ROLE OF PHARMACOKINETICS AND METABOLISM IN DRUG RESEARCH
`
`433
`
`required to displace the drug binding from the binding
`sites. Thus, in vitro studies designed to assess the pos-
`sibility of in vivo binding displacement must use undi-
`luted plasma and clinically relevant drug concentra-
`tions.
`
`The use of supratherapeutic drug concentrations or
`unusually low protein concentrations may produce bind-
`ing displacement in Vitro, but not in vivo. Zini et al.
`(1979) showed that indomethacin markedly decreased
`warfarin binding to human serum albumin in vitro at an
`indomethacin concentration of 100 ,u.M. However, Vesell
`et al. (1975) found no clinically significant displacement
`interaction between indomethacin and warfarin in vivo
`
`where the indomethacin concentration ranged from 0.08
`to 1.0 ,uM. Bupivacaine caused a 109% increase in the
`free fraction of mepivacaine in a solution of 011-acid gly-
`coprotein, but only a 9% increase in the free fraction of
`mepivacaine in plasma containing the same :11-acid gly-
`coprotein concentration (Hartrick et al., 1984). Both bu-
`pivacaine and inepivacaine are highly bound to high-
`aiiinity and low-capacity 0:1-acid glycoprotein and low-
`affinity and high-capacity albumin in plasma.
`Similar to metabo1ism—based drug interaction, the in-
`terpretation and extrapolation of in vitro displacement
`interaction data requires a good understanding of phar-
`macokinetic principles. Rowland and Aarons (Rowland,
`1980; Aarons and Rowland, 1981) have reviewed the
`theoretical and clinically relevant issues regarding drug
`displacement interactions. Depending on whether it is a
`low- or high-clearance drug, displacement interaction
`will cause different alterations in pharmacokinetics. As
`shown in equation [10], changes in the free fraction (fp)
`in plasma caused by displacement binding will affect
`drug distribution. As seen in equations [6] and [10], an
`increase in the fp of high—clearance drugs caused by
`binding displacement interaction will have little change
`in the clearance (cL), but will lead to an increase in the
`volume of distribution (Vd); hence, the elimination ti/4
`will increase. The tv; is related to both the cL and Vd as
`follows:
`
`0.693 X Vd
`CL
`
`.
`
`to,
`
`[12]
`
`For low—clearance drugs, both CL and Vd will increase
`with an increase in fp as shown in equations [5] and [10].
`Although the changes in cL and Vd may not exactly
`balance, the tn/1 will be affected to a much smaller degree
`compared with that of highly cleared drugs.
`Because only unbound drug is responsible for phar-
`macological effect, it is important to make a clear dis-
`tinction of the effects of displacement interaction on
`unbound and total drug concentrations in plasma. The
`simplest way of considering the effect of protein binding
`on the unbound and total drug concentration profiles is
`to examine the AUC. For low—clearance drugs, the AUC
`
`of unbound and total drug after intravenous dosing can
`be expressed as:
`
`AUC
`
`dose
`dose
`‘°“*‘ ‘ cL ‘ £1,-cL,,,,
`
`13
`
`1
`
`[
`
`and
`
`AUCunbound : AUCtotal ' fp 7
`— CI-‘int.
`
`dose
`
`On the other hand, the AUC of unbound and total
`drug of high-clearance drugs after intravenous adminis-
`tration can be expressed as:
`
`AUC
`
`_ dose _ dose
`total — CL — Qh
`
`[15]
`
`and
`
`dose - fp
`AUCunbound = AUCtotal ‘ fp = F
`Qh
`
`From equations [13] and [14], it is evident that the
`AUC of unbound drug for low—clearance drugs is inde-
`pendent of any change in fp if cLim is unaffected by
`displacement interaction, whereas an increase in the fp
`caused by binding displacement interactions will result
`in a decrease in the AUC of total drug. On the other
`hand, exactly the opposite situation occurs with a high-
`clearance drug in which the clearance and, hence, total
`drug concentration is unaffected by changes in plasma
`protein binding, whereas the unbound drug concentra-
`tion increases as a result of increased fp as shown in
`equations [15] and [16]. Figure 7 depicts the effects of
`displacement from protein binding sites on the steady-
`state unbound and total drug concentrations of low— and
`high-clearance drugs during intravenous
`infusion
`(Aarons, 1986).
`After oral administration, the AUC of unbound and
`total drug, regardless of whether it is a high— or low—
`clearance drug, can be expressed as equation [17], which
`
`C
`
`3%
`'23:
`§
`
`(8)
`
`T _
`
`Total
`
`(bl
`
`\.
`
`./
`
`T Total
`
`T
`
`_,-—--"--Unbound
`
`l‘-._L,|_n_b_ound _____
`.. ._,
`
`Time
`
`Time
`
`FIG. 7. The effect of displacing a low—clearance drug ( a) or high-
`clearance drug (bl, given chronically, from plasma protein binding
`sites. Displacement is produced by infusing a drug that displaces the
`first drug, starting from the arrowed point. Reproduced with permis-
`sion from Aarons (1986).
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2028 - 0031
`
`

`
`434
`
`LIN AND LU
`
`is similar to equation [8]:
`
`AUCt0ta1 =
`
`F ' dose
`CL
`
`dose
`= [17]
`
`and
`
`A-Ucunbound = AI-lctotal ' fp =
`
`dose
`
`From equations [17] and [18], the AUC of unbound
`drug after oral dosing is insensitive to the changes in the
`fp, whereas the AUC of total drug will decrease when the
`fp increases as a result of displacement interactions.
`Because a significant change in the unbound AUC of
`drugs after oral dosing is not expected, and because most
`drugs are given orally, the displacement interactions
`rarely have significant clinical effects
`(Mackichan,
`1984,1989; Sellers, 1979). When changes in binding are
`associated with clinical effects, it has almost always
`been found that this is the result of a change in the cLm,
`caused by a mechanism quite independent of plasma
`protein binding as indicated in equation [18]. Warfarin—
`phenylbutazone interaction is a good example. When
`concomitantly administered with warfarin, phenylbuta-
`zone caused profound potentiation of a hypoprothrom—
`binemic response (Sellers, 1986). Although phenylbuta-
`zone is known to displace warfarin from plasma
`proteins, it is clear from equation [18] that the hypopro-
`thrombinemic effect was not caused by binding displace-
`ment of phenylbutazone, because the unbound concen-
`tration of warfarin should not be changed. Later, it was
`found that phenylbutazone stereoselectively inhibited
`the metabolism of S—warfarin (Lewis et a1., 1974;
`O’Reilly et al., 1980). Thus, the metabolism inhibition,
`rather than binding displacement, causes the serious
`hemorrhagic complications of warfarin-phenylbutazone
`interaction. Similarly,
`although sulfaphenazole
`is
`known to displace tolbutamide from plasma proteins,
`the inhibitory effect of sulfaphenazole on the metabo-
`lism of tolbutamide is responsible for the serious hypo-
`glycemic reactions (Christensen et al., 1963).
`Whereas the unbound concentration after oral dosing
`is unaffected by displacement interaction, the transient
`increase in the unbound drug concentration occurring
`immediately after introduction of the displacing drug
`sometimes may be of clinical significance (Levy, 1976).
`Elie and Levy (1979a,b) reported that rapid intravenous
`infusion of salicylic acid or sulfisoxazole resulted in a
`transitory increase of unbound bilirubin concentration
`in rats. This suggests that the fatal kernicterus seen in
`the newborn after administration of sulfonamides may
`be due to a transitory increase in unbound bilirubin in
`the brain. In addition, the displacement interactions will
`be of clinical significance for high-clearance drugs after
`intravenous dosing. As shown in figure 7, a substantial
`increase in the unbound concentration may occur.
`
`V. Interindividual Variability: A Critical Issue in
`Drug Development
`
`From the market point of view, it is desirable that the
`dosage can be generalized to provide drugs for the treat-
`ment of a large number of patients. In reality, the gen-
`eralization may work for most patients, but not for all.
`The standard dosage regimen of a drug may prove to be
`therapeutically effective in most patients, ineffective in
`some patients, and toxic in others. Variability in drug
`response becomes an important problem in drug therapy
`for drugs that have a narrow therapeutic window. War-
`farin is a good example. There is a wide range of daily
`dose requirements (<2 mg—>11 mg‘) of warfarin needed
`to produce a similar prothrombin time (Koch-Weser,
`1975). Variability in drug response can be broadly di-
`vided into pharmacokinetic and pharmacodynamic
`bases. Sources of pharmacokinetic variability include
`genetics, disease, age, and environmental factors (Brei-
`mer, 1983).
`
`A. Pharmacokinetic Variability
`
`The patient’s exposure to drug is a crucial determi-
`nant of the drug’s actions, and therefore its efficacy and
`safety. The term “drug exposure” is defined as the time
`course of the concentration of the drug and its active
`metabolites in plasma. The time course of drug concen-
`tration is governed by absorption, distribution, metabo-
`lism, and excretion. All these processes can contribute to
`pharmacokinetic variability.
`I. Variability in absorption. Variation in absorption is
`one of the major sources of pharmacokinetic variability.
`An impression prevails that the degree of variability in
`the amount of drug reaching the systemic blood circula-
`tion is minimized if a drug‘ has high bioavailability,
`whereas the risk of greater variation in the amount
`taken up is increased if a drug has low bioavailability.
`However, all too often, the degree of variability in ab-
`sorption is similar for drugs of high and low bioavail-
`ability. The causes of absorption variability include
`pharmaceutical formulation, gastrointestinal physiol-
`ogy, and first—pass metabolism.
`Being absorbed primarily from the upper part of the
`small intestine, oral absorption of drugs is often affected
`by the gastric emptying time and small intestinal motil-
`ity, which vary considerably between individuals
`(Meyer, 1987; Weisbrodt, 1987). Usually, rapid gastric
`emptying results in rapid drug absorption. Changes in
`gastric emptying normally affect the rate of absorption
`but do not affect the amount of drug absorbed unless the
`drug is chemically unstable in the stomach or associated
`with saturable first-pass metabolism (Nimmo, 1976).
`Dietary factors are also important sources of absorp-
`tion variability that can be accounted for. The influence
`of food on the absorption of drugs is largely unpredict-
`able. Food may enhance or reduce the absorption of some
`drugs while having no effect on others, depending not
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2028 - 0032
`
`

`
`ROLE OF‘ PHARMACOKINETICS AND METABOLISM IN DRUG RESEARCH
`
`435
`
`only on the composition and volume of the meal or the
`drink, but also on the physicochemical properties of
`drugs. For example, absorption of the lipophilic drugs
`griseofulvin and sulfamethoxydiazine increased consid-
`erably when given with a high-fat meal (Crouse, 1961;
`Kaumeier, 1979). Amoxicillin, a poorly soluble antibi-
`otic, was absorbed to a greater extent when swallowed
`with 250 mL water (Welling et al., 1977). In addition,
`dietary factors have been shown to alter drug-metabo-
`lizing enzyme activity, leading to changes in first—pass
`metabolism and bioavailability. Both charcoal—broiled
`beef and a high-protein, low-carbohydrate diet cause an
`increase in theophylline and antipyrine metabolism
`(Kappas et al., 1978, 1976). Certain vegetables, includ-
`ing brussel sprouts, cabbage, broccoli, and cauliflower,
`contain chemicals that induce drug-metabolizing en-
`zyme activities (Pantuck, 1979). Because the diet is so
`different among patients, it is conceivable that the ef-
`fects of food account for a substantial part of the absorp-
`tion variability.
`Ironically, most clinical studies de-
`signed to address the question as to whether food intake
`affects drug absorption were conducted in healthy vol-
`unteers with or without a more or less standardized
`
`meal. Thus, such information may not be meaningful,
`sometimes even misleading.
`The problem of absorption variability is complicated
`further by diseases. Hepatic disease may influence the
`oral bioavailability of drugs highly metabolized by the
`liver. The bioavailability of propranolol was increased
`significantly from 35% in normal subjects to 54% in
`cirrhotic patients, and the steady-state unbound pro-
`pranolol concentration increased from 7.5 ng/mL to 22
`ng/mL (Wood et al., 1978). The increased bioavailability
`was due mainly to a decrease in hepatic first—pass me-
`tabolism.
`
`2. Variability in binding. As discussed earlier, plasma
`protein binding is an important determinant of the
`drug’s disposition and actions. The fp varies widely
`among drugs, and often (for highly bound drugs) among
`individuals. Differences in binding among drugs arise
`primarily from differences in their affinities for binding
`proteins, whereas differences in binding among individ-
`uals are due mainly to qualitative or quantitative differ-
`ences in binding proteins. Nevertheless, interindividual
`variability in drug binding is generally less as compared
`with that in other pharmacokinetic processes such as
`absorption and metabolism (Yacobi and Levy, 1977;
`Barth et al., 1976).
`a1-Acid glycoprotein is a major determinant for the
`binding of basic drugs in plasma (Piafsky and Borga,
`1977; Piafsky et al., 1978). Several inflammatory states
`(infections, rheumatic disorders, and surgical injury)
`and pathological conditions (myocardial infarction, ma-
`lignancies, and nephritis) elevate the plasma concentra-
`tion of al-acid glycoprotein (Abramson, 1982; Freilich
`and Giardini, 1984). Furthermore, ac,-acid glycoprotein
`is known to be inducible. Treatment with phenobarbital
`
`resulted in a substantial increase in plasma concentra-
`tion of oz1—acid glycoprotein (Abramson, 1991). Because
`there is a strong correlation between the binding of basic
`drugs and the plasma levels of 0:1-acid glycoprotein
`(Lunde et al., 1986; Sjtiqvist and Koike, 1986), an eleva-
`tion of this protein will increase the binding of basic
`drugs.
`In contrast to the elevation of 041-acid glycoprotein,
`hypoalbuminemia is always associated with a large va-
`riety of pathological conditions, including liver cirrhosis,
`renal failure, nephrotic syndrome, chronic inflamma-
`tion, malignancies, and sepsis (Gugler and Jensen,
`1986). In hypoalbuminemia, the binding of acidic drugs
`is reduced, and the decrease is related to a decrease in
`the plasma albumin concentration. Although normal
`subjects have a plasma albumin concentration of at least
`35 mg/mL, plasma albumin concentrations can be as low
`as 10 mg/mL in patients with nephrotic syndrome.
`In addition to the quantitative changes in plasma
`protein concentrations, qualitative structural changes of
`plasma proteins also alter the binding of drugs. High
`doses of acetylsalicylic acid can acetylate serum albumin
`and modify its binding sites (Hawkins et al., 1968).
`Cyanate, spontaneously formed from urea, carbamy-
`lates lysine residues on the albumin molecules and de-
`creases the binding of acidic drugs in uremic patients
`(Erill et al., 1980). Furthermore,
`in uremic patients,
`retained endogenous acids that are highly protein bound
`can displace the binding of drugs from proteins. Collier
`et al. (1986) have identified one of these acids, 3—car—
`boxy—4-methyl-5-propyl-2-furanpropanic acid, as a po-
`tent displacer of drug binding. From these data, it is
`clear that disease states also are the main sources of
`
`binding variability.
`Genetically determined variations in amino acid se-
`quences of serum albumin and 0:1-acid glycoprotein also
`can contribute to binding variability. To date, more than
`30 apparently different genetic variants of human se-
`rum albumin have been identified. Only approximately
`half of these variants have been absolutely character-
`ized by peptide mapping and sequence determination
`(Eap and Baumann, 1991). Kragh—Hansen et al. (1990)
`have compared the binding affinities (association con-
`stants) of warfarin, salicylate, and diazepam to five vari-
`ants of human serum albumin with known mutations.
`
`The association constants of all three drugs to albumin
`Canterbury (313 Lys—>Asn) and to albumin Parkland
`(365 Asp—>His) decreased substantially by a factor of 4-
`to 10-fold, whereas the binding affinity to albumin Ve-
`rona (570 Glu—>Lys) was unchanged. These results sug-
`gest that the region 313-365 seems to exert important
`effects on the binding of drugs, whereas the mutation
`570 near the C-terminus does not affect drug binding.
`Three main variants of a1—acid glycoprotein, namely
`ORM1 F1, ORM1 S, and ORM2 A, have been fully char-
`acterized (Eap and Baumann, 1991). Among the three
`variants, ORM2 A is the most important variant associ-
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2028 - 0033
`
`

`
`436
`
`LIN AND LU
`
`ated with the binding of basic drugs. Eap et al. (1990)
`have determined the in vitro binding of d-methadone,
`L—methadone and dl—methadone in plasma samples from
`45 healthy subjects. The concentrations of oil-acid glyco-
`protein variants also were measured. Using multiple
`stepwise regression analysis, significant correlations
`were obtained between the binding of methadone and
`the total 0:1-acid glycoprotein or ORM2 A concentrations,
`but only a weak correlation between the binding and
`ORM1 S concentrations, and no correlation between the
`binding and ORM1 F1 concentrations were found. The
`frequencies for the three phenotypes, i.e., ORM1 Fll
`ORM2 A, ORM1 FUORM1 S/ORM2 A, and ORM1
`S/ORM2 A were found to be 33.7, 50.5, and 15.2%, re-
`spectively,
`in a Swiss population (Eap et al., 1988).
`These results suggest genetically determined variations
`in O!1'3.C1d glycoprotein could be a major source of vari-
`ability in the binding of basic drugs.
`3. Variability in excretion. Although metabolism is the
`major route of elimination for most drugs, some drugs
`are excreted mainly as unchanged drug via the kidneys
`and liver. Both biliary and renal excretion correlate to
`their function. Ceftazidime, a cephalosporin antibiotic,
`is excreted mainly by the kidneys. The total clearance of
`ceftazidime correlated linearly with creatinine renal
`clearance in patients with varying degrees of renal func-
`tion (Van Dalen et al., 1986). Similarly, a strong corre-
`lation should exist between the clearance and hepatic
`function if a drug is excreted mainly by the liver. The
`biliary excretion of indocyanine green correlated well
`with hepatic function in cirrhotic patients (Kawasaki et
`al., 1985).
`Many endogenous organic acids are accumulated in
`the plasma of patients with renal dysfunction. These
`endogenous organic acids may inhibit the transport of
`certain drugs in the liver. The hepatic uptake and biliary
`excretion of bromosulfophthalein and dibromosulfoph-
`thalein are decreased in rats with acute renal failure
`
`(Silberstein et al., 1988). These data demonstrate that
`variations in hepatic and renal function, particularly in
`patients with hepatic and renal disorders, contribute
`significantly to pharmacokinetic variability.
`Reabsorption is one of the important factors governing
`renal clearance of drugs. Lipophilic drugs tend to be
`extensively reabsorbed, whereas hydrophilic drugs do
`not. Urine flow and pH have a substantial effect on the
`renal clearance of a drug that is mostly reabsorbed. An
`increase in the urine flow will result in a decrease in
`
`reabsorption, leading to an increase in renal clearance.
`The renal clearance of theophylline increases with in-
`creasing urine flow rate (Tan-Liu et al., 1982). Similarly,
`the renal clearance of phenobarbital is also dependent
`on the urine flow rate (Linton et al., 1967).
`Unlike plasma that has a narrow pH range of 7.3 to
`7.5, urine pH ranges from 4.5 to 8.5. Thus, the urine pH
`is an additional factor that influences the reabsorption
`of drugs that are weak acids and bases. The renal ex-
`
`cretion of salicylic acid is markedly pH-dependent. Re-
`nal excretion of salicylate increases more than ten-fold
`as the urinary pH increases from 5 to 8 (Macpherson et
`al., 1955). In contrast, the renal clearance of quinidine
`has been shown to diminish with increasing urinary pH
`(Gerhardt et al., 1969). Drugs that show pH—sensitive
`reabsorption also generally Show flow-rate dependence.
`Clearly, variations in urine flow and pH also contribute
`significantly to excretion variability.
`
`B. Pharmacogenetics of Drug Metabolism
`
`All enzymes involved in the metabolism of drugs are
`regulated by genes and gene products. Because of evo-
`lutionary and environmental factors, there is a remark-
`able degree of genetic variability built into the popula-
`tion. Thus, the genetic factor represents an important
`source of interindividual variation in drug metabolism.
`Mutations in the gene for a drug—metabolizing enzyme
`result in enzyme variants with higher, lower, or no ac-
`tivity or may lead to a total absence of the enzyme.
`Therefore, it is not unusual to find a ten-fold or as much
`as a 50-fold difference in the rate of drug metabolism
`among patients.
`With the technological breakthroughs in molecular
`biology, significant progress has been made in under-
`standing the role of genetic polymorphisms in drug me-
`tabolism. The major polymorphisms that have clinical
`implications are those related to the oxidation of drugs
`by CYP2D6 and CYP2C19 (Meyer et al., 1990b, 1992;
`Meyer, 1994; Wilkinson et al., 1989; Broly and Meyer,
`1993; Alvan et al., 1990), acetylation by N-acetyltrans-
`ferase (Evans, 1992), and S-methylation by thiopurine
`methyltransferase (Weinshiboum, 1992; Creveling and
`Thakker, 1994). Individuals who inherit an impaired
`ability to catalyze one or more of these enzymatic reac-
`tions may be at an increased risk of concentration-re-
`lated adverse effects and toxicity.
`1. Polymorphism in drug oxidation. CYPZD6 polymor-
`phism is perhaps the most studied genetic polymor-
`phism in drug metabolism. Since its discovery in 1977
`(Mahgoub et al., 1977), hundreds of studies have been
`carried out to investigate the nature of CYP2D6 poly-
`morphism,
`the mode of inheritance, and the conse-
`quences of the deficient trait on drug disposition and
`pharmacological effects. This polymorphism divides the
`populations into two phenotypes: EM and PM. Approx-
`imately 5 to 10% of individuals in Caucasian populations
`are the PM phenotype, compared with only 1 to 2% of
`individuals in Asian populations. To date, more than 50
`drugs,
`including antidepressants, antipsychotics, and
`cardiovascular drugs, are known to be catalyzed primar-
`ily by CYP2D6 (Parkinson, 1996).
`Clinical studies have demonstrated that the PMs of
`
`CYP2D6—mediated drugs represent a high—risk group
`with a propensity to develop adverse effects. The dispo-
`sition of haloperidol, a potent neuroleptic, was studied in
`a panel of six EMS and six PMs of debrisoquine (Llerena
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2028 - 0034
`
`

`
`ROLE OF‘ PHARMACOKINETICS AND METABOLISM IN DRUG RESEARCH
`
`437
`
`et al., 1992). The PMS that received 4 mg of haloperidol
`developed neurological side effects, whereas at the same
`dose, the EMS experienced only mild side effects, such as
`tiredness, difficulty concentrating, and some restless-
`ness. The PMS eliminated haloperidol significantly
`slower than the EMS, and the high plasma concentra-
`tions of haloperidol might, therefore, be associated with
`the side effects observed in the PMS. Similarly, an in-
`creased risk of side effects also was observed in the PMS
`
`of debrisoquine when taking other neuroleptics, such as
`perphenazine (Dahl—PuuStinen et al., 1989) and thiorid—
`azine (Meyer et al., 1990a). Both drugs also are metab-
`olized by CYP2D6.
`I antiarrhythrnic
`Similarly, propafenone, a class
`agent, is metabolized by CYP2D6. The relationship be—
`tween debrisoquine phenotype and pharmacokinetics
`and pharmacodynamics of propafenone was studied in
`28 patients (22 EMS and 6 PMS) with chronic ventricular
`arrhythmias (Siddoway et al., 1987). Steady—state con-
`centrations of propafenone in plasma were found to be
`significantly higher in PMS than EMS. These higher
`concentrations were associated with a greater incidence
`of CNS side effects in the PMS (67%), relative to the EMS
`(14%).
`
`The effects of CYP2D6 polymorphism on pharmaco-
`logical responses can be quite complex, depending on
`whether the parent drug or metabolites, or both, are
`pharmacologically active. Encanide, a class I antiar-
`rhythmic, is a good example. CYP2D6 O-demethylates
`encanide to a metabolite that is 6 to 10 times more
`
`potent than the parent drug in blocking sodium chan-
`nels. In both PMS and EMS, standard doses of this drug
`tend to produce similar therapeutic responses, because
`relatively high parent drug concentrations in the former
`are matched by relatively high active metabolite concen-
`trations in the latter (Buchert and Woosley, 1992). Sim-
`ilarly, both propafenone and its 5-hydroxy propafenone
`metabolite are pharmacologically active. The metabo-
`lism of propafenone to 5-hydroxy propafenone is grossly
`impaired in the PMS, resulting in very low or no levels of
`this active metabolite. However, as with encanide, there
`were no significant differences between EMS and PMs in
`an effective propafenone dosage or frequency of antiar-
`rhythmic response (Siddoway et al., 1987). This again
`can be explained by the compensatory effect of the active
`metabolite of 5—hydroxy propafenone, present in the
`plasma of EMS but not in that of PMS.
`Codeine is metabolized extensively by glucuronida-
`tion; the O-demethylation of codeine to morphine is a
`minor pathway that is mediated by CYP2D6 (Chen et
`al., 1988). As only a small fraction of the drug is metab-
`olized by the O-demethylated pathway, PMS are not
`expected to have an altered disposition of codeine rela-
`tive to EMS. As anticipated, plasma concentrations of
`codeine were similar in PMS and EMS, but measurable
`concentrations of morphine, its more analgesic O-dem-
`ethylation product, were only detected in EMS (Sindrup
`
`et al., 1991). Consequently, codeine increased the pain
`thresholds to copper vapor laser stimuli in EMS, but not
`in PMs, affirming the functional importance of the co-
`deine-morphine biotransformation for codeine analge-
`sia.
`
`CYP2C19 also exhibits genetic polymorphism in drug
`metabolism. The incidence of the PM phenotype in pop-
`ulations of different racial origin varies; approximately 2
`to 6% of individuals in the Caucasian populations are
`the PM phenotype, as are 14 to 22% in the Asian popu-
`lations (Wilkinson et al., 1992; Kalow and Bertilsson,
`1994). Although it is expected that PMS will have higher
`plasma
`concentrations
`of drugs metabolized by
`CYP2C19 than EMS and experience an increase in ad-
`verse effects, the clinical implications of CYP2C19 poly-
`morphism have not been thoroughly characterized. Con-
`trary to CYP2D6, CYP2C19 has been studied far less,
`which is reflected by the much shorter list of known
`drugs characterized by CYP2C19 than by CYP2D6 (Par-
`kinson, 1996).
`Diazepam is demethylated by CYP2C19 in humans
`(Anderson et al., 1990). The disposition of diazepam has
`been Studied in 13 Caucasians of the EM phenotype and
`3 Caucasians of the PM phenotype (Bertilsson et al.,
`1989). The plasma clearance of diazepam in the EMS
`was more than 2 times that in the PMS (11.0 and 5.0
`
`mL/min, respectively), whereas the iv; in the EMS was
`shorter than that in the PMS (59 and 128 h, respective-
`ly). The difference in the plasma clearance appeared to
`be related to formation of the desmethyl metabolite.
`Omeprazole, a proton pump inhibitor, is metabolized
`(by CYP2C19) by hydroxylation and oxidation of the
`sulfoxide group to a sulfone (Anderson et al., 1990). The
`metabolism of omeprazole has been studied in the EMS
`and PMS of S-mephenytoin selected from phenotyped
`healthy Swedes and Chinese (Andersson et al., 1992).
`The plasma concentrations of omeprazole and its metab-
`olites were determined after a single oral dose (20 mg).
`The AUC of omeprazole was substantially higher in PMS
`than in EMS in both Swedes (11.1 and 0.94 p'.M'h) and
`Chinese (13.3 and 2.6 MM-h). Although the AUC was not
`different between Swedish and Chinese PMS, there was
`a significant interethnic difference in EMS. The fact that
`the AUCS in Chinese EMS were 3 times higher than
`those of the Swedish EMS might be due to the higher
`proportion of heterozygotes in the Chinese.
`From a genetic point of view, the different enzyme
`polymorphisms in drug metabolism are inherited inde-
`pendently. However, an inherited deficiency of different
`drug—metabolizing enzymes could occur simultaneously
`on the basis of probability. A population study of mephe-
`nytoin hydroxylation and debrisoquine hydroxylation
`was carried out in 221 unrelated normal volunteers
`
`(Kiipfer and Preisig, 1984). Twelve (5%) of them exhib-
`ited defective hydroxylation of mephenytoin, and 23
`(10%) could be identified as PMS of debrisoquine. Among
`these 35 subjects, 3 (1 female and 2 males) displayed
`
`Patent Owner, UCB Pharma GmbH — Exhibit 2028 - 0035
`
`

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`438
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`LIN AND LU
`
`simultaneously both defects of mephenytoin and de-
`brisoquine hydroxylation.
`Propranolol is hydroxylated by CYP2D6 and N—deal—
`kylated by CYP2C19. The relative contributions of these
`two isoforms to propranolol metabolism have been stud-
`ied in a panel of phenotyped normal volunteers (Ward et
`al., 1989). Six subjects were EMS of both mephenytoin
`and debrisoquine. Four subjects were PMs of debriso-
`quine but rapid metabolizers of mephenytoin. Five sub-
`jects were PMs of mephenytoin but rapid metabolizers of
`debrisoquine, and one subject had a deficiency for both
`debrisoquine and mephenytoin. PMs of either mepheny-
`toin or debrisoquine had a similar disposition of pro-
`pranolol to that of EMs, whereas the subject with both
`mutations had a tie 2 times longer than the other sub-
`jects’.
`In view of the examples presented above, it is clear
`that genetic polymorphism in drug metabolism could
`lead to clinically significant differences in pharmacoki—
`netics and pharmacological responses of some patients
`and therefore might result in adverse effects or thera-
`peutic failure. Thus, drugs metabolized by enzymes ex-
`hibiting genetic polymorphism are considered to be un-
`desirable. However,
`the development of
`a drug
`sometimes is prematurely terminated based solely on
`the fact that its metabolism is polymorphic. To avoid
`premature termination, the clinical relevance of genetic
`polymorphism must be assessed carefully. Pharmacoki-
`netic differences between phenotypes are most relevant
`for drugs with narrow therapeutic indices. For com-
`pounds with a variability of plasma concentrations out-
`side the therapeutic range that is not associated with
`adverse effects, polymorphic metabolism will be of less
`or little concern. Propranolol is a typical example. De-
`spite the critical involvement of CYP2D6 and CYP2C19
`polymorphism in the metabolism of propranolol, this
`drug is quite safe clinically. Another important factor in
`determining the go/no—go decision is the overall benefit-
`to-risk ratio. If the benefit of a drug is significantly
`greater than its risk, and dosage can be titrated by direct
`clinical monitoring, then polymorphic metabolism is of
`less consequence.
`2. N~Acetylo;tion polymorphism. Acetylation is an im-
`portant route of elimination for a large number of hydr-
`azine and arylamine drugs (Weber et al., 1990). The
`N—acety1transferase (NAT) polymorphism in humans
`was discovered as a result of studying the rate of isoni-
`azid elimination in tuberculous patients in 1960 (Evans
`et al., 1960). The patients could be classified as slow and
`rapid acetylators based on their plasma concentrations
`of isoniazid. In addition to isoniazid, sulfamethazine,
`hydralazine, procainamide, dapsone, and nitrazepam
`also are polymorphically acetylated (Evans, 1992, 1989).
`The proportions of rapid and slow acetylators vary con-
`siderably between ethnic groups. For example, the per-
`centage of slow acetylators in Egyptians and Mideast-
`erners is 80 to 90%, whereas in Asian populations, it is
`
`only 10 to 20%, with European and North American
`Caucasians having an intermediate value of 40 to 70%
`(Evans, 1989). On the other hand, other N-acetylated
`compounds, such as p-aminobenzoic acid and p-amin-
`osalicylic acid, were unable to distinguish rapid and slow
`acetylators in vivo and in vitro (Evans, 1989). These
`compounds are,
`therefore, classified as monomorphic
`substrates.
`
`Although the acetylation polymorphism was sus-
`pected for nearly 40 years, the molecular mechanics
`underlying this polymorphism were not known until
`recently. Meyer and his colleagues (Blum et al., 1990;
`Grant et al., 1991) have successfully cloned three human
`genes: NAT1, NAT2, and a related pseudogene, NATP.
`The discovery of two separate genes encoding NAT1 and
`NAT2 resolved the old question on monomorphic and
`polymorphic substrates. NAT2 has a high affinity for
`polymorphic substrates, whereas NAT1 has a high af-
`finity for monomorphic substrates. Mutations of the
`NAT2 gene result in slow acetylation. The most common
`acetylator allele in Ca