Drug metabolism, as pictured above, is of a d v a n t a g e to t h e animal. I t has, however, its pitfalls, especially if chemists a n d pharmacologists set out in a combined effort t o fool n a t u r e .
The chemical structure of t h e drug molecules offered to t h e animal m a y be chosen so t h a t t h e y fit t h e metabolic enzymes, b u t cannot be altered by t h e m . An interaction between drug a n d enzyme takes place, b u t without immediate result. The drug resists metabolic changes a n d breakdown. The result is t h a t t h e enzymes are bound by these drug molecules and, therefore, fail in complet
ing their other tasks in drug metabolism or in normal metabolic processes.
The drug acts as an inhibitor in t h a t it blocks t h e enzymes. The more essential t h e enzymes t o t h e animal, or t h e more essential t h e metabolite normally processed by t h e m , t h e more serious will be t h e consequences.
Drugs closely related chemically t o essential metabolites will t e n d t o block t h e enzymes concerned with t h e processing of these metabolites. The blockade by t h e inhibiting drug m a y result i n : (2) a delay in t h e metabolic inactivation and, therefore, a prolongation of t h e action of a d r u g ; (2) a delay in t h e meta
bolic activation and, therefore, a decrease in t h e activity of a drug. Not only t h e so-called enzymic inhibitors influence drug metabolism. Because of " s u b strate c o m p e t i t i o n " m a n y drugs can m u t u a l l y influence their metabolism.
I AAA. Inhibition of Bio-inactivation and Detoxication
One of t h e best-known examples is t h e blockade of t h e acetylcholinesterase by certain congeners of acetylcholine which are called antiacetylcholinesterases (116).
Slight changes in t h e structure of a drug m a y change it t o an inhibitor for t h e
90 Ε. J. ARIENS AND A. M. SIMONIS
enzymes which normally metabolize t h a t drug. This is emphasized by t h e antagonism of steric isomers in this respect. The sparing action, resulting from t h e inhibition of t h e breakdown of t h e drug, has as a consequence t h e attain
m e n t of an effective concentration of t h e drug in t h e biophase with lower doses.
The effect of a certain dose is prolonged. The phenomenon appears t o be a
°/o contraction rect. abd. frog—| i n t e s t i n e rat 100 -i
10'* 10"J 10"'
m M ACh
% contraction sem. ves. rabbit-ι
m M Adrenaline
10"J 10"
m i v i A d r e n a l i n e
FIG. 12. Experimental log concentration-response curves for agonistic compounds tes
ted in the presence of enzyme-inhibitors: A. Eserine;B. Ephedrine; C. Diisopropylfluoro-phosphate (DFP); D. Cocaine. Note the shift of the curves to the lower concentration in the presence of the inhibitors, possibly as a result of a "sparing" effect. Chihara (46).
sensitization of t h e organism t o t h e drug. The dose-response curve shifts t o lower levels.
Other examples are t h e inhibition of amine oxidase by its specific inhibitors.
Figure 12 (46) represents t h e increase in t h e effectiveness of acetylcholine (ACh) and adrenaline for various isolated organs after addition of anti-ACh esterases and amine-oxidase inhibitors, respectively. I n t h e presence of these inhibitors t h e log dose-response curves for ACh a n d adrenaline shift to lower concentrations. There is strong experimental support for t h e supposition t h a t
Ι , Ι . Β . DRUG TRANSFERENCE: DRUG METABOLISM 91 a t least a p a r t of this sensitization has t o be a t t r i b u t e d to a direct action of t h e
" i n h i b i t o r " on t h e effector cells. After irreversible blockade of ACh esterase with organic phosphates such as diisopropylfluorophosphate, compounds like neostigmine a n d prostigmine still increase t h e sensitivity of isolated organs t o acetylcholine. The fact t h a t these compounds increase t h e sensitivity of iso
lated organs for acetylcholinomimetics, which are n o t split by t h e esterase, is strong evidence for direct sensitization (49,160,197).
I n t h e case of t h e amineoxidase inhibitors, e.g., ephedrine a n d cocaine, too, a direct sensitization of t h e effector cells is probable (22, 83).
An epinephrine-sparing action of ephedrine, etc. is n o t probable because t h e physiological disposition of epinephrine is mainly based on methylation of t h e meia-OH-group in t h e catechol nucleus u n d e r formation of metanephrine.
I n studies with H3-labeled epinephrine, Axelrod (13) demonstrated a rapid O-methylation in most tissues. Monoamine-oxidase inhibitors such as iproni
azid hardly influence t h e effects of epinephrine in vivo. The inhibition of t h e O-methylating enzyme, e.g., b y pyrogallol, however, prolongs t h e action and delays t h e inactivation of epinephrine (222).
Many of t h e inhibitors of drug metabolism are related chemically t o t h e compounds t h e y protect against enzyme action. This relation in structure leads t o a certain specificity in t h e inhibiting action. Most of t h e antiacetylcholin-esterases, especially t h e reversibly blocking agents, are related t o ACh (see Table X I ) (117)—pyrogallol is related t o t h e catechol nucleus of t h e catechol
amines, etc. Such a specificity, however, is n o t t h e rule.
The reversible acetylcholinesterase inhibitors are relatively specific. They inhibit acetylcholinesterase a n d pseudo-acetylcholinesterase. The alkylphos-phates block a great variety of esterases a n d amidases. Their action is, how
ever, still restricted t o these two types of hydrolysis (56, 96, 98, 144). For a consideration of t h e relations between structure a n d activity of t h e various compounds, t h e structure m u s t be judged a n d compared in a functional sense.
The physicochemical properties, including t h e steric structure, are essential (74, 173a, 216); the usual structural formulas are a relatively poor means of expressing t h e m .
An interesting aspect of t h e chemistry of enzyme inhibitors is t h e reactiva
tion of esterases irreversibly blocked by alkylphosphates, by means of al-doximes like PAM (pyridine aldoxime methiodide) (137, 216). The hydrolysis of acetylcholine, t h e irreversible blockade by paraoxon, a n d t h e reactivation of acetylcholinesterase blocked by paraoxon is schematized in Fig. 13.
The relations in t h e structure of t h e various compounds is represented in Table X I (72, 90, 114, 155). The introduction of a q u a t e r n a r y a m m o n i u m group in t h e alkyl phosphates, as realized by Fredriksson (72), increases t h e acetylcholinesterase-inhibiting action, probably because such compounds interact with both t h e esteratic and anionic site of t h e receptor (see Table X I ) .
TABLE XI
RELATIONSHIP IN STRUCTURE OF DRUGS WHICH INTERACT WITH ACETYLCHOLINESTERASE0
ACh Esterase Inhibitors
Ι,Ι.Β. DRUG TRANSFERENCE: DRUG METABOLISM 93 Not only phosphate esters b u t also esters of m e t h a n e sulfonic acid in which a q u a t e r n a r y ammonium group is located a t a suitable distance from the ester group, act as irreversible blockers of t h e acetylcholinesterase (see Table X I ) . Here, too, the formation of a relatively stable acid-enzyme derivative is probable. The enzyme can be reactivated b y iV^-methyl-3-pyridine aldoxime (114a). An interesting aspect of t h e irreversible blockade of acetylcholin
esterase and the reactivation by the aldoximes is t h e inhibition of t h e
reactiva-acetylcholine
C—N—C—C—OH
C — C - O H II
ο
ACh-esterase ACh-esterase ACh-esterase
O^ Ο—C—C
PC II o—c— c
HO ο
ACh-esterase ACh-esterase irreversibly blocked
0 2N —V V-OH paranitrophenol
P. A. M.
ACh-esterase ACh-esterase irreversibly blocked reactivated
FIG. 1 3 . Blockade and reactivation of acetylcholinesterase.
tion by small q u a t e r n a r y a m m o n i u m compounds. These probably compete with the onium group of the aldoximes for t h e negative site on t h e enzyme which is blocked irreversibly on the esteratic site (215a).
Some of t h e irreversible blockers of acetylcholinesterase such as malathion, are hydrolyzed a n d therefore detoxified by aliphatic esterases relatively quickly. A combination of malathion with inhibitors of these esterases results in an inhibition of t h e bio-inactivation a n d therefore in a potentiation of t h e
94 Ε. J. ARIENS AND A. M. SIMONIS
neurotoxicity of malathion (42c). F u r t h e r examples of the potentiation of drugs by inhibition of the degrading enzymes is the potentiation of 6-sub-stituted purines by inhibitors of xanthine oxidase (65a).
Inhibitors of drug metabolism which have a very large spectrum of inhibitive actions are known. Of special interest in this respect is t h e fact t h a t m a n y types of bio-transformation are blocked by diethylaminodiphenylpropyl acetate (SKF-525-A) (Fig. 14). This substance blocks various microsomal catabolic reactions possibly as a true multipotent enzyme inhibitor. This compound,
- O — C - C —Ν c — c C—C
// \ \ /
Ν )—C—Ο—Ν—Ν—C
II Η Η \
Ο c
Sch 5705 iproniazid
C I
c c — c C — C — O — C - C — C - O - C - CI / - N
II I II \ ο c ο xc — c
c I c I c I Sch 5 7 1 2
FIG. 14. Multipotent inhibitors of drug metabolism.
if applied alone, does little or no harm t o t h e animal. This is an argument for t h e supposition t h a t t h e enzyme systems concerned are not essential t o normal metabolism b u t are mainly used for t h e removal of compounds foreign to t h e body (32,37,69). SKF-525- A increases t h e toxicity or pharmacological activity
Ι,Ι.Β. DRUG TRANSFERENCE: DRUG METABOLISM 95
of m a n y drugs by decreasing t h e speed of inactivation. A method for measuring this is the determination of t h e half-life (defined in Section I.B.2.5), of the drug.
After p r e t r e a t m e n t with SKF-525-A, t h e sleeping time of r a t s after hexo
barbital is increased to m a n y times t h e normal duration. The half-life of hexo
barbital was also greatly prolonged (34). SKF-525-A also prolongs t h e action of a m p h e t a m i n e as a central nerve s t i m u l a n t and delays t h e demethylation of pethidine (Demerol) and aminopyrine. A practical application of SKF-525-A may be a potentiation of insecticidal action of drugs (88a). I n those cases in which resistance of insects to insecticides is based on an increased metabolic inactivation, SKF-525-A possibly can resensitize t h e insects.
Inhibitors of drug metabolism which increase the duration of action of a drug by decreasing the rate of degradation, m a y in a second phase, as a result of an a d a p t i v e increase in the activity of degrading enzyme systems (see Section I.B.2.4) cause a shortening of t h e action of t h e drug. Such relations are reported for the drug hexobarbital a n d the change in its duration of action by inhibitors such as SKF-525-A a n d iproniazid (173b, 113g,i). Iproniazid, too, is a m u l t i p o t e n t inhibitor for drug metabolism. Another example is t h e compound Lilly 18947 (Fig. 14) (69, 138a, 139a).
The great variety of inhibitions performed by these compounds evokes t h e question of whether t h e y really block t h e various enzymes. The reactions blocked are deaminations, dealkylations, side-chain oxidations, and ring hydroxylations, performed by microsomal enzymes a n d requiring N A D P H2* (15). Possibly these m u l t i p o t e n t inhibitors interfere with t h e access of t h e drugs to t h e microsomes, with t h e supply of N A D P H2, or with some other factor common to t h e various metabolic reactions with which t h e y interfere (38a, 107). Besides t h e inhibition of this N A D P H2- r e q u i r i n g microsomal system for drug metabolism, SKF-525-A also inhibits certain esterases in plasma. So, for instance, t h e procaine hydrolysis is inhibited in a competitive way by S K F -525-A. This also obtains for cholinesterase (135), a n d for amide-splitting enzymes extracted from microsomes.
The inhibition of drug metabolism by these m u l t i p o t e n t compounds has therapeutic consequences, especially because t h e y are frequently used.
Iproniazid has a multiple inhibitive action. I t not only is an inhibitor of amine oxidase, b u t it also inhibits t h e oxidative-enzyme systems in liver microsomes which t a k e care of dealkylations, side-chain oxidations, etc. (69). If combined with other drugs it may cause a strong increase in t h e actions of these drugs (130a), sometimes it may be a decrease. Dangerous potentiation of pethidine by iproniazid has been observed in patients (175). After iproniazid, small doses of a m p h e t a m i n e may cause intense headaches (53b) a n d small a m o u n t s of alcohol may lead to severe intoxication (154). Iproniazid has been t a k e n off the m a r k e t ; however, for other hydrazides such as phenelzine, analogous phenomena are reported (52a, 175).
* For nomenclature, see footnote to Section LB. 1.1.
96 Ε. J . ARIENS AND A. M. SIMONIS
/.β.4.2. Inhibition of Bio-Activation
Drugs closely related chemically t o precursors of biogenous amines, are of particular interest in this respect. The catecholamines, serotonin a n d hist
amine, are biosynthesized by decarboxylation of dihydroxyphenylalanine, 5-OH-tryptophan, and histidine, respectively. If, in these precursors, alkyl groups are introduced on the α-carbon atom in t h e side-chain (the carbon a t o m next to t h e carboxyl group), t h e elimination of t h e carboxyl group by the decarboxylase m a y be hindered (179). The carboxyl group is " p a c k e d " more or less. If t h e chemical properties of the compounds are not severely altered by such a procedure, the affinity or binding t o t h e active site on t h e enzyme m a y be maintained. Then these compounds act as blocking agents of t h e decarb
oxylases. Take for instance α-methyldihydroxyphenylalanine (a-methyl-DOPA), an agent blocking t h e aromatic Z-amino-acid decarboxylase. This compound inhibits t h e biosynthesis of norepinephrine and epinephrine, (Fig. 10) as well as t h a t of serotonin (182, 183, 184, 211).
A great variety of α-substituted amino acids, such as a-methyldopa, α-methylmetatyrosine, and a-methyl-5-hydroxytryptophan, have been tested and found to act as inhibitors of t h e decarboxylation of n a t u r a l substrates, or a t least to inhibit the formation of certain biogenous amines (norepinephrine;
serotonin) from their n a t u r a l substrates (184a, b, 152a, 88b). One should be aware of the fact t h a t t h e inhibition of t h e decarboxylation of a n a t u r a l substrate m a y be t h e result of a substrate competition. The α-substituted derivatives are possibly decarboxylated themselves. Nevertheless, t h e y will act as inhibitors for the decarboxylation of other substrates. The rate of decarboxylation of t h e α-substituted derivatives appears to be low as compared to t h a t of the n a t u r a l substrates (210b). Decarboxylation of the α-substituted derivatives results in the formation of the corresponding α-substituted amines. These, in their t u r n , m a y possibly act as inhibitors of t h e dopamine-/3-hydroxylase, t h e enzyme which converts dopamine to norepinephrine.
Amphetamine and ^-hydroxyamphetamine act as inhibitors of dopamine-β-hydroxylase, the enzyme which converts dopamine to norepinephrine (81).
Finally the metabolic products of t h e α-substituted amino acids possibly act as functional or afunctional substituents for endogenous catecholamines in stores and a t neurotransmission. Interesting developments can be expected in this field in t h e near future. The procedure is closely related to the α-methyl substitution in bio-active amines (Fig. 10).
Another example of blockade of metabolism by chemically related com
pounds is t h e impairment of t h e acetylations catalyzed by coenzyme A by means of α-phenyl-substituted acetic acids. The acetylation of sulfanilamides (bio-inactivation), the acetylation of choline to acetylcholine (bio-activation), a n d cholesterol synthesis, all are dependent on t h e action of coenzyme A. They are inhibited by compounds like α-phenyl-α-ethylacetic acid and a-phenylyl-a-ethylacetic acid (75a, 76) (Fig. 15).
Ι,Ι.Β. DRUG TRANSFERENCE: DRUG METABOLISM 97 For m a n y compounds, a bio-activation and a bio-inactivation occur simul
taneously. Take, for instance, organic phosphates like parathion. Oxidation and bio-activation to paraoxon as well as hydrolysis and bio-inactivation take place. Combination of such compounds with an inhibitor, interfering with bio-activation as well as bio-inactivation, m a y give a rather complex
Η I
-ο
ι ΗC—OH acetic acid
ο
IIΗ
C — C — O H a p h e n y l a
-\ — / ' jl ethyl acetic acid I 2
CH,
a p h e n y l y l a -ethyl acetic acid
FIG. 15. Acetic acid and acetic acid derivatives that block coenzyme A. Garattini etal. (76).
p a t t e r n of synergism and antagonism (224a). An interesting example of t h e inhibition of a bio-activation is the antagonistic action of certain quaternary phenothiazines with respect to tremors induced by tremorine (66c, 141a). This antitremorine action is used as a test for anti-Parkinsonian activity in drugs.
The tremors are induced in the central nervous system where also the anti
parkinsonian drugs have their point of a t t a c k . The fact t h a t quaternary phenothiazines had an antitremorine activity seemed to invalidate the rule t h a t q u a t e r n a r y compounds are devoid of direct central actions because of the inability to pass the blood-brain barrier in an adequate way (3a, 141a) (see Section LA.1.1).
A closer s t u d y of the action of tremorine revealed t h a t the tremors are caused mainly by an oxidation product of tremorine, oxotremorine (210a).
The q u a t e r n a r y phenothiazines which have an antitremorine activity did not antagonize the action of oxytremorine. These results indicate t h a t these quaternary compounds probably act by an inhibition of the bio-activation of tremorine (141a, 113j).
98 Ε. J. ARIENS AND Α. Μ. SIMONIS