ToxicoLoGxc PATHOLOGY ISSN:0192-6233
`Copyright © 1995 by the Society of Toxicologic Pathologists
`
`Volume 23, Number 2, 1995
`Printed in U.S.A.
`
`An Introduction to Drug Disposition: The Basic
`Principles of Absorption, Distribution,
`Metabolism, and Excretion*
`
`JOHN CALDWELL, IAIN GARDNER, AND NICOLA SWALES
`
`Department of Pharmacology and Toxicology, St. Mary’s Hospital Medical School,
`Imperial College of Science, Technology and Medicine, London W2 1 PG, United Kingdom
`
`ABSTRACT
`
`A knowledge of the fate of a drug, its disposition (absorption, distribution, metabolism, and excretion,
`known by the acronym ADME) and pharmacokinetics (the mathematical description of the rates of these
`processes and of concentration-time relationships), plays a central role throughout pharmaceutical research
`and development. These studies aid in the discovery and selection of new chemical entities, support safety
`assessment, and are critical in defining conditions for safe and effective use in patients. ADME studies provide
`the only basis for critical judgments from situations where the behavior of the drug is understood to those
`where it is unknown: this is most important in bridging from animal studies to the human situation. This
`presentation is intended to provide an introductory overview of the life cycle of a drug in the animal body
`and indicates the significance of such information for a full understanding of mechanisms of action and
`toxicity.
`Keywords.
`
`Xenobiotics; human and animal exposure; predictive value
`
`INTRODUCTION
`
`Humans and other animals are exposed on a daily
`basis to many xenobiotics, that is, compounds that
`are foreign to the normal energy-yielding metabo-
`lism of the body. Exposure to these xenobiotics may
`occur deliberately, as in the case of drugs and food
`additives; accidentally, as in the case of food con-
`taminants and pesticides, or coincidentally, as in
`the case of industrial chemicals and environmental
`
`pollutants. In this paper, the terms drug, xenobiotic,
`and foreign compound will be used interchangeably.
`In the present context, the importance of ADME
`(absorption, distribution, metabolism, and excre-
`tion) principles in drug development will be em-
`phasized, but it should be appreciated that these
`have comparable applicability in the safety assess-
`ment of all types of chemicals to which humans
`might be exposed.
`To achieve its effect, whether therapeutic or toxic,
`a drug and/or its metabolites must be present in
`appropriate concentrations at its sites of action. The
`
`* Address correspondence to: Professor J. Caldwell, Depart-
`ment ofPharmacology and Toxicology. St. Mary’s Hospital Med-
`ical School, Imperial College of Science, Technology and Med-
`icine. Norfolk Place. London W2 IPG, United Kingdom.
`
`concentration of xenobiotic attained will depend on
`the dose, formulation, and route of administration,
`the rate and extent of absorption, its distribution
`through the body and binding to tissues, biotrans-
`formation, and excretion. It is the purpose of this
`presentation to give an overview of these processes
`and to comment upon the factors influencing them
`and their biological significance.
`
`ABSORPTION
`
`The processes of absorption are those that lead to
`the entry of a xenobiotic into the systemic circula-
`tion of the body. The most important site of ab-
`sorption is the gastrointestinal tract, although ab-
`sorption through the skin, the main barrier between
`the internal milieu and the external environment,
`and the respiratory tract, which is important for
`volatile compounds and materials present in aero-
`sols and dust particles, can also occur. Regardless
`of the site of absorption, xenobiotics must cross cell
`membranes to enter the systemic circulation. Mech-
`anistically this can occur in 1 of 2 ways (4). Small,
`lipophilic compounds can cross the cell membrane
`by passive diffusion along a concentration gradient.
`This transfer is directly proportional to the mag-
`nitude of the concentration gradient across the
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`BASIC PRINCIPLES OF ADME
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`103
`
`membrane and the lipid : water partition coefficient
`of the drug (3). Large. highly polar or charged xe-
`nobiotics cannot cross the cell membranes by simple
`diffusion and. hence. are dependent on the presence
`of active carrier-mediated transport mechanisms.
`
`The Efleet of pH and pKa on Absorption
`from the Gastrointestinal Tract
`
`Many xenobiotics are weak acids or bases and are
`thus present in solution in both non-ionized and
`ionized forms. The non-ionized molecules tend to
`
`be lipid-soluble and cross membranes by passive
`diffusion. whereas the ionized forms have low lipid
`solubility and cannot cross the cell membrane (3).
`The partition of weak electrolytes across mem-
`branes will thus be a function of the pKa of the
`xenobiotic and the pH gradient across the mem-
`brane.
`
`The low pH in the stomach favors absorption of
`weak acids. Weak bases are ionized and, thus, gen-
`erally not absorbed from the stomach. In the intes-
`tine, absorption is rapid for weak acids (pK > 3) or
`weak bases (pK < 7.8). The longer transit time and
`increased surface area of the intestine mean that,
`for the majority of drugs, intestinal absorption is
`quantitatively more important even if it would be
`predicted to be less favorable on pH grounds (7).
`
`First—Pass Elimination
`
`Following absorption, drugs can be metabolized
`in the gut wall, prior to being transported to the liver
`via the hepatic portal vein (78). The hepatocytes of
`the liver are the major site of metabolism for the
`majority of drugs, and compounds can be exten-
`sively metabolized in the liver before reaching the
`systemic circulation. That portion of the dose that
`is absorbed from the lumen of the gastrointestinal
`tract but eliminated by metabolism in the gut wall
`and/or the liver on the way to the heart is said to
`have undergone f1rst—pass elimination (3). The ex-
`tent to which xenobiotics undergo first—pass elimi-
`nation will have a major influence on the exposure
`to the compound following oral administration. The
`enzymes contributing to the metabolism of xeno-
`biotics are also found in organs other than the liver,
`such as the lung and skin. albeit usually at a lower
`level. Thus. xenobiotics entering the body by routes
`other than the gastrointestinal tract can also be sub-
`ject to first-pass metabolism.
`
`DISTRIBUTION
`
`Following entry of a xenobiotic to the systemic
`circulation. its distribution into the various tissues
`
`of the body will be influenced by tissue hemody-
`namics. passive diffusion across lipid membranes.
`the presence of carrier-mediated active transport
`
`processes recognizing the xenobiotic. and protein
`binding in the blood and tissues.
`The majority oftissue membranes behave as typ-
`ical
`lipid barriers allowing small lipophilic mole-
`cules to cross cell membranes. Equilibrium drug
`concentration ratios are maintained by diffusion of
`drugs into and out oftissues. Drugs can accumulate
`in tissues at a higher concentration than predicted
`by simple diffusion under the influence of pH gra-
`dients. binding to intracellular constituents. or par-
`titioning into lipid depots. Larger or more polar
`substances do not cross lipid membranes by passive
`diffusion and require specific transporters to enter
`the tissues (44). If a drug does enter a tissue by an
`active transport mechanism. its concentration in the
`tissue may be many times greater than its plasma
`concentration.
`
`Active uptake processes tend to show stereosc-
`lectivity and can be particularly important for xe-
`nobiotics that may be analogs of nutrients (5 l). The
`operation of specific uptake mechanisms for xeno-
`biotics may play an important role in the toxicity
`ofsome compounds. For example. amantanide and
`phalloidin are toxic cyclopeptides of the fungus Am-
`anita phalloides (21). The toxins enter the liver via
`an active transport system involved in the transport
`of bile acids (23). Once inside the cells. the toxins
`bind to microfilamentous F-actin and destroy the
`mechanical stability ofthe liver cell membrane. This
`results in hemorrhagic liver swelling and animals
`die within 2-3 hr of intravenous dosing with the
`peptides (22). Co-administration of bile salts with
`the toxins reduces their hepatic uptake by this active
`transport mechanism and thereby limits the toxicity
`of the compounds. Distribution of xenobiotics can
`also be limited by binding to plasma proteins. Acidic
`drugs tend to bind to albumin, and basic drugs tend
`to bind to a,—acid glycoprotein. As only unbound
`drug is in equilibrium across membranes. a drug
`that is extensively and strongly bound to plasma
`proteins has only limited access to the tissues.
`
`Drug Reservoirs
`
`Accumulation ofa drug within a tissue can act as
`a reservoir serving to prolong its duration of action.
`Ifthe stored xenobiotic is in equilibrium with that
`in plasma and is released as its plasma concentration
`falls. then the concentration ofxenobiotic in plasma
`will be sustained and the pharmacological effect of
`the xenobiotic will be prolonged (3). Thus. the stor-
`age of a drug can prolong its action either within
`the tissue where the drug is held or at a distant site
`reached following redilfusion into the systemic cir-
`culation (29).
`The concepts of drug reservoirs and how they
`influence the concentration of a xenobiotic at
`its
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`104
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`CALDWELL ET AL
`
`TOXICOLOGIC PATHOLOGY
`
`target tissue are well illustrated by the behavior of
`the lipophilic anesthetic thiopental, which is given
`by bolus intravenous injection (2). As a consequence
`of the high blood flow to the brain and its lipid
`solubility, thiopental reaches its maximum concen-
`tration in its target tissue within 1 min of intrave-
`nous injection. When the injection is stopped, the
`plasma concentration falls as the drug distributes
`into tissues such as muscle. As thiopental is not
`tightly bound to brain lipid, its concentration in the
`brain changes in parallel with changes in the plasma
`concentration, leading to a rapid termination of an-
`esthesia by redistribution rather than elimination.
`A third distributive phase for thiopental occurs as
`the result of a slow, blood flow—limited uptake into
`poorly perfused tissues such as fat (3).
`On repeated administration, fat and other poorly
`perfused tissues can accumulate large amounts of
`thiopental. These reservoirs are then capable of
`maintaining plasma and, hence, brain concentra-
`tions of thiopental at levels above those needed for
`anesthesia. Thus, a compound whose duration of
`action is limited by rapid redistribution from its site
`of action to storage sites can become long acting if
`storage deposits of suflicient size are established. At
`this point, termination of drug action becomes de-
`pendent on biotransforrnation and excretion of drug.
`The pharmacological consequence of these changes
`in tissue distribution is that the sleeping time after
`dosing of thiopental is changed from a few minutes
`following a single administration to a few hours fol-
`lowing multiple dosing (29).
`Toxicity testing is often performed using much
`higher doses of xenobiotics than humans are ex-
`posed to. As well as leading to saturation of meta-
`bolic pathways, it must be appreciated that these
`high doses can lead to changes in tissue distribution
`similar to those seen following multiple dosing of
`thiopentone.
`
`METABOLISM
`
`Drugs and other xenobiotics that gain access to
`the body may undergo 1 or more of 4 distinct fates,
`as follows (12):
`
`l. Elimination unchanged
`2. Retention unchanged
`3. Spontaneous chemical transformation
`4. Enzymic metabolism
`
`Each of these fates are of importance but, in quan-
`titative terms it is enzymic metabolism, often also
`referred to as biotransformation, that predominates.
`The main site of metabolism of foreign com-
`pounds is the liver, although extrahepatic tissues,
`frequently the site of entry to or excretion from the
`body (e.g., lungs, kidneys, gastrointestinal mucosa),
`
`also play a role in the metabolism of xenobiotics
`(24 and references therein).
`Compounds eliminated unchanged are generally
`either (a) highly polar such as strong carboxylic or
`sulfonic acids (e.g., sodium cromoglycate) or qua-
`ternary amines (e.g., pancuronium), which if ab-
`sorbed are rapidly cleared into the urine or bile, or
`(b) volatile and hence readily lost via the lungs. In
`contrast, nonpolar, highly lipophilic compounds may
`be retained for long periods in tissue lipids, as occurs
`with chlorophenothane and many polyhalogenated
`aromatics. For a small number ofcompounds, spon-
`taneous chemical transformation within the tissues
`
`of the body can be important: this may involve
`hydrolysis at the appropriate pH (e.g., thalidomide
`with its numerous breakdown products) or reaction
`with nucleophilic or electrophilic centers in tissue
`macromolecules, most notably the nucleophilic — SH
`of glutathione (71).
`The scope of drug metabolism is immense, and
`this is reflected in the range of chemical reactions
`that are involved in the metabolism of substrates,
`including oxidation, reduction, hydrolysis, hydra-
`tion, conjugation, and condensation. Typically, the
`process of metabolism of xenobiotics is biphasic,
`whereby the compound first undergoes a function-
`alization reaction (oxidation, reduction, or hydro-
`lysis), which introduces or uncovers a functional
`group (—OH,—NH2,—SH) suitable for subsequent
`conjugation with an endogenous conjugating agent.
`By far the most important enzyme system in-
`volved in Phase 1 metabolism is cytochrome P-450,
`the terminal oxidase component of the microsomal
`electron transfer system, which is responsible for
`the oxidation of many xenobiotics. The required
`electrons are supplied by the closely associated en-
`zyme NADPH cytochrome P-450 reductase, a fla-
`voprotein that transfers 2 electrons to cytochrome
`P—450 from NAD(P)H.
`The cytochromes P-450 are an enzyme superfam—
`ily consisting of a number of related isoenzymes, all
`of which possess an iron protoporphyrin IX as pros-
`thetic group. The enzymes are named for the Soret
`band around 450 nm exhibited by the CO complex
`of the reduced form. The P-450 enzymes have been
`grouped together into families that share sequence
`identity. There are 10 mammalian gene families
`comprised of 18 subfamilies (52, 53). The most im-
`portant enzymes involved in xenobiotic metabolism
`belong to the 1A, 2B, 2C, 2D, and 3A subfamilies.
`Although the individual enzymes are thought to me-
`tabolize substrates via the same catalytic mecha-
`nism (27), they tend to show selectivity toward sub-
`strates. For individual isoforrns of P-450, the extent
`of this selectivity is highly variable with overlap of
`substrates and regio— and stereospecificities being
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`BASIC PRINCIPLES OF ADME
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`105
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`observed (26). In addition. substrates are often me-
`tabolized at more than I position. as in the case of
`testosterone. This is believed to be a function of
`
`hydroxylation of warfarin and phenytoin. Whereas
`CYPEC9 is involved in the metabolism of tolbu-
`tamide and a number of acidic nonsteroidal anti-
`
`both the binding characteristics ofthe enzyme and
`the ease with which the functional groups of the
`substrate undergo oxidation (66). A number of ac-
`tive site models have been proposed to explain the
`different substrate specificities of various P-450 iso-
`enzymes.
`
`CYPIAI
`
`The substrate binding site of CYP1A1 has been
`proposed to consist of a hydrophobic cleft asym-
`metrically disposed to the heme iron atom. The
`asymmetric position ofthe binding site restricts the
`number of faces of the substrate that can be exposed
`to the active oxygen species (32). CYP1A1 has been
`implicated in the metabolism ofa number of poly-
`cyclic aromatic hydrocarbon (PAH) compounds such
`as benzo(a)pyrene. The substrates tend to be large,
`rigid, planar molecules containing fused (het-
`ero)aromatic rings that are good electron acceptors.
`Lewis et al (42) proposed that the binding site of
`CYP1A1 contains a number of aromatic amino ac-
`
`ids that form a planar pocket to complement the
`(hetero)aromatic rings of the substrates. The me-
`tabolism of benzo(a)pyrene results in the preferen-
`tial production of the bay region 7,8—diol-9, lO—epox-
`ide, which is a potent DNA-reactive ultimate car-
`cinogen (33).
`In addition to PAH metabolism,
`CYP1A1 can metabolize a number of smaller non-
`
`PAH compounds in a regio- and stereoselective
`manner (62). It has been suggested that these sub-
`strates are positioned in the active site via hydrogen-
`bonding interactions between the substrate and an
`active site residue of CYP1A1 (38).
`
`CYPZBI/2
`
`The P-450 2B isozymes are involved in a number
`ofbiotransformations in the rat and are induced by
`phenobarbital. The substrates for CYPZB tend to
`be bulky, nonplanar molecules with greater confor-
`mational flexibility than CYPIA substrates (39). The
`substrates tend to have functional groups of similar
`size and hydrophobicity to isopropyl and to be poor
`electron acceptors (30, 39). It has been proposed
`that the binding site ofCYP2B contains hydrophilic
`amino acids that are capable of forming hydrogen
`bonds with carbonyl and/or amine groupings ofthe
`substrate and hydrophobic nonaromatic residues
`that complement the isopropyl function (42).
`
`CYPBC
`
`The CYPZC subfamily appears to be important
`in metabolizing a number of xenobiotics particu-
`larly in humans (66). CYPZC8 effects the aromatic
`
`inflammatory drugs and is potently inhibited by sul-
`faphenazole. CYPZCIS is subject to a genetic poly-
`morphism manifest in the hydroxylation of S-me-
`phenytoin and is not inhibited by sulfaphenazole
`(26). CYPZC substrates tend to have areas ofstrong
`hydrogen bond—forming potential positioned 5-10
`A from the site of oxidation. and a number are also
`charged at physiological pH (66). This has lead to
`the suggestion that hydrogen-bonding potential and
`possibly ion pair interactions are important in de-
`termining the substrate structure activity relation-
`ships of the P-4502C isozymes (66).
`
`C YPZD
`
`The CYPZD isozymes have been extensively in-
`vestigated, as they are involved in the genetic poly-
`morphic metabolism ofdebrisoquine. spaneine. and
`some 30 other substrates (17. 48). CYP2Dl (rat)
`and ZD6 (human) have similar substrate selectivi-
`ties, but inhibition studies with quinidine (more po-
`tent in humans than rats) and its diastereoisomer
`quinine (more potent in rats than humans) dem-
`onstrate that differences in the enzyme active site
`must exist (65).
`Substrates for CYPZD enzymes possess a basic
`nitrogen grouping that is mainly ionized at physi-
`ological pH. a hydrophobic region and a functional
`group capable of P-450 oxidation 5-7 A from the
`basic nitrogen (69). Reactions catalyzed include ar-
`omatic hydroxylation (propranolol). aliphatic hy-
`droxylation (metoprolol). and N—dealkylation (ami-
`fiamine) (17). The substrate binding site of CYPZD
`appears to contain a carboxyl group that binds and
`neutralizes the basic nitrogen of the substrate and a
`hydrophobic domain. The carboxylate group is as-
`sumed to serve as an anchoring site on the protein.
`Substrates can interact with either of the oxygen
`atoms of the carboxylate group (which are 2.2 A
`apart). explaining why for some substrates the dis-
`tance between basic nitrogen and site of oxidation
`is 5 A. typified by debrisoquine. and for other sub-
`strates it is 7 A. 1} pified by dextromethorphan (38).
`The ionic bonding between substrate and enzyme
`means that the en/_\ me tends to have a high affinity
`for substrates and. thus. a low Km (65). Many sub-
`strates also exhibit a coplanar conformation near
`the oxidation site and have a negative molecular
`electrostatic potential in a part ofthis planar domain
`approximately 3 A away from the oxidation site
`(38).
`The predictive value of the model was assessed
`by measuring the CYPZD6-mediated metabolism
`of-1 compounds. showing among them at least 14
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`CALDWELL ET AL
`
`Tox1coLoG1c PATHOLOGY
`
`TABLE I.—The 8 classical conjugation reactions.
`
`Reaction
`
`Conjugating agent
`
`A. Reactions involving activated conjugating agents
`Glucuronidation
`Glucose conjugation
`Sulfation
`Methylation
`Acetylation
`Cyanide detoxication
`B. Reactions involving activated foreign compounds
`Glutathione conjugation
`Amino acid conjugation
`
`UDP glucuronic acid
`UDP-glucose
`3’-Phosphoadenosine-5 ’-phosphosulfate
`S-adenosyl methionine
`Acetyl coenzyme A
`Sulfane SU.lf1lI'
`
`Glutathione
`Glycine, ornithine, taurine
`
`oxidative metabolic routes. From the model, 4 routes
`were predicted to be 2D6—mediated. In vivo and in
`vitro data from humans demonstrated that 3 of the
`
`4 predicted metabolic routes were in fact mediated
`by CYP2D6 (40).
`
`C YP3A
`
`The CYP3A family tends to be involved in the
`metabolism of large, structurally diverse, fairly li-
`pophilic compounds. Although substrates are bulky,
`metabolism tends to occur in small exposed func-
`tional groups that undergo reactions such as N-deal-
`kylation and aliphatic hydroxylation. Substrates in-
`clude the immunosuppressant cyclosporin A, nifed-
`ipine, and verapamil (66).
`It has been suggested that the binding site for
`CYP3A is dominated by hydrophobic interactions
`and that, in contrast to CYP2D, which is governed
`by ionic bonding, this allows for a degree of flexi-
`bility in the position of substrate binding (67).
`
`Conjugation Reactions
`
`Phase 2 conjugation reactions may be divided into
`2 distinct groups, depending on the source of energy
`for the process (10). In most instances, the energy
`is derived from the activated endogenous conjugat-
`ing agent, as is the case for the glucuronic acid,
`sulfate, methylation, and acetylation reactions. In
`other examples, the energy is derived by prior met-
`abolic activation of the xenobiotic, as is the case for
`glutathione and amino acid conjugations. Of the
`Phase 2 conjugation reactions listed in Table I, gluc-
`uronic acid conjugation ranks as highest importance,
`and many drugs (e.g., indomethacin, paracetamol,
`dapsone, clofibrate, morphine) are metabolized via
`this pathway. The conjugations are performed by a
`family of glucuronyl transferase enzymes located
`within the endoplasmic reticulum of the cells of the
`liver. intestine and kidney. These enzymes catalyze
`the conjugation of uridine diphosphate—a-l-gluc-
`uronic acid with nucleophilic O, N, C, and S atoms:
`during the reaction, C-1 of the sugar ring is inverted
`
`so that the products are 1-0-substituted B-D-glu-
`copyranosiduronic acids. The enzymes have a mo-
`lecular weight of between 50 and 60 kDa and exist
`as oligomers of between 1 and 4 subunits in vivo
`(60). At least 9 difierent isozymes in 2 different sub-
`families are known to exist (8). Glucuronidation oc-
`curs in most mammalian species with the cat and
`related felines and the Gunn rat being notable ex-
`ceptions.
`Glutathione-S-transferases catalyze the conjuga-
`tion of a number of functional groups (aryl and alkyl
`halides, lactones, epoxides, and quinones) with glu-
`tathione, the tripeptide 7-glutamylcysteinylglycine.
`The glutathione-S-transferases have a very exten-
`sive tissue distribution and are principally found in
`the cytosol of the cell. The proteins have a molecular
`weight of 24-28 kDa and exist as dimers in vivo
`(77). The dimeric proteins possess binding sites for
`glutathione and the electrophilic substrate, which
`brings the reactants close together (47). The mam-
`malian glutathione transferase enzymes have been
`divided into 5 evolutionary classes: oz, u, 7:-, 0, and
`microsomal (77). Typical substrates include para-
`thion, urethane, ethacrynic acid, and 1-chloro-2,4-
`dinitrobenzene. The glutathione transferase en-
`zymes are also very abundant in the liver cytosol
`(4—5°/o of total cytosolic protein). Thus, as well as
`having major significance in drug metabolism, these
`enzymes are also important in intracellular binding.
`This is particularly true for glutathione-S-transfer-
`ase B (Ligandin). Compounds that bind to gluta-
`thione-S-transferases include bilirubin, estradiol,
`cortisol, testosterone, tetracycline, penicillin, and
`indocyanine green (43).
`A number of catechol, phenol, and alcohol com-
`pounds are excreted as sulfate conjugates. This re-
`action between substrate and sulfate donor, 3’—phos-
`phoadenosine-5’-phosphosulfate, is catalyzed by a
`family of sulfotransferase enzymes (18). The sul-
`fotransferases have a cytosolic location and are found
`in many tissues including the liver, adrenals, lung,
`brain, jejenum, and blood platelets. The proteins
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`BASIC PRINCIPLES OF ADME
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`107
`
`have molecular weights between 32 and 34 kDa and
`exist as homodimers in viva. The sulfotransferase
`
`These are important in determining the biological
`effect of a xenobiotic. The intracellular concentra-
`
`enzymes are normally classified into subfamilies
`based on their substrate specificity. although there
`is overlap among the different isozymes (18). Six
`different phenol transferases and 7 different steroid.
`bile acid sulfotransferases have been characterized
`
`in the rat. In humans. only 2 phenol transferases
`(M-PST and P-PST) and l steroid/bile acid sulfo-
`transferase (DHEA-ST) have been characterized.
`Typical substrates of the human liver cytosolic
`transferases are dehydroepiandrosterone. pregnen-
`olone, and testosterone (DHEA-ST), dopamine and
`acetaminophen (M-PST), and phenol and minoxidil
`(P-PST). As well as exhibiting species differences. it
`is known that phenol sulfotransferase activity varies
`among individuals over a 15-fold range (35).
`N-acetyltransferase catalyzes the addition of an
`acetyl group to the amino group of amine, amino
`acid, and sulfonamide compounds using acetyl co-
`enzyme A as a cosubstrate. N-acetyltransferase is a
`26.5 kDa cytosolic enzyme found in the liver and
`intestine. Typical substrates include isoniazid and
`sulfanilamide. A well-defined polymorphism for
`N-acetyltransferase exists in many species including
`humans (61), which can have profound effects on
`toxicity. For example, isoniazid accumulates in slow
`acetylators and so may predispose these individuals
`to drug-induced neuropathy. On the other hand, the
`toxicity of isoniazid is related to the formation of
`its N-acetyl derivative, which is further metabolized
`to a reactive intermediate. Rapid acetylators are
`more susceptible to this type of side effect. The dog
`and related canine species do not possess this poly-
`morphic enzyme.
`Epoxide hydrolase enzymes catalyze the trans ad-
`dition of water to a variety of epoxide compounds.
`Microsomal epoxide hydrolase is found in the liver,
`testes, kidney, ovary, and lung. It
`is an approxi-
`mately 50-kDa protein, and typical substrates in-
`clude styrene oxide, vinyl chloride epoxide. ben-
`zo(a)pyrene-4,5-oxide, phenytoin epoxide, and car-
`bamazepine epoxide (63). Cytosolic epoxide hydro-
`lase is a 60-kDa protein that exists as a homodimer
`of 120 kDa in viva. Typical substrates ofthis enzyme
`include trans-stilbene oxide. epoxymethyl stearate.
`and arachadonic acid epoxide. Species differences
`occur in the activity of cytosolic epoxide hydrolase
`in that the mouse has high activity. the rabbit. guin-
`ea pig. and humans have intermediate activity. and
`the rat has very low activity (46).
`
`Factors .-lfliecting .lIerab0li.sm ofX'en0bi0lics
`
`Factors influencing the rate and extent of metab-
`‘V
`olism via Phase 1 or _ reactions m vim can be
`
`physiological. endogenous. or exogenous (Table ll).
`
`tion of a chemical is primarily dependent on dose
`size and its physicochemical and structural prop-
`erties. Because the metabolism of most compounds
`is enzymatic. any factor that can influence the ac-
`tivity of these enzymes can alter metabolism.
`
`Species Dt'[ferem'cs In llelabo/1fs‘nz and 7'0.\‘1't‘iIy
`
`Species differences in metabolism are of most sig-
`nificance. but variability can also occur in absorp-
`tion, distribution. and excretion of foreign com-
`pounds. Species differences in drug metabolism
`reflect differences in the activities of the enzymes
`responsible for the various transformations. Such
`variations arise primarily from differences in the
`absolute activities of the enzymes. but the amounts
`of any endogenous inhibitors present, or the extent
`of any reverse reactions, are also relevant. Species
`differences occur in both Phase I and 2 metabolism
`
`and can be either quantitative (same metabolic route
`at different rates) or qualitative (different metabolic
`routes) (12). Given that species differences do occur,
`it is possible that they arise from 1 or more of 3
`origins:
`
`1. Deficiencies in certain enzymes that lead to a
`defect in a metabolism reaction that is otherwise
`
`widespread in occurrence. For example, the glu-
`curonidation deficiency in the cat leads to in-
`creased toxicity ofglucuronidogenic compounds
`in this and related species. The lack ofacetylation
`ofaromatic amines in dogs may explain why they
`are more susceptible to p-aminobenzoic acid and
`various hydrazines. The guinea pig has a defi-
`ciency in N-acetylation and is unable to N-acety-
`late S-substituted cysteines to form acid conju-
`gates (10). This is not due to defective formation
`of glutathione conjugates (the first step in the
`mercapturic acid pathway), indicating that the
`defect lies in the final step of transformation of
`the glutathione conjugate to a mercapturic acid
`(15). The pig and opossum are defective in their
`ability to conjugate phenolic compounds with
`sulfate (10).
`Although most ofthe deficiencies involve con-
`jugation reactions (13). there are a few examples
`of defects in oxidative metabolism. These in-
`
`clude the inability ofthe guinea pig and steppe
`lemming to .\'-hydroxylate the ;ir_\l-acetamide,
`2—acet_\lamidolluorene. and the inability of the
`rat and marmoset to .\'-hydroxylate the aliphatic
`amine chlorphentermine (l0).
`. Restricted species occurrences. For instance. these
`are seen with the particular amino acid (glycine,
`glutamine. taurine. or ornithine) utilized in the
`
`Ix)
`
`|nnoPharma Exhibit 1025.0006
`
`

`

`108
`
`CALDWELL ET AL
`
`TOXICOLOGIC PATHOLOGY
`
`TABLE II.—Physicochemical, endogenous, and exogenous factors affecting the rate and extent of metabolism of xe-
`nobiotic chemicals in viva.
`
`Physicochemical
`
`Electrophilicity
`Nucleophilicity
`Lipophilicity
`Polarity
`Protein binding
`
`Endogenous
`
`Age
`Sex
`Species
`Strain
`Pathology
`Genetic deficiencies
`Cofactor availability
`
`Exogenous
`
`Dose
`Nutrition
`Route of administration
`Time of day
`Enzyme inhibition
`Enzyme induction
`
`conjugation ofacids. Most species conjugate ben-
`zoic acid and heterocyclic and cinnamic acids
`with glycine, whereas bird species use ornithine.
`Of special interest are the reactions that are re-
`stricted to primates such as the glutamine con-
`jugation of phenyl acetic acid (10).
`3. Most common, the relative rates of competing
`pathways of metabolism rather than the route of
`metabolism. Amphetamine undergoes either ar-
`omatic hydroxylation, producing 4’-hydroxyam-
`phetamine, or side-chain degradation to benzoic
`acid. The compound is metabolized extensively
`in most species, but the nature of metabolites
`formed is highly variable. In the rat, ring hy-
`droxylation predominates, in the guinea pig chain
`breakdown is the major route, and in other spe-
`cies both routes are significant (10, 11, 13). Di-
`azepam is extensively metabolized in humans,
`dog, and rat. In humans, the major metabolites
`are 3-hydroxydiazepam, N'-desmethyldiaze-
`pam, and 3—hydroxydesmethyldiazepam (oxa-
`zepam). In the dog, oxazepam is the major prod-
`uct, and in the rat ring hydroxylations predom-
`inate with 4’—hydroxy—3-hydroxydiazepam, 4’-
`hydroxy—N‘ —desmethyldiazepam, oxazepam, and
`3—hydroxydiazepam all being formed. Oxazepam
`is further metabolized to the 0-glucuronide in
`humans and dog but to a number of ring hy-
`droxylated compounds in the rat (10). Phenol
`can be conjugated on the hydroxyl group with
`either sulfate or glucuronide. Humans and Old
`World monkeys excrete phenol principally as the
`sulfate, whereas New World monkeys excrete
`phenol principally as the glucuronide and the rat
`and mouse excrete approximately equal amounts
`of both conjugates (10).
`
`Sex Dzflerences in Metabolism and Toxicity
`
`Sex differences have been noted in the absorption
`(cephradine). protein binding (diazepam, warfarin),
`and biliary excretion (indocyanine green, tanrazine)
`of drugs and xenobiotics (9). However, the sex dif-
`ferences found in metabolic pathways have proba-
`bly the most significance with regard to xenobiotic
`
`toxicity. Sex differences have been reported in both
`Phase 1
`(e.g., N—demethylation of morphine and
`oxidation of pentobarbital) and Phase 2 metabolism
`(e.g., glutathione conjugation of l,2-dichlorodini-
`trobenzene, glucuronic acid conjugation of p-nitro-
`phenol, sulfation of N-hydroxy-2-acetylaminoflu-
`orene). The exact nature of