Mechanisms of Ageing and Development, 64 (1992) 189-199
189
Elsevier ScientificPublishers Ireland Ltd.
HEPATIC CYTOCHROME P-4503A (CYP3A) ACTIVITY IN THE ELDERLY
CHRISTINE M. HUNT *a, W I L L I A M R. WESTERKAM a, G R E G G M. STAVE b and JOANNE A.P. WILSON a aDepartment of Medicine and bDepartment of Family and Community Medicine, Duke University Medical Center, Durham, NC 27710 (USA)
(Received December4th, 1991) (Revision receivedJanuary 27th, 1992)
SUMMARY Elderly patients exhibit decreased clearance of multiple drugs biotransformed by the hepatic cytochromes P-450. The cytochromes P-450 are a superfamily of enzymes, which comprise a central component of phase I drug metabolism. Distinct isoforms metabolize specific drugs. In human liver microsomes, the glucocorticoidinducible cytochrome P-450IIIA, CYP3A, catalyzes the N-demethylation of erythromycin. To examine the activity of hepatic CYP3A in elderly males and females, erythromycin N-demethylation was examined, as reflected by the recently described [14C]erythromycin breath test in 24 healthy volunteers, age 70-88. The [14C]erythromycin breath test was measured in normal elderly males and females to: (a) determine persistence of the gender-related dimorphism (evident in younger subjects) of CYP3A activity in the elderly population, (b) examine the effect of % ideal body weight, age, diet, and medication use on the activity of human hepatic CYP3A, and (c) compare breath test results obtained in normal geriatric volunteers with published results obtained in younger subjects, to determine aging-related alterations in CYP3A enzyme activity. Erythromycin N-demethylation varied fivefold among these patients. Similar to earlier studies examining erythromycin N-demethylation in younger subjects, CYP3A activity was found to vary with gender in the geriatric cohort. [14C]Erythromycin N-demethylation at 60 min was 3.14% 4- 0.75 (n = 13) in females and 2.15% 4- 0.77 (n = 11) in males (P = 0.005). In evaluating the role of % ideal body weight and % dietary fat using multivariable linear regression analyses, [lac]erythromycin N-demethylation, was found to decline significantly as % ideal body weight increased (P = 0.001). This was not Correspondence to: Christine M. Hunt, Department of Medicine, Box 3064, Duke UniversityMedical Center, Durham, NC 27710, USA.
004%6374/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
190 confounded by gender. [14C]Erythromycin N-demethylation was not related to dietary fat intake (P < 0.13). [14C]Erythromycin N-demethylation in the elderly volunteers was similar to values reported for subjects aged 20-60. Performance of a new non-invasive test of the human hepatic glucocorticoid-inducible CYP3A in a geriatric cohort suggests that: (a) the gender-related heterogeneity in function of the glucocorticoid inducible human CYP3A persists during normal aging, (b) that the activity of CYP3A may decrease in obesity, and (c) that the activity of CYP3A is stable throughout normal aging.
Key words: Cytochromes P-450; Drug metabolism in aging; Erythromycin Ndemethylation INTRODUCTION In 1980, 11% of the population was estimated to be over age 65 [1] this age group spent 21% of the US national total of $15.1 billion for drugs [2]. It is predicted that 40% of national drug expenditures will be made by those over age 65 in 2030 [2]. This widespread use of medications in the elderly is associated with a 24% incidence of adverse drug reactions [3]. The high incidence of adverse drug effects raises the possibility that changes in the activity of the hepatic cytochromes P-450 with normal aging may result in changes in drug metabolism [4]. The hepatic cytochromes P-450 are a supergene family of hemoproteins, located in the endoplasmic reticulum, which oxidize lipophilic compounds to polar metabolites for excretion into bile and urine. These enzymes metabolize drugs, carcinogens, steroid hormones, and prostaglandins, and therefore play an important role in toxicity as well as physiologic processes. Characterizations of the human liver cytochromes P-450 have revealed at least ten distinct gene families [5]. At least five of these gene families are important in drug metabolism [6]. Animal studies have revealed aging-related decline in the function of the hepatic cytochromes P-450 [4]. Alteration of the activity of the human hepatic cytochromes P-450 with age has not been extensively studied, to date. Elderly subjects exhibit decreased clearance of multiple drugs biotransformed by the hepatic cytochromes P-450, such as: diazepam [1], verapamil [8], midazolam [9], propanolol, and quinidine [1]. Antipyrine clearance, an activity which has been classically used to determine the activity of the human hepatic cytochromes P-450, also decreases significantly with age [10,11]. The glucocorticoid-inducible human hepatic CYP3A family consist of multiple forms of closely-related hemoproteins [12-19]. The hepatic CYP3A catalyse the oxidation of clinically important drugs including erythromycin [16,20,21], nifedipine [13,22], cyclosporine [23], midazolam [24], quinidine [25] triacetyloleandomycin [26], and clotrimazole [27]. Endogenous substrates such as testosterone and
191 glucocorticoids undergo CYP3A-catalysed metabolism to their respective 6/~hydroxylated derivatives [15,18,21]. The [14C]erythromycin breath test has been shown to non-invasively profile the activity the major human hepatic cytochrome CYP3A family member [20]. Previous clinical studies, evaluating the [14C]erythromycin breath test reveal significantly higher CYP3A activity in females than males [20]. Therefore, this study sought to define the activity of human hepatic CYP3A in vivo in male and female geriatric patients age 70-90. These results will be compared to a published series performed in younger subjects, age 20-60 [20], to determine potential aging-related activity alterations. In addition to possible age-related activity modulation of the hepatic cytochromes P-450, recent studies in the rat have revealed activity changes of specific cytochrome P-450 isoforms with dietary and weight alterations [28,29]. High-cholesterol diets in rats have been shown to increase the amount and activity of CYP3A [29]. Increases in the ethanol-inducible hepatic CYP2E1, as well as total hepatic cytochrome P-450 content, have been described in an obese rat model [28]. As total body fat increases with age in humans, this study will examine the interaction of % ideal body weight and the activity of the hepatic CYP3A. Human hepatic CYP3A activity, profiled with the [14C]erythromycin breath test, was found to decline with increasing % ideal body weight in a recent study in hypertensive subjects (Hunt, unpublished observations). This study will examine the effect of normal aging on the activity of human hepatic CYP3A in vivo using the [14C]erythromycin breath test [20]. Specific questions to be addressed include: (a) persistence of the gender-related dimorphism of CYP3A activity in the elderly population, which is evident in younger subjects, (b) examination of the effect of % ideal body weight, age, diet, and medication use on the activity of human hepatic CYP3A, and (c) comparison of [14C]erythromycin breath test results obtained in normal geriatric volunteers with published results obtained in a younger subjects, to determine aging-related alterations in CYP3A enzyme activity. MATERIALSAND METHODS
[N-methyl-14C]Erythromycin and Aquasol scintillation cocktail were obtained from Dupont/New England Nuclear Co. (Boston, MA). Methylbenzethonium hydroxide and thymolphthalein were obtained from Sigma Chemical Co. (St. Louis, MO). Patient selection Twenty-four healthy elderly outpatients (age 70-88), enrolled in the Duke Center for Aging, volunteered for the study. The Duke University Center for the Study of Aging and Human Development maintains a detailed registry of healthy elderly outpatient volunteers, who have expressed interest in participating in research
192 protocols. Biographical information on each subject is available in the Duke Aging Center registry, including birthdate, medical history, medications, etc.. Female and male volunteers, age 70-90, were recruited for the study by telephone solicitation. Patients exhibiting liver disease (as evidenced by abnormal transaminases or bilirubin) were excluded from the study. Informed consent was obtained from each patient in accordance with a protocol approved by the Duke University Medical Center Institutional Review Board. For the 2 days preceding the [laC]erythromycin breath test, the subjects were instructed to record a detailed dietary history. Using the Clinfo computer, a Clinical Research Unit dietitian analyzed the diet with respect to caloric content, % saturated and unsaturated fat, % dietary cholesterol, and % total fat for the two day period. Twenty-one of 24 patients were compliant with recording their diet in sufficient detail for computer analysis.
Statistical analyses Sample size calculations were performed to estimate the numbers of subjects, to determine whether the mean [14C]erythromycin clearance differed between elderly men and women. Sample size was calculated to yield an 80% power to detect a 25% difference in the means between the two groups, with alpha = 0.05 (one-tailed). A one-tailed t-test is appropriate because it is expected that if there is a difference in the means of the two groups, the mean for the elderly males would be lower. Sample size calculations indicated that 24 subjects were required for the proposed study. Differences between group means were assessed with the Student's t-test. Least-squares regression models were used to evaluate the effect of gender, age, % ideal body weight, and diet on the metabolism of 14[C]erythromycin [30].
[14C]Erythromycin breath test protocol Patients participating in the [N-methyl-14C]erythromycin breath test protocol presented to the Duke University Medical Center Clinical Research Unit following their morning meal. Height, weight, and medications were recorded (Table I). Ideal body weight was determined by comparison with charts obtained from the Society of Actuaries and Association of Life Insurance Medical Directors of America, 1979 Build Study [31]. Subjects were classified as obese when their body weight exceeded 20% of ideal body weight [31]. The [N-methyl-14C]erythromycin breath test protocol was performed in a manner identical to previously published methods [20]. RESULTS [14C]Erythromycin N-demethylation varied fivefold among subjects, age 70-88. The amount of 14CO2 exhaled after 1 h was 0.95-5.15% of the total administered dose (Table I). The [14C]erythromycin at 10 min was found to reflect [14C]erythromycin N-demethylation at 60 min (R = 0.96) and 120 min (R = 0.91). Although multiple patients were taking medications at the time of the protocol, none of the
77 72
83 80
70
86 80 72 72 73
88
85 81
72 77
74 75 84 82 77
72
71 73 75
1 2
3
5
6
8 9 10
11
12 13
14 15
16 17 18 19 20
21
22 23 24
7
4
Age
PT. no.
Male Male Female
Male
Male Female Female Female Female
Female Female
Male Male
Female
Female Male Female Male Male
Female
Male Male
Female Female
Sex
102 127 ! 25
140
156 119 94 116 102
109 96
142 105
107
125 161 129 120 160
104
86 ! 30
93 113
% ideal body weight
36.8 27.4 N/A
24.5
34.5 30.6 53.1 28.7 30.4
38.3 20.0
28.1 32.8
28.1
N/A 40.6 N/A 19.1 30.9
25.1
24.9 21.2
30.9 19.8
fat
% dietary
[14C]ERYTHROMYCIN BREATH TEST RESULTS
TABLE I
1076 206 877
91
204 2035 2603 1648 1016
1206 1315
697 1254
1195
1006 818 1057 888 355
1732
1013 955
1312 613
DPM of 1#C02 in breath collection at 10 min
2.84% 0.95% 2.62%
2.41%
0.95% 3.70% 5.15% 3.66% 2.60%
3.17% 3.18%
2.15% 3.13%
2.86%
1.29%
2.80% 2.81% 2.73% 2.34%
3.10%
2.76% 2.78%
2.83% 2.04%
60 min
4.56% 2.26% 4.59%
4.05%
2.05% 5.63% 8.08% 5.66% 4.52%
5.21% 5.27%
3.46% 4.98%
4.81%
4.91% 4.70O/O 4.58% 3.87% 2.42%
4.94%
4.27% 4.40%
4.68% 3.61%
120 min
% of total [14Clerythromycin dose N-demethylated
Multivitamins Hydrochiorothiazide, triamterene, vitamin D, calcium Loperamide, multivitamins Chlorthalidone, theophylline, aspirin, timolol maleate opthalmic solution Timolol maleate opthalmic solution Aspirin Verapamil None None lbuprofen, verapamil, multivitamins Captopril, furosemide, aspirin, potassium chloride Psyllium Aspirin, calcium, hydrocodone bitartrate, acetaminophen, temazepam None Hydrochiorothiazide medroxyprogesterone acetate, conjugated estrogens Meclofenamate sodium None None Multivitamins, calcium Conjugated estrogens, enalapril, levothyroxin sodium, hydrochlorothiazide Probenecid, captopril, pentoxyifyiline, colchicine, sulindac None None Levothyroxin sodium, acetaminophen
Medications
'qD
194
listed medications has been reported to affect the activity of hepatic cytochrome CYP3A [5,6]. No association of [14C]erythromycin N-demethylation and estrogen use was noted, however, only 2 females were receiving conjugated estrogens. Gender strongly influenced [14C]erythromycin N-demethylation in this geriatric cohort. [14C]Erythromycin N-demethylation at 60 min was 3.14% ± 0.75 (n = 13) in females and 2.15 4- 0.77 in males (P < 0.005). Females exhibited mean [14C]erythromycin N-demethylation values 46% higher than males. The effect of age, % ideal body weight, gender, and dietary intake (e.g. % dietary fat, % saturated fat, % unsaturated fat, and % cholesterol) were examined by linear regression analyses. A strong negative correlation of [14C]erythromycin N-demethylation and % ideal body weight was observed (P < 0.001). This relationship was not confounded by gender. Analysis of dietary histories revealed no association [14C]erythromycin N-demethylation and % dietary fat (P < 0.13), % saturated dietary fat, % unsaturated dietary fat, and % dietary cholesterol. DISCUSSION
[14C]Erythromycin N-demethylation values were significantly higher in elderly females than males. In healthy medical student volunteers, age 17-23, pharmacokinetic studies examining erythromycin clearance revealed significantly higher clearance in young females than males [32]. This raises the intriguing possibility that estrogen or other hormones may increase the activity of the glucocorticoid-inducible CYP3A in women. The steroid hormone, testosterone, is believed to regulate the expression of multiple mouse renal cytochrome P-450 enzymes [33]. A gender-related dimorphism in the hepatic cytochrome P-450-catalyzed metabolism of antipyrine and diazepam has been described [1,10]. These drugs exhibit marked age-related decline in clearance in males [1,10]; however, females exhibit little age-related change in clearance of these medications. Therefore, the [14C]erythromycin breath test results obtained in the healthy elderly male and female
T A B L E 11 % [J4C]ERYTHROMYCIN
N-DEMETHYLATLON
A T 60 M I N
Gender
Age 20-60
Age 70-90
Females
3.96% ± 1.42" (n = 8) 2.40% ~ 1.39" (n = 8) 3.02% ± 1.58"
3.14% + 0.75 ( P = 0.1) (n = 13) 2.15% ± 0.77 ( P = 0.6) (n = 11) 2.69% + 0.90 ( P = 0.38)
(n = 20)
(n = 20)
Males Males a n d females
Values reported as m e a n ± S.D. *Values for patients age 2 0 - 6 0 o b t a i n e d from W a t k i n s et al. [20].
195 subjects, age 70-88, were compared by gender to [14C]erythromycin N-demethylation in male and female subjects, age 20-60, profiled in other studies (Table II). A fivefold range of values was evident in the geriatric cohort, a range similar to that observed in other series [20]. The gender-related increase in [14C]erythromycin N-demethylation among females was persistent throughout adulthood and suggests that future pharmacokinetic studies examining potential aging-related alterations performed in one sex need to be reexamined with respect to the other sex. When the [~4C]erythromycin N-demethylation values of the geriatric volunteers were compared by Student's t-test to results obtained in an earlier study performed in patients age 20-60, a slight, but statistically non-significant decrease, in [14C]erythromycin N-demethylation was seen in females with age (P < 0.10). In the postmenopausal cohort, with ovarian involution resulting in a 60% decline in estrogen production [34], CYP3A function may be less strongly influenced by estrogen. Such a mechanism could explain the slight decline in CYP3A activity in the elderly females, as compared with the younger cohort. A larger number of subjects would be required to further study this possibility. In contrast, males exhibited unchanged [14C]erythromycin N-demethylation values throughout age 20-88 (P = 0.61). In view of the known interassay variation of the breath test [20], it is important to indicate that the values in patients age 20-60 were obtained in a separate laboratory [20], therefore, an aging-dependent change in CYP3A activity cannot be conclusively excluded. The activity of the CYP3A family in human fetal liver is similar to that seen in adult liver microsomes [35]. The high fetal activity of the CYP3A enzyme family is unusual, as most fetal isoforms of the cytochromes P-450 exhibit only 5-40% of the activity of adult isoforms [35]. This suggests a critical physiological role for the CYP3A family throughout ontogeny. This study reveals the activity of CYP3A remains stable throughout normal aging, supporting a central role of human hepatic cytochrome CYP3A throughout life. Prior pharmacokinetic data reveal conflicting information on the activity of the CYP3A gene family. The urinary excretion of 6fl-hydroxycortisol, a cortisol metabolite produced by CYP3A, remains stable in males throughout age 20-88 [36]. In contrast, midazolam, another CYP3A substrate, exhibits a more rapid induction time in patients over age 50 [37], attributed to a greater volume of distribution in the elderly. No aging-related difference in hepatic biotransformation of midazolam was described [37]. The study did not examine the effect of % ideal body weight or gender on midazolam kinetics. Pharmacokinetic studies reveal that both hepatic biotransformation and renal clearance of the CYP3A substrate, quinidine, declines with normal aging [38]. Gender-related differences in quinidine metabolism have not been described. In humans, the CYP3A gene family consists of at least four isoforms. The activity of the major isoform, profiled by the [14C]erythromycin breath test, appears unchanged over normal aging. The possibility remains that the activity of one or more other CYP3A isoforms may change with age.
196 In addition to potential alterations of the human hepatic cytochromes P-450, elderly individuals may experience: (a) an increase in adipose tissue and decrease in lean body mass relative to total body weight, which may alter the volume of distribution of lipophilic drugs; and (b) decreases in hepatic blood flow [1] which contribute to the reduced clearance of 'high-clearance' oxidized drugs, such as lidocaine, imipramine, and midazolam [1]. These physiological changes contribute to the impaired clearance of the CYP3A substrates, midazolam and quinidine, described in the elderly [1,37]. Direct measurement of human liver microsomal metabolism in vitro may more accurately predict true changes in the activity of the individual human hepatic isoforms [39]. This technique has been used to profile the activity of the ethanol-inducible human hepatic CYP2E1, which catalyzes the microsomal metabolism of ethanol, acetaminophen, and the activation of the procarcinogen, N-nitrosodimethylamine. The activity of human hepatic CYP2E1 was stable between age 30-75, and was not affected by gender or % ideal body weight [39]. [14C]Erythromycin N-demethylation was found to decline significantly in association with increasing % ideal body weight (P < 0.001). This relationship was not confounded by gender. The clearance of antipyrine and diazepam, drugs metabolized by the hepatic cytochromes P-450, also decline markedly with obesity [40] largely due to increases in the volume of distribution. Pharmacokinetic studies reveal the volume of distribution of erythromycin to be significantly higher in females [32]. However, females exhibit higher [14C]erythromycin N-demethylation than males. Therefore, changes in the volume of distribution would not explain the marked decrease in activity in subjects of higher body weight. A more likely explanation is that hormonal or metabolic changes associated with obesity, such as decreased plasma concentrations of growth hormone or testosterone [41], contribute to the repression of CYP3A enzyme activity. Modulation of other hepatic cytochrome P-450 isoforms have been described in obese rats [28]. These observations have important clinical ramifications, suggesting that weight modification may significantly alter the activity of the CYP3A family. One would postulate that such changes could significantly effect the metabolism of multiple drugs metabolized by the CYP3A family (e.g. cyclosporine, midazolam, erythromycin, and quinidine). Molowa et al. have found hepatic CYP3A enzyme activity to increase threefold in rats consuming a high cholesterol diet [29]. However, in humans, CYP3A enzyme activity was not related to consumption of a high fat diet (P < 0.13). No correlation between CYP3A enzyme activity and % saturated fat, % unsaturated fat, or % dietary cholesterol was noted. The results of the [laC]erythromycin breath test, a non-invasive determinant of human hepatic CYP3A activity, in a healthy ambulatory elderly population suggest that the activity of CYP3A remains stable throughout normal aging (although a slight decline in enzyme activity in women cannot be excluded). The elderly population may exhibit similarly preserved microsomal catalysis of the CYP3A substrates: nifedipine, cyclosporine, midazolam, quinidine, triacetyloleandomycin, and clotrimazole.
197 Recently developed non-invasive tests of specific human hepatic cytochrome P-450 isoforms will importantly contribute to our knowledge of aging-related alterations of the human hepatic cytochromes P-450. In recent years, specific drugs metabolized by multiple well-characterized human hepatic cytochrome P-450 isoforms have been characterized in human liver microsomal assays in vitro, using purified isoforms and immunoinhibition studies [6]. Hence, if aging-related changes in the activity of a specific isoform are discovered, these changes would be known to significantly alter the metabolism of multiple drugs. Results of the [ 14C]erythromycin breath test and other non-invasive tests will clearly refine and advance our knowledge of geriatric pharmacology. Non-invasive tests of specific human hepatic cytochrome P-450 isoforms can elucidate the role that age, obesity, gender, and other metabolic factors play in explaining interindividual variations in the expression of the human liver cytochromes P-450. Examining the [14C]erythromycin breath test in geriatric volunteers in vivo, hepatic CYP3A function was found to be significantly influenced by gender and body weight in normal aging. ACKNOWLEDGMENTS This study was supported, in part, by a grant from the John A. Hartford Foundation, N.I.H. 1-P01-HL-42A.A.A.-01-A1, and MO1-RR-30, National Center for Research Resources, General Clinical Research Program, N.I.H. REFERENCES 1 D.J. Greenblatt, E.M. Sellers and R.I. Shader, Drug disposition in old age. New Engl. J. Med., 306 (1982) 1081-1088.
2 R.F. Vestal, Pharmacology and aging. J. Am. Geriatr. Soc., 30 (1982) 191-200. 3 M.S. Anderson, A, Gilchrist and T. Mondeika et al., American Medical Association White Paper on Elderly Health; Report of the Council on Scientific Affairs. Arch. Int. Med., 150 (1990) 2459-2472. 4 D.J. Greenblatt, D.R. Abernethy and R.I. Shader, Pharmacokineticaspects of drug therapy in the elderly. Ther. Drug Monitoring, 8 (1986) 249-255. 5 F.J. Gonzalez, Molecular genetics of the P-450 supeffamily. Pharm. Ther., 45 (1990) 1-38. 6 D.E. Ryan and W. Levin, Purification and characterization of hepatic microsomal cytochrome P-450. Pharm. Ther., 45 (1990) 153-239. 7 D.J. Henderson and C.F.A. Van Bezooijen, The Effect of age on antipyrine metabolism by liver microsomes isolated from phenobarbital-treated rats. Mech. Ageing Dev., 49 (1989) 287-294. 8 D.R. Abernethy,J.B. Schwartz and E.L. Todd et al., Verapamilpharmacodynamicsand disposition in young and elderly hypertensive patients. Ann. Int. Med., 105 (1986) 329-336. 9 J.W. Dundee, N.J. Hailiday and P.G. Loughran et aL, The influence of age on the onset of anaesthesia with midazolam. Anaesthesia, 40 (1985) 441-443. 10 D.J. Greenblatt, M. Divoll and D.R. Abernethy et al., Antipyrine kinetics in the elderly: prediction of age-related changes in benzodiazepine oxidizing capacity. J. Pharm. Exp. Ther., 220 (1982) 120-126. 11 D.J.Greenblatt, D.R. Abemethyand A. Locniskar et al., Age, sex, and nitrazepam kinetics: relation to antipyrine disposition. Clin. Pharmacol. Ther., 38 (1985) 697-703.
198 12
13
14 15
16 17
18 19
20 21
22
23
24
25 26
27 28
29 30 31
32 33
D.T. Molowa, E.G. Schuetz and S.A. Wrighton et al., Complete cDNA Sequence of a cytochrome P-450 inducible by glucocorticoids in human liver. Proc. Natl. Acad. Sci. U.S.A., 83 (1990) 5311-5315. F.P. Guengerich, M.V. Martin and P.H. Beaune et al., Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem., 261 (1986) 5051-5060. M. Kitada, T. Kamataki and K. ltahashi et ai., Significance of cytochrome P-450 (P-450 HFLa) of hhuman fetal livers in the steroid and drug oxidations. Biochem. Pharm., 36 (1987) 453-456. D.J. Waxman, C. Attisano and F.P. Guengerich et al., Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6/~-hydroxylase cytochrome P-450 enzyme. Arch. Biochem. Biophys., 263 (1988) 424-436. P.B. Watkins, S.A. Wrighton and P. Maurel et al., Identification of an inducible form ofcytochrome P-450 in human liver. Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 6310-63t4. S.A. Wrighton, D.T. Molowa and P.S. Guzelian, Identification of a cytochrome P-450 in human fetal liver related to glucocorticoid-induciblecytochrome P-450 HLp in the adult. Biochem. Pharm., 37 (1988) 3053-3055. S.A. Wrighton, B.J. Ring and P.B. Watkins et al., Identification of a polymorphically expressed member of the human cytochrome P-450 III family. Mol. Pharmacol., 36 (1989) 97-105. T, Aoyama, S. Yamano and D.J. Waxman et al., Cytochrome P-450 hPCN3, a novel cytochrome P-450IIIA gene product that is differentially expressed in adult human liver. J. Biol. Chem., 264 (1989) 10388-10395. P. Watkins, S.A. Murray and L.G. Winkelman et al., The erythromycin breath test as an assay of glucocorticoid-inducible liver cytochromes P-450, J. Clin. Invest., 83 (1989) 688-697. M.P. Arlotto, A.J. Sonderfan, C.D. Klaassen et al., Studies on the pregnenolone-16 alpha carbonitrile form of rat liver microsomal cytochrome P-450 and UDP-glucuronosyltransferase. Biochem. Pharm., 36 (1987) 3859-3866. P.H. Beaune, D.R. Umbenhauer and R.W. Bork et al., Isolation and sequence determination of a cDNA clone related to human cytochrome P-450 nifedipine oxidase. Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8064-8068. T. Kronbach, V. Fischer and U.A. Meyer, Cyclosporine metabolism in human liver: identification of a cytochrome P-450III gene family as the major cyclosporine-metabolizing enzyme explains interactions of cyclosporine with other drugs. Clin. Pharm. Ther., 43 (1988) 630-635. G. Fabre, P. Crevat-Pisano and S. Dragna et al., Involvement of the macrolide antibiotic inducible cytochrome P-450 LM3c in the metabolism of midazolam by microsomal fractions prepared from rabbit liver. Biochem. Pharm., 37 (1988) 1947-1953, F.P. Guengerich, Muller-Enoch and I.A. Blair, Oxidation of quinidine by human liver cytochrome P-450. Mol. Pharmacol., 30 (1986) 287-295. S.A. Wrighton, E.G. Schuetz and P.B. Watkins et al., Demonstration in multiple species of inducible hepatic cytochromes P-450 and their mRNAs related to the glucocorticoid-inducible cytochrome P-450 of the rat. Mol. Pharm., 28 (1985) 312-321. J.K. Ritter and M.R. Franklin, Induction and inhibition of rat hepatic drug metabolism by Nsubstituted imidazole drugs. Drug Metab. Disp., 15 (1987) 335-343. D.E. Salazar, C.L. Sorge and G.B. Corcoran, Obesity as a risk factor for drug-induced organ injury. VI. Increased hepatic P-450 concentration and microsomal ethanol oxidizing activity in the obese overfed rat. Biochem. Biophys. Res. Comm., 157 (1988) 315-320. D.T. Molowa, J.M. Lu and G.M. Cimis, Effect of diet on the concentration of cytochrome P-450 isozymes in rat liver. J. Cell Biol., 107 (1988) All15. S.A. Glantz, Primter of Biostatistics, McGraw-Hill Book Co., New York, 1987, pp. 199-238. D.H. Alpers, R.E. Clouse and W.F. Stenson, In Manual of Nutritional Therapeutics, (Source of Basic Data: 1979 Build Study, Society of Actuaries and Association of Life Insurance Medical Directors of America), Little, Brown, and Company, Boston, MA, 1985, p. 150. K.L. Austin, L.E. Mather, and C.R. Philpot et al., lntersubject and dose-related variability after intravenous administration of erythromycin. Br. J. Clin. Pharmacol., 10 (1980) 273-279. C.J. Henderson, A.R. Scott. and C.S. Yang et al., Testosterone-mediated regulation of mouse renal cytochrome P-450 isoenzymes. Biochem. J., 260 (1990) 675-681.
199 34 35 36
37 38
39 40 41
G.T. Ross, Disorders of the Ovary and Reproductive Tract. In: J.D. Wilson and D.W. Foster (eds.), Williams Textbook o f Endocrinology, 7th edn., Philadelphia, W.B. Saunders Company, 1981, p. 227. M.G. Ladona, D.J.M. Spalding and L. Ekman et al., Human fetal and adult liver metabolism of ethylmorphine. Bk~chem. Pharmacol. , 38 (1989) 3147-3155. J.J. Crowley, B.J. Cusack and S.G. Jue et al., Aging and drug interactions. II. Effect of phenytoin and smoking on the oxidation of theophyUine and cortisol in healthy men. J. Pharm. Exp. Ther., 245 (1988) 513-523. J.W. Dundee, N.J. Halliday and P.G. Loughran et al., The influence of age on the onset of anaesthesia with midazolam. Anaesthesia, 40 (1985) 441-443. D.E. Drayer, M. Hughes and B. Lorenzo et al., Prevalence of high (3S)-3 hydroxyquinidine/quinidine ratios in serum, and clearance of quinidine in cardiac patients with age. Clin. Pharmacol. Ther., 27 (1980) 72-75. C.M. Hunt, S. Strater and G.M. Stave, Effect of normal aging on the activity of human hepatic cytochrome P-4501IEI. Biochem. Pharm., 40 (1990) 1666-1669. D.R. Abernethy, D.J. Greenblatt and M. DivoU et al., Alterations in drug distribution and clearance due to obesity. J. Pharmacol. Exp, Ther., 217 (1981) 681-685. W.F. Daniel, Eating disorders: obesity and anorexia. In: J.D. Wilson and D.W. Foster (eds.), Williams Textbook of Endocrinology, 7th edn., Philadelphia, W.B. Saunders Company, 1981, pp. 1093-1095.
189
Elsevier ScientificPublishers Ireland Ltd.
HEPATIC CYTOCHROME P-4503A (CYP3A) ACTIVITY IN THE ELDERLY
CHRISTINE M. HUNT *a, W I L L I A M R. WESTERKAM a, G R E G G M. STAVE b and JOANNE A.P. WILSON a aDepartment of Medicine and bDepartment of Family and Community Medicine, Duke University Medical Center, Durham, NC 27710 (USA)
(Received December4th, 1991) (Revision receivedJanuary 27th, 1992)
SUMMARY Elderly patients exhibit decreased clearance of multiple drugs biotransformed by the hepatic cytochromes P-450. The cytochromes P-450 are a superfamily of enzymes, which comprise a central component of phase I drug metabolism. Distinct isoforms metabolize specific drugs. In human liver microsomes, the glucocorticoidinducible cytochrome P-450IIIA, CYP3A, catalyzes the N-demethylation of erythromycin. To examine the activity of hepatic CYP3A in elderly males and females, erythromycin N-demethylation was examined, as reflected by the recently described [14C]erythromycin breath test in 24 healthy volunteers, age 70-88. The [14C]erythromycin breath test was measured in normal elderly males and females to: (a) determine persistence of the gender-related dimorphism (evident in younger subjects) of CYP3A activity in the elderly population, (b) examine the effect of % ideal body weight, age, diet, and medication use on the activity of human hepatic CYP3A, and (c) compare breath test results obtained in normal geriatric volunteers with published results obtained in younger subjects, to determine aging-related alterations in CYP3A enzyme activity. Erythromycin N-demethylation varied fivefold among these patients. Similar to earlier studies examining erythromycin N-demethylation in younger subjects, CYP3A activity was found to vary with gender in the geriatric cohort. [14C]Erythromycin N-demethylation at 60 min was 3.14% 4- 0.75 (n = 13) in females and 2.15% 4- 0.77 (n = 11) in males (P = 0.005). In evaluating the role of % ideal body weight and % dietary fat using multivariable linear regression analyses, [lac]erythromycin N-demethylation, was found to decline significantly as % ideal body weight increased (P = 0.001). This was not Correspondence to: Christine M. Hunt, Department of Medicine, Box 3064, Duke UniversityMedical Center, Durham, NC 27710, USA.
004%6374/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
190 confounded by gender. [14C]Erythromycin N-demethylation was not related to dietary fat intake (P < 0.13). [14C]Erythromycin N-demethylation in the elderly volunteers was similar to values reported for subjects aged 20-60. Performance of a new non-invasive test of the human hepatic glucocorticoid-inducible CYP3A in a geriatric cohort suggests that: (a) the gender-related heterogeneity in function of the glucocorticoid inducible human CYP3A persists during normal aging, (b) that the activity of CYP3A may decrease in obesity, and (c) that the activity of CYP3A is stable throughout normal aging.
Key words: Cytochromes P-450; Drug metabolism in aging; Erythromycin Ndemethylation INTRODUCTION In 1980, 11% of the population was estimated to be over age 65 [1] this age group spent 21% of the US national total of $15.1 billion for drugs [2]. It is predicted that 40% of national drug expenditures will be made by those over age 65 in 2030 [2]. This widespread use of medications in the elderly is associated with a 24% incidence of adverse drug reactions [3]. The high incidence of adverse drug effects raises the possibility that changes in the activity of the hepatic cytochromes P-450 with normal aging may result in changes in drug metabolism [4]. The hepatic cytochromes P-450 are a supergene family of hemoproteins, located in the endoplasmic reticulum, which oxidize lipophilic compounds to polar metabolites for excretion into bile and urine. These enzymes metabolize drugs, carcinogens, steroid hormones, and prostaglandins, and therefore play an important role in toxicity as well as physiologic processes. Characterizations of the human liver cytochromes P-450 have revealed at least ten distinct gene families [5]. At least five of these gene families are important in drug metabolism [6]. Animal studies have revealed aging-related decline in the function of the hepatic cytochromes P-450 [4]. Alteration of the activity of the human hepatic cytochromes P-450 with age has not been extensively studied, to date. Elderly subjects exhibit decreased clearance of multiple drugs biotransformed by the hepatic cytochromes P-450, such as: diazepam [1], verapamil [8], midazolam [9], propanolol, and quinidine [1]. Antipyrine clearance, an activity which has been classically used to determine the activity of the human hepatic cytochromes P-450, also decreases significantly with age [10,11]. The glucocorticoid-inducible human hepatic CYP3A family consist of multiple forms of closely-related hemoproteins [12-19]. The hepatic CYP3A catalyse the oxidation of clinically important drugs including erythromycin [16,20,21], nifedipine [13,22], cyclosporine [23], midazolam [24], quinidine [25] triacetyloleandomycin [26], and clotrimazole [27]. Endogenous substrates such as testosterone and
191 glucocorticoids undergo CYP3A-catalysed metabolism to their respective 6/~hydroxylated derivatives [15,18,21]. The [14C]erythromycin breath test has been shown to non-invasively profile the activity the major human hepatic cytochrome CYP3A family member [20]. Previous clinical studies, evaluating the [14C]erythromycin breath test reveal significantly higher CYP3A activity in females than males [20]. Therefore, this study sought to define the activity of human hepatic CYP3A in vivo in male and female geriatric patients age 70-90. These results will be compared to a published series performed in younger subjects, age 20-60 [20], to determine potential aging-related activity alterations. In addition to possible age-related activity modulation of the hepatic cytochromes P-450, recent studies in the rat have revealed activity changes of specific cytochrome P-450 isoforms with dietary and weight alterations [28,29]. High-cholesterol diets in rats have been shown to increase the amount and activity of CYP3A [29]. Increases in the ethanol-inducible hepatic CYP2E1, as well as total hepatic cytochrome P-450 content, have been described in an obese rat model [28]. As total body fat increases with age in humans, this study will examine the interaction of % ideal body weight and the activity of the hepatic CYP3A. Human hepatic CYP3A activity, profiled with the [14C]erythromycin breath test, was found to decline with increasing % ideal body weight in a recent study in hypertensive subjects (Hunt, unpublished observations). This study will examine the effect of normal aging on the activity of human hepatic CYP3A in vivo using the [14C]erythromycin breath test [20]. Specific questions to be addressed include: (a) persistence of the gender-related dimorphism of CYP3A activity in the elderly population, which is evident in younger subjects, (b) examination of the effect of % ideal body weight, age, diet, and medication use on the activity of human hepatic CYP3A, and (c) comparison of [14C]erythromycin breath test results obtained in normal geriatric volunteers with published results obtained in a younger subjects, to determine aging-related alterations in CYP3A enzyme activity. MATERIALSAND METHODS
[N-methyl-14C]Erythromycin and Aquasol scintillation cocktail were obtained from Dupont/New England Nuclear Co. (Boston, MA). Methylbenzethonium hydroxide and thymolphthalein were obtained from Sigma Chemical Co. (St. Louis, MO). Patient selection Twenty-four healthy elderly outpatients (age 70-88), enrolled in the Duke Center for Aging, volunteered for the study. The Duke University Center for the Study of Aging and Human Development maintains a detailed registry of healthy elderly outpatient volunteers, who have expressed interest in participating in research
192 protocols. Biographical information on each subject is available in the Duke Aging Center registry, including birthdate, medical history, medications, etc.. Female and male volunteers, age 70-90, were recruited for the study by telephone solicitation. Patients exhibiting liver disease (as evidenced by abnormal transaminases or bilirubin) were excluded from the study. Informed consent was obtained from each patient in accordance with a protocol approved by the Duke University Medical Center Institutional Review Board. For the 2 days preceding the [laC]erythromycin breath test, the subjects were instructed to record a detailed dietary history. Using the Clinfo computer, a Clinical Research Unit dietitian analyzed the diet with respect to caloric content, % saturated and unsaturated fat, % dietary cholesterol, and % total fat for the two day period. Twenty-one of 24 patients were compliant with recording their diet in sufficient detail for computer analysis.
Statistical analyses Sample size calculations were performed to estimate the numbers of subjects, to determine whether the mean [14C]erythromycin clearance differed between elderly men and women. Sample size was calculated to yield an 80% power to detect a 25% difference in the means between the two groups, with alpha = 0.05 (one-tailed). A one-tailed t-test is appropriate because it is expected that if there is a difference in the means of the two groups, the mean for the elderly males would be lower. Sample size calculations indicated that 24 subjects were required for the proposed study. Differences between group means were assessed with the Student's t-test. Least-squares regression models were used to evaluate the effect of gender, age, % ideal body weight, and diet on the metabolism of 14[C]erythromycin [30].
[14C]Erythromycin breath test protocol Patients participating in the [N-methyl-14C]erythromycin breath test protocol presented to the Duke University Medical Center Clinical Research Unit following their morning meal. Height, weight, and medications were recorded (Table I). Ideal body weight was determined by comparison with charts obtained from the Society of Actuaries and Association of Life Insurance Medical Directors of America, 1979 Build Study [31]. Subjects were classified as obese when their body weight exceeded 20% of ideal body weight [31]. The [N-methyl-14C]erythromycin breath test protocol was performed in a manner identical to previously published methods [20]. RESULTS [14C]Erythromycin N-demethylation varied fivefold among subjects, age 70-88. The amount of 14CO2 exhaled after 1 h was 0.95-5.15% of the total administered dose (Table I). The [14C]erythromycin at 10 min was found to reflect [14C]erythromycin N-demethylation at 60 min (R = 0.96) and 120 min (R = 0.91). Although multiple patients were taking medications at the time of the protocol, none of the
77 72
83 80
70
86 80 72 72 73
88
85 81
72 77
74 75 84 82 77
72
71 73 75
1 2
3
5
6
8 9 10
11
12 13
14 15
16 17 18 19 20
21
22 23 24
7
4
Age
PT. no.
Male Male Female
Male
Male Female Female Female Female
Female Female
Male Male
Female
Female Male Female Male Male
Female
Male Male
Female Female
Sex
102 127 ! 25
140
156 119 94 116 102
109 96
142 105
107
125 161 129 120 160
104
86 ! 30
93 113
% ideal body weight
36.8 27.4 N/A
24.5
34.5 30.6 53.1 28.7 30.4
38.3 20.0
28.1 32.8
28.1
N/A 40.6 N/A 19.1 30.9
25.1
24.9 21.2
30.9 19.8
fat
% dietary
[14C]ERYTHROMYCIN BREATH TEST RESULTS
TABLE I
1076 206 877
91
204 2035 2603 1648 1016
1206 1315
697 1254
1195
1006 818 1057 888 355
1732
1013 955
1312 613
DPM of 1#C02 in breath collection at 10 min
2.84% 0.95% 2.62%
2.41%
0.95% 3.70% 5.15% 3.66% 2.60%
3.17% 3.18%
2.15% 3.13%
2.86%
1.29%
2.80% 2.81% 2.73% 2.34%
3.10%
2.76% 2.78%
2.83% 2.04%
60 min
4.56% 2.26% 4.59%
4.05%
2.05% 5.63% 8.08% 5.66% 4.52%
5.21% 5.27%
3.46% 4.98%
4.81%
4.91% 4.70O/O 4.58% 3.87% 2.42%
4.94%
4.27% 4.40%
4.68% 3.61%
120 min
% of total [14Clerythromycin dose N-demethylated
Multivitamins Hydrochiorothiazide, triamterene, vitamin D, calcium Loperamide, multivitamins Chlorthalidone, theophylline, aspirin, timolol maleate opthalmic solution Timolol maleate opthalmic solution Aspirin Verapamil None None lbuprofen, verapamil, multivitamins Captopril, furosemide, aspirin, potassium chloride Psyllium Aspirin, calcium, hydrocodone bitartrate, acetaminophen, temazepam None Hydrochiorothiazide medroxyprogesterone acetate, conjugated estrogens Meclofenamate sodium None None Multivitamins, calcium Conjugated estrogens, enalapril, levothyroxin sodium, hydrochlorothiazide Probenecid, captopril, pentoxyifyiline, colchicine, sulindac None None Levothyroxin sodium, acetaminophen
Medications
'qD
194
listed medications has been reported to affect the activity of hepatic cytochrome CYP3A [5,6]. No association of [14C]erythromycin N-demethylation and estrogen use was noted, however, only 2 females were receiving conjugated estrogens. Gender strongly influenced [14C]erythromycin N-demethylation in this geriatric cohort. [14C]Erythromycin N-demethylation at 60 min was 3.14% ± 0.75 (n = 13) in females and 2.15 4- 0.77 in males (P < 0.005). Females exhibited mean [14C]erythromycin N-demethylation values 46% higher than males. The effect of age, % ideal body weight, gender, and dietary intake (e.g. % dietary fat, % saturated fat, % unsaturated fat, and % cholesterol) were examined by linear regression analyses. A strong negative correlation of [14C]erythromycin N-demethylation and % ideal body weight was observed (P < 0.001). This relationship was not confounded by gender. Analysis of dietary histories revealed no association [14C]erythromycin N-demethylation and % dietary fat (P < 0.13), % saturated dietary fat, % unsaturated dietary fat, and % dietary cholesterol. DISCUSSION
[14C]Erythromycin N-demethylation values were significantly higher in elderly females than males. In healthy medical student volunteers, age 17-23, pharmacokinetic studies examining erythromycin clearance revealed significantly higher clearance in young females than males [32]. This raises the intriguing possibility that estrogen or other hormones may increase the activity of the glucocorticoid-inducible CYP3A in women. The steroid hormone, testosterone, is believed to regulate the expression of multiple mouse renal cytochrome P-450 enzymes [33]. A gender-related dimorphism in the hepatic cytochrome P-450-catalyzed metabolism of antipyrine and diazepam has been described [1,10]. These drugs exhibit marked age-related decline in clearance in males [1,10]; however, females exhibit little age-related change in clearance of these medications. Therefore, the [14C]erythromycin breath test results obtained in the healthy elderly male and female
T A B L E 11 % [J4C]ERYTHROMYCIN
N-DEMETHYLATLON
A T 60 M I N
Gender
Age 20-60
Age 70-90
Females
3.96% ± 1.42" (n = 8) 2.40% ~ 1.39" (n = 8) 3.02% ± 1.58"
3.14% + 0.75 ( P = 0.1) (n = 13) 2.15% ± 0.77 ( P = 0.6) (n = 11) 2.69% + 0.90 ( P = 0.38)
(n = 20)
(n = 20)
Males Males a n d females
Values reported as m e a n ± S.D. *Values for patients age 2 0 - 6 0 o b t a i n e d from W a t k i n s et al. [20].
195 subjects, age 70-88, were compared by gender to [14C]erythromycin N-demethylation in male and female subjects, age 20-60, profiled in other studies (Table II). A fivefold range of values was evident in the geriatric cohort, a range similar to that observed in other series [20]. The gender-related increase in [14C]erythromycin N-demethylation among females was persistent throughout adulthood and suggests that future pharmacokinetic studies examining potential aging-related alterations performed in one sex need to be reexamined with respect to the other sex. When the [~4C]erythromycin N-demethylation values of the geriatric volunteers were compared by Student's t-test to results obtained in an earlier study performed in patients age 20-60, a slight, but statistically non-significant decrease, in [14C]erythromycin N-demethylation was seen in females with age (P < 0.10). In the postmenopausal cohort, with ovarian involution resulting in a 60% decline in estrogen production [34], CYP3A function may be less strongly influenced by estrogen. Such a mechanism could explain the slight decline in CYP3A activity in the elderly females, as compared with the younger cohort. A larger number of subjects would be required to further study this possibility. In contrast, males exhibited unchanged [14C]erythromycin N-demethylation values throughout age 20-88 (P = 0.61). In view of the known interassay variation of the breath test [20], it is important to indicate that the values in patients age 20-60 were obtained in a separate laboratory [20], therefore, an aging-dependent change in CYP3A activity cannot be conclusively excluded. The activity of the CYP3A family in human fetal liver is similar to that seen in adult liver microsomes [35]. The high fetal activity of the CYP3A enzyme family is unusual, as most fetal isoforms of the cytochromes P-450 exhibit only 5-40% of the activity of adult isoforms [35]. This suggests a critical physiological role for the CYP3A family throughout ontogeny. This study reveals the activity of CYP3A remains stable throughout normal aging, supporting a central role of human hepatic cytochrome CYP3A throughout life. Prior pharmacokinetic data reveal conflicting information on the activity of the CYP3A gene family. The urinary excretion of 6fl-hydroxycortisol, a cortisol metabolite produced by CYP3A, remains stable in males throughout age 20-88 [36]. In contrast, midazolam, another CYP3A substrate, exhibits a more rapid induction time in patients over age 50 [37], attributed to a greater volume of distribution in the elderly. No aging-related difference in hepatic biotransformation of midazolam was described [37]. The study did not examine the effect of % ideal body weight or gender on midazolam kinetics. Pharmacokinetic studies reveal that both hepatic biotransformation and renal clearance of the CYP3A substrate, quinidine, declines with normal aging [38]. Gender-related differences in quinidine metabolism have not been described. In humans, the CYP3A gene family consists of at least four isoforms. The activity of the major isoform, profiled by the [14C]erythromycin breath test, appears unchanged over normal aging. The possibility remains that the activity of one or more other CYP3A isoforms may change with age.
196 In addition to potential alterations of the human hepatic cytochromes P-450, elderly individuals may experience: (a) an increase in adipose tissue and decrease in lean body mass relative to total body weight, which may alter the volume of distribution of lipophilic drugs; and (b) decreases in hepatic blood flow [1] which contribute to the reduced clearance of 'high-clearance' oxidized drugs, such as lidocaine, imipramine, and midazolam [1]. These physiological changes contribute to the impaired clearance of the CYP3A substrates, midazolam and quinidine, described in the elderly [1,37]. Direct measurement of human liver microsomal metabolism in vitro may more accurately predict true changes in the activity of the individual human hepatic isoforms [39]. This technique has been used to profile the activity of the ethanol-inducible human hepatic CYP2E1, which catalyzes the microsomal metabolism of ethanol, acetaminophen, and the activation of the procarcinogen, N-nitrosodimethylamine. The activity of human hepatic CYP2E1 was stable between age 30-75, and was not affected by gender or % ideal body weight [39]. [14C]Erythromycin N-demethylation was found to decline significantly in association with increasing % ideal body weight (P < 0.001). This relationship was not confounded by gender. The clearance of antipyrine and diazepam, drugs metabolized by the hepatic cytochromes P-450, also decline markedly with obesity [40] largely due to increases in the volume of distribution. Pharmacokinetic studies reveal the volume of distribution of erythromycin to be significantly higher in females [32]. However, females exhibit higher [14C]erythromycin N-demethylation than males. Therefore, changes in the volume of distribution would not explain the marked decrease in activity in subjects of higher body weight. A more likely explanation is that hormonal or metabolic changes associated with obesity, such as decreased plasma concentrations of growth hormone or testosterone [41], contribute to the repression of CYP3A enzyme activity. Modulation of other hepatic cytochrome P-450 isoforms have been described in obese rats [28]. These observations have important clinical ramifications, suggesting that weight modification may significantly alter the activity of the CYP3A family. One would postulate that such changes could significantly effect the metabolism of multiple drugs metabolized by the CYP3A family (e.g. cyclosporine, midazolam, erythromycin, and quinidine). Molowa et al. have found hepatic CYP3A enzyme activity to increase threefold in rats consuming a high cholesterol diet [29]. However, in humans, CYP3A enzyme activity was not related to consumption of a high fat diet (P < 0.13). No correlation between CYP3A enzyme activity and % saturated fat, % unsaturated fat, or % dietary cholesterol was noted. The results of the [laC]erythromycin breath test, a non-invasive determinant of human hepatic CYP3A activity, in a healthy ambulatory elderly population suggest that the activity of CYP3A remains stable throughout normal aging (although a slight decline in enzyme activity in women cannot be excluded). The elderly population may exhibit similarly preserved microsomal catalysis of the CYP3A substrates: nifedipine, cyclosporine, midazolam, quinidine, triacetyloleandomycin, and clotrimazole.
197 Recently developed non-invasive tests of specific human hepatic cytochrome P-450 isoforms will importantly contribute to our knowledge of aging-related alterations of the human hepatic cytochromes P-450. In recent years, specific drugs metabolized by multiple well-characterized human hepatic cytochrome P-450 isoforms have been characterized in human liver microsomal assays in vitro, using purified isoforms and immunoinhibition studies [6]. Hence, if aging-related changes in the activity of a specific isoform are discovered, these changes would be known to significantly alter the metabolism of multiple drugs. Results of the [ 14C]erythromycin breath test and other non-invasive tests will clearly refine and advance our knowledge of geriatric pharmacology. Non-invasive tests of specific human hepatic cytochrome P-450 isoforms can elucidate the role that age, obesity, gender, and other metabolic factors play in explaining interindividual variations in the expression of the human liver cytochromes P-450. Examining the [14C]erythromycin breath test in geriatric volunteers in vivo, hepatic CYP3A function was found to be significantly influenced by gender and body weight in normal aging. ACKNOWLEDGMENTS This study was supported, in part, by a grant from the John A. Hartford Foundation, N.I.H. 1-P01-HL-42A.A.A.-01-A1, and MO1-RR-30, National Center for Research Resources, General Clinical Research Program, N.I.H. REFERENCES 1 D.J. Greenblatt, E.M. Sellers and R.I. Shader, Drug disposition in old age. New Engl. J. Med., 306 (1982) 1081-1088.
2 R.F. Vestal, Pharmacology and aging. J. Am. Geriatr. Soc., 30 (1982) 191-200. 3 M.S. Anderson, A, Gilchrist and T. Mondeika et al., American Medical Association White Paper on Elderly Health; Report of the Council on Scientific Affairs. Arch. Int. Med., 150 (1990) 2459-2472. 4 D.J. Greenblatt, D.R. Abernethy and R.I. Shader, Pharmacokineticaspects of drug therapy in the elderly. Ther. Drug Monitoring, 8 (1986) 249-255. 5 F.J. Gonzalez, Molecular genetics of the P-450 supeffamily. Pharm. Ther., 45 (1990) 1-38. 6 D.E. Ryan and W. Levin, Purification and characterization of hepatic microsomal cytochrome P-450. Pharm. Ther., 45 (1990) 153-239. 7 D.J. Henderson and C.F.A. Van Bezooijen, The Effect of age on antipyrine metabolism by liver microsomes isolated from phenobarbital-treated rats. Mech. Ageing Dev., 49 (1989) 287-294. 8 D.R. Abernethy,J.B. Schwartz and E.L. Todd et al., Verapamilpharmacodynamicsand disposition in young and elderly hypertensive patients. Ann. Int. Med., 105 (1986) 329-336. 9 J.W. Dundee, N.J. Hailiday and P.G. Loughran et aL, The influence of age on the onset of anaesthesia with midazolam. Anaesthesia, 40 (1985) 441-443. 10 D.J. Greenblatt, M. Divoll and D.R. Abernethy et al., Antipyrine kinetics in the elderly: prediction of age-related changes in benzodiazepine oxidizing capacity. J. Pharm. Exp. Ther., 220 (1982) 120-126. 11 D.J.Greenblatt, D.R. Abemethyand A. Locniskar et al., Age, sex, and nitrazepam kinetics: relation to antipyrine disposition. Clin. Pharmacol. Ther., 38 (1985) 697-703.
198 12
13
14 15
16 17
18 19
20 21
22
23
24
25 26
27 28
29 30 31
32 33
D.T. Molowa, E.G. Schuetz and S.A. Wrighton et al., Complete cDNA Sequence of a cytochrome P-450 inducible by glucocorticoids in human liver. Proc. Natl. Acad. Sci. U.S.A., 83 (1990) 5311-5315. F.P. Guengerich, M.V. Martin and P.H. Beaune et al., Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem., 261 (1986) 5051-5060. M. Kitada, T. Kamataki and K. ltahashi et ai., Significance of cytochrome P-450 (P-450 HFLa) of hhuman fetal livers in the steroid and drug oxidations. Biochem. Pharm., 36 (1987) 453-456. D.J. Waxman, C. Attisano and F.P. Guengerich et al., Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6/~-hydroxylase cytochrome P-450 enzyme. Arch. Biochem. Biophys., 263 (1988) 424-436. P.B. Watkins, S.A. Wrighton and P. Maurel et al., Identification of an inducible form ofcytochrome P-450 in human liver. Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 6310-63t4. S.A. Wrighton, D.T. Molowa and P.S. Guzelian, Identification of a cytochrome P-450 in human fetal liver related to glucocorticoid-induciblecytochrome P-450 HLp in the adult. Biochem. Pharm., 37 (1988) 3053-3055. S.A. Wrighton, B.J. Ring and P.B. Watkins et al., Identification of a polymorphically expressed member of the human cytochrome P-450 III family. Mol. Pharmacol., 36 (1989) 97-105. T, Aoyama, S. Yamano and D.J. Waxman et al., Cytochrome P-450 hPCN3, a novel cytochrome P-450IIIA gene product that is differentially expressed in adult human liver. J. Biol. Chem., 264 (1989) 10388-10395. P. Watkins, S.A. Murray and L.G. Winkelman et al., The erythromycin breath test as an assay of glucocorticoid-inducible liver cytochromes P-450, J. Clin. Invest., 83 (1989) 688-697. M.P. Arlotto, A.J. Sonderfan, C.D. Klaassen et al., Studies on the pregnenolone-16 alpha carbonitrile form of rat liver microsomal cytochrome P-450 and UDP-glucuronosyltransferase. Biochem. Pharm., 36 (1987) 3859-3866. P.H. Beaune, D.R. Umbenhauer and R.W. Bork et al., Isolation and sequence determination of a cDNA clone related to human cytochrome P-450 nifedipine oxidase. Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8064-8068. T. Kronbach, V. Fischer and U.A. Meyer, Cyclosporine metabolism in human liver: identification of a cytochrome P-450III gene family as the major cyclosporine-metabolizing enzyme explains interactions of cyclosporine with other drugs. Clin. Pharm. Ther., 43 (1988) 630-635. G. Fabre, P. Crevat-Pisano and S. Dragna et al., Involvement of the macrolide antibiotic inducible cytochrome P-450 LM3c in the metabolism of midazolam by microsomal fractions prepared from rabbit liver. Biochem. Pharm., 37 (1988) 1947-1953, F.P. Guengerich, Muller-Enoch and I.A. Blair, Oxidation of quinidine by human liver cytochrome P-450. Mol. Pharmacol., 30 (1986) 287-295. S.A. Wrighton, E.G. Schuetz and P.B. Watkins et al., Demonstration in multiple species of inducible hepatic cytochromes P-450 and their mRNAs related to the glucocorticoid-inducible cytochrome P-450 of the rat. Mol. Pharm., 28 (1985) 312-321. J.K. Ritter and M.R. Franklin, Induction and inhibition of rat hepatic drug metabolism by Nsubstituted imidazole drugs. Drug Metab. Disp., 15 (1987) 335-343. D.E. Salazar, C.L. Sorge and G.B. Corcoran, Obesity as a risk factor for drug-induced organ injury. VI. Increased hepatic P-450 concentration and microsomal ethanol oxidizing activity in the obese overfed rat. Biochem. Biophys. Res. Comm., 157 (1988) 315-320. D.T. Molowa, J.M. Lu and G.M. Cimis, Effect of diet on the concentration of cytochrome P-450 isozymes in rat liver. J. Cell Biol., 107 (1988) All15. S.A. Glantz, Primter of Biostatistics, McGraw-Hill Book Co., New York, 1987, pp. 199-238. D.H. Alpers, R.E. Clouse and W.F. Stenson, In Manual of Nutritional Therapeutics, (Source of Basic Data: 1979 Build Study, Society of Actuaries and Association of Life Insurance Medical Directors of America), Little, Brown, and Company, Boston, MA, 1985, p. 150. K.L. Austin, L.E. Mather, and C.R. Philpot et al., lntersubject and dose-related variability after intravenous administration of erythromycin. Br. J. Clin. Pharmacol., 10 (1980) 273-279. C.J. Henderson, A.R. Scott. and C.S. Yang et al., Testosterone-mediated regulation of mouse renal cytochrome P-450 isoenzymes. Biochem. J., 260 (1990) 675-681.
199 34 35 36
37 38
39 40 41
G.T. Ross, Disorders of the Ovary and Reproductive Tract. In: J.D. Wilson and D.W. Foster (eds.), Williams Textbook o f Endocrinology, 7th edn., Philadelphia, W.B. Saunders Company, 1981, p. 227. M.G. Ladona, D.J.M. Spalding and L. Ekman et al., Human fetal and adult liver metabolism of ethylmorphine. Bk~chem. Pharmacol. , 38 (1989) 3147-3155. J.J. Crowley, B.J. Cusack and S.G. Jue et al., Aging and drug interactions. II. Effect of phenytoin and smoking on the oxidation of theophyUine and cortisol in healthy men. J. Pharm. Exp. Ther., 245 (1988) 513-523. J.W. Dundee, N.J. Halliday and P.G. Loughran et al., The influence of age on the onset of anaesthesia with midazolam. Anaesthesia, 40 (1985) 441-443. D.E. Drayer, M. Hughes and B. Lorenzo et al., Prevalence of high (3S)-3 hydroxyquinidine/quinidine ratios in serum, and clearance of quinidine in cardiac patients with age. Clin. Pharmacol. Ther., 27 (1980) 72-75. C.M. Hunt, S. Strater and G.M. Stave, Effect of normal aging on the activity of human hepatic cytochrome P-4501IEI. Biochem. Pharm., 40 (1990) 1666-1669. D.R. Abernethy, D.J. Greenblatt and M. DivoU et al., Alterations in drug distribution and clearance due to obesity. J. Pharmacol. Exp, Ther., 217 (1981) 681-685. W.F. Daniel, Eating disorders: obesity and anorexia. In: J.D. Wilson and D.W. Foster (eds.), Williams Textbook of Endocrinology, 7th edn., Philadelphia, W.B. Saunders Company, 1981, pp. 1093-1095.
