Heterogeneity of CYP3A Isoforms Metabolizing Erythromycin and Cortisol

Heterogeneity of CYP3A isoforms metabolizing erythromycin and cortisol

The N-demethylation of erythromycin and 6P-hydroxylation of cortisol are both functions of the glucocorticoid-inducible CYP3A in human liver microsomes. To determine whether 6P-hydroxylation and erythromycin N-demethylation are catalyzed by similar or distinct CYP3A isoforms, erythromycin N-demethylase activity, as reflected by the recently described 14[C]-erythromycin breath test, was compared with urinary 6P-hydroxycortisoYcortiso1 ratios, a measure of cortisol 6P-hydroxylase activity, in nine patients. Erythromycin N-demethylation varied fourfold and 6P-hydroxycortisoVcortiso1 ratios varied sevenfold among the subjects; no correlation was found between these activities (3= 0.065). New noninvasive tests of CYP3A strongly suggest cortisol 6P-hydroxylation and erythromycin N-demethylation are performed by distinct CYP3A isoforms. (CLINPHARMACOL THER 1992;51:18-23.)

Christine M. Hunt, MD," Paul B. Watkins, MD, Paul Saenger, MD, Gregg M. Stave, MD, JD, MPH, Neal Barlascini, MD, Charles 0.Watlington, MD, Jackson T. Wright, Jr., MD, and Philip S. Guzelian, MD~ Richmond, Va., Ann Arbor, Mich., and Bronx, N.T.

The hepatic cytochromes P450 are a supergene fam- pine, cyclosporine, midazolam, quinidine, troleando- ily of microsomal hemoproteins that oxidize lipophilic mycin, and c1otrimazole.l Important endogenous compounds to polar metabolites for excretion into bile substrates, such as testosterone and glucocorticoids, and urine. Characterizations of the human liver cy- also undergo CYP3A-catalyzed metabolism to their tochromes P450 have revealed at least nine distinct respective 6P-hydroxylated derivatives in humans495 gene families.' The glucocorticoid-inducible human and rats.''-'* hepatic CYP3A gene family consist of at least four The two major human liver CYP3A isoforms, closely related hem~~roteins.~.~The hepatic CYP3A CYP3A3 and CYP3A4, are encoded by distinct genes; superfamily includes family members CYP3A3 (for- however, these two genes and proteins are so similar merly known as HLp), CYP3A4 (hPCN1 or P450NF), that these forms cannot be reliably distinguished by CYP3A5 (hPCN3), and CYP3A6 (HLp2 or HFLa). oligonucleotide hybridization ~tudies'~"~or immuno- The isoforms CYP3A3 and CYP3A4 metabolize clini- chemical detection. l5 By use of noninvasive tests of cally important drugs, including erythromycin, nifedi- human hepatic CYP3A function, we sought to determine whether identical or distinct isoforms of the CYP3A gene family performed cortisol 6P-hydroxylation and erythromycin N-demethylation. These activi- From the Department of Medicine, Medical College of Virginia, Richmond; the Department of Medicine, University of Michigan, ties were examined by (I) urinary 6P-hydroxycorti- Ann Arbor; and the Department of Pediatrics, Montefiore Medi- sol/cortisol ratios and (2) the ['4~]-erythromycin cal Center, Bronx. breath test, respectively. The urinary 6P-hydroxycorti- Supported by grants AM-18976 and ES-03699 from the National In- sol/cortisol ratio corrects for changes in glucocorticoid stitutes of Health (Bethesda, Md.). production rates and therefore more closely reflects Received for publication July 1, 1991; accepted Aug. 28, 1991. Reprint requests: Christine M. Hunt, MD, Box 3064, Duke Univer- rates of hepatic cortisol 6~-hydroxylation.'6.'7 On the sity Medical Center, Durham NC 27710. basis of the observation that erythromycin is a specific "Supported by an Associate Investigator's Award from the Veterans substrate for the hepatic CYP~A,~.'~Watkins et a1.18 Administration (McGuire VA Medical Center, Richmond, Va.) recently showed that measurement of exhaled 14c02 and by an American Liver Foundation Post-Doctoral Fellowship after administration of ['4~]-erythromycinprovides a Award. hRecipient of the Burroughs Wellcome Toxicology Scholar Award specific and reproducible noninvasive measurement of (Richmond, Va.). the amounts of the hepatic immunoreactive CYP3A 1311133447 and erythromycin N-demethylase activity in liver mi- VOLUME 51 NUMBER 1 Heterogeneity ofCYP3 isofm 19

Table I. Measurement of urinary 66-hydroxycortisol/cortisol ratios and [14c]-erythromycin metabolism at 60 minutes in subjects with hypertension

24-Hr urine % Total 14C- 6P-OH Ratio of erythromycin Patient Age Ideal body cortisol urinary 6P-OH metabolism No. (yr) Gender Race weight (%) (mglday) cortisollcortisol (60 min) Medications 1 45 Female Black 110 222.7 8.7 4.0 None 2 65 Female Black 121 158.1 5.3 3.1 Ibuprofen, glyburide hy - drochlorothiazide, chlorazepate dipotassium 3 57 Female White 139 443.5 7.4 2.6 Atenolol, triamterene, hydrochlorothiazide, calcium 4 47 Female Black 147 360.0 14.2 2.3 Gemfibrozil, atenolol 5 52 Female Black 174 222.7 28.6 2.0 Propanolol, hydrochlorothiazide 6 50 Female White 223 386.4 14.6 1 .O Chlorthalidone 7 72 Male White 97 219.8 9.6 2.1 P-Blocker study drug 8 67 Male White 115 363.6 6.6 1.9 P-Blocker study drug 9 50 Male White 185 198.0 3.9 1.7 Diltiazem, atenolol, aspirin

crosomes. Moreover, because erythromycin decreases state before inclusion in the study.) Informed consent the clearance of methylprednisolone in humans,19 was obtained from each patient in accordance with a it seemed reasonable to propose that cortisol 6P-hy- protocol approved by the Medical College of Virginia droxylation and erythromycin N-demethylation may Committee for the Conduct of Human Research (Rich- be performed by the same or similar CYP3A iso- mond, Va.). Each volunteer had a plasma free cortisol form(s). To examine this possibility, the erythromycin determination at 8 AM and a 24-hour urine collection N-demethylation activity was measured by the for cortisol, 6P-hydroxycortisol, and creatinine deter- [14~]-erythromycinbreath test in a convenience sam- mination. All antihypertensive medications were dis- ple of nine patients with hypertension whose cortisol continued for at least 1 month before plasma and urine 6P-hydroxylase activity was previously characterized. cortisol and 66-hydroxycortisol collections. After suc- Despite the abundant evidence for a common catalyst cessful completion of these tests, patients received for these reactions, we conclude that erythromycin antihypertensive medications as deemed appropriate N-demethylation and cortisol 6P-hydroxylation pro- by their private physician. The patients were subse- ceed by distinct pathways in humans. quently asked to participate in the [14~]-erythromycin breath test protocol. A convenience sample of nine MATERIAL AND METHODS of the 28 volunteers consented to participate in the [14~]-~-~ethyl-erythromycinand Aquasol scintilla- ['4~]-erythromycinbreath test protocol. tion cocktail were obtained from Du Pont Diagnostic Measurement of urinary 6P-hydroxycortisol. Uri- Imaging Division (North Billerica, Mass.). Methyl- nary free cortisol was measured at Hazelton Labora- benzethonium hydroxide and thymolphthalein were tory, Vienna, Va., by radioimmunoassay. Urinary 6P- obtained from Sigma Chemical Co. (St. Louis, Mo.). hydroxycortisol values were determined by the Patient selection. A group of 28 patients (age laboratory of P. Saenger by use of a radioimmunoas- range, 45 to 72 years) recently diagnosed with hyper- say.I6 A single patient's urine (patient 9) underwent tension were prospectively enrolled in the study as urinary 6P-hydroxycortisol and cortisol profiling by paid volunteers. All patients had diastolic blood pres- HPLC analysis, following the method of Kishida and sures between 90 and 110 mm Hg on at least two sep- ~ukushima.~' arate occasions. Patients who exhibited cardiac dis- ['4~]-~rythromycinbreath test protocol. The ease, pulmonary disease, or diabetes mellitus were [14~]-~-methyl-erythromycinbreath test protocol was excluded from the study. (All patients underwent de- performed in a manner identical to previously pub- termination of plasma renin and aldosterone to ex- lished methods.18 Ideal body weight was determined clude primary hyperaldosteronism or hyperreninemic by comparison with charts obtained from the Society CLIN PHARMACOL THER 20 Hunt et al. JANUARY 1992

Urinary 6D-OH Cortisol/Cortisol Ratio

Fig. 1. Nine volunteers underwent a 24-hour urine collection for cortisol, 6P-hydroxycortisol, and creatinine determination. Urinary free cortisol was measured at Hazelton Laboratory, Vienna Va., by radioimmunoassay. Urinary 6P-hydroxycortisol values were determined by the laboratory of P. Saenger by radioimmunoassay. l8 Results were expressed as the ratio of urinary 6P-hydroxycortisol/cortisol. The ['4C]-N-methyl-erythromycinbreath test protocol was performed as described previou~ly.~~Regression analyses were used to assess the relationship between 6P-hydroxycortisol/cortisol ratios and ['4C]-erythromycin clearance. of Actuaries and Association of Life Insurance Medi- among the patients (Table I). This range is similiar to cal Directors of America, 1979 Build previously measured control values in adults.26 No Statistical analyses. Regression analyses were used significant association was found between age, gen- to assess the relationship between 6P-hydroxycorti- der, or race and the ratio of urinary 6P-hydroxycortisol/cortisol ratios and ['4~]-erythromycinclearance sol/cortisol. and to evaluate the effect of the potential confounders The patients then underwent ['4~]-erythromycin of age, gender, race, and percentage of ideal body breath test analyses between 9 and 11 AM (within 2 weight. Differences between group means were as- hours of breakfast). ['4~]-~~thromycinN-demethyla- sessed with the Student t test. tion curves revealed a fourfold difference in the l4co2 production among patients. Of the total administered RESULTS dose of ['4~]-~-methyl-erythromycin,1.O% to 4.0% Plasma free cortisol (8 AM) was normal in the nine was detected as l4co,exhaled in the first hour (Table patients with newly detected hypertension participat- I). The amount of '4~~,exhaled after 2 hours was ing in this study (results not shown). From measure- 2.0% to 4.9% of the total administered dose. This ments of 6P-hydroxycortisol and of cortisol in 24-hour range of values is not significantly different from pub- urine collections obtained in these patients, 6P- lished results of ['4~]-erythromycinbreath tests car- hydroxylase activity was estimated as the ratio of uri- ried out on subjects without hypertension.'8 The nary 6P-hydroxycortisol and cortisol (6P-hydroxy- [14~]-erythromycinbreath test results, expressed as a cortisol/cortisol). As described previously, this ratio percentage of total dose of ['4~]-~-methyl-erythromy- corrects for changes in glucocorticoid production rates cin metabolized at 60 minutes, and the urinary 6P- and therefore more closely reflects rates of cortisol hydroxycortisol/cortisol ratio showed no statistical as- 6P-hydro~ylation.'~~'~The urinary 6P-hydroxycorti- sociation (Fig. 1; r2 = 0.065). sol/cortisol ratios ranged from 3.9 to 28.6 (sevenfold) Of the medications being taken by the participants, VOLUME 51 NUMBER 1

4 Males

% Ideal Body Weight

Fig. 2. The ['4C]-N-methyl-erythromycin breath test protocol was performed as described previ- ous~~~~in nine volunteers. 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 .23 Regression analyses were used to assess the relationship between ['4C]-eryth- rornycin N-dernethylation and percentage of ideal body weight, when examined by gender. only the diltiazem ingested by patient 9 could poten- However, when urinary 6P-hydroxycortisol/cortisol tially interfere with this study by competitively inhib- ratios were compared with percentage of ideal body iting CYP3A and reducing 14c02prod~ction.~~ Pa- weight, this ratio was not found to show an associa- tient 9 displayed the lowest [14~]-erythromycin tion (r2 = 0.15). metabolism of the men tested and the lowest urinary 6P-hydroxycortisol/cortisol ratio. DISCUSSION No significant gender-dependent difference in eryth- The most striking finding was the lack of correla- romycin N-demethylation was detected in this small tion (3 = 0.065) between our estimates of 6P- patient population (p = 0.36). However, it was ob- hydroxylation and of erythromycin N-demethylation in served that percentage of ideal body weight was sig- each subject, even though these catalytic activities are nificantly negatively associated with the metabolism supported by CYP3A-related isoforms in human liver of [14~]-~-methyl-erythromycinat 60 minutes in both micro some^.^^^^'^ This suggests that closely related men and women (Fig. 2; 3 = 0.91 in women; r2 = but separate CYP3A isoforms in the liver mediate 0.90 in men). When the effect of percentage of ideal these two activities. Studies of an anhepatic man body weight was compared with the [14~]-~-methyl- strongly support the conclusion that the [14c]-erythro- erythromycin metabolism at 60 minutes in the group mycin breath test largely reflects CYP3A4-catalyzed as a whole, the correlation was less remarkable (r2 = erythromycin N-demethylation in the liver.24 How- 0.46). Even though the single obese male patient was ever, extrahepatic CYP3A-related cytochromes have also receiving treatment with diltiazam, a calcium been detected immunohistochemically in human kid- channel blocker metabolized by CYP~A,~~exclusion ney, small intestine, polymorphonuclear leukocytes, of this subject from the analyses still left a negative pancreas, gall bladder, skin, testes, and ovaries25 and correlation between [14~]-erythromycinN-demethyla- could account for a significant amount of urinary ex- tion and percentage of ideal body weight (r2 = 0.42). cretion of 66-hydroxycortisol. CLIN PKAItMACOL THER 22 Hunt et al. JANUARY 1992

The ['4C]-erythromycin breath test, performed in by glucocorticoids in human liver. Proc Natl Acad Sci this small group of patients with hypertension exhib- USA l986;83:53 11-5. ited similiar rates of [14~]-~-methyl-erythromycinme- Guengerich FP, Martin MV, Beaune PH, Kremers P, tabolism at 60 and 120 minutes to those obtained for a Wolff T, and Waxman DJ. Characterization of rat and human liver microsomal cytochrome P-450 forms in- hospitalized population." A fourfold range of values volved in nifedipine oxidation, a prototype for genetic was seen in both patient groups. Patients with hyper- polymorphism in oxidative drug metabolism. J Biol tension exhibited a range of values (sevenfold varia- Chem 1986;261:5051-60. tion) in urinary 6P-hydroxycortisollcortisol ratios sim- Kitada M, Kamataki T, Itahashi K, Rikihisa T, ilar to results from patients without hypertension.26 Kanakubo Y. Significance of cytochrome P-450 (P-450 Preliminary studies had revealed increased hepatic HFLa) of human fetal livers in the steroid and drug ox- cortisol 6P-hydroxylase activity, as reflected by uri- idations. Biochem Pharmacol 1987;36:453-6. nary 6P-hydroxycortisol/cortisol ratios,I6 in a subset Waxman DJ, Attisano C, Guengerich FP, and Lapen- of patients with essential hypertension, most of whom son DP. Human liver microsomal steroid metabolism: were black (Watlington CO, Wright JT, Grogan WM. identification of the major microsomal steroid hormone Unpublished observations, July 1990). However, de- 6P-hydroxylase cytochrome P-450 enzyme. Arch Bio- spite the suggestion that 6P-hydroxycortisol evokes chem Biophys 1988;263:424-36. Watkins PB, Wrighton SA, Maurel P, et al. Identifica- hypertension,27'29 the measured parameters of 6P- tion of an inducible form of cytochrome P-450 in hu- hydroxycortisol production did not support this hy- man liver. Proc Natl Acad Sci USA 1985;82:6310-4. pothesis within this group of patients with hyperten- Wrighton SA, Molowa DT, Guzelian PS. Identification sion. This may be attributable to the wide variety of of a cytochrome P-450 in human fetal liver related to etiologic factors that contribute to hypertension, in ad- glucocorticoid-inducible cytochrome P-450 HLp in the dition to 6P-hydroxycortisol, as well as a sampling ar- adult. Biochem Pharmacol 1988;37:3053-5. tifact attributable to the small size of our patient pop- Wrighton SA, Ring BJ, Watkins PB, Vandenbranden ulation. M. Identification of a polymorphically expressed mem- The [14~]-erythromycinN-demethylation activity ber of the human cytochrome P-450111 family" Mol showed a strong negative association with percentage Pharmacol 1989;36:97-105. of ideal body weight but no association with 6P- Aoyama T, Yamano S, Waxman DJ, et al. Cytochrome P-450 hPCN3, a novel cytochrome P-450 IIIA gene hydroxylase activity. This finding suggests the intrigu- product that is differentially expressed in adult human ing possibility that specific CYP3A isoforms may de- liver. J Biol Chem 1989;264:10388-95. crease in obese subjects. It would be desirable to con- Cheng KC, Schenkman JB. Testosterone metabolism firm this novel observation with additional techniques by cytochrome P-450 isozymes RLM3 and RLM5 and to exclude such alternative explanations as an expan- by microsomes. Metabolite identification. J Biol Chem sion of the body's formaldehyde pool, resulting in a 1983;258:11738-44. lower apparent rates of l4co2production. Increases in Waxman DJ, Dannan GA, Guengerich FP. Regulation total hepatic cytochrome P450 content, as well as the of rat hepatic cytochrome P-450: age-dependent expres- ethanol-inducible CYP2E1, have recently been de- sion, hormonal imprinting, and xenobiotic inducibility scribed in a strain of obese rats.30 The possibility that of six specific isoenzymes. Biochemistry 1985;24: obesity resulted in the reciprocal repression of other 4409- 17. 12. Waxman DJ, KO A, Walsh C. Regioselectivity and isoforms, a commonly observed effect of induction, stereospecificity of androgen hydroxylations catalyzed was not examined. The present approach of using by cytochrome P-450 isozymes purified from pheno- noninvasive methods holds promise as a way to eluci- barbital-induced rat liver. J Biol Chem 1983;258: date the roles that obesity and other previously unrec- 1 1937-47. ognized factors play in explaining interindividual vari- 13. Schuetz JD, Molowa DT, Guzelian PS. Characteriza- ations in the expression of the human liver tion of cDNA encoding a new member of the glucocor- cytochromes P450. ticoid-response cytochromes P450 in human liver. Arch Biochem Biophys 1989;274:355-65. 14. Bork RW, Muto T, Beaune PH, et al. Characterization References of mRNA species related to human liver cytochrome 1. Gonzalez FJ. Molecular genetics of the P-450 super- P-450 nifedipine oxidase and the regulation of catalytic family. Pharmacol Ther 1990;45:1-38. activity. J Biol Chem 1989;264:910-9. 2. Molowa DT, Schuetz EG, Wrighton SA, et al. Com- 15. Beaune PH, Umbenhauer DR, Bork RW, Lloyg RS, plete cDNA sequence of a cytochrome P-450 inducible Guengerich FP. Isolation and sequence determination of VOLUME 51 NUMBER 1 Heterogeneity of CTP3 isofm 2 3

a cDNA clone related to human cytochrome P-450 ni- enzymes involved in the N-demethylation of diltiazem. fedipine oxidase. Proc Natl Acad Sci USA 1986; Drug Metab Dispos l99O;l8:7 11-9. 83:8064-8. 24. Watkins PB, Kolars JC, Merion RM, Campbell DA, 16. Saenger P. 6P-Hydroxycortisol in random urine sam- Fishman K, Saenger P. Erythromycin and 6P-OH corti- ples as an indicator of enzyme induction. CLINPHARMA- sol: studies in 32 patients and in an anhepatic man" Clin COL THERl983;34:8 18-21, Res 1989;32:342A. 17. Roots I, Holbe R, Hovennann W, Nigam S, Heinem- 25. Murray GI, Barnes TS, Sewell HF, et al. The immuno- eyer G, and Hildebrandt AG. Quantitative determina- cytochemical localisation and distribution of cyto- tion by HPLC of urinary 6P-hydroxycortisol, an indica- chrome P-450 in normal human hepatic and extrahe- tor of enzyme induction by rifampicin and antiepileptic patic tissues with a monoclonal antibody to human drugs. Eur J Clin Pharmacol 1979; 16:63-71. cytochrome P-450. Br J Clin Pharmacol 1988;25: 18. Watkins P, Murray SA, Winkelman LG, Heuman 465-75. DM, Wrighton SA, Guzelian PS. The erythromycin 26. Ohnhaus EE, Breckenridge AM, Park BK. Urinary ex- breath test as an assay of glucocorticoid-inducible cretion of 6P-hydroxycortisol and the time course mea- liver cytochromes P-450. J Clin Invest 1989;83:688- surement of enzyme induction in man. Eur J Clin Phar- 97. macol 1989;36:39-46. 19. LaForce CF, Szefler SJ, Miller MF, Ebling W, Brenner 27. Watlington CO, Atkins JL, McNeal JS, Grogan WM, M. Inhibition of methylprednisolone elimination in the Johnson JP. Corticosterone is converted to 6P-hydroxy- presence of erythromycin therapy. J Allergy Clin Im- corticosterone in rat: effects of the metabolite on uri- munol 1983;72:34-9. nary electrolyte excretion. J Steroid Biochem 1988; 20. Kishida S, Fukushima DK. "Radioimmunoassay of 6P- 31 :947-54. hydroxycortisol in human plasma and urine. Steroids 28. Yamaji T, Motohashi K, Morakara S, et al. Urinary ex- 1977;30:741-9. cretion of 6P-hydroxycortisol in states of altered thyroid 21. Alpers DH, Clouse RE, Stenson WF. Manual of nutri- function. J Clin Endocrinol Metab 1969;29:801-6. tional therapeutics. (Source of basic data: 1979 Build 29. Grogan W, Fidelman M, Newton D, Duncan R, Study, Society of Actuaries and Association of Life In- Watlington CO. A corticosterone metabolite produced surance Medical Directors of America). Boston: Little, by A6 (toad kidney) cells in culture: identification and Brown, 1985: 150. effects on sodium transport. Endocrinology 1985; 22. Austin KL, Mather LE, Philpot CR, McDonald PJ. In- 116: 1189-94. tersubject and dose-related variability after intravenous 30. Salazar DE, Sorge CL, Corcoran GB. Obesity as a risk administration of erythromycin. Br J Clin Pharmacol factor for drug-induced organ injury. VI. Increased he- 1980;10:273-9. patic P-450 concentration and microsomal ethanol oxi- 23. Pichard L, Gillet G, Fabre I, et al. Identification of the dizing activity in the obese overfed rat. Biochem Bio- rabbit and human cytochromes P-450IIIA as the major phys Res Commun 1988;157:315-20.

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