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nhibition of human intestinal walletabolism by macrolide antibiotics: Effect

f clarithromycin on cytochrome P450A4/5 activity and expression

Background: Clarithromycin increases both hepatic and intestinal availability of the selective cytochrome P450(CYP) 3A probe midazolam. This study was designed to identify determinants of variability in the extent ofintestinal wall CYP3A inhibition by clarithromycin, such as CYP3A5 genotype, and the mechanism of inhibition.Methods: Ten healthy volunteers received 500 mg oral clarithromycin twice a day for 7 days. Before and afteradministration of clarithromycin, small-bowel mucosal biopsy specimens were obtained endoscopically.Intestinal CYP3A activity was determined from the rate of 1�-hydroxymidazolam and 4-hydroxymidazolamformation by incubation of small-bowel homogenate with midazolam (25 �mol/L) and NADPH for 5minutes. Intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid was quantified by real-time reversetranscriptase–polymerase chain reaction. Intestinal CYP3A4 and CYP3A5 protein concentrations were de-termined by immunoblotting. Serum and homogenate concentrations of midazolam, clarithromycin, andmetabolites were determined by liquid chromatography–mass spectrometry. CYP3A5 genotype was deter-mined by real-time polymerase chain reaction.Results: The formation of 1�-hydroxymidazolam (1.36 � 0.46 pmol · min�1 · mg�1 at baseline versus 0.35� 0.16 pmol · min�1 · mg�1 after administration) and 4-hydroxymidazolam (0.39 � 0.12pmol · min�1 · mg�1 at baseline versus 0.12 � 0.05 pmol · min�1 · mg�1 after administration) wassignificantly (P < .001) reduced after clarithromycin administration. Clarithromycin administration did notresult in a significant change in intestinal CYP3A4 and CYP3A5 messenger ribonucleic acid and proteinexpression. All subjects had detectable serum clarithromycin concentrations after 7 days of clarithromycin(3.71 � 2.43 �mol/L). The mean concentration of clarithromycin in the intestinal biopsy homogenate was1.2 � 0.7 nmol/L (range, 0.42-2.39 nmol/L). Compared with CYP3A5 nonexpressers, subjects with at least1 CYP3A5*1 allele (CYP3A5 expressers) had greater inhibition of intestinal CYP3A activity after treatmentwith clarithromycin. There was a strong linear relationship between the decrease in intestinal CYP3A activityand baseline catalytic activity (R2 � 0.9).Conclusion: Baseline intestinal activity of CYP3A4 was a key determinant of variability of the inhibitory effectof clarithromycin among individuals. CYP3A5*1 alleles were associated with greater baseline intestinalCYP3A activity and, therefore, greater extent of inhibition. The primary in vivo mechanism was not rapidlyreversible competitive or irreversible inhibition but was likely formation of metabolic intermediate com-plexes. (Clin Pharmacol Ther 2005;77:178-88.)

Amar G. Pinto, MD, Ying-Hong Wang, PhD, Naga Chalasani, MD, Todd Skaar, PhD,Dhanashri Kolwankar, PhD, J. Christopher Gorski, PhD, Suthat Liangpunsakul, MD,
Mitchell A. Hamman, MS, Million Arefayene, MS, and Stephen D. Hall, PhD Indianapolis, Ind
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Clarithromycin is a macrolide antibiotic that haseen shown to exhibit a broad in vitro antibacterial

rom the Department of Medicine, Indiana University School of Med-icine.

his study was supported by National Institutes of Health grantsT32GM08425, AG 13718, and MO1-RR00750 and Food and DrugAdministration Cooperative Agreement FDT-001756.

eceived for publication June 4, 2004; accepted Oct 5, 2004.
eprint requests: Stephen D. Hall, PhD, Indiana University School of d
78

pectrum that includes staphylococci, streptococci, le-ionella, Haemophilus influenzae, Neisseria gonor-

Medicine, Department of Medicine, Division of Clinical Pharma-cology, Wishard Memorial Hospital, Indianapolis, IN 46202.

-mail: [email protected]/$30.00opyright © 2005 by the American Society for Clinical Pharmacologyand Therapeutics.

oi:10.1016/j.clpt.2004.10.002

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CLINICAL PHARMACOLOGY & THERAPEUTICS2005;77(3):178-88 Clarithromycin and intestinal CYP3A4/5 activity 179

hoeae, chlamydia, and many anaerobes.1 It has pri-arily been used in the treatment of respiratory tract

nfections, such as pharyngitis, sinusitis, acute bronchi-is, and community-acquired pneumonia. In combina-ion with amoxicillin (INN, amoxicilline) and a protonump inhibitor such as omeprazole, clarithromycin isndicated for the treatment of Helicobacter pylori–ssociated duodenal and gastric ulcers.2 The cyto-hrome P450 (CYP) 3A enzymes are abundantly ex-ressed in the liver and small-intestinal epithelia ofdults3 and are responsible for the metabolism of aarge proportion of the drugs in current clinical use,ncluding macrolide antibiotics, benzodiazepines, cal-ium channel blockers, immunosuppressants, and anti-onvulsants.4 In humans clarithromycin is a potent in-ibitor of both hepatic and intestinal CYP3A activity.5

xamples of drugs that are metabolized by humanYP3A enzymes and whose elimination is inhibited bylarithromycin include cyclosporine (INN, ciclos-orin),6 midazolam, omeprazole,7 and tacrolimus.8

The mechanism underlying clinically important in-ibition of CYP3A catalytic activity in vivo is usuallyssumed to be both reversible and competitive. How-ver, several clinically important CYP3A4 inhibitors,uch as clarithromycin, diltiazem, and verapamil, areeak competitive inhibitors in vitro (inhibition con-

tant, �10 �mol/L) and would not be predicted toause significant drug interactions in vivo. These inhib-tors have the capability to efficiently form metabolicntermediate (MI) complexes in human liver micro-omes in vitro, and consequently, it has been suggestedhat this phenomenon may be responsible for the inhi-ition of CYP3A activity in vivo.5,9,10 MI complexormation is important because it causes slowly revers-ble inhibition of CYP3A4, resulting in prolonged re-uction of activity of this enzyme under physiologiconditions and may explain the unexpected potency ofany of the CYP3A inhibitors and the delayed onset

nd offset of the inhibitory effect.4,5,9 However, there iso evidence for MI complexes with human CYP3A inhe liver and intestine in vivo. Therefore we designedhis study to test the hypothesis that clarithromycinnhibits intestinal wall CYP3A activity by a non–apidly reversible mechanism. We reasoned that 2ours after the last dose the concentration of clarithro-ycin in the intestinal biopsy homogenate (50� dilu-

ion) would be negligible and any observed reduction inatalytic activity would reflect a non–rapidly reversibleechanism of inhibition. The potential contribution of

ecreased intestinal wall CYP3A messenger ribonu-leic acid (mRNA) and protein concentration was also
xamined to assess whether the mechanism might in- q
olve irreversible inhibition. The extent of inhibition ofYP3A activity in the liver and gut wall is greater in

ndividuals who have a higher baseline activity.5 Thussecondary goal of our study was to determine whether

he capability of clarithromycin to inhibit intestinalYP3A was dependent on baseline CYP3A activity, asell as to quantify the impact of the genetically poly-orphic expression of CYP3A5.10-14

ETHODSSubjects. The study was performed in 10 healthy

ubjects (7 men and 3 women). All subjects were aged8 years or older and received no prescription or over-he-counter medications for 2 weeks before the study.ndividuals with intolerance to macrolide antibiotics orenzodiazepines or a significant medical history, smok-ng, or alcohol intake were excluded from the study.ne week before and during the study, subjects ab-

tained from consuming grapefruit or juice, apple juice,itrus products, or vegetables from the mustard greenamily. The Clarian and Indiana University Purdueniversity Indianapolis (IUPUI) Institutional Reviewoard approved this study. All subjects provided writ-

en informed consent.Experimental design. After an overnight fast, all

ubjects underwent upper intestinal endoscopy and 4ucosal biopsy specimens were obtained from the sec-

nd portion of the duodenum close to the ampullaefore and after administration of 500 mg clarithromy-in for 7 days. Subjects received intravenous midazo-am (Roche Pharmaceuticals, Nutley, NJ) to achieveonscious sedation for esophagogastroduodenoscopy,nd the dose of midazolam used to achieve consciousedation varied from subject to subject (range, 2-13g). Blood samples were drawn at 1-hour intervals forhours for the determination of serum midazolam and

�-hydroxymidazolam concentrations. Beginning onhe next day, all subjects received 500 mg clarithromy-in twice daily by mouth for 7 days. After the last dosef clarithromycin (day 8), subjects returned to the en-oscopy suite and were sedated as before and proximalmall bowel biopsy and blood collection were repeated.ll biopsy specimens were immediately snap frozen in

iquid nitrogen and stored at �80°C until the time ofnalysis. An additional blood sample was obtained justefore esophagogastroduodenoscopy after clarithromy-in administration for 7 days to measure serum concen-rations of clarithromycin and its metabolites. Compli-nce with clarithromycin dosing and diet, alcohol, andrug restrictions was assessed by pill count and patient
uestioning.

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CLINICAL PHARMACOLOGY & THERAPEUTICS180 Pinto et al MARCH 2005

Duodenal homogenates. Duodenal biopsy speci-ens (approximately 20 �g wet tissue weight) were

omogenized with 20 strokes by use of a handheldlass homogenizer in an ice jacket with 1 mL iceold buffer consisting of 50-mmol/L Tris(hydroxym-thyl)aminomethane, 2-mmol/L ethylenediaminetet-aacetic acid, 1-mmol/L phenylmethylsulfonyl fluoride,-mmol/L benzamidine, 50 �g aprotinin, and 20%lycerol (pH 7.4). The resultant homogenate was thennap frozen in liquid nitrogen and stored at �80°C untilssayed. Homogenate protein concentrations were de-ermined by the microassay of Lowry et al15 by use ofovine serum albumin.Midazolam hydroxylation activities. An aliquot of

.5 mL of duodenal homogenate was diluted with 0.5L of 100-mmol/L sodium phosphate buffer (pH 7.4)

nd incubated at 37°C with 25 �mol/L midazolam, andhe reaction was started with the addition of 1 �molADPH. The reaction was quenched after 10 minutesith 1 mL of ethyl acetate/hexane (50:50 [vol/vol]) and

hen 1 mL of 1N sodium hydroxide/1-mol/L glycinepH 11.3) buffer on ice. N-desmethyldiazepam washen added as an internal standard. The mixture washen extracted with an additional 3 mL of ethyl acetate/exane (50:50 [vol/vol]). The organic phase was driednder vacuum. N-desmethyldiazepam and 1�- and 4�-ydroxymidazolam were analyzed by HPLC–masspectrometry (Navigator; Finnigan, San Jose, Calif).he samples were reconstituted in 100 �L of mobilehase (20-mmol/L ammonium acetate [pH 7.4]/aceto-itrile/methanol [40:40:20, vol/vol/vol]). The separa-ion was achieved with an isocratic flow of 1 mL/min to

Phenomenex Luna C18 column (5 �m, 150 �.6–mm internal diameter; Phenomenex, Torrance,alif). The eluent was analyzed by atmospheric pressurehemical ionization (APCI) in the positive mode withone voltage of 25 V and source and probe temperaturesf 200°C and 550°C, respectively. N-desmethyldiazepamnd 1�- and 4�-hydroxymidazolam were detected withelected ion monitoring at mass-to-charge ratios of 271nd 342, respectively. The limit of quantification of theydroxylated metabolite of midazolam was 0.2 ng, andhe precision at 2 ng was 3.2%.

CYP3A4 and CYP3A5 mRNA. Total ribonucleiccid (RNA) from the samples was prepared by use ofrizol reagent (Invitrogen Corp, Carlsbad, Calif) ac-ording to the manufacturer’s instructions. RNA yieldas determined by spectrophotometry (Beckman DU40; Beckman Coulter, Inc, Fullerton, Calif), and qual-ty was assessed by the calculated 260/280-nm ratio.fter RNA isolation, all samples were stored at �80°C
ntil complementary deoxyribonucleic acid (cDNA) a
reparation. In a 20-�L reaction containing randomexamers, 2 �g of total RNA was reverse-transcribednto cDNA by use of the Promega Reverse Transcrip-ion System (Promega Corp, Madison, Wis) accordingo the manufacturer’s instructions.

Real-time polymerase chain reaction analysis. Aeal-time polymerase chain reaction (PCR) method wassed to measure the amount of CYP3A4 mRNA inntestinal biopsy specimens by use of a previouslyescribed method16 with minor modifications. Primerspecific to CYP3A4 transcripts were purchased fromntegrated DNA Technologies (Coralville, Iowa). TheYP3A4 forward primer sequence was 5�-CAT TCCCA TCC CAA TTC TTG AAG T-3�, and the reverserimer sequence was 5�-CCA CTC GGT GCT TTTTG TAT CT-3�. As a control, each sample was as-

ayed for the expression of the housekeeping genelyceraldehyde 3-phosphate dehydrogenase (GAPDH)y use of the forward primer 5�-GAA GGT GAA GGTGG AGT C-3� and the reverse primer 5�-GAA GATGT GAT GGG ATT TC-3�. The cDNA samples were

ssayed for the expression of CYP3A4 and GAPDH byse of SYBR Green Core Reagents (Applied Biosys-ems, Foster City, Calif) on a BioRad iCycler Thermalycle (Applied Biosystems). The running conditions

nclude 3 steps as follows: 95°C for 10 minutes (acti-ation of Taq), 95°C for 15 seconds (denaturation), and8°C for 1 minute (annealing step and extension). Stepsand 3 are repeated for 40 cycles. Melt-curve analysis

s used to ensure that the expected PCR products areeing generated continuously and reproducibly in eacheal-time PCR reaction. The presence of a single in-ection point on the melt curve, at the previouslystablished temperature, indicates that a single PCRroduct is generated. CYP3A5 mRNA expression wasetermined by use of a previously described method.17

dilution series (200 femtograms to 0.2 attograms)ontaining the cDNA for CYP3A4 and GAPDH wassed as a positive control and to generate a calibratoreries. These controls are required to determine theCR efficiencies and were included with every assay. A

ine was obtained by plotting cycle threshold (CT) val-es as a function of starting cDNA. The slope of thisurve was used for efficiency calculation by use of theollowing formula: E � 10(1/Slope) � 1. The relativexpression of CYP3A4 and CYP3A5 genes, expresseds fold variation over control, was calculated afteretermination of the difference between CT of theYP3A4 and CYP3A5 gene and that of the calibratorene GAPDH in the intestinal biopsy samples before
nd after treatment with clarithromycin.

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Immunoblotting. An aliquot (45 �g for CYP3A5nd 20 �g for CYP3A4) of small-bowel homogenateas mixed with Laemmli sample buffer containing

odium dodecyl sulfate (SDS) and 2-mercaptoethanolnd heated to 95°C for 4 minutes. The samples under-ent electrophoresis in 9% polyacrylamide/0.1% SDSels until the bromphenol blue dye front had run off theel. The samples were transferred onto Hybond-P hy-rophobic polyvinylidene difluoride membrane (Amer-ham Pharmacia Biotech, Piscataway, NJ) with a 25-mol/L Tris and 192-mmol/L glycine buffer at 100 V

or 20 minutes. The blots were blocked for 1 hour inhosphate-buffered saline solution containing 0.1%ween 20 and 5% nonfat dry milk. The membrane was

hen probed for 1 hour with polyclonal antibody toYP3A4 (Gentest, Woburn, Mass), anti-CYP3A5olyclonal antibody (gift from Steve Leeder, Kansasity, Mo), and a mouse monoclonal antibody to chickillin that is known to cross-react with human villinChemicon, Temecula, Calif). After extensive washingith phosphate-buffered saline solution containing.1% Tween 20, the membranes were probed with aorseradish proxidase secondary antibody and devel-ped with an enhanced chemifluorescence kit (Amer-ham Pharmacia Biotech). The membrane was exposedo BioMax film (Kodak, Rochester, NY), and opticalensities of the bands on the film were converted touantitative numbers by use of Kodak Electrophoresisocumentation and Analysis System 290 (EDAS 290)

nd Kodak 1D image analysis software. The levels ofillin, CYP3A4, and CYP3A5 in the biopsy specimensere obtained by comparison with villin standard and

erial dilutions of recombinant human CYP3A4 andYP3A5, respectively. The standards were run on the

ame gel as the biopsy samples. The relative levels ofYP3A4 and CYP3A5 in the enterocytes were ex-ressed as a ratio with the villin level of the sameample.

Correction for interbiopsy variation of enterocyteontent. CYP3A4 and CYP3A5 proteins are expressedxclusively in mature enterocytes. Enterocytes representnly a small portion of tissue obtained in the pinch biopsyf the intestine.18 It is well known that there is significantnterbiopsy variation in the content of enterocytes amongiopsy specimens obtained from single individuals, andifferences in percent enterocyte content in intestinal bi-psy specimens will alter the amount of immunoreactiverotein observed on a blot. Villin, an enterocyte-specificrotein, is able to control for the variation in biopsyontent of mature enterocytes.18 Therefore biopsy levelsf CYP3A4 were expressed as a ratio with the villin level
n the same sample. These villin-corrected values provide S
relative measure of enterocyte CYP3A4/CYP3A5oncentration.

Midazolam and clarithromycin assay. Midazolamnd 1�-hydroxymidazolam were quantified in human se-um by use of desmethyldiazepam as the internal standard,s previously described in detail.19 Compounds of interestere separated by use of a Luna C18 column (5 �m, 4.6

150–mm internal diameter; Phenomenex) equippedith a Brownlee RP-18 guard column (PerkinElmer,helton, Conn). The mobile phase (acetonitrile/20-mol/L ammonium acetate [pH 7.4]/methanol [40:40:20,

ol/vol/vol]) was delivered isocratically at 1 mL/min.esmethyldiazepam, midazolam, and 1�-hydroxy-idazolam were quantified by use of a mass spectrometer

Navigator; Finnigan) equipped with an APCI interface.he limit of quantification in serum was 0.25 ng/mL foridazolam and 1�-hydroxymidazolam; the respective cor-

esponding coefficient of variation and relative error foridazolam and 1�-hydroxymidazolam at 1.4, 10, and 40

g/mL were less than 7% and 10%.Clarithromycin, 14-hydroxyclarithromycin, and

-desmethylclarithromycin serum concentrations wereetermined by use of troleandomycin as the internaltandard, as previously described in detail.20 In brief,he compounds of interest were separated by use of a8 column (5 �m, 100 � 4.6–mm internal diameter;rownlee) and a mobile phase of 20-mmol/L ammo-ium acetate (pH 7)/methanol (20:80 [vol/vol]). Theobile phase was delivered isocratically at 1 mL/min.etection was achieved by use of a mass spectrometerith an APCI inlet (Navigator; Finnigan). The limit ofuantitation in serum and homogenate was 2.5 ng/mLor clarithromycin and its metabolites. The respectiveorresponding coefficients of variation and relative er-or for clarithromycin and its metabolites at 10 ng/mLere less than 8% and 9%.CYP3A5 genotype. Genomic deoxyribonucleic acid

as extracted from whole blood by use of the Qiagenidi Kit according to the manufacturer’s instructions

Qiagen, Valencia, Calif). CYP3A5*3, CYP3A5*6, andYP3A5*7 genotyping was carried out by use of aethod described previously21,22 with PCR conditions

s follows: 5 minutes at 95°C; 35 cycles of 0.5 minutet 94°C, 0.5 minute at 55°C, and 1 minute at 72°C; and,nally, 10 minutes at 72°C. As appropriate, the PCRroduct (5 �L) was then digested with DdeI restrictionnzyme (Roche Pharmaceuticals) in a 15-�L reactionor 8 hours at 37°C and subsequently analyzed byapillary electrophoresis by use of an Agilent Bioana-yzer (Agilent Technologies).

Statistical analysis. Data are reported as mean and
D. The effect of clarithromycin was determined by

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ESULTSEffect of clarithromycin on intestinal CYP3A cata-

ytic activity. This fixed-order study was completed by0 healthy adult volunteers (7 men and 3 women)ithout endoscopy- or sedation-related side effects.he rate of formation of 1�-hydroxymidazolam and-hydroxymidazolam in intestinal biopsy homogenatefter incubation with 25 �mol/L midazolam for 10inutes is shown in Fig 1. There was a significant

eduction (P � .001) in the rate of formation of 1�-ydroxymidazolam after 7 days of treatment with clar-thromycin (0.35 � 0.16 pmol · min�1 · mg�1) comparedith that at baseline (1.36 � 0.46 pmol · min�1 · mg�1)

Fig 1). Similarly, there was a significant reduction (P �001) in the rate of formation of 4-hydroxymidazolamfter 7 days of treatment with clarithromycin (0.12 � 0.05mol · min�1 · mg�1) compared with that at baseline0.39 � 0.12 pmol · min�1 · mg�1) (Fig 1). Theseifferences corresponded to 74% and 62% reductionsn the rate of formation of 1�-hydroxymidazolam and-hydroxymidazolam, respectively, after treatmentith clarithromycin compared with baseline. Thereas a strong correlation (R2 � 0.89) between base-

ine intestinal CYP3A activity as measured by theate of formation of 1�-hydroxymidazolam andhange (Baseline � Treatment) in CYP3A activityfter treatment with clarithromycin (Fig 2). Theean concentration of clarithromycin in the intesti-

al biopsy homogenate was 1.2 � 0.7 nmol/L (range,.42-2.39 nmol/L). One subject had an undetectablelarithromycin concentration, and another subject hadnsufficient biopsy homogenate for analysis. Of 10 sub-ects, 3 had detectable 14-hydroxyclarithromycin (range,4.0-70.7 nmol/L) and N-desmethylclarithromycinrange, 46.4-96.3 nmol/L) concentrations in the intestinalomogenate.

Effect of clarithromycin on intestinal CYP3A4 ex-ression. Representative immunoblots of intestinalYP3A4 and villin proteins before and after treatmentith clarithromycin for 7 days are shown in Fig 3, A.he concentrations of CYP3A4 protein in the intestinaliopsy homogenates expressed as the ratio of integratedensity values of CYP3A4 to villin proteins at baselinend after treatment are shown in Fig 3, B. There was noignificant change (P � .18) in intestinal CYP3A4rotein concentration ratio at baseline (20.05 � 14.03)hen compared with the concentration after 7 days of

reatment with clarithromycin (27.4 � 9.37). There was
o change (P � .87) in the ratio of intestinal CYP3A4 d
o GAPDH mRNA concentration before (0.18 � 0.20)nd after (0.19 � 0.18) treatment with clarithromycinFig 4).

Effect of clarithromycin on hepatic CYP3A activ-ty. A single-point dose-normalized midazolam con-entration and the ratio of serum 1�-hydroxymidazolamo midazolam have been shown to predict hepaticYP3A activity when intravenous midazolam is useds a probe of hepatic CYP3A in vivo.23,24 There was aignificant increase in dose-normalized serum midazo-am concentration after 7 days of treatment with clar-thromycin at 120 minutes (7.6 � 2.9 ng/mL, P � .001)nd 180 minutes (6.9 � 2.5 ng/mL, P � .001) afteridazolam dosing when compared with corresponding

oncentrations at baseline (4.53 � 1.3 ng/mL and 3.0 �.6 ng/mL, respectively) (Fig 5). There was a signifi-ant decrease (P � .001) in the ratio of serum 1�-ydroxymidazolam to midazolam after treatment withlarithromycin at 120 minutes (0.07 � 0.05) and 180inutes (0.05 � 0.03) compared with corresponding

atios at baseline (0.21 � 0.09 and 0.24 � 0.11, re-pectively) (Fig 5). The 2-fold increase in dose-ormalized serum midazolam concentration after intra-enous administration is consistent with results of ourrevious study, in which intravenous midazolam areander the curve was increased by 2.7-fold after admin-stration of clarithromycin for 7 days.5 This serves as annternal control to verify that the systemic effects oflarithromycin are comparable between this study andur previous study, which demonstrated increased gutall availability of midazolam after the same clarithro-ycin dosing regimen.Effect of CYP3A5 genotype on intestinal CYP3A

ctivity and expression. Of 10 subjects, 3 had at least 1YP3A5*1 allele (CYP3A5 expressers) and 6 had 2YP3A5*3 alleles (CYP3A5 nonexpressers) (Table I).ne subject was characterized as having CYP3A5*1/*3

nd CYP3A5*1/*7 and was presumed to be CYP3A5*3/*7n the basis of the absence of CYP3A5 mRNA androtein expression in the intestinal biopsy homogenate,nd consequently, this subject was included in theYP3A5 nonexpresser group (Table I). All subjects in theYP3A5 expresser group were of African American de-

cent. One of the subjects with the CYP3A5*1/*1 geno-ype possessed a CYP3A5*6 variant allele characterized asYP3A5*1/*6. After clarithromycin administration for 7ays, CYP3A5 expressers had a significantly greaterhange (P � .01) in the rate of formation of 1�-ydroxymidazolam (1.53 � 0.25 pmol · min�1 · mg�1)ompared with CYP3A5 nonexpressers (0.83 � 0.4mol · min�1 · mg�1) (Fig 1). There was no significant
ifference in the observed serum concentrations of clar-

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thromycin (5.9 � 3.6 �mol/L versus 3.0 � 1.8 �mol/L,� .18), 14-hydroxyclarithromycin (1.1 � 0.6 �mol/L

ersus 0.7 � 0.3 �mol/L, P � .13), or N-desmethyl-larithromycin (0.7 � 0.4 �mol/L versus 0.6 � 0.3mol/L, P � .44) between the CYP3A5 expresser andYP3A5 nonexpresser groups (Table I).There was no difference in the intestinal CYP3A4

rotein concentration ratio between CYP3A5 express-rs and CYP3A5 nonexpressers at baseline (22.11 �7.9 versus 19.16 � 13.6, P � .77) or after treatmentith clarithromycin (22.0 � 14.1 versus 29.8 � 6.5, P

.24). One subject with both CYP3A5*3 andYP3A5*7 alleles failed to express CYP3A5 protein in

he intestine. One of the subjects with theYP3A5*1/*1 genotype also failed to express CYP3A5rotein in the intestine despite expressing wild-typeYP3A5 mRNA. This discrepancy in the expression of

ntestinal CYP3A5 protein in a subject withYP3A5*1/*1 genotype may be a result of secondaryegradation of CYP3A5 protein.There was no significant difference (P � .64) in the

atio of CYP3A4 to GAPDH mRNA expression ataseline between CYP3A5 expressers (0.13 � 0.15)nd CYP3A5 nonexpressers (0.20 � 0.22). AllYP3A5 expressers had detectable intestinal CYP3A5ild-type mRNA expression compared with only 3 of 7YP3A5 nonexpressers. There was no significant dif-

erence (P � .94) in the ratio of intestinal CYP3A5 toAPDH mRNA before (0.17 � 0.20) and after (0.16 �.21) treatment with clarithromycin. The ratio of intes-inal CYP3A5 to GAPDH mRNA was 6-fold higher inhe CYP3A5 expresser group (0.25 � 0.24) when com-

Fig 1. Intestinal CYP3A activity before and affor 7 days). CYP3A activity was determined b�mol/L midazolam (MDZ) for 10 minutes an

ared with the CYP3A5 nonexpresser group (0.04 � i

.05), but this difference was not statistically differentP � .22).

ISCUSSIONWe found a significant (P � .001) reduction in

ntestinal CYP3A activity after treatment with 500 mglarithromycin for 7 days (Fig 1). There was a signif-cant (P � .001) 74% reduction in the rate of formationf 1�-hydroxymidazolam after treatment with clarithro-ycin compared with baseline. This reduction in intes-

inal CYP3A activity represents a reduction in intrinsiclearance of gut wall CYP3A (CLint). On the basis ofur previous study, administration of clarithromycin fordays increased gut wall bioavailability of midazolam

rom 0.4 (baseline) to 0.8 (after treatment).5 With thessumption that the absorption rate constant of the gutall (AG) remains unchanged, a 75% reduction in in-

estinal CYP3A intrinsic clearance (CLint,G) of mida-olam accurately predicts doubling of gut wall bioavail-bility (FG) of midazolam by use of the followingquation10: FG � AG/(AG CLint,G). Thus the site ofntestinal mucosal sampling used in this study is rep-esentative of the overall effect of clarithromycin onntestinal wall availability of midazolam.

The loss of CYP3A activity in the intestinal wallfter clarithromycin treatment could reflect altered ex-ression of enzymes and inhibition of activity by clar-thromycin and its metabolites. We were able to ruleut altered enzyme expression (see later), but the mech-nism of intestinal CYP3A inhibition by clarithromycinould involve (1) reversible, competitive inhibition; (2)ovalent modification of CYP apoprotein and irrevers-

ent with clarithromycin (500 mg twice dailytion of intestinal biopsy homogenate with 25f hydroxymidazolam formation.

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Fig 2. Relationship between intestinal CYP3A activity as measured by rate of formation of1�-hydroxymidazolam at baseline and decrease (Baseline � Treatment) in CYP3A activity after 7days of treatment with clarithromycin. Solid squares represent CYP3A5 expressers, and open
squares represent CYP3A5 nonexpressers.
Fig 3. A, Representative immunoblots of intestinal CYP3A4 and villin proteins before (B) and after(A) 500 mg clarithromycin twice daily for 7 days. Immunoblots used 50 �g of homogenate proteinprepared from intestinal biopsy specimens. B, Intestinal CYP3A4 protein concentration before andafter treatment with 500 mg clarithromycin twice daily for 7 days. Concentrations are expressed as
ratio of integrated density values of intestinal CYP3A4 to villin protein.

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omplex with CYP heme (MI complex).4 Competitivenhibition cannot explain our data because the non–nzyme-bound inhibitor is essentially absent from thentestinal tissue and our catalytic activities were deter-ined in tissue homogenates that further dilute the

nhibitor by approximately 50-fold. The mean concen-ration of clarithromycin in the intestinal homogenateas 1.2 � 0.7 nmol/L, which is approximately 10,000-

old lower than the reported inhibition constant (10mol/L) for reversible competitive inhibition ofYP3A4 by clarithromycin in vitro.25 Similarly, theoncentrations of clarithromycin metabolites were inhe low nanomolar range, and this is not consistent withignificant reversible inhibition. Thus the inhibition ofntestinal wall CYP3A by clarithromycin in vivo mustnvolve either irreversible modification of the apopro-ein or MI complex formation, and these 2 possibilitiesan be distinguished in vitro. We have previously dem-nstrated that when human liver microsomes are pre-ncubated with clarithromycin a spectrophotometricallyetermined MI complex is formed with CYP3A, result-ng in a loss of catalytic activity.9 Indeed, the formationf the MI complex accounts for all of the loss ofYP3A activity in vitro, and consequently, this phe-omenon is the most likely mechanism underlying thentestinal CYP3A inhibition in vivo. However, themall quantity of biopsy tissue available precluded airect demonstration of MI complex formation in vivo.Our conclusion that clarithromycin inhibits intestinal

YP3A in vivo by MI complex formation is supportedy the findings that clarithromycin formed MI complexith hepatic CYP3A when administered alone and toexamethasone-pretreated rats.26,27 In humans a relatedacrolide antibiotic, troleandomycin, formed MI com-

lex with hepatic CYP3A in vivo, but the amount ofYP3A protein in the liver was increased by 5-fold andrythromycin demethylation activity was reduced com-ared with that in controls.28 Similarly, 500 mg eryth-omycin 3 times a day for 7 days formed MI complexith hepatic CYP3A in vivo and increased CYP3Arotein by 2-fold in the livers of patients undergoinglective surgery.29

Clarithromycin had no effect on intestinal CYP3A pro-ein expression after administration for 7 days comparedith baseline (Fig 3, B). Several studies have demon-

trated that erythromycin and troleandomycin administra-ion results in increases in hepatic CYP3A content in bothumans and rodents by protecting the enzyme from deg-adation (ie, substrate stabilization).28-31 In rodents clar-thromycin administration for 7 days increased hepaticYP3A protein content by 3-fold compared with controls.
owever, the effect of clarithromycin or any other mac- s
olide on intestinal CYP3A protein expression has noteen studied in humans or animals. Loss of intestinalYP3A can result from a diminished protein concentra-

ion in small-intestinal mucosa biopsy specimens, as notedor grapefruit juice. In one study administration of thentestinal CYP3A inhibitor grapefruit juice (8 oz 3 times aay for 6 days) to healthy human subjects reducedYP3A4 concentration by 62% in the intestinal homog-nate, and the authors32 subsequently demonstrated thatomponents of grapefruit juice modify CYP3A4 throughuicide inhibition, resulting in accelerated degradation ofYP3A4 enzyme. However, MI complex formation be-

ween clarithromycin and CYP3A protein does not resultn stabilization or accelerated degradation of CYP3A pro-ein in the human intestine.

In rodents treatment with troleandomycin for 5 daysncreased the expression of hepatic CYP3A mRNA.30

n contrast to the up-regulation of CYP3A mRNA inhe rat liver after troleandomycin administration, ourtudy did not demonstrate a significant change in intes-inal CYP3A4 or CYP3A5 mRNA expression afterreatment with clarithromycin compared with baselineFig 4). Thus long-term administration of clarithromy-in does not result in a change in the rate of transcrip-ion of intestinal CYP3A4/CYP3A5 mRNA comparedith baseline. This difference in findings may reflectifferences in the way CYP3A mRNA is regulated inumans and rodents, as well as differences in the ef-ects of individual macrolides on CYP3A mRNA ex-ression within a given species. In addition, there isvidence to suggest that intestinal CYP3A4 and hepaticYP3A4 are regulated differentially by the tissue-

ig 4. Ratio of intestinal CYP3A4 to glyceraldehyde-phosphate dehydrogenase (GAPDH) messenger ribonucleiccid (mRNA) before and after treatment with 500 mg clar-thromycin twice daily for 7 days.

elective expression of nuclear factors.33

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CYP3A activity in the intestine and liver reflects theombined effects of CYP3A5 and CYP3A4, andYP3A5 is polymorphically expressed in the human

iver and intestine. There are several known variants inhe human CYP3A5 gene that affect CYP3A5 enzymexpression and activity in the liver and intestine. TheYP3A5*3 and CYP3A5*6 variant alleles result in im-roperly spliced CYP3A5 mRNA that fails to producefunctional CYP3A5 protein.13 The CYP3A5*7 variantllele causes single nucleotide insertion, results inrame-shift mutation, and produces a nonfunctional

Fig 5. Dose-normalized midazolam (MDZMDZ)/MDZ concentration ratio at 120 minadministration in 10 healthy volunteers before500 mg clarithromycin twice daily for 7 days

able I. Volunteer demographics and serum midazola-desmethylclarithromycin concentrations, with all subdays

SubjectNo. Race

Bodymassindex

(kg/m2)

Midazolam intravenousdose (mg)

cBaseline

Afterclarithromycin

1 Black 37.5 13 122 Black 35.1 10 123 White 26.1 10 124 Black 28.0 10 125 White 21.5 6 56 White 30.0 2 77 Black 27.5 10 118 Black 31.4 6 79 White 25.1 5 4

10 Black 30.0 11 8

†Genotyped by use of restriction fragment length polymorphism (Liu et al‡Subject was *1/*3 and *1/*7.

YP3A5 protein.14 In a study of 500 white subjects t

ho donated blood, the respective frequencies ofYP3A5*3, CYP3A5*6, and CYP3A5*7 alleles were1%, 0.1%, and 0.0%.22 The respective frequencies ofYP3A5*3, CYP3A5*6, and CYP3A5*7 alleles amonglack subjects are 27%, 15%, and 10%.13,22,34 Thetudies correlating CYP3A5 genotype and pharmaco-inetic parameters have yielded mixed results. In atudy limited to CYP3A5*3/*3 and CYP3A5*1/*3 in ahinese population, midazolam area under the curveas not different in CYP3A5*3/*3 and CYP3A5*1/*3

ubjects after oral midazolam administration.35 In con-

ntration and 1�-hydroxymidazolam (1�-OH180 minutes after intravenous midazolam

bars) and after (solid bars) administration ofk, P � .001 compared with baseline.

ithromycin, 14-hydroxyclarithromycin, andaving received 500 mg clarithromycin twice daily for

mmycinl/L)

Serum14-hydroxy-clarithromycin

(�mol/L)

SerumN-desmethyl-

clarithromycin(�mol/L)

CYP3A5genotype†

0.77 0.61 *3/*7‡1.1 0.92 *1/*30.75 0.57 *3/*30.75 0.61 *3/*31.0 0.99 *3/*30.71 0.64 *3/*30.4 0.28 *3/*31.9 0.98 *1/*10.2 0.15 *3/*30.44 0.26 *1/*6

Schaik et al22).

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rast, CYP3A5*1 genotype positively correlated with

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epatic CYP3A activity and tacrolimus dose require-ent.36 The influence of CYP3A5 genotype on the

xtent of inhibition by CYP3A inhibitors such as clar-thromycin in humans is unknown. In our studyYP3A5 expressers displayed a significantly greaterhange (1.53 � 0.24 pmol · min�1 · mg�1) in CYP3Activity after treatment with clarithromycin for 7 daysompared with the CYP3A5 nonexpresser group (0.83

0.4 pmol · min�1 · mg�1) (P � .01) (Fig 2). Theikely explanation for this phenomenon is that close-to-omplete inhibition by a strong CYP3A inhibitor suchs clarithromycin causes a greater absolute change inctivity in individuals with high baseline CYP3A ac-ivity and CYP3A expressers fall into the high activityange. This phenomenon is consistent with findings inur previous study, in which a significant correlationetween initial midazolam intestinal bioavailability andncrease in intestinal bioavailability was observed afterdays of treatment with clarithromycin.5 Subjects with

igher baseline CYP3A activity such as CYP3A5 ex-ressers may be more vulnerable to severe drug inter-ctions compared with CYP3A5 nonexpressers when atrong CYP3A inhibitor such as clarithromycin is ad-inistered. However, further studies are needed to fully

haracterize the influence of CYP3A5 genotype on theegree of intestinal CYP3A activity inhibition by cla-ithromycin.

In conclusion, long-term administration of clarithro-ycin causes slowly reversible inhibition of intestinalYP3A activity, most likely as a result of formation ofn MI complex. Subjects with a CYP3A5*1 allele tendo have higher baseline intestinal CYP3A activity and aignificantly greater inhibition of intestinal CYP3A ac-ivity after clarithromycin administration.

None of the authors reports any personal or financial conflicts ofnterest relevant to this report.

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inhibition of human intestinal wall metabolism by macrolide antibiotics: effect of clarithromycin on...

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