearity may be occur-O'Callaghan, 1993; O'CaUaghan and Miller, ring simultaneously at high doses and at dif-1993). These differences may be the result of ferent times of dosing, confounding thethe ability of MDMA, but not fenfluramine, to interpretation of the data. The metabolism ofdeplete dopamine acutely, and if these findings fenfluramine becomes rapidly saturated in theare confirmed, it would appear that much of rat at 4 rog/kg and this continues until 24the previous screening that has been under- rog/kg, but thereafter remains constant (Zac-taken to examine the toxicity of the putative zek et al., 1990). Exposure in these animalsmetabolites, using only 5-HT as a biochemical becomes five to six times higher than would bemarker, may need to be repeated, expected from simple dosage considerations.

NonlinearityFig.3. (opposite)The metabolic chromatographic

During toxicological investigations, doses patterns 2 h after the administration of (+) _4C-are normally increased until a maximum toler- fenfluramine (1 rog/kg) in the brains of mouse, rat,ated dose is reached or a desired effect shown, and squirrel monkey (F = fenfluramine, NF =It is often assumed that the kinetics is linear noffenfluramine).


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MolecularNeurobiology Volume11, 1995

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204 Campbell

This saturation in metabolism is reflected by a ministration of these drugs could lead tofourfold increase in half-life, from 2.3 to 9.6 h significant kinetic interactions. A similar con-

found in the rat when the dose is increased from sideration would hold for the equivalent5 to 40 mg/kg (Caccia et al., 1981). However, enzyme CYP2D1, which is responsible for thethere appears to be less of a saturation of the demethylenation of MDMA in rats (Kumagaimetabolism of norfenfluramine, such that the et al., 1992b). Suggestions that this may be theAUC only increases by a factor of 1.5-1.9 over case come from the work of Hashimoto and

the same range of doses. There are also species Goromaru (1990), who showed that coadmin-

differences in the saturation of metabolism, istration of MDMA and paroxetine to rats leadsand longer half-lives are found in the dog at to a significant reduction of paroxetine clear-higher doses, but not in the mouse (Caccia et ance with a subsequent elevation of blood andal., 1982). Although little data are available for brain levels. In addition, it is known in humans

other substituted phenylethylamines, it can be that CYP2D6 exhibits genetic polymorphism,expected that similar saturation does occur, and it has been suggested that the severe reac-suggesting that investigators should be cau- tions that occur in some individuals may be thetious when extrapolating doses within and result of a reduction in activity of this enzyme,across species, which occurs in 8%of caucasians (Tuckeret al.,

1994). Another example of kinetic interactionis found with the coadministration of d-

Associated Treatments amphetamine and fenfluramine. Prior pretreat-ment of fenfluramine (Jori et al., 1978) or

It is common practice for various com- amphetamine (Hunsinger et al., 1985) willpounds to be coadministered with a test drug, increase plasma and brain concentrations offor example, to block receptors, enzymes, or the other compound by as much as 100%, per-uptake, and if a positive result is found, to use haps because of the competition for the cata-this effect to suggest a mechanism of activity, bolic enzymes of deamination. Without theUnfortunately, there may be kinetic or meta- measurement of plasma and brain drug levelsbolic consequences of such an interaction that during such pharmacological interaction stud-may well provide an alternative and more ies, it is not known which of several hypoth-valid interpretation. For example, it has been eses may be correct.assumed that animals that have been pre- The handling of the animals may also infiu-treated with the 5-HT uptake blockers, such as ence the kinetics, as can the time in the dayfluoxetine or paroxetine, show a reduction in when the drug is administered, since clearancethe 5-HT depleting effects of MDMA and other is for certain drugs under circadian controlsubstituted amphetamines owing to inhibition / leading to different toxicities (L_vi et al., 1994).of 5-HT uptake into the neuron via the seroto- [ Thus, amphetamine levels may be fourfoldnih transporter (Schraidt, 1987). Although this] lower in stressed rats compared to rested onesmay be true, another explanation exists. These] (Pashko and Vogel, 1980), whereas anesthesiacompound_nhibit metabolic p_-_-_ can significantly alter the uptake and distribu-Ways (Fuller et al., 1992) s_____fic_ tion of drugs (Gordon et al., 1986; B/ich et al.,

. in humans (Br6s_ 1991). Recent findings have suggested thatS_91; Bergstrolllel al,_ Sin_ lowering the raised temperatures found afteralu 1992; Stevens and Wrighton, 1993), th_ large doses of the substituted amphetamines

e_e now known to-cle-methy_ienate__-_M_ reduces their toxicity with compounds thatto _p0teffti_IIYtoxic corn _S_(see-M_tib_ have a wide range of pharmacological activi-Pathways). Since both MDMA and MDA hav_ ties. It is interesting to speculate what part kin-high affinity for the CYP2D6 isozyme (Kin= 1-_3 etics may play in this effect, since it is wellg/VD (Tucker et al., 1994), it is likely that coad- known that temperature can have a marked


UseofToxicokinetics 205

effect on metabolic clearance and drug tram-

port, wRh, for example, a doubling of metha- .\c.,,.,done levels with a 10°C increase in temperature(Herr, 1989). m.,. ,.

Similarly, surgical manipulation can mark- "_ '"'_ _',_.:_._ '._,--. F.,edly alter the kinetics of drugs. Adrenalectomy, ,_. -._.,," _ ---for example, significantly augments the (,)weight-reducing properties of fenfluramine(York and Maclean, 1992), and supplementary '_. '"_' "'cortisone treatment reverses this effect. It has, i ,, "therefore, been surmised that in some way the r · iFm,m_,. c,. ,'q

pituitary-hypothalamic axis plays an impor-tant modulatory role in the anorectic activity Fig. 4. Relationship between brain levels ofof fenfluramine. However, when plasma and fenfluramine (F) and norfenfluramine (NF) and

corresponding changes in body weight following abrain levels of fenfluramine and norfenflu- dose of 10 rog/kg to rats, with or without adrena-famine were measured, it was shown that both lectomy and cortisone supplementation.plasma, and more importantly brain concen-trations, had increased approximately fivefoldafter adrenolectomy, and the additional activ- sooo

ity could be completely explained by the increase

in brain exposure. Indeed, when the combined _" 4m0 gl _ebrain levels of fenfluramine and norfenflu-

ramine were plotted against the reduction in _ -_ Fe,ulethe weight loss in these animals, irrespectiveof the modulatory influence of adrenolectomy _ 3000with or without cortisone, a direct curv'tlinear

relationship was found (Fig. 4). _ 2ooo

!Gender Differences

It is becoming well documented that rodentsand, in particular, rats often show gender- 0related differences in the metabolism of certain mm ma

drugs, which is thought to be under endocrine Fig. 5. The effect of gender on the levels of MDMAcontrol (Zaphiropoulos et al., 1989). This is, and its metabolite MDA, 4 h after 40 rog/kg schowever, rarely seen in larger animals. Theuse MDMA (n = 5). (Redrawn from Fitzgerald et al.of in vitro hepatic microsomal systems has [1989.])shown that this mainly occurs for drugs thatare metabolized by CYP3A, and that the femalerat is deficient in this isozyme. Other metabolic 1994b) are higher in females than males. Some-routes may also show this gender difference, times this difference is artificially lost, sinceThe enzymatic activity can be returned by the dosage is nearly always based on mg/kg, andadministration of testosterone or when the since males tend to be heavier, they actually get

females pass menopause (Kato, 1974). Thus, more drug. Since this is not necessarily takenplasma levels of both MDMA and MDA up into the larger volume of distribution, the(Fitzgerald et al., 1989) (Fig. 5), various narcot- consequence is higher than expected plasmaicg (Karo, 1974), and fenfluramine (Campbell, and brain levels in males compared with


206 Campbell

females, thereby reducing any inherent gender enzymes, or the saturation of biodispositiondifferences. Again, because of the complexity processes. There is much evidence to show thatof the situation, if gender differences in appar- repeated administration of the phenylethyl-ent sensitivity are observed, particularly in amines leads to a reduction in activity mea-rats, then kinetic evaluation should be under- sured by abnormal behavior or brain aminestaken to negate this possibility. (Rebec and Segal, 1980), which could be the

result of a reduction in plasma or brain levels.On the contrary, levels of amphetamine, for

Repeat Dosing Tolerance example, actually increase (Kuhn et al., 1978),and Sensitization as could be expected from normal accumula-

tion. It would seem that this reduction in effect

Depending of the half-life of the drug and is more likely to be a biochemical adaptationthe frequency of administration, it can be rather than some change in kinetics. Similarexpected that many drugs will accumulate considerations hold for cocaine, where levelsin blood and tissues after repeated dosing accumulate after repeat dosing, but the effectaccording to the formula: diminishes (Cass and Zahniser, 1993). How-

ever, using an unusual dosing paradigm withRac--(1.44 x ha_-l_/dosingfrequency) rats treated with methylamphetamine (MA)

every 6 h for alternate d for 7 d with escalatingwhere Rac is the accumulation index, doses, when compared to the first day of treat-

Thus, a drug with a half-life of only 2 h, but ment at the same final dose of 15 mg/kg, a 50%administered every 4 h will accumulate, but one reduction in brain amphetamine and methyl-with a half-life of 6 h given daily willnot. The time amphetamine levels was reported providing

plasma levels take to reach steady state nor- some rationale for the observed tolerance inmally occurs within 3-5 half-lives for all drugs CNS activity seen with prolonged administra-with linear kinetics if dosed at every half-life, tion (Schmidt et al., 1985). In contrast, for d-fen-but for most of the substituted phenylethyl- fluramine, although there were reductions inamines administered to rats once or twice a brain 5-HT levels, 15 and 28 d after semi-acute

day, steady-state levels of the unchanged drug high dosing (10 mg/kg) for 4 d, escalating theare reached after the first dose. However, the dose to the same final amount over a 28-d

half-lives of many primary amine metabolites period produced no change in brain amines,are longer than the secondary or tertiary amine and the brain levels of ferffluramine and nor-parent drug (Campbell et al., 1986; Caccia and fenfluramine were not significantly changedGarattini, 1992). Presumably, dealkylation is (Rose et al., 1993).

more rapid than deamination, and for thesewhere half-lives may be approx 10 h in rats,steady-state is reached in approx 2 d. In the Stereochemistrycase of fluoxetine, the half-lives of both parentdrug and metabolite are only slightly longer As many as 25% of all drugs used clinicallythan those observed with the phenylethyl- are thought to have at least one chiral carbonamines at lower doses, but at 15-20 mg/kg, atom and can exist as more than one enanti-doses that can reduce 5-HT levels, the half-lives omer (Campbell, 1990a). Similar consider-increase to 13 h or more (Caccia et al., 1990), ations are true for drugs of abuse, such as alland up to 14 d of dosing are required to attain the amphetamines, and those that are also usedsteady-state concentrations and maximal effect as pharmacological tools, such a paroxetine,(Gardier et al., 1993). Some drugs on repeated fluoxetine, and fenfluramine. There can beadministration can alter their own kinetics by important quantitative differences in theirauto-induction or auto-inhibition of hepatic kinetics and metabolism that can become


!


important when the activities of the corn- metabolize the S(+) active dextroenantiomerpounds and/or their active metabolites also more rapidly, at high doses in the rat, the clear-exhibit stereoselectivity. Thus, although in ance of R(-)laevo fenfluramine is faster (Cacciavitro, the enantiomers of fluoxetine have equal et al., 1982). This may not be important, sincepotency as an uptake inhibitor, the metabolites neither the l-enantiomer of the drug nor thehave different activities (Wong et al., 1985, metabolite is active at low doses. However, at1990; Fuller et al., 1992). In mice, S-fluoxetine high dosage (_40 rog/kg) there is a greateris more slowly converted to the active metabo- saturation in the kinetics of the 1-isomer of bothlite S-norfluoxetine in contrast to the more parent drug and metabolite and combined lev-extensive formation of the relatively inactive els are higher than the d-antipodes (Caccia etR-norfluoxetine from the R-enantiomer (Fuller al., 1981). At these very high levels, the 1-enan-and Snoddy, 1993). This could provide a ratio- tiomers start to produce an activity in reduc-nale regarding why the R-enantiomer has been lng 5-HT (Kleven et al., 1988). It is clear that ifshown to have a reduced protected activity on racemic mixtures are administered duringdepletion of 5-HT by PCA (Robertson et al., pharmacological or toxicological investigation,1988) owing to the relative temporal differ- particularly over a wide dosage range, andences in the kinetics of the active isomers of when active metabolites may be chirally selec-both drug and metabolites. Stereotaxic direct tive themselves, then a knowledge of the kinet-injection of (+) MDMA into the dorsal or ics of the individual isomers is importantmedial raphe does not change 5-HT levels in (Campbell, 1990a; Tucker and Lennard, 1990).the hippocampus or striatum (Paris andCunningham, 1991). However acute sc admin-istration with the (+) or (-) enantiomer of Extrapolation to HumansMDMA produces a reduction in 5-HT and5HIAA in the striatum (Schmidt et al., 1987) There have been many attempts to relate theand cortex (Schmidt, 1987) within 3 h, which general toxicity observed in animals with thatremain reduced after 7 d for the (+) isomer, but expected in humans (Kimmel, 1990; Doursonnot the antipode. Similarly, Johnson et al. and Derosh, 1991), and more recently the prob-(1988) found that at lower repeated doses (3-5 lem of neurotoxidty extrapolation has beenmg/kg), the d-isomers of either MDMA or addressed (Slikker, 1991; Federal Register,MDA were more active on reducing 5-HT com- 1993). There are many difficulties and unknowns,pared to the l-isomers, but this difference was including species differences in sensitivity, bio-lost at higher dosage (5-10 mg/kg). The disposition, environment, and so forth. Thereplasma profiles and kinetics are similar for is a need to evaluate a dose-response relation-both enantiomers of MDMA (Cho et al., 1990; ship in animals to determine the no effect dos-Hiramatsu et al., 1991; Lim et al., 1993), but the age (NOEL), to compare this with the exposurelevels of the metabolite MDA were approxi- measured by simulations or directly expectedmately three times higher after (+) MDMA, in humans, and divide this figure by an uncer-compared to (-) MDMA and these stereo- taintyfactorbringingtogetheralltheseunknowns.selective differences in pharmacokinetics and Thus, a 10-fold uncertainty factor (UF) is usedmetabolism (Hiramatsu et al., 1989a) may pro- when relevant animal data are compared tovide some explanation for these differences in known prolonged exposure in humans to accounttoxicological findings. Differences in the activ- for individual sensitivity differences; anotherity of the optical isomers of PCA have been 10-fold UF is used if available data are inad-reported, which in part may be explained also equate for humans; a further 10-fold UF is usedby differences in kinetics (Stekerke et al., 1975). for inadequate animal data, e.g., short-termFor fenfluramine, the situation is more corn- dosing. A final 10-fold factor is used when con-plex, since although most animals tend to verting from a NOEL to a LOEL, bringing a


1

208 Campbell

possible divisible factor to 100,000 (Barnes and ,40 _-"'-_--Dourson, 1988). Clearly, there are problems · .with this approach, and various authors have ,_ ·

attempted to improve these estimates by using ·//·_.._"_either a benchmark dose, which is the lowest { '. ·effective dose that would produce a measur- _ ,0oable change of, for example, 10% of maximal

observed response, which is used as a baseline ! eostarting point for factorization (Crump, 1984),

or quantal responses where the individual I 6o /dose-response data points are joined by some { /mathematical model to identify doses that can _ !cause an effect outside those that have been _ 4o /measured (Kimmel, 1990). This can be linked _ /

/to measurable biomarkers, such as 5-HT, 20 !dopamine, and their metaboHtes, after MDMA /administration (Ali et al., 1991; Gaylor and ,,Slikker, 1992). Although these approaches may o _-0 , 2 3 4 5 &

be improvements, they still do not take into ,_.,.. ,,_ __._,_, (_..x-1.consideration one of the largest uncertaintyfactors, that of kinetic differences. The use of Fig. 6. Relationship between changes in aminediscrete kinetic dynamic mathematical mod- efflux in striatal dialysate and plasma MD/viAels can overcome some of the limitations concentration. (--Line drawn in original paper,---

line drawn assuming a receptor-based saturabledescribed above, interaction assuming reversible Michaelis-/Vienton

kinetics). (Redrawn from Hiramatsu and Chu [1991.1)

Kinetics Dynamic Relationshipssimplest level of a reversible receptor or enzy-

Measurement of the plasma or brain levels matic interaction, a linear relationship can onlyis clearly a more useful assessment of exposure partially describe the binding, and then only atwhen comparing across dose, species route, low concentrations (Campbell, 1990b). Forand so on. However, it assumes that there is more complex interactions, a direct linear rela-some simple direct relationship between the tionship cannot be expected, particularly ifmeasured concentration and the activity. Often more than one receptor is involved or the reac-this relationship is presumed to be linear, and, tion is irreversible. An example of such pos-although never stated, implicit in this belief is sible complex interactions can be seen with thethat a doubling of the dosage will lead to a dou- effect of fenfluramine and norfenfluramine onbling of the effect. For example, in Fig. 6 where the change in 5-HT in the rat cerebral cortexMDMA plasma concentrations have been plot- (Campbell et al., 1991). This analysis is derivedted against percentage change in striatal 5-HT from the data published by Zaczek et al. (1990).dialysate levels, the authors (Hiramatsu et al., At the low pharmacological dose of 2 mg/kg1991) attempted to draw a straight line through administered in divided doses over 4 d, therethe data points whereas an asymtopic curve is an increase in brain 5-HT levels, but as thereflecting a saturable receptor binding would dose increases up to a maximum of 48 mg/kg,be more appropriate and provide more useful the 5-HT levels progressively decrease. In aninformation (Fig. 6), such as a maximal effect attempt to relate these changes to the brain(Emax) at approx 5 nM/mL, and a potency effect drug concentrations, combined peak levels ofat 50% of E_x (ECs0) of _,2 nM/mL. Even at the fenfluramine and the active metabolite norfen-


tL%


104 · Cla3

loo _--.. _,,.'"" .......... l_i'v'a'"' 242!a_. Cla3

75 ·

,' \

· -5o ' _'_' X '\'N 'X_ ¢1.t3

_ ._.

N._...__ -101 · Ci.U-loo Eahk_,'' 2_8u s · Cl.i5

10 100 1000 10,000 100,000

Fenfluramtne + Norfenfluramine cone. (ng.g -1)

Fig. 7. Brain-dynamic modeling showing computed line of best fit (--) through data points (lB) from threehypothesized "receptor" activities, E1 (uptake 1--), E2 (release -.-), E3 (feedback--), where C = brainconcentration of fenfluramine + norfenfluramine.

fluramine were related to the percentage (Gobbi et al., 1993). Assuming that these pro-change in 5-HT from controls at the same time cesses may be involved in the observed 5-HT(0.5 h), and a three-receptor mathematical changes, it is possible to obtain measures of themodel was found to describe best the resultant activity at half-maximum effect (potency), and

curve using the following formula for each these can be compared to the dissociation orreceptor interaction: binding constants found in in vitro studies

(Fig. 7). Although only six points were used forEffect(E)--(Emax' C_IEC_o+ C_) (3) this modeling, interestingly, the computed

ECs0 from the assumed uptake inhibitionwhere Emax is the maximum effect, C the con- receptor model (242 ng/mL), and that forcentration of drugs in the brain, ECs0 the con- release (2348 ng/mL) are indeed comparablecentration at 50%, and Em_x and y are the slope with those found directly from in vitro bindingof the relationship or Hill coefficient. In vitro studies, using rat synaptosomes and combinedreceptor binding studies in the rat indicate that, fenfluramine and norfenfluramine values ofat low levels, fenfluramine blocks the reuptake approx 200 and 1000 ng/mL, respectivelyof serotonin, whereas at higher levels, both (Garattini et al., 1988). Such multi-Ema ×modelsfenfluramine, and to a greater extent norfen- have also been used to explain the complexfluramine, release 5-HT from serotonergic ter- action of other CNS drugs, such as clonidine,minals (Mennini et al., 1991). In addition, at on central blood pressure control (Paalzow,higher doses, paroxetine binding, a measure of 1984). Using fids integrated mechanistic approach,the functional ability of uptake sites, is it is possible to investigate more confidentlydecreased, suggesting a reduced number, func- the exposure and the possible underlyingtion, or a feedback downregulation in the pres- mechanism that may occur in humans at thera-ence of high initial synaptosomal 5-HT levels peutic doses from data obtained in animals.


210 Campbell

The steady-state levels of more than 300 which chemical moiety is responsible for thepatients taking dexfenfluramine for up to 12 apparent toxicity, unchanged drug, an activemo (30 mg/d) are 20 and 12 ng/mL for metabolite, a free radical, or a catabolic prod-dexfenfluramine and dexnorfenfluramine, uct of 5-HT or dopamine. However, without

respectively (Gordon, 1991). Using these corn- some attempt to measure these entities andbined drug levels to calculate brain levels in then to relate their concentrations in somehumans assuming a brain/plasma ratio of 9 meaningful mathematical mechanistic method,(Fleisher and Campbell, 1969; Holmes and attempts to understand the underlying pro-Gordon, 1981), it can be seen from Fig. 7 at a cesses fully will come to naught.combined human brain leveI of 300 ng/mLthat the action of the drug in humans at the-

rapeutic doses is mainly uptake blockade Acknowledgmentswith little release, and that the levels are

approx 20-fold lower than the crossover point I would very much like to thank V_roniquewhere there is no change in 5-HT levels (_6000 Guly for not only deciphering my writing, butng/mL) and 350 times less than those that pro- translating the hieroglyphics into readableduce the maximum reduction in 5-HT (100,000 English and redrawing my sketches intong/mL). This approach makes many assump- understandable figures.tions, including that the response in the ratand humans is similar, but this is still a better

measure of comparative exposure than other Referencestechniques, and the results can be updatedwhen comparative in vitro binding con-

Ali S. F., Change L. W., and Slikker W. Jr. (1991)stants in humans are obtained.Biogenic amines as markers for neurotoxicity.Biomed.Environ. Sci. 4, 207-216.

Ames M. M., Nelson S. D., Lovenberg W., andConclusion Sasame H. A. (1977) Metabolic activation of para-

chloroamphetamine to a chemically reactiveIt is hoped that this discussion has high- metabolite. Commun.Psychopharmacol.1, 455-460.

lighted the problems of obtaining toxicological Barnes D. G. and Dourson M. (1988) Reference dosedata from one experiment and attempting to (RfD) description and use in health risk assess-

extrapolate the findings to another dose, spe- ments. Regul. Toxicol.Pharmacol.8, 471-486.Berger U. V., Grazanna R., and Molliver M. E. (1992)cies, or experimental condition without some The neurotoxic effects of p-chloroamphetaminesmeasure of drug and or metabolite exposure, in rat brain are blocked by prior depletion ofThe new worldwide harmonized discussion serotonin. BrainRes. 578, 177-185.

document on toxicokinetics (ICH2, 1993), Bergstrom R. F., Peyton A. L., and Lemgerger L.which when used with the draft guideline on (1992) Quantisation and mechanism of thethe assessment of neurotoxicity (Federal Reg- fluoxetine and tricyclic antidepressant inter-ister, 1993), may provide a more meaningful action. Clin. Pharmacol.Ther.51, 239-248.framework for future studies. It would be nice Boyce S., Kelly E., Reavill C., Jenner P., and Marsen

to think that once the compound is measured C.D. (1984) Repeated administration of N-in the appropriate body tissue, the kinetic methyl-4-phenyl-1, 2, 5,6-tetra-hydropyridine torats is not toxic to striatal dopamine neurons.aspect can be forgotten, but as suggested in the Biochem.Pharmacol.33, 1747-1752.text, toxicokinetics is still in its infancy, and Boxembaum H. (1982) Interspecies scaling,there is still much to be discovered. Most allometry, physiological time and the ground planimportant is what should be measured, and for of pharmacokinetics. J. Pharm.Biop. 10, 201-227.MDMA, which has undergone considerable Boxembaum H. and Fertig J. (1984) Scaling ofresearch, we are still far from understanding antipyrine intrinsic clearance of unbound drug



in 15 mammalian species. Eur. f. Drug Metab. Campbell D. B. (1994b) Are interspecies compar-Pharmacokinet. 9, 117-183. isons in the toxicity of centrally acting drugs

Britto M. R. and Wedlund E J. (1992) Cytochrome valid without brain concentrations?--A com-P-450 in the brain. Potential evolutionary and mentary. Neurochem. Int. 26(2), 103--110.therapeutic relevance of localization of drug- Campbell D. B. (1994c) Sizing up the problem ofmetabolizing enzymes. Drug Metab. and Dispo- exposure extrapolation: new directions insition 20(3), 446--450. allometric scaling, in Toxicology of Industrial

Br6sen K. and Skjelbo E. (1991) Fluoxetine and Compounds (Waechter F. and Thomas H., eds.),norfiuoxetine are potent inhibitors of P45011D6. Taylor and Francis, in press.Br. J. Clin. Pharmacol. 32, 136,137. Campbell D. B., Ings R. M. J., Gordon B. H. G, Zac-

Biich U., Altmayer E, Iserverg S. C., and Biich H. E zek R., and De Souza E. (1991) The use of kinetic(1991) Increase of thiopental concentrations in dynamic models in the interpretation of therat tissues due to anaesthesia with isoflurane, effects of fenfluramine on SHT turnover. Abstract

Methods Find. Exp. Clin. Pharmacol. 13, 687--691. presented at Serotonin Club, Birmingham.Caccia S. and Garattini S. (1992) Pharmacokinetic Campbell D. B. and Jochemsen R. (1994)

and pharmacodynamic significance of antide- Nonclinical pharmacokinetics and toxico-pressant drug metabolites. Pharmacol. Res. 26(4), kinetics, in International Pharmaceutical Product317-329. Registration, Aspects of Quality, Safety, and

Caccia S., Dagnino G., Garattini S., Madonna R., Efficacy--Chemistry, Pharmacy and Manufacturingand Zanini M. G. (1981) Kinetics of fenfluramine (Cartwright A. C. and Matthews B. R., eds.), Ellisisomers in the rat. Eur. J. Drug Metab. Horwood, Chichester, pp. 560-627.Pharmacokinet. 6, 297-301. Campbell D. B., Richards R. P., Caccia S., and

Caccia S., Ballabio M., Guiso G., Rocchetti M., and Garattini S. (1986) Stereoselective metabolism

Garattini S. (1982) Species differences in the and the fate of fenfluramine in animals andkinetics and metabolism of fenfluramine humans, in Development of Drugs and Modernisomers. Arch. Int. Pharmacodyn. 258, 15-28. Medicines (Gorrod J. W., Gibson G. G., and

Caccia S., Cappi M., Fracasso C., and Garattini S. Mitchard M., eds.), Ellis Horwood, Chichester,(1990) Influence of dose and route of admin- pp. 298-311.istration on the kinetics of fluoxetine and its Cass W. A. and Zahniser N. R. (1993) Cocaine levelsmetabolitenorfluoxetine in the rat. Psychopharm- in situation and nucleus acumens. Augmen-acology 100, 509-514. ration following challenge injection in rats with-

Caccia S., Fracasso C., Garattini S., Guiso G., and drawn from repeated cocaine administration.Sarati S. (1992) Effects of short- and long-term Neurosci. Lett. 152(1-2), 177-180.

administration of fluoxetine on the monoamine Cho A. K., Hiramatsu M., DiStefano E. W., Changcontent of rat brain. Neuropharmacology 31(4), A.J., and Jenden D. J. (1990) Stereochemical343-347. differences in the metabolism of 3,4,methylene-

Caldwell J., Dring L. G., and Williams R. T. (1972) dioxy methamphetamine in vivo and in vitro: aMetabolism of [14C]methamphetamine in man, pharmacokinetic analysis. Drug Metab. Dispos.the_ea Eig and therat,.Bi_!t _-.t,_9_Ll_. 18, 686.

Campbell"D/'B: -(19-7§) Pharmacokinetics 0f _-_Cho A. K., Hiramatsu M., Kumagai Y., and Patel N... amphetamine and fenfluramine after therapeutic _ (1993) Pharmacokinetic approach to the study of

' dosage and overclosage. PhD. Thesis. University _ drug action and toxicity in assessing neuro-· of London, Poisons Unit, Guy's Hospital, _ toxicity of drugs of abuse. Natural Institute on..... London, SE1. ._. Drug Abuse Res. Monograph Series, vol. 136 (Erinofcampi_il D.B, (lgo_a) Stereoselectivity in clinlZ_ ......... 1n.L., ed.), pp. 213--215.

pharmacokinetics and drug development. Eur. J. Chu M., Hiramatsu M., and Cho A. K. (1992) In vivoDrug Metab. Pharmacokinet. 15(2), 109-125. formation of catecholamines from methylene-

Campbell D. B. (1990b) The use of kinetic-dynamic dioxyamphetamine (MDA) and -methamphet-interactions in the evaluation of drugs. Psycho- amine (MDMA) FASEB J. 6(4), abstract 3668,pharmacology 100, 433-450. pA1569.

Campbell D. B. (1994a) Can allometric interspecies Clarke B. and Smith D. A. (1984) Pharmacokineticsscaling be used to predict human kinetics? Drug and toxicity testing. CRC Critical Rev. Toxicol. 12,In_.J. 28, 235--245. 343--385.


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Commins D. L., Vosmer G., Virus I_ M., Woolverton Fuller R. W., Snoddy H. D., and Perry K. W. (1988)W. L., Schuster C. R., and Seiden L. S. (1987) Metabolism of fenfluramine to norfenfluramine

Biochemical and histological evidence that in guinea-pigs. J. Pharm. Pharmacol. 40, 439-441.methylenedioxymethylamphetamine (MDMA) Garattini S., Bizzi A., Caccia S., Mennini T., and

is toxic to neurons in the rat brain. J. Pharmacol. Samanin R. (1988) Progress in assessing the roleExp. Ther. 241(1), 338-345. of serotonin in the control of food intake. Clin.

Cook C. E., Jeffcoat A. R., Sadler B. M., Hill J.M., Pharmacol. Il(Suppl. 1) S8-S32.Voyksner R. D., Pugh D. E., White W. R., and Gardier A. M., Lepoul E., Trouvin J. H., Chanut E.,Perez-Reyes M. (1992) Pharmacokinetics of oral Dessalles M. C., and Jacquot C. (1993) Changesmethamphetamine and effects of repeated daily in dopamine metabolism in rat forebrain regionsdosing in humans. Drug Metab. Dispos. Biol. Fate after cessation of long-term fluoxetine treatment:Chem. 20, 856-862. relationship with brain concentrations of

Crump K. S. (1984) A new method for determining fluoxetine and norfluoxetine. L_CeSci. 54, 51-56.available daily intakes. Fundam. Appl. Toxicol. 4, Gaylor D. and Slikker W. Jr. (1992) Risk assessment

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uncertainty factors in establishing safe levels of and Mennini T. (1993) Decreased serotonin

exposure, in Statistics in Toxicology (Krewski D. uptake carriers after chronic d-fenfluramine:and Franklin C., eds.), Grodon & Breach Science, neurotoxicity or down-regulation? PosterNew York, pp. 613-627. presented at the 23rd Annual Meeting Societyfor

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Federal Register (1993) EPA draft report: principles Chemoreceptors in Respiratory Control (Ribero J.of neurotoxicity risk assessment. $8, 41,556- H. and Pallor D. J., eds ), Croom Helm, London,41,599. pp.394-407.

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Fleisher M. R. and Campbell D. B. (1969) Fenflur- Herr F. (1989) Grundlagen der Pharmakologie. Jenaamine overdosage. Lancet 2, 1306,1307. Veb. p. 271.

Fuller R. W. and Perry K. W. (1992) Comparison of Hiramatsu M., DiStefano E. W., and Cho A. K.fluoxetine and norfluoxetine enantiomers as (1989a) Stereochemical differences in the in vivoinhibitors of hexobarbitone metabolism in mice and in vitro metabolism of MDMA. Fed Am. Soc.

J. Pharm. Pharmacol. 44, 1041,1042. Exp. Biol. J. 3, A1035.Fuller R. W., Snoddy H. D., Krushinski J. H., and Hiramatsu M. and Cho A. K. (1989b) Rapid forma-

Robertson D. W. (1992) Comparison of norflu- tion and disappearance of the MDMA metaboliteoxetine enantiomers as serotonin uptake inhib- 3,4-dihydroxymethamphetamine by rat liveritors in vivo Neuropharmacology 31, 997-1000. microsomes. Neurosciences Abs. 15, 1185.

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