I n t h e foregoing sections t h e change in t h e drug molecules as a result of meta
bolic processes had our attention. Now t h e assimilation or incorporation of t h e drug in t h e body constituents will be discussed. If t h e drugs given to t h e animal are very closely related t o t h e n a t u r a l metabolites, it m a y become very h a r d for t h e enzymes to differentiate between t h e endogenous and foreign com
pounds. The enzymes are fooled; t h e y erroneously accept t h e drug on t h e anabolic route with t h e consequence of assimilation of t h e drug. They are handled as if t h e y were t h e n a t u r a l metabolite (130). Taking into account t h e rather high degree of specificity usual for enzymes engaged in anabolic pro
cesses, it will be clear t h a t this will occur only with drugs very closely related to t h e metabolite in a physicochemical sense (24). Assimilation of t h e drug results in t h e biosynthesis of functional a n d adequate, or nonfunctional a n d inadequate, end-products :
1. When t h e drug substitutes for t h e metabolite in a morphological as well as in a functional sense, it takes t h e place of t h e metabolite in an effective way a n d m a y be called a " p a r a m e t a b o l i t e . "
2. When t h e drug substitutes for t h e metabolite only in a morphological b u t not in a functional sense, it acts as an antimetabolite, t h u s , blocking t h e normal biological process (180a, 219, 220, 221).
I n t h e case of neurotransmitters, instead of t h e t e r m " p a r a m e t a b o l i t e " a n d
" a n t i m e t a b o l i t e , " t h e suffix " m i m e t i c " a n d " l y t i c " is used, respectively, e.g., acetylcholinomimetics (parasympathomimetics) a n d acetylcholinolytics (parasympatholytics) (163). I t should be pointed out t h a t t h e antimetabolites a n d t h e lytics not only antagonize t h e n a t u r a l metabolite or t h e neurotrans
mitters, b u t also t h e corresponding parametabolites a n d mimetics. Likewise antihormones will antagonize both hormone a n d parahormone. This t y p e of antagonism is often called specific antagonism or competitive antagonism, because t h e agonist and t h e antagonist compete for t h e same active sites on t h e enzymes in t h e biological object. The active sites usually are referred to as t h e specific receptors for t h e drug. The fact t h a t these receptors m a y be located on enzymes or on some other effector constituent, does not make an essential difference. Much information on t h e theme, metabolite vs. antimetabolite, has accumulated in monographs and reviews (47, 48, 95, 128, 139, 147, 174, 1789
220,221).
I.B.5.1. Metabolites and Parametabolites
The parametabolite follows t h e metabolic route of t h e metabolite in an effec
tive way, a process t h a t might be called a vital synthesis. As a m a t t e r of fact, t h e term metabolite can be used in a broad sense, covering all n a t u r a l com
pounds processed in some way in t h e biological object. Therefore, t h e vitamins and t h e neurotransmitters, such as acetylcholine a n d arterenol, m a y also be included.
Ι,Ι.Β. DRUG TRANSFERENCE! DRUG METABOLISM 99 An example of a vital synthesis is t h e incorporation of certain vitamin analogs into coenzymes. For t h e p-aminobenzoic-acid-(PABA)-deficient strain of Escherichia coli (E. coli 273), 2-amino-5-carboxypyridine m a y be substituted for P A B A as a growth factor. A conversion of this compound to P A B A is practically impossible. P A B A is supposed t o be incorporated into folic acid, which in its t u r n serves in t h e synthesis of enzymes t h a t effect t h e transfer of
"one-carbon u n i t s " and are essential for amino acid and purine synthesis (12).
This makes it probable t h a t 2-amino-5-carboxypyridine is incorporated into folic acid analogs, which t a k e over t h e function of folic acid in t h e bacteria concerned. I n t h e fluid in which E. coli 273 is grown without P A B A b u t with 2-amino-5-carboxypyridine, a growth factor is found for t h e folic-acid-deficient strain Streptococcus lactis R. This growth factor is antagonized in a specific
OH
, . 2 - a m i n o - . ,. . . ,
para- aminobenzoic , nicotinic acid . j D c a rDOXY . j
acid . J amide
pyridine
FIG. 16. 2-Amino-5-carboxypyridine in its relation to vitamins p-aminobenzoic acid and nicotinic acid amide.
way by folic acid antagonists such as #-methylfolic acid. This makes t h e in
corporation of 2-amino-5-carboxypyridine into a folic acid analog still more probable (8).
An interesting feature of 2-amino-5-carboxypyridine as a substitute for P A B A in growth of E. coli 273, is t h a t a t higher concentrations of t h e compound a n autoinhibition or substrate inhibition is observed. This inhibition can be reversed by t h e addition of nicotinic acid amide. The compound 2-amino-5-carboxypyridine is not only closely related chemically t o PABA, b u t also t o nicotinic acid (Fig. 16). I n experiments with a nicotinic-acid-deficient strain of E. coli (E. coli 267), 2-amino-5-carboxypyridine was found t o behave as a specific antagonist for nicotinic acid amide in t h e same way as sulfonamides antagonize P A B A and 2-amino-5-carboxypyridine in t h e case of E. coli 273.
The conclusion is t h a t 2-amino-5-carboxypyridine probably acts as a para-metabolite, substituting for p-aminobenzoic acid, a n d as an antipara-metabolite, counteracting nicotinic acid amide.
Many vitamin analogs are known which, although t h e y cannot be converted by t h e body to t h e vitamin, m a y nevertheless, substitute for it. They act as
TABLE X I I
RELATIONSHIP IN THE STRUCTURE BETWEEN PARAMETABOLITES, METABOLITES, AND ANTIMETABOLITES
Parametabolite
Pyridoxine (B6) I Desoxypyridoxine
C )H ( 3H C )H ( )H
0 0
, j ^ N - C - C HII 3
I ^ . . . i Nicotinic acid #-acetylpyridine
// \
11H2N — ^
J—C—OH
0
H8N — ^ V—C—QH
Ο
H2N—I \ — S — N H2
\ / II
2-Fluoro-p-aminobenzoic acid p-Aminobenzoic acid \ f Q
Sulfanilamide
HOOC HOO<^5 HOO(j)
1
c
C Cin
H 0 0 C AH CAH
2 W H O O C H O O2-Fluoro folic acid Folic acid (PGA) Aminopterin C OHO
CH2OH—C—C—C—N—C—C—COOH
C OHO
CH2OH—C—C—C—N—C—C—COOH
2 I
C OHO Ο . ,
Γ
1 II II / / \CH 2OH—C—C—C—Ν—C—C—C—ζ J C—OH
Oxypantothenic acid
C . 1
Pantothenic acid
c
Phenylpantothenone
0 0
||
0 0
II II
l ^/j l ^/J - C - C = C - ( C - C - C - C )3- C
II
0
Menadion
A ° i
Vitamin
II II
0 0 Diphtiocol
1,1.B. DRUG TRANSFERENCE: DRUG METABOLISM 101
102 Ε. J. ARIENS AND Α. Μ. SIMONIS
para vitamins or parametabolites (see Table X I I ) . If a compound acts as para-metabolite or para vitamin, this implies t h a t it follows, a t least in p a r t , t h e anabolic p a t h w a y of t h e metabolite or t h e vitamin. This also obtains for hormone analogs with a hormone activity, etc.
An exception m u s t be made for t h e case t h a t vitamin- or hormone-analogs act indirectly, by protecting t h e n a t u r a l vitamins or hormones present in t h e body against metabolic degradation, or if t h e y act as liberators for n a t u r a l compounds in the body (see Section I.B.6).
/.B.5.2. Antimetabolites
There is no special reason why antivitamins (Table X I I ) or antimetabolites would not also follow t h e anabolic route of t h e vitamins or metabolites t o which t h e y are chemically related. Possibly, t h e antimetabolite follows t h e anabolic route of t h e metabolite for a certain number of steps resulting in t h e formation of afunctional intermediate products which block t h e rest of t h e route. This process is called a " l e t h a l s y n t h e s i s " (147, 148).
An example often cited is t h e assimilation of monofluoroacetic acid in citric acid synthesis, with t h e resultant formation of fluorocitric acid. This compound blocks t h e enzyme aconitase t h a t normally brings a b o u t t h e conversion of citric acid into isocitric acid. The supply with oxaloacetic acid is exhausted.
Consequently, a blockade of t h e tricarboxylic cycle occurs (44, 66, 153).
The action of antivitamins m a y be based on a blockade a t t h e entrance of t h e vitamin t o t h e anabolic p a t h w a y s , or perhaps t h e antivitamin follows t h e route for a number of steps. I t is debatable whether one should speak of a lethal synthesis only if t h e foreign compound has several metabolic steps in common with t h e natural metabolite, or whether one step, t h e first one, is sufficient t o classify t h e process as such. If t h e l a t t e r concept is held, most of t h e antimeta
bolites, antivitamins, a n d t h e so-called lytics should be classified as compounds leading t o a lethal synthesis (127,127a, 130).
Lethal synthesis a n d bio-activation both lead t o toxication of a drug. They are closely related, if n o t identical, principles. More essential is t h e question whether t h e assimilation of a drug a n d t h e formation of afunctional products lead t o a real fixation in t h e body constituents. I n t h a t case, t h e blocking action of t h e drug continues after t h e disappearance of t h e free drug from t h e biophase. Once t h e antimetabolite is assimilated, its action will not, or will hardly be, antagonized by t h e metabolite. There are two essentially different types of relations for metabolite and antimetabolite: a competition based on a simple dynamic equilibrium and a nonequilibrium competition (see Fig. 17).
The assimilation a n d incorporation of antimetabolites will proceed easily for t h e compounds t h a t are closely related t o t h e metabolite in t h e chemical properties essential for t h e incorporation, p-Aminobenzoic acid (PABA) is built into folic acid, which in its t u r n is t a k e n u p in coenzyme F . This enzyme plays a role in t h e introduction of t h e one-carbon unit in t h e biosynthesis of
Ι , Ι . Β . DRUG T R A N S F E R E N C E : DRUG METABOLISM 103
t
x - m e t a b o l i t e - y x - a n t i m e t a b o l i t e - y
metabolite antimetabolite metabolite antimetabolite EQUILIBRIUM COMPETITION NON-EQUILIBRIUM COMPETITION
FIG. 17. Two types of competitive interaction.
sulfathiazole 2 - m e t h y l - P A B A
folic acid
FIG. 18. Two antimetabolites of ρ-aminobenzoic acid (PABA) and their relation to folic acid.
PABA
100
ηmvfPABA nr\M PABA FIG. 19. Log dose-response curves for growth (turbidity) of the p-aminobenzoic acid (PABA)-requiring strain E. coli 273 in the presence of a series of concentrations of the antimetabolites sulfathiazole(ST)and 2-methyl-4-aminobenzoic acid(2-CH3PABA). Curves show growth for various concentrations of the metabolite. Metabolite and antimetabolite were applied simultaneously. Note that the sets of dose-response curves obtained are characteristic for the competitive relation between metabolite and antimetabolite.
purines, pyrimidines, and certain amino acids (206). Antimetabolites of PABA m a y be obtained by substitution in t h e ring. 2-Methyl-PABA for instance acts as an antimetabolite of PABA in t h e same way as t h e sulfonamides, e.g.,
sulfa-100 80 60 40 20 A
CH PABAlmvi
m M PABA mvr PABA
FIG. 20. Differences in growth of E. coli 273 after simultaneous and sequential addition of growth factor and antimetabolite. Log dose-response curves of Fig. 19 are here given for the same combinations of metabolite and antimetabolite but now with incubation time for the antimetabolite: right, 20 hr; left, 29 hr. Note in the case of sulfathiazole the incu
bation does not change the results obtained. In the case of 2-methyl-PABA, after incu
bation no growth is obtained anymore with the metabolite, or much higher concentrations of the metabolite are necessary. The action of this metabolite is less reversible or even irreversible as compared to the easily reversible action of sulfathiazole.
Ι,Ι.Β. DRUG TRANSFERENCE: DRUG METABOLISM 105 thiazole, do (178). A comparison of 2-methyl-PABA and sulfathiazole with the PABA-moiety of folic acid shows t h a t if one of t h e two compounds is to be built in, 2-methyl-PABA has t h e best chance (see Fig. 18). As long as metabolite and antimetabolite are applied simultaneously, t h e metabolite PABA will be able to protect t h e organism against t h e inhibiting action of 2-methyl-PABA as well as t h a t of sulfathiazole. If, however, t h e organism is incubated with antimetabolites for some time, while t h e metabolite is added subsequently, t h e antimetabolites get t h e chance of becoming assimilated and incorporated, and, t h u s , blocking metabolism on a level where t h e metabolite cannot displace it anymore (see Fig. 17).
Experiments with t h e PABA-requiring strain, E. coli 273, showed t h a t after incubation with 2-methyl-PABA t h e inhibition of growth is not reversible (or only with a great delay, reversible) with PABA, while after incubation with sulfathiazole, t h e inhibition is annihilated (Fig. 20) as easily as when metabolite and antimetabolite are added simultaneously (see Fig. 19).
For other antimetabolites, such as for 5-bromouracil (59, 223) and 8-aza-guanine (8, 88, 119, 130), support for t h e assumption of a biosynthetic incor
poration into t h e cell constituents has been obtained (127). For more detailed information, t h e reader is referred t o a review by Matthews (129) on t h e bio
synthetic incorporation of metabolite analogs and to symposia dealing with the subject (47, 128, and P . Emmelot, "Molecular P h a r m a c o l o g y , I I , P a r t 3).
I.B.6. D R U G S A C T I N G INDIRECTLY
A n u m b e r of drugs induce effects not because of an interaction with receptors related t o t h e effector, b u t on t h e strength of endogenous compounds. They act by virtue of a second compound.
I.B.6.1. Protection of Endogenous Compounds
The drug m a y protect neurohormones or other endogenous compounds against rapid inactivation and, therefore, increase t h e concentration of it in t h e biophase. The drug can in this way produce effects which, in fact, are caused by t h e endogenous compounds protected from enzymatic breakdown.
I n biological preparations with a functioning autonomic nerve supply, t h e in
hibitors of acetylcholinesterase produce effects similar t o those induced by acetylcholine and, therefore, simulate acetylcholinomimetics. In t h e same way, pyrogallol protecting epinephrine against O-methylation, simulates sympathomimetics (221).
I.B.6.2. Release of Endogenous Compounds
Other examples of drugs acting on t h e strength of endogenous components are t h e so-called " l i b e r a t o r s , " for instance, t h e histamine liberators. These drugs behave as histaminomimetics b u t produce their effect by liberating
106 Ε. J. ARIENS AND A. M. SIMONIS
endogenous histamine. The effects are antagonized in a specific way by anti-histaminics (146). Such a liberating effect can cause a depletion of t h e endo
genous substance. This happens when histamine liberators are administered repeatedly. After a high dose of t h e compound, which m a y lead to histamine shock, a second dose is much less effective, while a third dose will be definitely ineffective. The slower t h e synthesis of t h e endogenous compound, t h e more easily will a depletion manifest itself as a tachyphylaxis or even as a develop
m e n t of a tolerance for t h e drug.
Another example of t h e release of endogenous compounds—this time cate
cholamines—are t h e indirectly acting sympathomimetics (or pseudo-sympa-thomimetics) such as phenylethylamine and amphetamine. The peripheral effects induced by these drugs probably are due t o t h e release of catecholamines (40, 41, 94,107,170), as a result of a displacement of t h e catecholamines from intracellular granules by t h e pseudo-sympathomimetic drugs (171).
The release phenomenon is not restricted to biogenous amines. The hypo
glycemic action of t h e sulfonureides used as oral antidiabetics, is ascribed t o a release of endogenous insulin by these compounds (58, 125). Antidiuretic hormone is released by a variety of drugs like nicotine, morphine, and BAL (British Anti-Lewisite) (152).
When t h e release of t h e endogenous compounds proceeds slowly, not t h e effect of t h e compound which is released, b u t rather t h e depletion of its stores, becomes manifest. The depletion of t h e stores of catecholamines and serotonin after reserpine and guanethidine, m a y count for t h e decrease in t h e responsive
ness of t h e sympathetic nerve system by these drugs (37). As demonstrated by B u r n (40, 41), t h e indirectly acting sympathomimetics like phenylethylamine become inactive if t h e animals are reserpinized, probably because t h e stores of endogenous amines on which t h e action of t h e indirectly acting sympatho
mimetics is based, are exhausted. Administration of dihydroxyphenylalanine (DOPA), a precursor of t h e catecholamines or of norepinephrine, leads t o a return of t h e response t o indirect sympathomimetics. This is probably because t h e stores are refilled (41, 163b).
Repeated administration of pseudo-sympathomimetic drugs, phenylethyl
amine, amphetamine and tyramine, results in tachyphylaxis. This is caused possibly by a depletion of t h e stores of catecholamines. Administration of norepinephrine restores t h e response, a t least partially, or prevents t h e tachy
phylaxis (52).
Storage and release of various biogenous amines, like dopamine, norepine
phrine, epinephrine, and serotonin, are being intensively studied. Sites of storage are, e.g. granules in argentaffin and chromaffin cells and t h e blood platelets (89). The storage can be barred by t h e releasers*. Epinephrine ad
ministered to mice is metabolized in two phases—a rapid initial O-methylation of about two-thirds of t h e epinephrine present followed by a second phase of much slower metabolism of t h e rest. Pre t r e a t m e n t with reserpine leads t o a
Ι , Ι . Β . DRUG TRANSFERENCE: DRUG METABOLISM 107
disappearance of t h e second phase. Then, a m u c h higher fraction of t h e epine
phrine is rapidly metabolized. The slow phase in metabolism is interpreted as a t e m p o r a r y binding of epinephrine in stores or other sites of binding a n d a subsequent release a n d metabolism. As long as epinephrine is bound in t h e stores or a t other inert sites of adsorption, it is protected from metabolism.
Reserpine is assumed t o prevent this binding and, t h u s , to accelerate meta
bolism of epinephrine (14).
I.B.6.3. Release or Displacement of Drugs from Silent Receptors As mentioned, drugs m a y interact with active sites (receptors) related t o t h e effector system and, t h u s , induce effects. They m a y interact with t h e active sites on enzymes a n d become metabolized; t h e y m a y interact with t h e active sites on carriers a n d become t r a n s p o r t e d ; t h e y m a y interact with specific sites of storage from which a regulated release is possible; and, finally, t h e y m a y become adsorbed t o indifferent sites of binding—the silent receptors. On t h e silent receptors, t h e drug is out of reach of metabolic enzymes. The adsorption of sulfonamides t o plasma proteins, which results in a longer duration of t h e action, as mentioned before, is a n example (39).
The t e r m "silent r e c e p t o r " was introduced b y Goldstein (80). As early as 1925, Storm v a n Leeuwen (190, 191) distinguished between dominant recep
tors, or sites of action concerned with t h e p r i m a r y pharmacological effects of t h e drug, and secondary receptors, n o t concerned with t h e p r i m a r y effect. The t e r m "sites of l o s s " as used by Veldstra (200) includes t h e active sites on enzymes, t h e storage receptors, as well as t h e silent receptors.
On t h e silent receptors, a displacement of one drug b y t h e other m a y t a k e place. Drugs m a y compete for these sites of inert adsorption. P h e n y l b u t a z o n e can displace a n d release sulfonamides from their silent receptors on albumin (6). The competition for t h e silent receptors takes place when phenylbutazone is added previously t o , simultaneously with, or subsequently t o , t h e sulfon
amide. T h e antibacterial action of t h e sulfonamides is restricted t o t h e free drug molecules. P h e n y l b u t a z o n e , by releasing sulfonamide from t h e silent receptors, m a y simulate an antibacterial action. A related compound, sulfinpyrazone was found to be an even stronger displacer of protein-bound sulfonamides a n d other organic acids (6a).
The displacement of histamine from its silent receptors in plasma b y compounds such as antihistamines, which act as histamine liberators in this case, is another example of a release (145e, d). The histamine binding b y plasma is called " h i s t a m i n o p e x i e . "
Various steroid hormones, e.g., testosterone, estrone, a n d progesterone, compete for common binding sites on albumin (211a, 21a, 21b). Protein binding of hydrocortisone is decreased in t h e presence of synthetic corticoids (201a). These processes can play a p a r t in t h e m u t u a l influence of various steroid hormones in their action (53a, 133a, 145a, 211b).
108 Ε. J. ARIENS AND A. M. SIMONIS
The interference of reserpine with t h e slow phase of epinephrine metabolism is due possibly t o a competition with epinephrine for silent receptors. The in
directly acting sympathomimetic compounds, like phenylethylamine, ephe
drine and amphetamine, also augment catecholamine metabolism. The directly acting sympathomimetics, like synephrine a n d paredrine, have no influence.
The compounds mentioned have no effect on catechol-O-methyltransferase activity (16). The indirectly acting sympathomimetics probably act as releasers of catecholamine (40, 41). Their action on catecholamine metabolism m a y also result from prevention of protective binding of t h e catecholamines on silent receptors and, therefore, increase t h e rate of metabolism (16). The increase in t h e rate of excretion of morphine in animals t r e a t e d with nalorphine (1) pos
sibly has to be ascribed, too, to a competition of these compounds for inert sites of adsorption. Competition of two compounds for common sites of action and common silent receptors has interesting consequences (222a).
The potentiation of curariform drugs by various bases which are not curari-form themselves has been a t t r i b u t e d to a displacement of t h e curaricurari-form drug by these bases from nonspecific sites of binding, e.g., t h e sulfate groups in t h e acidic mucopolysaccharides (25, 43, 45). Recently Vane (198) suggested an analogous mechanism for t h e potentiation of catecholamines by various bases such as hexamethonium and bretylium.
One of t h e hypotheses p u t forward to interpret t h e difference in toxicity of certain acetylcholinesterase inhibitors for flies a n d mice, is based on t h e sug
gestion of a difference in concentration of silent receptors—non-esteratic proteins—binding t h e organic phosphorus derivatives in both species (10).
The storage receptors differ from t h e silent receptors. The release from t h e stores is regulated. The silent receptors are inert sites of adsorption, where t h e drug is adsorbed in a physical equilibrium with t h e free drug.
As a m a t t e r of fact, for one compound different types of storage a n d silent receptors m a y exist. For such, a differentiation in various types of blocking and releasing drugs m a y be expected (38b, 183b). The various types of binding of biogenous amines to tissues and release mechanisms have been summarized by Green (82a).
I n these chapters concerned with drug t r a n s p o r t a n d drug metabolism, t h e t e r m " r e c e p t o r s " and its equivalents have been used in a general sense.
More specific definitions of drug-receptor interaction will be discussed in Section I I .
To reiterate for t h e purpose of explanation, drug transport, metabolism, and dosage determine t h e concentration of t h e drug in t h e immediate vicinity of t h e specific receptors, t h a t is, those receptors which are essentially concerned with t h e effect produced. I n t h e various processes t h a t t a k e p a r t in t h e drug transport and metabolism, carriers, enzymes, silent receptors, storage recep
tors, etc. m a y be involved. The interaction of a drug with these receptors, although it influences t h e relation between dosage and effect, is not essential
Ι,Ι.Β. DRUG TRANSFERENCE: DRUG METABOLISM 109
for t h e induction of t h e effect. An exception m u s t be m a d e for releasing drugs a n d bio-activation.
C O N C L U D I N G R E M A R K S The conclusion here is a double one :
a. The action of a drug as observed in an animal is t h e resultant of a com
plex of processes and actions. E a c h of t h e m m a y require specific features in t h e drug molecule. D a t a obtained with whole animals are so complex, t h a t t h e y are of little use in a primary effort t o interpret pharmacological action on a molecular level. For t h a t purpose, t h e s t u d y of t h e interaction of t h e drug with much simpler systems is inevitable. Isolated organs or cells, microsomes a n d mitochondria, or single enzymes are t h e biological elements from which infor
mation useful in molecular pharmacology can be obtained.
b. The integrated pharmacological d a t a obtained with isolated organs, cells, etc. m a y give an indication of t h e action of t h e drug in t h e living animal.
B u t , because of t h e very complicated situation there, this detailed information will not suffice t o permit definite predictions of its action. Much less can be predicted of therapeutic potentialities of a drug in m a n .
Bio-activation m a y change negative results in t h e test with isolated organs to positive ones in an animal experiment. An activity found in an isolated system does not guarantee t h e activity in t h e whole animal. Although t h e drug might be active once it reaches an effective concentration in t h e biophase, t h e various steps in drug transference m a y raise insurmountable barriers t o its reaching such a concentration. Differences in species, as far as drug metabolism is concerned, introduce new difficulties. Some very illustrative examples of this were given by Brodie in his paper on clinical implications of drug metabolism (32).
The study of t r a n s p o r t a n d metabolism of drugs is essential for progress in pharmacology. The use in such studies of drugs marked b y radioactive tracers,
The study of t r a n s p o r t a n d metabolism of drugs is essential for progress in pharmacology. The use in such studies of drugs marked b y radioactive tracers,