effect of hyperlipidemia on ketoconazole–midazolam drug–drug interaction in rat

Effect of Hyperlipidemia on Ketoconazole–MidazolamDrug–Drug Interaction in Rat

DALIA A. HAMDY,1,2 DION R. BROCKS1

1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada

2College of Pharmacy, Qatar University, Doha, Qatar

Received 18 February 2011; revised 13 April 2011; accepted 3 June 2011

Published online 22 June 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22675

ABSTRACT: Hyperlipidemia (HL) was previously shown to lower liver uptake of the morepotent (−) enantiomer of ketoconazole (KTZ) in rat. The current study examined the possiblemodifying influence of experimental HL on a KTZ pharmacokinetic interaction with midazolam(MDZ). Normolipidemic and hyperlipidemic rats were administered a single intravenous doseof MDZ (5 mg/kg) with or without a single oral dose of racemic KTZ (40 mg/kg). Serial bloodsamples were collected over 8 h following MDZ injections via jugular vein cannulas. Plasmawas jointly assayed for MDZ and KTZ concentrations using a validated assay. MDZ meanclearance (CL) was unchanged by KTZ coadministration. HL caused a significantly 61% lowerMDZ-unbound fraction and decreases in volume of distribution (VD) but by itself had no effecton MDZ CL. This suggested that MDZ could bind to lipoproteins. With KTZ coadministered tohyperlipidemic rats, there were significant decreases in MDZ CL and VD. HL caused a decreasein unbound plasma fraction of oral KTZ but no significant difference in its pharmacokinetics.HL caused a more pronounced KTZ -associated inhibition of MDZ CL. This may be related tothe decrease of MDZ’s unbound fraction and perhaps to attenuation of CYP3A by HL in the rat.© 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4986–4992,2011Keywords: protein binding; CYP3A; pharmacokinetics; pharmacodynamics; enzymeinhibition; antifungal drugs

INTRODUCTION

(±)-Ketoconazole (KTZ) is a broad spectrum anti-fungal used clinically as racemate. As with otherazoles, its antifungal activity is attributable to theinhibition of the cytochrome P450 (CYP)-mediated14-"-demethylation of lanosterol in ergosterolbiosynthesis. This results in fungal ergosterol deple-tion, interruption of membrane integrity/activity, andinhibition of cell growth.1 These CYP inhibitory prop-erties in fungi extend to mammalian CYP, with stronginteractions being possible with both hepatic drug-metabolizing enzymes.2–4 Owing to the risk of KTZ-induced hepatotoxicity and serious drug–drug inter-actions, the current use of KTZ as an antifungal agenttends to be restricted to more serious infections thatare resistant to other safer azoles.5,6 However, KTZ isstill a widely used prototypical CYP3A inhibitor and

Correspondence to: Dion R. Brocks (Telephone: +780-492-2953;Fax: +780-492-1217; E-mail: [email protected])Journal of Pharmaceutical Sciences, Vol. 100, 4986–4992 (2011)© 2011 Wiley-Liss, Inc. and the American Pharmacists Association

P-glycoprotein (Pgp) modulator for in vitro and in vivodrug interaction studies.7–11

Stereoselectivity in KTZ pharmacodynamics ispresent with (−)-KTZ being the more potent anti-fungal and CYP3A inhibitory enantiomer.12 Recently,stereoselectivity in the pharmacokinetics and proteinbinding of KTZ was revealed in rats.13 (±)-KTZ is ex-tensively (∼97%–99%) bound to rat plasma proteins,and its high-log octanol/water partition coefficient14

of 4.4 suggested it to be a possible candidate for bind-ing to serum lipoproteins and hence altered pharma-cokinetics in the presence of hyperlipidemia (HL).15–17

Subsequently, it was reported that HL led to an in-crease in the volume of distribution (VD) of KTZ enan-tiomers after administration of racemate to rats. Itwas also noted that the liver concentrations of themore potent (−) enantiomer were significantly de-creased after oral doses of (±)-KTZ.18 Those datasuggested a possibly increased lipoprotein-mediatedtransport of the drug to some tissues, with lower drugconcentrations being attained in liver during its oralabsorption.

4986 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 11, NOVEMBER 2011

EFFECT OF HYPERLIPIDEMIA ON DRUG INTERACTIONS 4987

The decrease in the hepatic uptake of the morepotent CYP3A inhibitory (−) enantiomer raises aninteresting possibility that in HL, KTZ may have areduced ability to inhibit drug metabolism. Midazo-lam (MDZ) is a short-acting benzodiazepine, whichundergoes extensive hepatic and gastrointestinalpresystemic extraction,19 and is often used as a se-lective probe for measurement of CYP3A activity.20

Because MDZ clearance (CL) is primarily governed byCYP3A, and because it is not known to be a Pgp sub-strate, any interaction between MDZ and KTZ can belargely ascribed to inhibitory effects on CYP3A.20–22

The goal of the current study was to explore the pos-sibility that HL could enhance the magnitude of KTZ-related CYP3A inhibition, using MDZ as a probe.

Experimental

Materials and Reagents

General laboratory chemicals were purchased fromFisher Scientific (Fair Lawn, New Jersey). KTZ wasobtained from Sigma (St. Louis, Missouri). MDZ forinjection (5 mg/mL) (Sandoz, Boucherville, Quebec,Canada) and KTZ (200 mg tablets) (Nizoral R© McNeilConsumer Healthcare, Guelph, Ontario, Canada)were used for administration to rats.

Animals and Pre-experimental Procedures

Experimental protocols involving animals were ap-proved by the University of Alberta Health Sci-ences Animal Policy and Welfare Committee. MaleSprague–Dawley rats (Charles River, Canada) wereused for the pharmacokinetic interaction studies. Therats weighed between 250 and 350 g and were housedin temperature-controlled rooms with 12 h of light perday. The animals were fed a standard rodent chowcontaining 4.5% fat (Lab Diet R© 5001; PMI NutritionLLC, Richmond, Indiana). Free access to food and wa-ter was permitted prior to experimentation.

All rats were given a single 5 mg/kg intravenous(i.v.) dose of MDZ. This was administered to nor-molipidemic (NL) and HL rats in the presence andabsence of 40 mg/kg KTZ given orally. Each groupincluded four to seven rats. HL was induced by in-jection of 1 g/kg intraperitoneal (i.p.) doses of P407(0.13 g/mL) solution in normal saline; NL rats re-ceived the same volume of sterile normal saline i.p.The P407 doses were administered under light anes-thesia using isoflurane and surgical oxygen, andthen allowed to recover. Eighteen hours later, theright jugular vein of each rat was catheterized withSilastic R© Laboratory Tubing (Dow Corning Corpora-tion, Midland, Michigan) under isoflurane anesthesia.The cannula was filled with 25 U/mL heparin in 0.9%saline. After implantation, the rats were transferredto their regular holding cages and allowed free access

to water, but food was withheld overnight so that drugwould be administered in the fasted state. The nextmorning, rats were transferred to metabolic cages fordosing and blood sample collection.

Drug Administration and Sample Collection

A KTZ (44 mg/mL) suspension was prepared fromtablets. The tablets were weighed and ground to apowder using a mortar and pestle, then dispersed in1% methylcellulose. For i.v. dosing, MDZ injectablesolution (5 mg/mL) was used. On the morning of thepharmacokinetic study, NL and HL rat groups re-ceived the desired dose of either KTZ suspension inmethylcellulose or methylcellulose only by oral gav-age. Later (1.5 h), the MDZ dose was injected over1 min via the jugular vein cannula, immediately fol-lowed by 0.5 mL sterile normal saline. At the timeof first sample withdrawal after i.v. dosing, the first0.2 mL volume of blood was discarded.

Blood samples (0.15–0.25 mL) were nominallyscheduled for collection at 0.75 h before and then seri-ally at 0.08, 0.33, 0.67, 1, 1.5, 2, 3, 4, and 8 h after thei.v. doses into polypropylene microcentrifuge tubes.Heparin in normal saline (25 U/mL) was injected intothe cannula after each collection of blood. Plasma wasseparated promptly by centrifugation of the blood at2500g for 3 min. The samples were kept at −30◦Cuntil assayed for KTZ and MDZ concentrations.

Plasma Protein Binding

The unbound fractions of MDZ and KTZ in plasmawere determined using ultrafiltration (Centrifree R©,Amicon, Beverly, Massachusetts). Blood was obtainedfrom rats (about 12 mL per rat) by exsanguinationvia cardiac puncture into syringes. The blood was ob-tained from NL rats, and HL rats collected 36 h afteri.p. doses of P407. The blood was placed in heparinizedtubes and centrifuged at 2500g for 10 min. The NLand HL rat plasma was spiked with MDZ injectablesolution to allow for final concentrations of 10 mg/L.In some samples, (±)-KTZ methanolic solution wasalso added to allow a final concentration of approxi-mately 10 mg/L of both drugs. The volume of methanoladded to each tube did not exceed 0.05% (v/v). Tubeswere incubated for 1 h in a 37◦C water bath shaker. Avolume of 1 mL of each tube was transferred to an ul-trafiltration device, which was then placed in a fixedangle centrifuge rotor and spun at 2000g for 10 min at37◦C. The samples were then analyzed for MDZ andKTZ concentrations. Replicates of four were used foreach determination of unbound fraction.

For KTZ protein binding, preincubation of the ul-trafiltration devices with 5% Triton R© X-100 (SigmaLife Sciences, St. Louis, Missouri) for 12 h wasrequired23 in order to overcome the known bindingof KTZ to the filter.18 The devices were then usedafter being rinsed with double-distilled water.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 11, NOVEMBER 2011

4988 HAMDY AND BROCKS

Assay

A validated nonstereospecific high-performance liq-uid chromatographic method for the simultaneous de-termination of MDZ and KTZ, with diazepam as aninternal standard, was used for measurement of thedrug concentrations.24 The validated lower limit ofquantitation was 25 ng/mL for both drugs based on0.1 mL plasma or buffer. Volumes of plasma assayedin samples from the pharmacokinetic studies rangedbetween 75 and 150:L. Standard curves were pre-pared from similar drug-spiked matrices.

Data and Statistical Analysis

Noncompartmental methods were used to calculatethe pharmacokinetic parameters. The terminal elim-ination rate constant (λz) was calculated by subject-ing the plasma concentrations in the terminal phaseto linear regression. The terminal phase half-life (t 1

2)

was calculated by division of 0.693 by λz. The AUC0–∞was calculated using the combined log-linear trape-zoidal rule from time 0 h after dose to the time ofthe last measured concentration, plus the quotient ofthe last measured concentration divided by λz. Theconcentration at time 0 (C0) after i.v. dosing was esti-mated by back extrapolation of the log-linear regres-sion line using the first three measured plasma con-centrations to time 0 h. The CL was calculated as thequotient of dose to AUC0–∞ and the steady-state VD(VDss) as CL × AUMC/AUC, where AUMC is the areaunder the first moment plasma concentration versustime curve, from time of dosing to infinity. The VD ofthe central compartment (Vc) was calculated as thequotient of dose to C0. The KTZ maximum plasmaconcentration (Cmax) and the time at which it occurred(Tmax) were determined by visual examination of thedata.

The plasma-unbound fraction (fu) was determinedby dividing the MDZ or KTZ concentration in the fil-trate by that measured in the prefiltered plasma. Allcompiled data were reported as mean ±SD, unlessotherwise indicated. Significance of comparisons ofmeans for the interactions of lipoprotein status andKTZ were assessed by two way analysis of variance(ANOVA), followed by Bonferroni post-hoc analysis(Sigmaplot, Systat, San Jose, California). KTZ phar-macokinetic parameters were tested for significanceusing Student’s unpaired t-test. In all cases, the levelof significance was set at p = 0.05.

RESULTS

The MDZ plasma concentrations generally declinedmultiexponentially after i.v. doses of 5 mg/kg MDZ(Figure 1 and 2). Plasma concentrations of KTZ weremuch higher than those of MDZ throughout the studyperiod (Fig. 2).

Figure 1. Plasma midazolam concentration versus timeprofiles in normolipidemic (NL) and hyperlipidemic (HL)rats alone and with 40 mg/kg (±)-KTZ given 90 min beforethe intravenous midazolam dose.

Midazolam exhibited extensive (>98%) binding toplasma proteins. In the absence of KTZ, HL was as-sociated with a significant 61% reduction in MDZ fu(Table 1). In the presence of KTZ, there was a similardecrease in mean fu (43%). There was a statisticallysignificant effect of HL on reducing the unbound frac-tion of MDZ.

In the NL rats only given MDZ, there was littlenumerical different present in the mean pharmacoki-netic parameters from NL rats coadministered KTZ(Table 1). In HL rats, larger differences appeared tobe present between those rats only given MDZ fromthose also given KTZ. Statistical analysis confirmedthat in HL rats, oral administration of (±)-KTZ 1.5 hprior to the i.v. doses of 5 mg/kg MDZ caused signif-icant changes in MDZ pharmacokinetics. There wasa statistically significant effect of HL on reducing theVc and VDss. As for fu, KTZ did not affect the VD ofMDZ.

Two-way ANOVA detected an interaction betweenlipoprotein status and KTZ with MDZ CL and AUC.Specifically, the changes in these parameters in KTZ-treated rats were accentuated by the presence of HL.Although KTZ did not significantly alter the AUC orCL in NL rats, KTZ caused a 130% increase and 56%decrease, respectively, in the AUC and CL of MDZ inHL rats. Within the rats given KTZ, there was like-wise a significant increase of 50% and a 25% decrease,respectively, in the AUC and CL of MDZ in HL rats.Hence, HL was found to accentuate the inhibitory ef-fect of KTZ on MDZ pharmacokinetics.

No significant differences were noted in the phar-macokinetics of orally administered (±)-KTZ between

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 11, NOVEMBER 2011 DOI 10.1002/jps


Figure 2. Comparative mean ±SD plasma concentration versus time curves of oral (±)-ketoconazole (KTZ) and i.v. midazolam. Left panel, profiles of the drugs in normolipidemic(NL) rats; center panel, profiles of the drugs in hyperlipidemic (HL) rats; right panel, KTZplasma concentrations in NL and HL rats.

NL and HL rats (Fig. 2, Table 1). The exception to thiswas in the plasma protein binding, where HL was as-sociated with a significant decrease of almost 70% inthe (±)-KTZ-unbound plasma fraction.

DISCUSSION

Much of the knowledge regarding drug interactionsis gained through the administration of drugs tohealthy volunteers during drug development. The ex-ceptions to this are observations from clinical ex-perience, including case reports in patients, whichmay prompt more formal studies, again most ofteninvolving healthy volunteers. One aspect of interac-tions often overlooked is the possibility of concur-rent conditions in patients, which may modify, ei-ther by accentuating or attenuating, the strength of

the interaction. This is something that cannot be de-termined from studies involving healthy volunteers,but is of relevance in dictating the variability indrug response and measures of effect expected in pa-tients receiving drug treatment. One clinical condi-tion that may be present in patient populations is HL,which poses a major risk factor for other cardiovas-cular diseases.25–27 Most often, the clinically relevantlipoprotein elevations in HL involve those of low den-sity and very-low-density lipoprotein classes such asthat found in P407-treated rats. The observation ofdifferential uptake of KTZ in HL rats prompted thequestion: can HL influence the strength of the CYPinhibiting potency of KTZ?

To explore our hypothesis that HL would cause anincrease in the strength of KTZ inhibitory potency,MDZ (the CYP3A probe) was injected 1.5 h after KTZ

Table 1. Pharmacokinetics Parameters of Midazolam and Ketoconazole in Normolipidemic (NL) andHyperlipidemic (HL) Rats

Midazolam Alone Midazolam Plus Ketoconazole

NL (n = 6) HL (n = 4) NL (n = 6) HL (n = 7)

MidazolamAUC0–∞ [mg/(h L)] 2.41 ± 0.597 2.06 ± 0.338a 3.23 ± 0.450b 4.78 ± 0.979t 1

2(h) 4.12 ± 1.93 2.34 ± 1.08 3.13 ± 1.16 4.23 ± 2.36

CL [L/(h kg)] 2.17 ± 0.458 2.48 ± 0.445a 1.57 ± 0.200b 1.08 ± 0.223VDss (L/kg) 4.47 ± 2.78 2.95 ± 1.80c 3.50 ± 1.03 1.82 ± 0.576c

Vc (L/kg) 1.32 ± 0.263 0.791 ± 0.128c 1.17 ± 0.269 0.695 ± 0.197c

fu (%)d 1.97 ± 0.38b 0.760 ± 0.29c 1.78 ± 0.15b 1.02 ± 0.21c

KetoconazoleCmax (mg/L) – – 23.5 ± 6.44 19.6 ± 8.05Tmax (h) – – 2.67 ± 0.589 2.27 ± 0.818AUC0–∞ [mg/(h L)] – – 84.0 ± 25.2 69.1 ± 25.1fu (%)d – – 2.03 ± 0.34e 0.65 ± 0.22e

aSignificantly different from KTZ in the same lipoprotein status group.bSignificantly different from HL in the same KTZ-treatment group.cSignificantly lower in the HL compared with NL rats but with no interaction with KTZ treatment.dDetermined in vitro using drug-spiked plasma, n = 4 per determination.eDetermined in absence of midazolam.



oral pretreatment. This approach was taken basedon the data of a previous study in which the largestdifference between liver concentrations of drug be-tween NL and HL rats occurred at about 1.5 h afteran oral dose of KTZ.18 This design was also in line withdata illustrating the need to consider the liver uptakeof enzyme inhibitors for the quantitative predictionof drug–drug inhibitory interactions.28 Oral dosingof KTZ was chosen to maximize KTZ liver uptake,wherein the drug would first pass through the liverduring entry to the systemic circulation. The studydesign also matched well with other experimental de-signs previously reported in literature having an av-erage time interval between the two doses rangingfrom 0.5 to 2 h.28–31

The CL of MDZ in NL rats (Table 1) was similar toprevious reports,29–32 although the t 1

2appeared to be

longer. The underlying reason behind may be that inthe current study, MDZ was measured for up to 8 hafter i.v. dose, whereas most of the reported studieshave followed the MDZ profile for only 2 or 4 h. Thismay have prevented measurement of the true termi-nal phase t 1

2(Figure 1 and 2).29,30 The only other pa-

per reporting this drug interaction up to 8 h showeddata from only two rats, thereby preventing statisti-cal comparisons.21

In NL rats, it was found that oral administration ofKTZ caused a modest but nonsignificant decrease inMDZ mean CL, with no effect on t 1

2(Table 1). Using

the reported MDZ blood-to-plasma ratio of 133 andhepatic blood flow of 13.8 mL/min in a 250 g rat,34

the estimated hepatic extraction ratio of MDZ in NLis that of a moderately high extraction ratio (0.66),meaning that its hepatic CL is relatively highly de-pendent on hepatic blood flow. Given this, it might notbe entirely surprising that the change in CL for NLrats was relatively insensitive to the coadministrationof KTZ.

In the absence of KTZ, there was neither a dif-ference in CL nor AUC between NL and HL rats(Table 1, Fig. 1). The decreases in MDZ fu and Vc inHL, however, suggest that MDZ has the ability to bindto lipoproteins, something that has not been reportedpreviously.

On the basis of the knowledge that there was lowerhepatic uptake of (−)-KTZ in HL,18 we anticipatedthat there might be an attenuated inhibitory responseof MDZ metabolism with KTZ coadministration in theHL rats. Specifically, a higher CL of MDZ in HL ratscompared with NL rats given KTZ was expected. Incontrast, in KTZ-treated rats, HL appeared to accen-tuate the KTZ–MDZ drug interaction, as evidenced by31% significant decrease in MDZ CL compared withNL rats (Fig. 1 and Table 1). On the basis of the liveruptake of KTZ, this was not expected, although twofactors were present or identified, which could have

worked to enhance the inhibitory potency of KTZ onMDZ CL. The first factor is attributed to the unex-pected decrease in the fu of MDZ in HL rats. The sec-ond one is related to the fact that certain CYPs—in-cluding CYP3A—have been shown to be downregu-lated in HL rats,35 which might have strengthenedthe inhibitory response to KTZ.

The KTZ plasma concentrations measured in thisstudy in the presence of MDZ matched the previ-ously reported sum of KTZ enantiomers when givenalong after oral racemate doses of 40 mg/kg. The av-erage of the sum of the two enantiomers was 21.6 mg/L for Cmax, and between 75 and 101 mg/(h L) forAUC0–∞,13,18 which conformed well with the cur-rent results (Table 1). As expected,28 KTZ exhibitedhigher plasma concentrations than MDZ at all times(Fig. 2). HL did not significantly affect the AUC0–∞,Cmax or Tmax of (±)-KTZ after its oral administration(Table 1).

We were able to determine the effect of HL on fu ofKTZ, where HL decreased the fu of KTZ by 68%. Sev-eral attempts were previously made to determine thefu of KTZ in HL plasma using the erythrocyte-bindingtechnique, although these yielded inconsistent mea-sures. Ultrafiltration techniques were associated withKTZ binding to the filter. Attempts to pretreat the fil-ter with 5% Triton for 12 h23 overcame the problem ofmembrane binding, but interference with the stere-ospecific KTZ assay proved problematic.18,36 Here, themethod was reapplied and fortunately this did not in-terfere with the nonstereospecific assay of MDZ andKTZ.24 The sum of the unbound fraction of the twoenantiomers in NL rats using the erythrocyte-bindingtechnique closely matched the current values for theracemate using the ultrafiltration method.13

The results of this study cannot be directly extrapo-lated to the clinical population because it employed ananimal model of HL known to cause marked increasesin lipoproteins beyond which would be seen in hu-mans. Despite the sizable increases in plasma lipidsafter P407 injection, it is considered a useful model ofHL with little toxicity,37 noninflammatory action,38

absence of other metabolic abnormalities such asdiabetes, and short time for induction of HL.39

CONCLUSION

In conclusion, in divergence to the decrease of (−)-KTZ noted in liver of HL rats, the condition was as-sociated with a greater inhibition of MDZ CL by oralKTZ. This may have been due in part to the involve-ment of a decrease in MDZ fu, and perhaps to thepossible reduction in CYP3A in the HL state. Thedata does provide some evidence that the potency ofa drug interaction can be influenced by HL, whichcould potentially contribute to variability in pharma-codynamic properties of drugs.



ACKNOWLEDGMENTS

Funded in part by a grant from the CanadianInstitutes of Health Research (MOP 87395). DAH wasthe recipient of a studentship award from the govern-ment of Egypt, and a University of Alberta Disserta-tion Fellowship.

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effect of hyperlipidemia on ketoconazole–midazolam drug–drug interaction in rat

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