EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1984, Vol. 9, No 3, pp. 195-200

Cimetidine and ranitidine : Their interaction withhuman and pig liver microsomes and with purifiedcytochrome P-450

S. RENDIC, H.H. RUF *, P. WEBER ** and F. KAJFEZInstitute for the Control of Drugs, Zagreb, Yugoslavia

Received for publication: April 5, 1983

Key words: cimetidine, ranitidine, human liver microsomes, cytochrome P-450, interactions.

SUMMARY

Cimetidine and ranitidine interact with microsomes from human and pig liver and with purified cytochrome P-450 in the ligand­type manner. The affinity for cimetidine is about 10 times as high as that for ranitidine. Accordingly amplitudes of the specta aremuch higher for cimetidine. These results are in accordance with those obtained earlier with rat liver microsomes. The inhibitory pot­ency of either compound with regard to dealkylation of 7-ethoxycoumarin appears to be less in the human preparation.

*

INTRODUCTION

In our previous papers (1-3) we have shown thatHj-receptor antagonists cimetidine and ranitidineinteract with cytochrome P-450 as ligands. Theseagents inhibited microsomal drug-metabolisingactivity in rat liver in vitro. In these studies cimeti­dine was shown to interact with higher affinity andto inhibit microsomal activity to a greater extent aswell. It was concluded that cimetidine and raniti­dine interact with different functional groups andwith different forms of the enzyme. For cimetidineinteractions of its imidazole and its cyano group,whereas for ranitidine interactions of its nitronicoxygen and one of the amino groups were sug­gested (3). In clinical experiments cimetidine, andnot ranitidine, was shown to decrease the biotrans­formation of a number of drugs (4-6). The inhibi­tory activity of cimetidine was attributed to itsligand binding properties (I).

In this study interactions of cimetidine and rani­tidine with human liver microsomes, pig liver mic-

Department of Physological Chemistry, University ofSaarland, Homburg-Saar, F.R. Germany.

** Faculty of Biology, University of Constance, Konstanz,F.R. Germany.

Send reprint requests to: Dr. S. Rendic, Institute for theControl of Drugs, Mosa Pijade 160, Yu-41000 Zagreb,Yugoslavia.

rosomes and purified pig liver cytochrome P-450(PLM IV) are reported. Model studies of this kindas well as those made with rat liver microsomes,may prove useful for the understanding and predic­tion of clinical drug interactions.

CIMETIDINE

RANITIDINE

MATERIALS AND METHODS

Animals and preparation of liver microsomalfractions.

A human liver microsomal fraction was pre­pared using tissue from a donor who died after atraffic accident (healthy, female 18 years old, non-

196 European Journal of Drug Metabolism and Pharmacokinetics. 1984. No 3

smoker, having taken no alcohol immediatelybefore accident). The liver was removed perfusedwith ice cold isotonic saline, and the preparation ofthe microsomal fraction by differential centrifuga­tion (7) started within I hr after death. All experi­ments, except EPR, were performed using thefreshly prepared microsomal fraction. Pig livermicrosomes were prepared from a phenobarbital­pretreated pig (a dutch 18-rib pig, 16 kg bodyweight received phenobarbital, 80 mgfkg for5 days). The microsomal fraction was preparedaccording to the method described by van der Hoe­ven and Coon (8). Purified pig liver cytochrome P­450, prepared by H. Graf et al. (9), was a generousgift from V. Ullrich. Pig and human liver micro­somal fractions, and purified cytochrome P-450were stored under nitrogen at -80°C.

RESULTS AND DISCUSSION

Figure I shows the optical difference spectra ofhuman liver microsomes after addition of cimeti­dine (Fig. IA) and ranitidine (Fig. IB). Similar topreviously reported results, obtained with rat livermicrosomes (1-3), both compounds producedspectra indicating a ligand-type interaction withcytochrome P-450. With cimetidine a peak occuredat 431 nm, a trough at about 393 nm, and a shoul­der at about 408 nm (Fig. 1A) and an isosbesticpoint at 418 nm was noted. These data correspondvery well to those reported earlier (1) for rat livermicrosomes. Ranitidine produced a peak at 426 nmand a trough at 407 nm (Fig. 1B and Table I),which is closely similar to results obtained withmicrosomes from untreated rats (2).

500400 450

X/nm

Bs 0.002cl:l-e0

01:«<I I

-0.002 407

400 450 500X/nm

Fig. 1 : Optical difference spectra of human liver micro­somes in the presence of ranitidine and cimetidine.Difference spectra were recorded using tandem cells,each compartment containing 1.25ml of either 0.1 MTris/Hel or microsomal suspension in the same buf­fer. Either cimetidine dissolved in methanol, or rani­tidine dissolved in buffer, were added to the com­partment of the sample cell containing microsomalsuspension, and to the compartment with buffer ofthe reference cell. The corresponding volume of thesolvent was added to the other compartments. Themicrosomal suspension contained 1.25 mg of pro­tein/ml.

Key: Fig. 1A: Difference spectra in the presence ofcimetidine. Final concentrations of cimetidinefor each curve were: 1,0.08; 2, 0.24; 3, 0.4; 4,0.72 ; 5, 1.36 mM.

Fig. 1B : Difference spectra in the presence ofranitidine. Final concentrations of ranitidinewere: 1,1.2; 2,3.2; 3, 8.4 mM.

cull.005oce.a~ 0r--------:J<:..------=~-------1.a«<I

-0.00

Measurements and analysesRecording of spectra: Difference spectra were

recorded on an Aminco DW-2 spectrophotometer.The spectral dissociation constants were obtainedas follows: the reciprocal of the difference ofabsorption between the peak and trough in eachdifference spectrum was plotted against the recip­rocal of the concentration used. The value of thedissociation constant K, was determined by linearregression, as the reciprocal of the abscissal inter­cept.

Electron paramagnetic resonance (EPR) wasmeasured with a Varian E-9 spectrometer con­nected to a digital computer (Data General Nova820) which performed signal averaging, doubleintegration and superposition of the spectra. The g­values were calculated by using magnetic fieldreadings from the «Varian Fieldial », DPPH (di­phenylpicrylhydrazine) was used as g-marker (g =12.0036). Human liver microsomes were sedi­mented by centrifugation and resuspended at aconcentration of about 65 mg protein/mi. Bufferblank values were substracted from all spectra.

Inhibition of 7-ethoxycoumarin O-dealkylation :The inhibition was measured fluorometrically bythe method of Ullrich and Weber (10). The resultswere plotted both as double reciprocal (I/sp.act. vsljS) and Eadie-Hofstee graphs (sp.act. vs sp. act./S)from which type of inhibition, slopes and inter­cepts for the inhibition constants were calculatedby linear regression.

Determination of cytochrome P-450: Cytoch­rome P-450 was determined by the method ofOmura and Sato (II).

Determination of protein: Protein concentra­tion was determined by the biuret method (12).

Synthesis of compounds: Cimetidine and raniti­dine were synthesized by methods described earlier(13, 14, 15).

S. Rendic et al., Cimetidine, ranitidine-cytochrome P-450 interaction 197

curve 1); the trough was shifted to 411 nm at higherconcentrations of ranitidine (Fig. 2A, curve 2).With purified cytochrome P-450 a peak at 428,5 nmwas produced by both compounds. A broad troughbetween 392 nm and 407 nm was produced withcimetidine (Fig. 2B, curve 2).

At higher concentration of ranitidine (> 0.4mM) in the presence of purified cytochrome P-450the base Iinie changed markedly. Such an effectwas not observed with cimetidine at concentrationsup to 2.0 mM. The nature of this phenomena, pos­sibly caused by destruction of cytochrome P-450, isnot known.

Apparent dissociation constants, Ks, and ampli­tudes of spectra were calculated from the doublereciprocal plots of the spectral change at differentconcentrations of each compound (Table I). Usinghuman liver microsomes, straight lines wereobtained with both compounds. A similar resultwas obtained previously with ranitidine and ratliver microsomes (2). Cimetidine added to the ratmicrosomal preparations always produced a dis­tinct break in the plot, and this was explained byassuming an interaction with different forms ofcytochrome P-450 (I). The affinity of both, humanand rat liver microsomal preparations for cimeti­dine is about 10 times as high as that for ranitidine,when comparing the second low-affinity bindingconstant, Ks2'

Accordingly, maximal amplitudes, as well asamplitudes at lower concentrations of the com­pounds, are much higher for cimetidine. The samerelationship was found with pig liver microsomes,and purified cytochrome P-450 (Table I, Fig. 1)(1-3). In experiments with purified cytochrome P­450, cimetidine again produced a break in thedouble reciprocal plot. Two dissociation constantswere evaluated with pig liver microsomes as well.The existence of two dissociation constants sug­gests that different functional groups in the cimeti­dine molecule should interact with the same formof cytochrome P·450, but these groups differ inaffinity. Our recent findings (3) which show thatcimetidine interacts with cytochrome P-450through the imidazole as well as the cyano groupsupports this suggestion.

The interaction of cimetidine and ranitidinewith rat liver microsomes also has been detected byEPR (1-3). Cimetidine shown a greater effect thanranitidine : The cytochrome P-450 bound to cimeti­dine showed g-values shifted from 2.44 to 2.53 and1.92 to 1.88 indicating binding of its imidazole orcyano group to the heme iron (1,3). With raniti­dine a very small interaction of a heterogeneoustype is obtained (2, 3). With human ·liver micro­somes the interaction of the compounds was quali­tatively similar to that observed in rat liver micro­somes (Fig. 3a, b) although the lower content of

0.005

-0.00s.

450

450

428.5

I

A/nm

3

400

400

B0.02

-0.Q1

Atflm

Fig. 2: Difference spectra of pig liver microsomes and puri­fied cytochrome P-450 in the presence of cimetidineand ranitidine.

Key: Fig. 2A : Difference spectra of pig liver micro­somes in the presence of cimetidine and raniti­dine. Microsomal suspension contained 2.5 mgof protein/rnl, All other experimental detailsare the same as in Fig. I. The curves' number­ing means: I, ranitidine 0.08 mM; 2, raniti­dine 0.56 mM ; 3, cimetidine 0.04 mM.

Fig. 2B: Difference spectra of purified cytoch­rome P-450 in the presence of cimetidine andranitidine. The solution of cytochrome P-450in the buffer contained 0.06 mg of protein/ml,cytochrome P-450 content was 10 nmol/mg ofprotein, I mI and 0.5 mI cells were used forrecording the spectra in the presence of cimeti­dine and ranitidine, respectively. The curves'numbering means: I, ranitidine 0.4 mM; 2,cimetidine 0.3 mM. In both figures, 2A and 2B,the ordinate on the left correspond to cimeti­dine and on the right to raniditine.

A 432I

002 0.01

ea0.01 0.005.cL-a11-c 0 0<J

-0.01 -0.005

~a0.01i!aen.c« 01--,------''-".:=o-=i-4- _-=:::.:..:.::.=.=....--=:::::::==10<J

A ligand type of interaction was also observedwhen the above compounds were added to the mic­rosomal suspension from the phenobarbital pre­treated pig (Fig. 2A), and to the solution of purifiedpig liver cytochrome P-450 (PLM IV), (Fig. 2B).With pig liver microsomes cimetidine produced apeak at 432 nm and a trough at 407 nm, with ashoulder at about 392 nm (Fig. 2A, curve 3). Withthe same preparation, ranitidine produced a peakat 430 nm and a broad trough at 410 nm (Fig. 2A,

198 European Journal of Drug Metabolism and Pharmacokinetics. 1984, No 3

Table I: Data derived from optical difference spectra of human and pig liver microsomes and of purified cytochrome P-450 (PLMIV), recorded in the presence of cimetidine and ranitidine.

Cytochrome P-450 Absorbance Absorbance difference Ksl Ks2max. min. t. Amax. 1 t. Amax. 2

Compound (nmole/mg protein) (nm) (nm) t.A/nmole cytochrome P-450 (mM) (mM)

Human livermicrosomes 0.69

Cimetidine 431 393,408* .1. 0.022 .1. 0.75Ranitidine 426 407 .1. 0.004 .1. 6.6

Piglivermicrosomes 2.68

Cimetidine 432 407,385* 0.0053 0.013 0.009 0.14Ranitidine 430 411 0.002 0.0044 0.34 2.26

Purified pig livercytochrome P-450 10.0

Cimetidine 428.5 392-407 0.055 0.1 0.1 0.38Ranitidine 427-429 407-410 .1. 0.014 .1. 2.5**

Experimental details are the same as in Figs. 1,2,4. K, and fj. Amax values were calculated from double reciprocal plots using linearregression analysis. Each value is the mean of at least three measurements.

* Shoulder in the spectrum.

** Low intensities of the spectra and instable base line made the calculation uncertain.

cytochrome P-450 yielded spectra with higher noiseand background signals. The g-values of humanliver cytochrome P-450 g = 2.42, 2.25 and 1.91(spectra A), were close to rat liver cytochrome P­450. The low-field peak at g = 2.42 showed aremarkable heterogeneity. Small shifts in g-valuesare known for the different forms of the cytoch­rome from various species.

With cimetidine (Fig. 3a) a form with g = 2.39and 1.93 was converted to a form with g-valuesprobably at g = 2.51 and 1.88. Here the reactingform had a lower anisotropy than in rat liver. Withranitidine (Fig. 3b) only very minor changes in theEPR spectrum could be detected indicating a weakinteractions with a form at g = 2.38 and 1.92. Theinteraction was so weak that the resulting spectrumcould not be detected.

Cimetidine and ranitidine habe been shown toinhibit dealkylation of 7-ethoxycoumarin in ratliver microsomes, and the former compound is themore potent inhibitor. Cimetidine was found to bea competitive inhibitor, whereas ranitidine was amixed-type inhibitor (1-3). Both cimetidine andranitidine behaved in the same manner with humanliver microsomes, except that with this preparationcimetidine also causes mixed-type inhibition atconcentrations ~ 3mM (Fig. 4A, B). However,Eadie-Hofstee plots suggest that cimetidine causedmixed-type inhibition even at lower concentration(Table II).

When comparing the 150 and Ki values forcimetidine and ranitidine given in Table II to thecorresponding values obtained with rat liver micro­somes (1-3), the inhibitory potency of either com­pound appears to be less with the human prepara­tion.

CONCLUSIONS

The results show that cimetidine and ranitidineinteract in the ligand - type manner with micro­somes from human and pig liver, and purified pigliver cytochrome P-450. With both microsomal pre­parations and with purified pig liver cytochrome P­450 (PLM IV) higher affinity of interaction wasobtained with cimetidine and this is in accordancewith results obtained earlier with rat liver micro­somes. The ligand type of interaction of both com­pounds, the difference in binding affinity as well asbinding with different functional groups to cytoch­rome P-450 in human liver! microsomes was alsoshown by EPR. These results as well as those per­taining to the inhibition of microsomal drug metab­olising activity in vitro (15, 16) support our sugges­tion that a ligand-type interaction of cimetidinewith cytochrome P-450 should interfere with the invivo metabolism of other drugs in humans as well(I, 2). Furthermore, a good correlation of the inhi-

S. Rendic et al., Cimetidine, ranitidine-cytochrome P-450 interaction 199

Q)

.~

iii>.;:Q)

oe.2C....oII)

.D-cIXQ.W

240

2.42I

I I2.51 2.39

2.25I

300

1.91I

I I1.93 1.88

360 mT

Table II: K, and Iso values for the inhibition of 7-ethoxy­coumarin dealkylation by human liver micro­somes.

Compound ISO Kt Kli*(mM) (mM) (mM)

Cimetidine 2 1 14.5 ***Ranitidine 3.2 2.1 15

Concentrations of the substrate for Kj determinations werefrom 5 x 10.6 M to 5 x 10-4 M and that for lsewas 5 x 10-4 M.Linear regression analysis was used to determine the inter­cepts and slopes. The results are the mean of at least threemeasurements and are calculated from double reciprocalplots.

* Slope inhibition constant.

** Intercept inhibition constant.

*** Calculated from Eadie-Hofstee plots.

Magnetic Field

Fig. 3: Interaction of human liver microsomes with cimeti­dine (3a) and ranitidine (3b) as detected by EPRspectra. Traces A - native human liver microsomes(about 40 ~M cytochrome P-450 and 65 mg protein/ml).

Traces B - after addition of saturating amounts ofthe solid compound.

Traces C - computed difference spectra 2* (B-A).EPR was measured at 9.18 GHz microwave fre­quency, 40 mW microwave power, 2.5 mT modula­tion amplitude, a gain of 2000 and temperature 90 K.

100

2

8060

3

o

40

40 60 80 100

1 / [7 - Ethoxycoum<:,rin] (mtvf1)

20

20

8

>.~ 15

~t:l

U~ 10III

20

~ 15.~

~u~ 10VI---..

-20

-20

A

Fig. 4: Double reciprocal plots of the inhibition of O-deal­kylation of 7-ethoxycoumarin in human liver micro­somes with cimetidine (Fig. 4A) and ranitidine(Fig. 4B). The straight lines' numbering represents: I,without addition of inhibitor; 2, inhibitor added,I mM ; inhibitor added, 3 mM.

I1.92

300

Magnetic Field

I2.38

,240

Q)>;;III>.;:Q)

oeo;;Co...oIII.D-cIXQ.W

200 European Journal of Drug Metabolism and Pharmacokinetics. 1984. No 3

bitory potency of cimetidine ~ ranitidine in vitrousing microsomal preparation from differentexperimental models with inhibition of drugmetabolising activity in clinical trials confirm theusefulness of this model for the understanding andprediction of drug interactions.

ACKNOWLEDGMENTS

We wish to thank Professor Dr. V. Ullrich (Faculty ofBiology, University of Constance, Konstanz, F.R. Germany)for helpful discussions and for providing the necessary facili­ties for this work.

This work was supported in part by Alexander von Hum­boldt Fundation, Bonn, F.R. Germany. During part of thisstudy Dr. S. Rendic was a receiver of Alexander von Hum­boldt Fellowship.

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