Pharmacological Research Communications, VoI. 9, No. 9, 1977

IN VITR 0 BINDING AND METABOLISM OF IOPRONIC ACID BY RAT LIVER MICROSO~S

Elisabetta Raffaeli= Roberto Maffei Facino*~ Mario Salmona**= and Davide Pitt,***.

Istituto di Chimica FarmaceuticajeJTdssicologica viale Abruzzi, 42

201311~i~ano, Italy.

Istituto di Ricerche Farmacologiche "Mario Negri" via Eritrea 62, 20142 Milano, Italy.

***

Laboratori Ricerche Bracco Industria Chimica via Folli 50, 20134 Milano= Italy.

R~eived ~ final form 17 ~ r

S ~ Y

1977

lopronic acid (2-~2-[(3-acetamido-2,4,6-

triiodophenoxy)-ethoxy]- methyl}- butyric acid), a cholecystographic agent= is metabolized by rat liver microsomes to hydroxyethyl-(3-acetamido-2,4=6-triiodophenyl)ether and to 3-acetamido-2~4=6-triiodophenoxy-acetic acid. This metabolism is oxygen- and NADPH-dependent,,and it is inhibited by CO. The addition of iopronic acid to microsomes causes a type II spectral change. The in vitro results are in good agreement with the in vivo data already available showing that iopronic acid is metabolized to a limited extent and that the three iodine atoms present in the parent compound are also found in the metabolites.

833

Pharmacological Research Comrnunications,,Vol: 9, No, 9, 1977

834

INTRODUCTION

The hepatic microsomalmixed-function oxidase system is known to metabolize a wide variety of substrafes bo>h of endogenous and exogenous origin~ most of whiQh combine with hepatic microsomal P-450 to produc~ spectral changes of type I or II (Mannering~ 197~; Mailman et al.~ 1974). Although the ability of c o m p o u n d s to bind with cytochrome P-450 does n o t guarantee their metabolism and v!.ceversa , it is always interesting to investigate whether or not chemicals which are clearly metabolized through an oxidative pathway might be capable of combining with cytochrome P-450 (Imai and S a t o s 1967; Jefcoate and Gaylor, 1969; Jefcoate, Gaylor and Calabrese, 1969). lopronic acid (IA) (Pitr~ and Maffei Facino, 1976), an orally active cholecystographic agent widely used in radiological diagnostic examination of biliary ducts, has been shown to be metabolized by rat liver microsomal preparations. The two metabolites found, hydroxyethyl-(3-acetamido-2,4,6-triiodophenyl)-ether (HPE) and 3-acetamido-2~4~6-triiodophenoxy-acetic acid (PAA), arise from oxidative cleavage of the side chain of IA (Fig.l). In this paper we present data confirming that cytochrome P-450 is directly involved in the biotransformation of IA.

MATERIALS AND M E T H O DS All the organic solvents used were of analytical grade. The iopronic acid(IA) was batch N. 46/c (Bracco S.p.A.) NADP~ glucose-6-phosphate (G-6-P) and glucose-

Pharmacological Research Communications, Do/. 9, No. 9, 1977 -6-phosphate dehydrogenase

and phospholipase C were

purchased from Boehringer~ Mannheim, Germany. Homogenization was carried out in a Potter Elvehjem glass tube with a Teflon pestle. Centrifugation was at IO~O00 X g in a Sorvall Rc-5 centrifuge and at iO5~OO0 x g in a Spinco model L ultracentrifuge. Incubations were performed in a Dubn0ff metabolic ~ shaker at 37°C.

Ti ssue preparation ~le

Wistar rats (200-250 g) were used through-

out this study. The animals were killed by a blow to the head and the livers were excised and placed in cold 1.15% KCI. The livers were then minced and homogenized with 3 vol. of a 2:1 solution of 1.15% KCI and 0.2 M phosphate buffer~ pH 7.4. The homogenates were centrifuged at IO,O00 x g for 20 min. tO remove cell debris~ nuclei and mitochondria~ and the supernatants recentrifuged at IO5~OOO x g for 1 h. The microsomal pellet was suspended in a volume of O.I M phosphate Duffer pH 7.4 to give a suspension containing 8.5 mg of protein/ml. Protein was determined by the method of Lowry et al. (1951)~ using bovine serum albumin (Fluka-Swiss) as standard.

For studies of druK metabolism (Fig. i-3) Incubation mixtures Samples of the microsomal suspension were incubated for 60 min., with shaking, at 37°Cj in an atmosphere of oxygen. Each ml of incubation mixture contained 8,5 mg of microsomal protein, 2.5 }/tool of NADP, 20 nmol of MgCI2, 40~=nol of G-6-P, 5 units of G-6-P dehydrogenase

835

836

Pharmacological'Research CommUnications,. VoL 9, No. 9, 1977

and 62.O2~noi of IA. Triplicate

10 ml aliquots were incu-

bated in each experiment. Control samples conlsisted of microsomes inactivated by heating at 60°C per 5 min. At zero time the flasks were placed in the metabolic shaker and allowed to preincubate for 5 min.~ with shaking to ensure adequate starting concentrations

CI2H5 .OCH2CH2OCH2-CH-COOH

't@I'

of NADPH.

2 1~[(3-Acetamido- 2. 4. 6- tr,,odophenoxy} -ethoxy]-methy{ l-butyric ac,d

NHCOCH3

I

OCH2CH20H

't@'

Hydr oxyet hy|- ( 3-Acetamtdo-2,4.6-triiodop~enyl) ether (HPE)

NHCOCH3

!

OCH2COOH

I.~. I

3 - a c e t a m i d o - 2,4.6-lr,iodophenoxy -acetic acid (PAA)

"NHCOCH3 1 Figure 1 :

Structure of iopronic acid and its metabolites.

Extraction The incubation mixtures were adjusted to pH 1.5 with conc. HCI and extracted 4 times for 5 min. with 4

volumes of diethylether. The ether e×tracts were concentrated under vacuum

and the residuess

taken up in 0.5 ml of methanol s were

assayed.for quantitative determination of iopronlc acid and its metabolites

(Pitr~ and Felder s 1976).

Pharrnacologica/ Research Communications, L/o/. 9, No. 9, 1977

837

Aqueous residues from the incubation mixtures were tested for the presence of inorganic iodide (Pitr~ and Maffei Facino,1976).

elm

•~

75 6o

E

C

0

30

60

90

120 rain.

-o c

E'S

3~.

20-

oE ,o -

"

0

..............

I

.

.

.

.

.

30

Figure 2 :

'

I

"

--

60

-

|

.

.

.

.

.

.

.

.

90

-

•

.

.

.

.

.

.

120 min

Time course of IA metabolism by rat liver cromosomes° Experimental

conditions are described

in the text. Each point representsthe mean + S.E. of four different determinations. x

x

= unchanged IA = HPE

•

For~studies

•

=PAA

of substrate binding (Fig. 4,5)

I) Gytochrome P-450 content Microsomal

cytochrome P-450 was determined by

its carbon monoxide difference

spectrum after reduction

Pharmacologicat~Research Communications, Vol. 9, No. 9, 1977

838

with Na2S204. Cytochrome P-450 contenO was measured on a model 25 Beckman spectrophotometer ence in absorbance

by the differ-

between 450-490 nm 3 Using the

extinction coefficient

of 91 mM -1 cm -1 (Schenkman

and Sato,1968),In all the experiments~

the concentration

of microsomal P-450 was 0,522± 0.070 nmol/mg of protein (Curve 1 Fig. 5),

20"

10

0 Fi gure 3 :

•

|

30

60

1

|

120 min.

90

Dependence of IA metabolism on liver microsomal monooxygenases.

Each point

representsthe mean ~ S.E, of four different experiments. x

x

= incubation in the presence of NADPH and 02

A

A

= incubation in the absence of NADPH and 02

•

= incubation in the pres-~ ence of NADPH after replacement of 02 with CO.

" 'a Pharmacotogm I Research Communications, VoL 9, No. 9, 1977

839

2) S__ubstrate bindin~ a) Aerobic substrate binding Spectra were determined between 500 and 350 n m w i t h the Beckman model 25 spectrophotometer.Difference

binding

spectra were obtained at room temperature.All cuvettes contained 2.5 ml of a suspension of 8.5 mg of microsomal protein/ml in O.l M phosphate buffer.The sample cuvette also contained 2.5~Jmol of

IA in lO~ul of methanol and

the reference cuvette contained IO ~I of methanol. B6th cuvettes were

gently inverted 5 times and then

left to equilibrate in

the sample chamber for I min.

before scanning (Fig.4).

U

J~

,i

i

i

lie

soo w A w u ~ ' ~ (n,n)

Figure 4:

Spectral change observed agter addition of 2.5 ~mol of IA to the sample cuvette.

(X max

425, /(

395)

Experimental conditions as in the text.

Pharmacological'Research Communications,...VoL 9, No. 9;. 1977

840

b) Dependence on substrate concentration The study of the dependence of the binding on substrate concentration was performed as described above~ adding to the sample cuvette 1.25, 2.5, 5.0 or

7.5 ~nol

of IA (Table I), i

TABLE I Intensity of IA difference spectra at various drug concentrations

~A(425_500)/mgof

Amount of IA added to the sample

protein

cuvette

0=oi ) ,

,,,

_

,,,,

1.25

0.0025 ÷ 0.00022

2.5

0.0030 ~0.00031

5.O

0.0042 + 0.00015

7.5

0.0045 + 0.00097

Each figure is the mean ~ S. E. of three different determinations.Details are given under

of experimental

conditions

"Materials and Methodos".

c) Substrate binding to reduced cytochrome The 2.5 ml of microsomal cuvettes were reduced with an excess of dithionite. then added IA ( 7 . 5 ~ o i ) ,

suspension in both

7.5~moI

NADPH or with

To the sample cuvette was as described in section 2a.

d) Interaction of IA with dithionite-reduced

P-450-CO

complex 2.5)amol of IA in methanol were added to 2.5 ml microsomal

suspension in O.I M phosphate buffer.

Pharmaco/ogical Research Communications, Vol. 9, No. 9, 1977 The cytochrome

841

P-450 had been reduced by addition of

an excess of Na dithionite. CO was bubbled through

the

sample cuvette for 2 min. The reference cuvette contained dithionite but no CO (curve 2~ fig. 5). e) Binding to phospholipase C-treated microsomes The methodology used for phospholipase C incubation

Opticat density i

0.3.

1 0.2" 2

0.1

|

400 Figure 5:

-

i

w

WAVELENGTH

i

•

(nm)

600

Effect of IA on the dithionite-reduced P-450

spectrum.

Curve I, without drug;curve 2, after addition of 2 . 5 ~ o i

of IA.

Experimental conditions as in the text.

Pharmacological" Research.Communications, VoL 9, No. 9, 1977

842

was that of Chaplin and Mannering (1970). At the end of the incubation period £he cytochrome P-450 content was again determined: phospholipase C treatment caused a loss of about 25% of cytochrome 2-450. ,Binding studies were performed as described in section (2a) above.

RESULTS The time course of iopronic acid metabolism by t

rat liver microsomes is shown ~ in Fig. 2. Time studies of incubatidn o~ iopr6nic acid with rat liver microsomes showed a linear increase ,in the amount of HPE and PAA formed between i anti 90 min. After 90 min. ~ whenl the amount of IA metabolites represent 22% of the unchanged drug~ no further increase occurred. Replacement of 02 with an 02 + CO atmosphere (IO :' 90), Which is known to inhibit the hepatic microsomal m i x e d - f u n c , t i o n

oxidase

(Conney ed a l . ,

1957), result-

1

ed i n a c o n s i d e r a b l e

decrease

in the metabolism

of

t

iopronic

acid

(Fig.,' 3).

I n t h e same way, t h e a b s e n c e

i

in the incubation

mixture

of an

NADPH-generating p

produced a marked decrease

'system

~

in the metabolism

of iopronia

acid.

Binding of IA to hepatic micros0ma ~ P-450

Type of spectra

The addition of IA as substrate for the microsomal mixed-function oxidase system to aerobic liver microsomes causes a type II spectral change, characterized by the appearance of an absorption peak at 425 nm and a trough at 395 nm (Fig. 4).

Pharmacological Research Communications, 1/ol. 9, No. 9, 1977 DI SC,,USSI ON The dependence of the IA metabolism on 02 and NADPH and its inhibition by CO demonstrate the involvement of the NADPH-dependent mixed-function oxidase system. The molecular specificity of type II binding exhibited by IA also confirms the role of the mixed-functlon oxidase system in the biotransformation of this compound. !n vitro experiments carried out with IA added to rat liver microsomal preparations indicate that the drug is metabolized only to a limited extent. The metabolites~ HPE and PAA~ are formed at the same rates, although larger quantities of HPE are found. This is in agreement with data already available in man. Pitr~ and Felde= (1976)~ in fact~ reported that only 8% of the administered dose is excreted as PAA and HPE in urine in the first 48 hours. The fact that the two metabolites still contain the three iodine atoms present in the parent compound indicates that the C-I bonds of the molecule are not involved in the oxidative pathway. Furthermore, our own analyses for the presence of free iodide show that during incubation no iodine leakage occurred in the incubation mixture. This fact~ together with the limited extent of the metabolism, indicate from a pharmacological point of view that IA seems to fulfil some of the requirements for a cholecystographic agent. As reported in table I~ the magnitude of the spectral change observed depends on the concentration of substrate added to the microsomal suspension. Like aniline~ a type II substrate (Schenknmn et al.~

843

Pharmacological'Research Communications, Vo/. 9, No. 9, 1977

844

TABLE

II

Effect of reducing agents (NADPH and dithionite) and phospholipase C-treatment on iopronic acid binding to P-450.

CUVETTE CONTENT Reference

Microsomal

suspension

Sample

~A(425-500)/rag of protein

Microsomal suspension

0.0045 + 0.O01

+ 7,5 2~mol IA

Microsomal suspension + 7.5 ~mol NADPH

Mi crosomal suspension

O. 0020 + O. 0002

+ 7.5 ~umol NADPH + 7.5)xnol IA

Microsomal suspension

Microsomal suspension

+ Na2S204 in excess

+ Na2S204 in excess + 7.5 )~nol IA

Phospholipase C-treated

Phospholipase C-treated

microsomes

microsomes + 7.5~moI

0.0025 + 0.0003

IA

The data are the means + S.E. of three different determinations. Details of experimental conditions are given under "Materials

and Methods".

1973), IA interacts with ferricytochrome P-450 and displaces carbon monoxide from the dithionite-reduced P-450. The extent to which-IA interacts with dithionite-reduced P-450 is evident in Fig. 5" from the magnitude

Pharmacological Research Communications, Vol..9, No. 9, 1977 of the decrease in the CO-complex spectrum. Equally,the addition af an equimolecular amount of NADPH to micra_ somes in the presence of IA causes a decrease in the absorption peaks of the binding spectra~ while the addition of an excess of the chemical reductant sodium dithionite obliterates the IA-induced spectral changes (Table II)• as previously described by Schenkman et al. (1967)for other type II substrates. As further proof that IA give s type II binding spectra• we studied the binding of IA to phospholipase C-treated microsomes•

since this agent is known to

destroy type I binding spectra but not type II (Chaplin and Mannering•

1970).

The results obtained show that IA binding fz not destroyed by such treatment ~Table Ill• even though the strength of binding is less than the initial strength because of partial denaturation of the cytochrome P-450 by phosphollpase C.

REFERENCES CHAPLIN M.D., and MANNERING G.J., Mol. Pharmacol. 6, 631 (1970). CONNEY A.H. ~ BROWN R.R. ~ MILLER J.A. and MILLER E.C., Cancer Res., 17~ 628 (1957). IMAI Y. and SATO R.~ J. Biochem.

(Tokyo)• 62•

239, (1967). JEFCOATE C.R. and GAYLOR J.L.~ Biochemistry N.Y., _Sj 3464 (1969). JEFCOATE C.R. 3 GAYLOR J.L. • CALABRESE R.L. • Biochemistry N.Y.• 8• 3455 (1969).

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Pharmacological'Research Communications, VoL :9, No. 9, 1977

LOWRY O.H.,ROSEBNOUOH N.J., FARR A.L. and RANDALL R.J., Jo Biol. Chem., 193, 265 (1951). MAILMAN R.B., KULKARNI A.P., BAKER R.C. and HODGSON E., Drug Metab. Dispos., 2~ 301 (1974). MANNERING G.J., Fundamental of Drug Metabolism and Drug Disposition, (Eds. La Du B°N.~ Mandell H. G. and Way E.L.) p. 206, Williams and Wilkins, Baltimore (1971).

PITRE D. and FELDER E., Farmaco ed. prat., 31~ 540 (1976). PITRE D° and MAFFEI FACINO R.~ Farmaco ed. sci°~ 31, 755 (1976). SCHENKMAN J.B., CINTI D.L., MOLDEUS P.W. and ORRENIUS S.~ Drug Metab. Dispos., _I, IIi (1973). SCHENKMAN J.B., REMMER H., ESTABROOK R.W. ~ Mol. Pharmacol.~ 3, 113 (1967). SCHENKMAN J.B. and SATO R., Mol. Pharmacol., _4~ 613 (1968).