Section LB

Section LB

Drug Transference: Drug Metabolism*

Introduction . . . . . . . . . . . . 54

I.B.I. Dissimilation of Drugs . . . . . . . . . 54

1.1. Oxidation . . . . . . . . . . 54

1.2. Reduction . . . . . . . . . . 58

1.3. Hydrolysis 58 1.4. Conjugation . . . . . . . . . . 60

1.5. Chemical Antagonism or Antagonism by Neutralization. . . 64

1.6. Multiple Metabolic Pathways . . . . . . . 65

I.B.2. Factors Influencing Drug Metabolism . . . . 6 6 2.1. Chemical Properties . . . . . . . . . 66

2.2. Effect of Dosage 68 2.3. Route of Application . . . . . . . . 69

2.4. Diet and Drugs 69 2.5. Species Effect 70 2.6. Effect of Sex Differences 73 2.7. Individual Variations . . . . 7 4 I.B.3. General Aspects of Drug Metabolism . . . . . . . 75

3.1. Bio-Inactivation and Detoxication . . . . . . 76

3.2. Bio-Activation . . . . . . . . . . 77

3.3. Evolutionary Aspects . . . . . . . . 82

3.4. Some Practical Consequences . . . . . . . 83

I.B.4. Inhibitors of Drug Metabolism . . . . . . . . 89

4.1. Inhibition of Bio-Inactivation and Detoxication . . . . 89

4.2. Inhibition of Bio-Activation . . . . . . . 96

I.B.5. Assimilation of Drugs . . . . . . . . . . 98

5.1. Metabolites and Parametabolites . . . . 9 8 5.2. Antimetabolites . . . . . . . . . 102

* By E. J. Ariens and A. M. Simonis.

5 3

54 Ε . J . A R I E N S AND Α. Μ. SIMONIS

I N T R O D U C T I O N

The general introduction to this volume outlined t h e factors which govern t h e concentration of a drug in t h e biophase. The preceding chapter discussed t h e processes of t r a n s p o r t of drugs across membranes. This present section will discuss processes of equal importance in t h e maintenance of a d e q u a t e concen­

trations of drug in t h e biophase—the effects of metabolism.

Metabolism is usually divided into catabolism and anabolism. Catabolic processes lead to a dissimilation, t h e degradation or breakdown of t h e meta­

bolites, which in this way are eliminated. Anabolic processes lead to an assimi­

lation, t h e incorporation of t h e metabolites in t h e body constituents. For drug metabolism a similar differentiation is useful: (a) Dissimilation leads t o bio­

chemical changes in t h e drug molecule with, as a final result, t h e elimination of t h e drug; (b) Assimilation leads to an incorporation of t h e drug in t h e body constituents and, therefore, a fixation of t h e drug.

Dissimilation as a rule leads t o a decrease in t h e effective concentration of t h e active drug. However, a temporal increase as a result of bio-activation of originally inactive, or less active, compounds can t a k e place. Assimilation of t h e drug molecules means t h a t there will be a prolonged action even after t h e free drug has disappeared from t h e biophase. Assimilation of t h e drug and change in, or inhibition of, enzyme action by t h e drug in a number of cases is responsible for its pharmacological action (17a, 47,128,129).

I.B.1. DISSIMILATION OF D R U G S

The main routes of drug catabolism will be stressed. They can be categorized into 4 general types of chemical alteration: (a) oxidation; (b) reduction;

(c) hydrolysis; and (d) conjugation or synthesis.

I.B.1.1. Oxidation

Many of t h e oxidative changes of drugs are brought a b o u t by r a t h e r non­

specific enzymes. These are located in t h e endoplasmatic reticulum of t h e liver cells. After homogenization of t h e cells, t h e microsomes are obtained from this reticulum (145). I n some reactions t h e more specific oxidoreductase systems of t h e mitochondria t a k e p a r t . Many of t h e rather nonspecific oxidative sys­

tems, such as " h y d r o x y l a s e s " in t h e microsomes of t h e liver cells, require

I.B.6. Drugs Acting Indirectly . . . . . . . . . 105

6.1. Protection of Endogenous Compounds. . . . 1 0 5 6.2. Release of Endogenous Compounds . . . . . . 105 6.3. Release or Displacement of Drugs from Silent Receptors . . 107 Concluding Remarks . . . . 1 0 9

References . . . . . . . . . . . . . 109

Ι , Ι . Β . DRUG TRANSFERENCE: DRUG METABOLISM 55 oxygen a n d reduced triphosphopyridine nucleotide (NADP). * An intermediate formation of activated oxygen in t h e form of a peroxide, which m a y serve as a

" h y d r o x y l donor," is assumed (33, 37, 38a, 51, 77, 78a).

The most common t y p e of oxidation of a drug molecule is t h e a t t a c k on t h e alkyl chains, including those bearing carbonyl, aldehyde, carboxyl, a n d amino groups. As a rule, t h e oxidation starts as an ω-oxidation, resulting in a conver­

sion of t h e terminal carbon a t o m to a carboxyl group, followed by shortening of t h e chain b y j8-oxidation, also called (ω — 1) or penultimate oxidation (76b, 214.)

I n t h e case of amines, oxidative deamination occurs (17, 22). This deamina- tion is catalyzed by different types of enzymes. Phenylethylamine, t y r a m i n e , dopamine, normethanephrine a n d 5-hydroxytryptamine are deaminated by monoamine oxidase. The α-methyl-substituted amines, like a m p h e t a m i n e a n d ephedrine, are resistant against t h e action of this enzyme. They m a y be de­

aminated by a microsomal deaminase (11,15). Histamine is deaminated b y a diamine oxidase.

A second t y p e is t h e oxidative dealkylation. B y this process, m e t h y l a n d ethyl groups are eliminated from amines a n d alkyl ethers. Examples are t h e conversion of morphine into normorphine, of codeine into morphine, a n d of phenetidine into p-aminophenol. Also, various iV-methylated cyclic ureides, e.g., iV-methylbarbiturates are demethylated. An example of this t y p e of reac­

tion is t h e conversion of mephobarbital t o phenobarbital (73,133b, 150).

A t h i r d t y p e of oxidation is t h e introduction of O H groups into aromatic rings, for instance, t h e formation of p-aminophenol from aniline.

I n drug catabolism, t h e oxidative reactions seem t o prevail. As far as ali­

phatic chains are concerned, oxidation runs most smoothly when straight chains are involved. Branching brings on difficulties, while tertiary-substituted carbon atoms form a nearly insurmountable barrier t o t h e oxidative process (108, 212). Secondary a n d t e r t i a r y alcohols or alcohols with t h e carbonyl group on a t e r t i a r y carbon a t o m are h a r d t o oxidize. They are usually conjugated t o glucuronides (see Table I). F u r t h e r examples are pinacol, trichloro- a n d tri- bromoethanol, a n d 2,2,3-trichlorobutanol. The analogous aldehydes t e n d t o be reduced instead of oxidized. Chloralhydrate a n d butylchloralhydrate are con­

verted t o trichloroethanol a n d 2,2,3-trichlorobutanol, respectively. Analogous, too, is t h e reduction of 1,3-dichloroacetone t o 1,3-dichloroisopropyl alcohol a n d t h e reduction of other ketones (212).

The conclusion m a y be t h a t , as a rule, " p a c k e d " alcohols are less vulnerable t o oxidative processes. I n regard t o this, it is interesting t o note t h a t m a n y of

* In accordance with the recommendations of the Commission on Enzymes of the International Union of Biochemistry, 1960, NAD (nicotinamide-adenine dinucleotide) has been substituted for DPN (diphosphopyridine nucleotide); NADH2, the reduced form, for DPNH; NADP (nicotinamide-adenine dinucleotide phosphate) for TPN (triphospho­

pyridine nucleotide; NADPH2, the reduced form, for TPNH.

56 Ε . J . A R I E N S AND A. M. SIMONIS

the so-called minor tranquilizers are alcohols of t h e t y p e just mentioned or their carbamates (see Table I I ) .

E t h y l alcohol also is a tranquilizer. I t is difficult, however, to maintain an effective ataractic level of this drug in body fluids. This can only be attained by frequent medication with small doses. If one tries t o cover a longer period with a single dose, t h e required concentration is exceeded a n d instead of being

T A B L E I

GLUCURONIDE CONJUGATION OF PRIMARY, SECONDARY, AND TERTIARY ALCOHOLS IN RABBITS"

Primary G.C.⁶ Secondary G.C.⁶ Tertiary G.C.*

alcohol (%) alcohol (%) alcohol (%)

—If:— OH 10 C—(ί—C C—C—OH 0.5 C—G—OH 10 C—C—OH 24

J — C — O H 14 C — C — C C—C—G—OH 0.9 C—C—G—OH 14 C—C—<J—OH 58

I

c

— O H 45 C — C — C — C C—C—C—C—OH 1.8 C—C—C—G—OH 45 C—C—C—C—OH 57

C—C—C—C —C —OH 6.7 C—C—C—C—G— OH 54 — c — c — c — C

a Selected from data presented by Williams (214).

b G.C. = Glucuronide conjugation.

tranquilized, t h e biological object becomes intoxicated. The more oxidation- resistant alcohols, which are also more fat-soluble t h a n ethyl alcohol, seem t o be more suitable in this tranquilizing ability.

For drugs with chains bearing carboxyl groups, blockade of β-oxidation, which can be realized by α-substitution, decreases t h e vulnerability of t h e drug to oxidation. Substitution on t h e α-carbon atom m a y be considered as a

" p a c k i n g " of t h e carboxyl group. A higher fraction of t h e acid then appears in t h e conjugated form in urine. Examples are trimethylacetic acid (61), α-ethylbutyric acid, and α-ethylhexanoic acid (109), benzoic acid, a-phenyl- acetic, and α-diphenylacetic acid (212). I n the case of phenylalkyl derivatives,

Ι , Ι . Β . DRUG TRANSFERENCE: DRUG METABOLISM 57 depending on an odd or even n u m b e r of carbon atoms in t h e chain, benzoic acid or phenylacetic acid will be t h e respective end-products.

For t h e amines, an analogous situation exists. While most aliphatic amines are deaminated oxidatively, heavily " p a c k e d " amines, as for instance mec- amylamine a n d pempidine, are excreted largely unchanged in t h e urine (4, 135,183a, 185,196,199) (see Table I I I ) .

TABLE II

"PACKING" OF THE CARBINOL GROUP IN VARIOUS MINOR TRANQUILIZERS

Ϊ

C I — C — C — O H

h

Trichlorethanol

Η

?

C

C—C—C—A—C—OH h

Meprobamate (carbarn.)

C — C — ^ — O H I

c

Amylenehydrate Phenaglycodol

— O H

C — C — £ — O H

Methylparafynol

III c

O H

Ethinamate (carbarn.)

0 —C \ C — ( J — O H

h III Br

c

Repocal

I n all t h e examples given, a steric hindrance of t h e enzymes concerned with drug catabolism is feasible. The principle of " p a c k i n g " m a y be tried as a means of preparing more stable drugs, a t least as far as oxidative breakdown is concerned. An objection is t h a t t h e steric hindrance m a y concern n o t only the catabolic enzymes, b u t also t h e specific receptors with which t h e drugs have t o interact in order t o produce their effect (193a). For certain biological actions, a " p a c k i n g " of t h e active group in t h e drug seems t o be of a d v a n t a g e . Examples are t h e blood-pressure-lowering a n d t h e ganglion-blocking amines represented in Table I I I (185,196,199) a n d t h e minor tranquilizers represented in Table I I .

58 Ε . J . ARIENS AND Α. Μ. SIMONIS

T A B L E I I I

"PACKING" OF THE AMINO GROUP IN VARIOUS HYPOTENSIVE DRUGS

c

c

Mecamylamine Dimecamine

C — Ν — C

c

4

Penbutamine Pempidine

I.B.1.2. Reduction

The organism makes use of reduction in some cases where t h e oxidative enzymes fail. The conversion of chloralhydrate and butylchloralhydrate into t h e corresponding alcohols are examples. Many ketones, especially cyclic ketones, are reduced, for instance, progesterone into pregnanediol (105). For some molecular configurations, reduction seems to be t h e only possible cata- bolic route, viz., for t h e azo configuration, e.g., t h e conversion of prontosil into sulfanilamide, and for t h e nitro groups, e.g., t h e conversion of nitrophenols into aminophenols.

Many esters foreign t o t h e body are hydrolyzed by t h e enzymes of blood plasma and cells. Where serum esterases are concerned, t h e corresponding amides are frequently more stable t h a n t h e esters. Procainamide is more stable t h a n procaine, for instance. Substitutions on t h e α-carbon atom of t h e alcohol and/or the acid portion of t h e ester, tend t o stabilize it against hydrolysis by plasma esterases (123). This is again an example of packing of t h e essential group (see Fig. 1). Here, too, a steric hindrance with respect t o t h e active site on t h e esterase is feasible.

The resistance of lidocaine (Xylocaine) and 2,6-dimethoxybenzoylcholine against enzymatic hydrolysis is a further example of t h e influence of packing of t h e vital points in drugs on their stability (124, 194).

Thomas and Stoker (194) studied t h e enzymatic (acetylcholinesterase) and nonenzymatic (OH ions) hydrolysis of various or^o-substituted benzoyl- cholines and t h e inhibition of t h e hydrolysis of acetylcholine by acetylcholin­

es.*.3. Hydrolysis

Ι , Ι . Β . DRUG T R A N S F E R E N C E ; DRUG METABOLISM 59 esterase b y means of these benzoylcholines. This s t u d y gives a good basis for t h e discussion of some factors t h a t play a role in t h e relation between t h e chemical structure a n d t h e r a t e of hydrolysis. The change in t h e nonenzymatic hydrolysis as a result of t h e various substitutions in benzoylcholine is due t o a change in t h e stability of t h e ester bond as such. I t m a y result from inductive effects a n d other effects leading t o a change in t h e intramolecular distribution of charges. The enzymatic hydrolysis is also dependent on t h e ester bond stability. Here a steric hindrance on t h e enzyme surface, especially with respect to t h e approach between t h e vital spot (the ester group in t h e drug) a n d its complement on t h e enzyme, a n d a change in t h e affinity between t h e drug a n d its receptors on t h e enzyme, have t o be t a k e n into consideration.

C ι C— C — O - C - C — N - C

II ι I

Ο A C

acetylcholine j ACh esterase

ι ι

c — c —o- c II I

ο

c I - N - C

I c

methacholinermore resistant to the esterase

H2N I

c- o —c

11 ι I

•C—Ν ' L Jj

c — c

c - c ι

rate of hydrolysis in human serum

R2 y / m l / h r

Η Η 500

Η CH3 15

CH3 CH3 0

FIG. 1. Influence of "packing" of the ester group on the stability of drugs.

F r o m Table IV.A it m a y be seen t h a t t h e nonenzymatic hydrolysis, t h e en­

zymatic hydrolysis, and t h e affinity of t h e benzoylcholines t o t h e enzyme acetylcholinesterase, as represented by anti-acetylcholinesterase activity, are not strictly correlated. This means t h a t each of t h e three processes depends on t h e structure in its own way. The decrease in t h e r a t e of hydrolysis does not necessarily lead t o a decrease in cholinergic a c t i v i t y ; this m a y even increase (194a, 194b).

Possibly also t h e resistance of certain penicillins against penicillinase is based on such factors as mentioned for t h e benzoylcholines. 2,6-Dime- thoxyphenylpenicillin is not only penicillinase-resistant b u t it is also found t o act as a competitive inhibitor of t h e B. cereus penicillinase (161). The bond in penicillin split by penicillinase is t h e lactam bond which, although situated closely t o t h e 2,6-substituted ring, is not directly adjacent t o it as in t h e case of t h e benzoylcholines mentioned. The bis-or^o-substituted derivative, 5-methyl-3-phenyl-4-isoxazolyl-penicillin is also a competitive inhibitor of

60 Ε. J . A R I E N S AND A. M. SIMONIS

penicillinase. Experiments of Citri etal. (48a, b, 75b) m a k e it probable t h a t as a result of the or^o-substitution in t h e side chain of penicillin t h e conformation of t h e active site on t h e penicillinase is changed in such a way t h a t t h e enzymatic hydrolysis cannot take place. I n general t h e character of t h e side-chain in t h e various penicillins, especially α-substitution, clearly influences the rate of hydrolysis by penicillinase (10a, 48a, 54a, 75b).

T A B L E I V . A

NONENZYMATIC HYDROLYSIS, ENZYMATIC HYDROLYSIS ( A C H ESTERASE), AND ANTI- A C H ESTERASE ACTIVITY IN VARIOUS Ortho-SUBSTITUTED BENZOYLCHOLINE COMPOUNDS'*

^ T J- O - O - C - C .

?

—Ν—C 1 c

Nonenzymatic

hydrolysis⁶ Enzymatic hydrolysis⁶

Anti-ACh esterase activity^c

2-H 1.00 1.00 1.00

2-CH3 0.65 0.24 3.10

2-C1 1.40 1.30 8.81

2-Br 1.02 0.70 4.20

2-J 0.74 0.24 14.50

2 - N 02 1.40 0.19 8.58

2-OCH3 0.77 0.73 6.64

2,6-diCl 0 0 6.26

2,6-diCH3 0 0 —

2,4,6-triBr 0 0 5.80

2,4,6-triN02 3.40 0 8.23

° The ratio enzymatic: nonenzymatic hydrolysis for benzoylcholine is 5.30.

After Thomas et al. (194)

b Relative rate; benzoylcholine = 1.00.

c Relative activity tested on horse serum cholinesterase; benzoylcholine = 1.00.

Interesting studies on t h e relations between t h e chemical structure of amides and t h e rate of hydrolysis by amidases from r a b b i t liver were performed by B r a y et al. (27). Some of their results are summarized in Table I V . B .

The decrease in t h e velocity of hydrolysis for compounds with higher values for n, m a y be ascribed t o a decrease in t h e concentration of free molecules.

This results from t h e increasing tendency of these compounds t o form micelles (91).

I n addition to esterases and amidases, glucosidases also t a k e p a r t in enzym­

atic hydrolysis.

I.B.1.4. Conjugation

I n conjugation, drug molecules are bound t o other molecules by elimination of water a n d formation of esters, amides, and other substitution products (26).

Ι , Ι . Β . DRUG T R A N S F E R E N C E : DRUG METABOLISM 61

C H3( C H2)NC O N H2 / \ _ ( C H2)N— C O N H2

η (%) (%)

0 2 4 4

1 3 1 7

2 1 3 1 0 5

3 5 5 5 8

4 9 0 2 6

5 9 0

6 6 3

7 3 8

8 1 8

9 8

1 0 8

1 1 3

1 2 8

1 3 2

1 4 3

a From Bray (27).

One or both reacting molecules t a k e p a r t in a n activated state. Usually, t h e active molecule is derived from metabolic processes of t h e organism. Well- known examples are *' active " acetate, sulfate, a n d glucuronic acid. Sometimes t h e drug or its metabolites, e.g., benzoic acid a n d phenylacetic acid, are acti­

vated, t h a t is, bound t o coenzyme A (CoA). The active products are usually formed b y mitochondrial intervention in t h e liver cells. A transferase accom­

plishes t h e transfer of t h e activated molecule t o t h e other members of t h e conjugation pair.

These transferases sometimes demonstrate a certain degree of specificity with respect t o t h e drug a n d are also located in t h e mitochondria of t h e liver ceUs (35, 60, 121a,b).

1. Conjugation with glucuronic acid occurs with alcohols resisting oxidation (demonstrated in Table I) a n d with phenolic OH-groups. Carboxyl groups, especially if t h e y are located on a n aromatic nucleus or close t o it, are in some cases converted t o glucuronides, e.g., benzoic acid, phenylacetic acid a n d diphenylacetic acid. T h e same holds t r u e for carboxyl groups with a heavy a-carbon-substitution. Take, for instance, trimethylacetic acid a n d t e r t i a r y butylacetic acid (61). " A c t i v e " glucuronic acid, uridine diphosphoglucuronic acid, is t h e source of glucuronic acid (Fig. 2) (60, 87). Some glucuronic acid

T A B L E I V . Β

PERCENTAGE OF AMIDES HYDROLYZED IN 5 H R AT P H 7 . 4 BY RABBIT-LIVER EXTRACT"

62 Ε. J . A R I E N S AND A. M. SIMONIS

transferases in liver microsomes effect t h e transfer under formation of ether-, ester-, and other glucuronides, depending on t h e substrate (60, 60a, 214).

2. Like glucuronic acid, amino acids are conjugated with acids. The foreign

COOH n N

Κ ° ° V Y

(oh Y — O — P — O — P — O — C ^ ^ O ^ ^ N ^ ^ HtT f OH OH

OH

HO OH uridine diphosphoglucuronic acid

OH CI

+ H O - C - C — C I I I

ci

trichloroethanol

COOH

Μ i¹ jfoH ) 2- 0 - C — c— CI + uridine-diphosphate

HO ( CI OH

trichloroethyl glucuronide

FIG. 2. Glucuronic acid conjugation of trichloroethanol.

acids, activated as acyl-CoA, react with t h e amino groups of an amino acid.

Mammals usually conjugate with glycine, b u t m a n a n d ape sometimes with glutamine. Examples are t h e formation of hippuric acid, phenaceturic acid,

Ο

// \\

Ο , V Ο

C - S — R + H?N — C — C O O H γ \ ) C~ N H — C — C O O H + H S — R

benzoyl-coenzyme A glycine hippuric acid coenzyme A FIG. 3. Glycocol conjugation of benzoic acid.

nicotinuric acid from t h e corresponding acids, benzoic acid, phenylacetic acid, and nicotinic acid (Fig. 3). Birds use ornithine instead of glycine; t h e y convert benzoic acid to ornithuric acid, which is t h e analog of hippuric acid. Certain spiders use glutamic acid and arginine for conjugation of aromatic acids (166a).

Ι,Ι.Β. DRUG TRANSFERENCE: DRUG METABOLISM 63

Ο Ο II II HO—S—Ο—Ρ—Ο—C

Ο OH I I

Ο OH HO—l>-OH

ο

adeno sine - 3' - phosphate - 5' - phospho sulfate

HO—S— Ο - γ y + a d e n o s i n e - 3 ' , 5'-diphosphate Ο

\=s

phenol phenolsulfate

FIG. 4. Sulfate conjugation of phenol.

CH3

u

ooc—c—c - C —

S —

COOH

HO OH

S-adenosyl methionine nicotinic acid

COOH

+ adenosine + homocysteine

trigonelline

FIG. 5. Methylation of nicotinic acid.

64 Ε . J . ARIENS AND Α. Μ. SIMONIS

3. Acetylation is another t y p e of conjugation. Foreign compounds bearing amino groups not suitable for oxidative deamination, mainly t h e aromatic amines, e.g., t h e anilides, are conjugated with acetic acid ( " a c t i v e " acetate) derived from acetyl-CoA. The acetylation of various sulfonamides m a y serve as an example (120a).

4. Phenolic hydroxyl groups m a y be conjugated with glucuronic acid b u t also with sulfuric acid, which results in t h e formation of ethereal sulfates.

" A c t i v e " sulfate, 3'-phosphoadenosine-5'-phosphosulfate serves as a source for sulfate (Fig. 4) (23,82b). A sulfuric acid transferase brings about t h e transfer to t h e foreign compound. The formation of paracresylsulfuric acid from para- cresol is an example.

/ \

iOOH Ο iodobenzol c y s t e i n e acetyl c o e n z y m e A

I ft \ \ — S — C — C — N H — C — C H3 + R—SH COOH Ο

/>-iodophenylmercapturic acid c o e n z y m e A FIG. 6. Cysteine conjugation of iodobenzol.

5. Methylation is another t y p e of bio-transformation. I t takes place with nitrogen in aromatic heterocyclic rings, for instance, t h e formation of iV¹- methylnicotinamide from nicotinamide (165). Phenolic OH-groups in t h e catecholamines, e.g., epinephrine (13,14, 82), and in steroids, e.g., estrone a n d estradiol, m a y be methylated t o m e t h o x y compounds (71). #-Adenosylmeth- ionine probably serves as a source for t h e methyl groups in this case (Fig. 5) (12, 16a).

6. Finally, mercapturic acid synthesis m a y be mentioned, whereby cysteine is substituted for hydrogen on aromatic rings, or for halogen atoms on aromatic rings or aliphatic chains (Fig. 6). This is of importance in t h e dehalogenation of foreign compounds, for example, halogenobenzene derivatives (18, 26b, 30).

I.B.1.5. Chemical Antagonism or Antagonism by Neutralization This t y p e of antagonism has something in common with conjugation. Here, too, t h e inactivation of a drug takes place as a result of its coupling with other molecules. This time t h e molecules are not of endogenous origin, as were glucuronic acid a n d acetic acid, nor are enzymes concerned with their binding

Ι,Ι.Β. DRUG TRANSFERENCE: DRUG METABOLISM 65 t o t h e drug. B o t h reacting compounds are drugs; one as a rule is a toxin, t h e other an antidote. The t y p e of reaction is often chelation. I n t h e paragraph concerned with chelation as a mechanism of t r a n s p o r t (Section I.A.1.3.C), various examples of this t y p e of bio-inactivation are mentioned: Ca++<-» cit­

r a t e , Hg++<->BAL,* Pb++<->EDTA,f Cu++<->penicillamine. Some more ex­

amples, curare<-> congo red, a n d curare<->germanin, will be mentioned in t h e following chapters. A q u a n t i t a t i v e theory on this t y p e of bio-inactivation is proposed by Gaddum (75) a n d Ariens (9). Because of t h e similarity of t h e dose- response curves for t h e competitive antagonism a n d for t h e chemical antagon­

ism, it will get special a t t e n t i o n in Section I I . B . 3 . I.B.1.6. Multiple Metabolic Pathways

Compounds foreign t o t h e body m a y be catabolized simultaneously along different roads.

1. One chemical group in t h e drug m a y be processed in different ways. Take, for instance, phenol. I t is p a r t l y converted t o its glucuronide a n d t o its sulfuric acid ether. Benzoic acid given orally t o h u m a n s , dogs, or pigs m a y be recovered from t h e urine p a r t l y conjugated with glycine t o hippuric acid a n d p a r t l y as t h e glucuronide (212).

2. A drug molecule m a y be a t t a c k e d a t different points. An example is

^-aminosalicylic acid. This compound contains 3 groups suitable for conjuga­

tion ; an amino group bound t o t h e ring; a carboxyl group bound t o t h e ring;

a n d a phenolic OH-group. After oral administration, a large fraction (42%) is excreted conjugated a t t h e carboxyl group with glycine, 2 9 % is found in t h e urine as an iV^-acetylated derivative, 2 5 % is excreted unchanged, while little if a n y of the drug is conjugated a t t h e phenolic OH-group (120).

N o t only conjugations b u t also different types of metabolic processes m a y t a k e place simultaneously. Salicylic acid is excreted in t h e urine, mainly conjugated with glycine a t t h e carboxyl group, as salicyluric acid ( 5 0 - 6 0 % ) ; a fraction is conjugated with glucuronic acid (20-25%). Two types are found, one probably conjugated in t h e carboxyl group, t h e other in t h e phenolic OH- group. A small fraction appears as oxidation products. One of these is gentisic acid (2,5-dihydroxybenzoic acid) (5, 84, 181). Also, for morphine and related compounds, a variety of metabolic changes occur in t h e body (121). Blockade of one metabolic p a t h w a y can lead to an increased metabolism of t h e drug along other routes (165a).

For more detailed and extensive information on drug metabolism t h e reader is referred t o William's well-known monograph (212, 214) a n d t o various review articles (15, 26a, 34, 35, 68, 68a, 78b, 121,122, 138a,b, 164,189, 208). A valuable discussion of t h e kinetics of metabolism of foreign organic compounds is given by Teorell (193) a n d by B r a y (28).

* British Anti-Lewisite.

t Ethylenediaminetetraacetic acid.

66 Ε . J . A R I E N S AND Α. Μ. SIMONIS

I.B.2. FACTORS I N F L U E N C I N G D R U G METABOLISM The relation between t h e dose of a drug and t h e effective concentration of t h e active drug is t h e resultant of a n u m b e r of detail processes. The various catabolic routes for a drug can be considered as parallel flow systems. The drug will be divided among these systems according to t h e capacity of each of t h e m . The relative capacity of t h e various catabolic systems for a drug, a n d therefore t h e fraction of t h e drug processed by each of t h e m , depends on various factors, as for instance: (a) t h e chemical properties of t h e d r u g ; (b) t h e dose; (c) the route of administration; (d) t h e diet a n d drugs given t o t h e a n i m a l ; (e) t h e animal's species; (/) its sex; a n d (g) its individual variations.

I.B.2.1. Chemical Properties

The way in which a certain group in a compound, e.g., a phenolic OH-group, is processed varies with t h e place of such a group in t h e molecule. Table V gives

T A B L E V

INFLUENCE OF RING SUBSTITUTIONS ON METABOLISM OF HYDROXYBENZALDEHYDE AS S E E N BY THE EXCRETION PRODUCTS IN URINE OF RABBITS"

% of dose* excreted as:

Ether-soluble Ester Ether Ethereal acid glucuronide glucuronide sulfate

2-Hydroxybenzaldehyde 75 18 9 3 3-Hydroxybenzaldehyde 75 9 9 7 4-Hydroxybenzaldehyde 67 4 16 9

* From Bray (28).

b Dose level 0.4 gm/kg.

examples of t h e influence of t h e substitution of OH-groups on t h e ortho, meta or para place on t h e t y p e of conjugation of hydroxybenzaldehyde in t h e r a b b i t (28). Table V I demonstrates t h e influence of ortho-, meta- a n d para-chloro- substitution in acetanilide on metabolism of t h e drug in r a t s (141).

Differences in charge distribution on the drug molecules often play a role in drug metabolism (95a). I t has been observed, for instance, t h a t t h e r a t e of acetylation of amines is related to the electronic charge on the amino nitrogen (if not protonated). The rate of deacetylation of iV^-acetyl amines is reported as correlating with the dipositivity (the positive charge on the nitrogen atom and on the carbonyl carbon atom) of the iV-acetyl bond (146c).

Differences in t h e biological activity of t h e stereoisomers of a compound strongly suggest t h e implication of drug-receptor interactions. Such a stereo-

Ι , Ι . Β . DRUG T R A N S F E R E N C E : DRUG METABOLISM 67 specificity m a y have its origin not only in t h e interaction of t h e drug with t h e receptors essential t o t h e effect studied, b u t in t h e processes concerned with t h e drug transference as well. Serum esterases only hydrolyze t h e L( + ) isomer of acetyl-jS-methylcholine (79). After a dose of racemic mepacrine, an optically active mepacrine appears in t h e urine (85). The antipodes in d^glutethimide are degraded in different ways (42b, 113m). Certain permeases, specific penetra­

tion systems found in bacteria, exhibit stereo-specificity (50). The same m a y be t r u e for t h e displacement of drugs from silent receptors (19).

T A B L E V I

INFLUENCE OF RING SUBSTITUTION ON METABOLISM OF CHLOROACETANILIDES AS SEEN IN THE EXCRETION PRODUCTS IN THE U R I N E OF RATS AFTER INJECTION OF 2-, 3-,

AND 4-CHLOROACETANILIDE ( C l^{8 e})^a

Products

-C—CH8

ο

2-chloroacetanilide 3-ehloroacetanilide 4-chloroacetanilide (%) (%) (%)

4-OH-Derivatives⁶ 45.10 57.32 —

2-OH-Derivatives* — — 61.96 6-OH-Derivatives* 8.53 26.46 — Deacetylation 28.28 8.26 2.52 Unchanged 3.18 1.34 4.63 Chloride ions 0.62 1.98 1.18

"From Newell (141).

b After acid hydrolysis of the glucuronides, ethereal sulfates, etc.

The relatively high, in vivo amine-oxidase-inhibiting activity of isopropyl- hydrazine with respect t o n a t u r a l amino acids, as compared with t h e u n n a t u r a l , is t h o u g h t t o be related t o specific t r a n s p o r t mechanisms n o t present in in vitro experiments. I n t h e latter, t h e differences in amine-oxidase-inhibiting activity toward n a t u r a l a n d u n n a t u r a l compounds are m u c h smaller (15).

The d-isomers of various amino-acid derivatives inhibit, in a competitive way, t h e hydrolysis of t h e Z-isomers b y a-chymotrypsine (208). The d-isomer of a-phenoxyethyl phenylpenicillin inhibits t h e hydrolysis of t h e Z-isomer by staphylococcal penicillinase (186). The breakdown of Z-histidine b y liver histi- dase is inhibited by d-histidine (65). There is a competition between t h e enan- tiomorphs of methionine during intestinal absorption (9p, 100).

I n t h e isomer which is not or only slowly metabolized, one of t h e groups concerned m a y have a wrong orientation with respect to t h e active site on t h e enzyme. A group essential for the reaction m a y point in t h e wrong direction, a

68 Ε. J . ARIENS AND Α. Μ. SIMONIS

group not essential for the reaction m a y cause a steric hindrance, etc. As mentioned before, a " p a c k i n g " around the vital group in a drug molecule like α-methyl substitution and or£/io-substitution often strongly influences drug metabolism (see Table I, Fig. 1, a n d Table IV.A). The cause of this m a y be steric hindrance; however, in oxidative processes the absence of α-Η atoms which possibly play an essential role in the process m a y be the cause as well (20).

Most catabolic changes of drugs, especially oxidations, reductions, and con­

jugations, t a k e place intracellularly. Many types of hydrolysis occur mainly in t h e plasma. Penetration of t h e cell and t h e functional units of t h e cell, microsomes and mitochondria, is essential for m a n y metabolic processes. This means t h a t a certain degree of lipid solubility will be an advantage t o these processes. Take, for instance, t h e compounds chlorothiazide and flumethiazide as compared t o t h e more lipophilic hydrogenated compounds, dihydrochloro- thiazide and dihydroflumethiazide. These lipophilic compounds are absorbed more completely from the g u t ; t h e volume of distribution is larger for dihydro- chlorothiazide t h a n for chlorothiazide. The " d i h y d r o " compounds are reab­

sorbed in t h e tubules and, therefore, are excreted less rapidly in t h e urine.

Chlorothiazide is excreted in urine completely unchanged, while t h e " d i h y d r o "

compounds are excreted partly metabolized (132).

Many strong bases, e.g., a, ω-bis-trimethylammonium compounds (hexa- methonium, decamethonium, etc.) a n d strong acids, e.g., sulfonic acids, ethereal sulfates, and phthalic acid are slowly absorbed from t h e gut, while t h e fraction reabsorbed in t h e tubules is small. These drugs are excreted mainly unchanged in t h e urine.

/.β.2.2. Effect of Dosage

The fraction of t h e dose of a drug subjected t o bio-transformation by a certain metabolic route m a y v a r y with t h e dose. I n h u m a n s when a dose of 400 mg andosterone is given orally, 4 . 4 % is recovered as t h e sulfate, a n d 4 8 % as t h e glucuronide. When a dose of 4000 mg is given, t h e recoveries are 2 1 % and 47 %, respectively (167). An increase in t h e doses of phenol given t o rabbits results in a decrease in the ratio sulfate conjugation .glucuronide conjugation.

This is probably because t h e sources of sulfate are restricted (29).

At a certain dosage the optimal capacity of a metabolic system m a y be reached. A further increase of t h e dosage will lead t h e n t o a switch t o other systems.

I t m a y be expected when combinations of drugs are given which are proc­

essed by t h e same metabolic system, t h a t a m u t u a l interference will occur on t h e basis of competition for active sites on enzymes, etc. p-Aminobenzoic acid is found t o compete with salicylic acid and with benzoic acid for t h e glycine conjugation system (181).

Drugs can compete for common metabolic systems. Probenecid reduces

Ι , Ι . Β . DRUG T R A N S F E R E N C E : DRUG METABOLISM 69 in vitro conjugation with glycine of p-aminobenzoic acid (PABA). The reduc­

tion in excretion of t h e PABA-glycine conjugate by probenecid m a y be p a r t l y due to t h e interference with t h e conjugation (20a).

I.B.2.3. Route of Application

This will be influential on drug metabolism for drugs which are rapidly metabolized in t h e liver, especially if oral a n d parenteral administration are compared. The liver plays an i m p o r t a n t role in drug metabolism. Examples of differences in drug metabolism dependent on t h e route of application have been described for ^-aminosalicylic acid (134), for cortisone a n d Prednisone (149), a n d for p a r a t h i o n a n d paraoxon (93). If drug metabolism in t h e liver leads t o a bio-inactivation, t h e parenteral application will be t h e most effective one. If bio-activation takes place in t h e liver, t h e oral route m a y be preferable (Section I.B.3.2).

Ι.Β.2Λ. Diet and Drugs

The fractions of benzoic acid excreted as hippuric acid, a n d as benzoylglu- curonide by t h e pig, vary with t h e supply of glycine in t h e diet. W i t h a protein- free diet, 6 0 % of t h e excreted benzoic acid is in t h e hippuric acid form; t h e addition of gelatin, rich in glycine, increases t h e hippuric acid fraction t o 8 9 % (53).

Animals on diets poor in sulfur-containing compounds, have difficulty in t h e bio-transformation of materials usually excreted as mercaptans. The ad­

dition of cysteine, cystine, or methionine leads t o an increased excretion of mercapturic acid conjugates. Sulfate conjugation of phenols given in high doses is increased by feeding t h e animals sulfate precursors like I-cystine a n d sodium sulfite. Glucuronide conjugation is t h e n decreased (29). A counterpart of these dietary requirements is t h e induction of a deficiency in cysteine as a result of t h e administration of high doses of compounds t h a t require cysteine for their conjugation. An instance of this process is demonstrated when bromo- benzene is given t o young animals on diets poor in t h e sulfur-containing amino acids (187,188).

Injection of glycine or glucuronic acid strongly decreases t h e toxicity of salicylic acid in dogs; t h e excretion of salicylglucuronic acid increases (78).

Administration of glucose, fructose, or glucuronolactone can accelerate glucuronide formation a n d excretion (18a, 94a).

Adaptive responses of t h e body t o drugs by increasing t h e metabolizing enzymes are common (114c, 134a). Such an a d a p t a t i o n is " crossed" for drugs processed along t h e same metabolic way. P r e t r e a t m e n t of r a t s with pheno- barbital increases t h e liver activity with respect to t h e metabolism of hexo- barbital and other drugs. A decrease in duration of anesthesia for barbiturates like hexobarbital is t h e result (50a, 113b, 115a, 118,158,158b). This is found to be due t o an increased inactivation of t h e drug.

70 Ε. J . ARIENS AND Α. Μ. SIMONIS

Changes in drug metabolism induced by p r e t r e a t m e n t with drugs can cause a tolerance for other drugs (113d, e,f, g,j, k, 114b, 158a, c). This will be the case if the metabolic process results in a bio-inactivation of these drugs. If, however, a bio-activation takes place, the p r e t r e a t m e n t m a y result in an increased sensitivity (see Sections LB.3.1 and 3.2.) As a m a t t e r of fact, application of drugs can lead to a change in metabolism of endogenous compounds, too, e.g., steroid hormones (76a; see also 21 and 156).

An adaptive increase of drug metabolism induced by drugs such as pheno­

barbetal and methylcholanthrene can be suppressed by inhibitors of protein synthesis like ethionine (50b, 113h).

Administration of various drugs stimulates t h e conversion of glucose t o glucuronic acid. This is often associated with increased excretion of ascorbic acid. This phenomenon is absent in t h e guinea pig, monkey, and m a n , species which lack t h e ability to convert glucuronic acid to ascorbic acid (42).

I n r a t s t h e increased excretions of ascorbic acid after drugs like Chloretone is attended with an increase in t h e concentration of lactonase (substrate D-glucuronolactone) in liver microsomes (113c).

Induction of enzyme synthesis by substrates is a common phenomenon in biochemistry. N o t only drugs, b u t also biological substrates are known to lead to adaptive increase in enzyme production. An interesting aspect is the specifi­

city of the induction (208a). I t appears t h a t the inducer does not necessarily have to be a substrate for the enzyme whose production is induced. For the induction of ^-glucuronidase, it is found t h a t bad substrates m a y be good inducers a n d t h a t good substrates m a y nevertheless lack t h e capacity of induction (84a). Enzyme-inducing drugs have often also a direct inhibiting action on t h e enzymes concerned. B u t there is no direct relation between the two properties (113g).

/.β.2.5. Species Effect

There are wide variations between species in t h e metabolism of substances.

Knoefel et al. (115) made extensive studies of t h e metabolic changes, especially t h e conjugation with glycine and glucuronic acid of ο-, πι-, and p-aminobenzoic acid salts. Table V I I summarizes some of their results for species differences in renal clearance. The hippuric acids have a high, t h e glucuronides a lower, clearance in t h e rabbit. The latter results in a larger circulating fraction of t h e glucuronide formed.

Chemical analogs of nicotinic acid, e.g., 3-acetylpyridine, are known t o act as antimetabolites. I n certain enzyme reactions, however, these analogs can substitute for nicotinic acid in a functional way. N A D , substituted with 3-acetylpyridine or thionicotinic acid amide is effective in t h e case of lactic acid dehydrogenase of various sources. The efficacy varies with t h e animal species and t h e organ used as a source for t h e enzyme (42a, 110, 111).

Acetylation of p-aminobenzoic acid, sulfonamides, a n d other aromatic

Ι , Ι . Β . DRUG TRANSFERENCE: DRUG METABOLISM 71 amines, occurs in most mammals with t h e exception of t h e dog. This animal excretes t h e drugs with t h e amino groups unchanged. A m p h e t a m i n e is deamin­

ated by t h e rabbit, while in t h e dog a n d r a t , ring hydroxylation is t h e principle metabolic route (11,12). The well-known a n t i r h e u m a t i c drug, phenylbutazone, is metabolized very rapidly in t h e mouse, r a b b i t , a n d dog. Therefore, large doses are required in these animals in order t o demonstrate anti-inflammatory

T A B L E V I I

CONJUGATION OF AMINOBENZOATES AND EXCRETION OF THEIR CONJUGATED DERIVATIVES IN RABBITS AND DOGS°

Conjugated products of aminobenzoate (/xM/kg/4.5 hr)

Ortho Meta Para

Hipp* Gluc^c Hipp Glue Hipp Glue

Rabbit

Excreted in urine 108 84 93 21 32 16

Excreted in bile 3 0.03 1 0 1 0

Circulated in body 0 24 0 34 6 0

Total conjugated 111 108 94 55 39 16

% of the dose 25 24 21 12 9 4

)og

Excreted in urine 23 140 119 53 10 36

Excreted in bile 0 10 0.1 1 0.03 1

Circulated in body 0 21 4 4 0 117

Total conjugated 23 171 124 58 10 154

% of the dose 5 38 28 13 2 34

° From Knoefel (115).

b Hippuric acid product.

c Glucuronide product.

effects. I n m a n , t h e compound is metabolized slowly a n d t h e anti-inflammatory action is demonstrable with much lower doses. Procaine is readily hydrolyzed b y m a n b u t appears unchanged in t h e urine of horses (106). On t h e other hand, procaine is reported to inhibit succinylcholine hydrolysis. This probably occurs as a result of a substrate competition for t h e esterase concerned (164a).

Often t h e differences are n o t qualitative b u t q u a n t i t a t i v e . An interesting study on comparative drug metabolism has been m a d e b y Sheppard et al. (176).

They studied t h e degradation of reserpine labeled with C^{1 4} in t h e 4-methoxy carbon position of t h e trimethoxybenzoic acid moiety of t h e drug, b y slices of the liver of various animals, under aerobic a n d anaerobic conditions. Among

72 Ε . J . ARIENS AND A. M. SIMONIS

the degradation products studied were reserpine-like substances, trimethoxy- benzoic-acid-like substances, and C 02. The results, summarized in Table V I I I , demonstrate t h a t O-demethylation (the production of C 02) occurs especially in t h e rat, mouse, and dog. The guinea pig has a remarkable hydrolytic capacity (the formation of trimethoxybenzoic-acid-like substances). The mouse also has such properties. The sleeping time of various species following t r e a t m e n t with hexobarbital varies greatly. I t is about proportional t o t h e half-life of t h e

T A B L E V I I I

DEGRADATION OF RESERPINE C¹⁴ BY THE LIVER OF VARIOUS ANIMALS"

No. of Gas Total

Species animals phase A^b B^C recovery

Pigeon 5

o

1 N2 0 86.0 1.28 87.3

Dog 5

o

1 N2 0 96.2 0.34 96.5

Rabbit 5

o

1 N2 0 93.8 10.3 104.1

Mouse 5

o

Rat 5 02 ² 19.7 58.1 1.37 79.27

1 N2 1.3 92.8 2.09 96.25

Guinea pig 5 o2 2.67 3.19 80.6 87.97

1 N2 3.08 2.25 91.8 97.75

a Liver sections (500 mg) incubated with reserpine-C¹⁴ in Krebs-Ringer bicarbonate buffer at 37° C for 3 hr.

From Sheppard (176).

b Fraction A : C 02

c Fraction Β: reserpine-like substances

d Fraction C: trimethoxybenzoic acid-like substances.

drug in these species. "Half-life" is t h e t i m e necessary t o reduce t h e concen­

tration of t h e compound in plasma t o 5 0 % of its maximal concentration. The half-life is found t o be inversely proportional t o t h e activity of those enzymes in t h e liver-cell microsomes t h a t bring a b o u t inactivation (34, 35, 37, 38a).

H e a r t glucosides are degraded a t different rates in various species. These differences correlate well with t h e differences in duration of action in these species (158d).

Even in one species, variations of drug metabolism are found. I n a number of cases this appears t o be genetically determined. Certain strains of r a b b i t s have enzymes in their blood t h a t can rapidly hydrolyze t h e alkaloid atropine.

This property is probably inherited as a partially dominant factor (205, 213).

Ι , Ι . Β . DRUG T R A N S F E R E N C E : DRUG METABOLISM 73

Relative Sleeping time Plasma level enzyme activity'

Sex (min) at 60 min (μ%)

Male 22 23 682 Estradiol-treated male 84 62 177 Female 90 65 134 Testosterone-treated female 38 37 543

a From Brodie (34).

b /xg of drug metabolized by liver microsomes per hour under standard conditions.

one longer branched side-chain (92, 131). The difference in sleeping time is reflected by a more rapid disappearance of t h e drug in t h e males. P r e t r e a t m e n t of t h e males with estrogens eliminates t h i s difference between t h e sexes (35,37).

Castration of t h e males increases their sensitivity to b a r b i t u r a t e action; in­

jection of testosterone decreases it again (34,92,113,113a, 131) (see Table I X ) . Interesting in this respect is t h e prolonged action of hexobarbital after treat­

m e n t with various malonic acid derivatives (Sch-5712 a n d -5715) (Fig. 14) which inhibit b a r b i t u r a t e metabolism (118). The difference between males a n d females is intriguing in this respect. I n t h e males, t h e sleeping time with a given dose of hexobarbital is increased 2 - 3 times after Sch-5712 b u t in t h e females, 10-15 times. Castration of t h e males a n d females vitiates this difference; t h e Great differences in drug metabolism are reported for certain inbred strains of r a t s (154a). Another well-known example is t h e inability of t h e Dalmatian dog t o reabsorb uric acid in t h e kidney tubules, while other dogs reabsorb it t o a great extent. The result t h e n is t h a t most dogs excrete allantoin as t h e meta­

bolic end-product while the Dalmation excretes uric acid (205). (For reviews on pharmacogenetics, see Kalow (106a,b,c) a n d Clark (48c).

N o t only does drug metabolism as such v a r y for various species, b u t also drug-induced a d a p t i v e changes in this metabolism vary. According t o H a r t et al. (86b) adult rabbits, unlike r a t s , have drug-metabolizing hepatic enzymes which can be strongly stimulated b y phenobarbital.

I.B.2.6. Effect of Sex Differences

Not only differences in species, b u t also differences in sex affect drug meta­

bolism. The sleeping time induced by b a r b i t u r a t e s is much shorter in male t h a n in female rats. This is especially t r u e for t h e barbiturates with one shorter a n d

T A B L E I X

SEX DIFFERENCES IN DURATION OF ACTION AND IN METABOLISM OF HEXOBARBITAL IN RATS'*

74 Ε . J. ARIENS AND A. M. SIMONIS

males become more, t h e females less, sensitive. P r e t r e a t m e n t of t h e males with estradiol increases, and p r e t r e a t m e n t of t h e females with testosterone de­

creases, t h e prolongation of t h e hexobarbital sleeping time produced by Sch- 5712. The difference in sensitivity t o Sch-5712 is clear in vivo b u t absent in liver homogenates.

I n certain strains of mice, t h e males are much more sensitive t o t h e nephro­

toxicity of chloroform t h a n t h e females. Castration of male mice abolishes this difference (207).

For male r a t s procaine toxicity is smaller t h a n for females (60 days old). A corresponding sex difference was found in procaine esterase activity in liver homogenates. This is larger in males. Castration of t h e males eliminates t h e difference, it is not restored by testosterone. I n younger animals, too, there was a sex difference in procaine esterase activity, b u t no difference in procaine sensitivity (135a). Sex differences for toxicity, etc., m a y vary for various species. The antibiotic acetoxycycloheximide is more toxic in female r a t s and mice t h a n in males. I n dogs no sex differences in toxicity occur (145b).

Sex and species differences are observed for drug-induced a d a p t a t i o n s in enzyme activity, too. Glucuronide formation from the substrate ortho- aminophenol is greater in male t h a n in female r a t liver microsomes. I n male r a t s estradiol decreases, in female r a t s testosterone increases glucuronide transferase activity (95b).

I.B.2.7. Individual Variations

Besides t h e biological variations, there are particular individual variations in drug metabolism. I n newborns t h e enzyme systems for drug metabolism are not fully developed yet (63a). Conjugations with glucuronic acid are im­

paired (202, 203). This results in a relatively high toxicity of certain drugs, such as chloramphenicol, progesterone, and iV-acetyl-^-aminophenol. Prob­

ably, neonatal bilirubinemia is also due t o insufficient conjugation with glucuronic acid (63, 209). Also, acetylation of sulfanilamides is impaired (67).

Jondorf (101) proved t h a t t h e newborn mouse and guinea pig show an insuffi­

ciency in glucuronide conjugation as well as in a number of oxidative degra­

dations of drugs. Fouts (215) found t h a t t h e newborn r a b b i t is unable to metabolize drugs such as hexobarbital, amphetamine, acetanilide and p-nitrobenzene.

An adaptive increase of hepatic microsomal drug metabolism in fetal a n d newborn rabbits can be induced by injecting the pregnant animal or t h e new­

born animal with phenobarbital. No increase was observed in fetuses t a k e n from the pre treated doe for 4-8 days prior to term. Concurrent application of ethionine blocks the increase in enzyme activity (86b). The absence of drug metabolism in newborn animals is due possibly to t h e absence of inducing stimulants as a result of the relatively protected situation in utero. The lack of drug metabolism in early fetal life can be ascribed to such factors as a lack of the

Ι , Ι . Β . DRUG T R A N S F E R E N C E : DRUG METABOLISM 75

systems for enzyme synthesis or a lack of t h e mechanism necessary for the induction of t h e enzyme synthesis by t h e drug (86b, 114c).

I n this respect m a y be mentioned t h e extreme differences in sensitivity to drugs, found for maternal a n d fetal tissues. P o t e n t teratogens, such as folic acid antagonists a n d other antivitamins, are often relatively harmless to t h e mother. I n fetal tissue t h e need for vitamins is probably relatively high (207b, 216a). The teratogenic action (66b) of t h e drug thalidomide is supposed to be due to the formation of metabolites with an a n t i vitamin activity.

Occasionally, individual hypersensitivity, an idiosyncracy, for a particular drug is observed, which is due t o a deficiency in t h e formation of degradative enzymes. Such deficiencies m a y be caused b y liver damage (70a). Sometimes t h e y appear t o be genetically determined: '' inborn errors of m e t a b o l i s m ' ' (48c, 66a, 106b). The excessively prolonged action of succinylcholine, observed in some individuals, is correlated with a low serum-cholinesterase activity (77a, 86a), which is probably genetically determined.

Chronic administration of drugs m a y lead t o changes in t h e metabolism of t h e drug concerned. During tolerance developed against narcotics like mor­

phine, jV-demethylation of morphine a n d other narcotic drugs is strongly reduced (15). This might suggest t h a t iV^-demethylation leads t o a bio-activa­

tion of t h e drug a n d t h a t tolerance is t h e result of an impairment of it. The nor compounds, however, have little or no analgesic or narcotic activity. Although there is a correlation between tolerance a n d t h e decrease in i^-demethylation, this relation is n o t causal (121).

I.B.3. G E N E R A L ASPECTS OF D R U G METABOLISM

Summarizing, it m a y be concluded t h a t :

1. A certain lipid-solubility is necessary for a drug in order t o reach t h e intracellular enzyme systems for drug metabolism.

2. As a rule, chemical changes in a drug molecule will t a k e place on t h e sites of great reactivity. Oxidative processes, for instance, will t a k e place a t spots where alcoholic hydroxyl groups or amino groups are present, especially if t h e y are situated on a terminal carbon of alkyl chains. For reductions, hydrolysis, a n d conjugations, analogous conditions are required for t h e appropriate reactions.

3. Oxidations prevail; reductions t a k e over where oxidations fail. Hydroly­

sis is common for esters a n d glucosides; amides, as a rule, are more resistant.

4. The oxidation and reduction processes change t h e molecule. New polar groups are introduced which, as a rule, are suitable for conjugation. Hydrolysis liberates polar groups suitable for conjugation or for oxidation followed b y conjugation.

5. Conjugation is, as a rule, a final step in drug metabolism.

76 Ε. J . ARIENS AND A. M. SIMONIS

6. Oxidation often results in a decrease of the number of atoms in the mole­

cule and consequently in a reduction in size. Metabolic processes generally increase t h e hydrophilic character of t h e drug molecule.

7. Packing of t h e " v i t a l " groups by adjacent methyl substituents, as a rule, protects such groups against metabolic a t t a c k .

8. Reactive spots in a drug molecule are often essential t o its pharmaco­

logical properties. The specificity of action is closely related t o t h e distribution in t h e molecule of spots with a high or low electron density and their spatial relations. Consequently metabolic changes in t h e drug molecule will frequently result in a change in its pharmacological properties, not only in a quantitative, b u t also in a qualitative sense.

Drug metabolism, especially oxidations, reductions, and hydrolyses, may lead to a bio-activation as well as to a bio-inactivation. As most of t h e studies on drug metabolism are done with pharmacologically active compounds, inacti- vation studies dominate the literature. A closer study of t h e metabolism of pharmacologically active compounds reveals t h a t a number of t h e m are only active because t h e y are converted in t h e body into active compounds (bio- activation) .

I.B.3.1. Bio-Inactivation and Detoxication

The oxidation of fat-soluble compounds will often result in t h e formation of acids with a decreased number of atoms. Smaller, more polar molecules are formed. I t is evident t h a t an increase in water-solubility will usually be t h e result of t h i s t y p e of bio-transformation; it effects a conversion of lipophilic compounds into more hydrophilic ones. The formation of carboxylic acids results, as a rule, in a loss of action. Among t h e m a n y drugs in use, such acids are relatively scarce. Ammonium compounds and amines are much more frequent.

The hydrolysis of various drugs also results in more water-soluble products.

The acids and alcohols derived from esters, and t h e acids a n d amines derived from t h e amides, are generally more water-soluble t h a n t h e esters and amides themselves. They become more easily metabolized to products t h a t are still more water-soluble. A loss of activity is t h e rule. Acetylcholine, procaine, suxamethonium, etc., are examples.

Deamination of a drug, too, as a rule, brings a b o u t bio-inactivation. Hista­

mine, mescaline, dopamine, etc., are examples.

I n cases of reduction, especially if nitro groups are converted t o amines, hydrophilic properties also increase. The same is true for m a n y conjugations, especially coupling with glucuronic acid, sulfuric acid, and amino acids, which often leads to products with a greater polarity and, t h u s , to a better solubility in water t h a n t h e original compounds. An exception to this rule is t h e acetyl- ation of aromatic amines, for instance, t h e production of acetanilides. Here, t h e polarity of t h e compounds decreases as a result of t h e coupling. Conjugation