Phosphodiesterase Inhibitors: Their Comparative Effectiveness in Vitro in Various Organs

PHOSPHODIESTERASE

INHIBITORS:

EFFECTIVENESS

IN

THEIR

VITRO

IN

COMPARATIVE

VARIOUS

ORGANS

Kenji ADACHI* and Fujio NUMANO *Department of Dermatology , University of Miami Medical School, Miami, Florida 33152, U.S.A. and Department of Medicine, Tokyo Ika-Shika National University, Faculty of Medicine, Bunkyo-ku, Tokyo 113, Japan Accepted September 16, 1976

Abstract-The inhibitor GMP-phosphodiesterase

constants of several inhibitors for cyclic AMP and cyclic from various organs are compared. The inhibitors were

classical theophylline, papaverine, and some of newly developed inhibitors: dazolidinone compound, R020-1724, and two phthalazinol compounds, and EG 626. Among the inhibitors the most potent. Both compounds

an imi EG 467

tested, papaverine and EG 626 were found to be were extremely inhibitory to platelet and arterial

phosphodiesterases. EG 626 was much more inhibitory terase than to cyclic GMP phosphodiesterase in platelet

to cyclic AMP and brain-extract

phosphodies and R020

1724 was inhibitory to cyclic AMP but not cyclic GMP-phosphodiesterase in brain extract. When the skin adenyl cyclase was activated by AMP, the addition of theophyl line blocked this activation, but EG 626 or EG 467 further potentiated the activation. These in vitro studies may serve as basic specific phosphodiesterase inhibitors.

Amer et al. (1, 2) recently aorta

of hypertensive

stress-and

rats.

desoxycorticosterone-induced)

of cyclic AMP and the increased appeared

et al. have more recently rich diet (3).

sensitivity

cyclic AMP

practical parameter

mechanism

in humans

level

and Numano

aorta.

since receptor A practical

of an inhibitor

of cyclic AMP.

of inhibition

in atherogenesis.

with hypertension,

adenylate

perhaps beneficial.

cyclase in the aorta

sites for the agonist way to control

are

the cyclic

for cyclic nucleotide-phosphodies We have compared

for the cyclic nucleotide-phosphodiesterase The determination

agonist.

may be therapeutically

by stimulating

this may be hazardous

terase, which inhibits the degradation

an in vitro system.

decreased

changes in aorta of rabbits fed a cholesterol

levels in the aorta

and weak in the diseased

inhibitors

cyclase to 3-adrenergic

are present

AMP level in a tissue is the administration

of several

the

The decrease in cyclic AMP in aorta

similar biochemical

cyclic AMP levels can be elevated

abnormal

were

in

(spontaneous,

activity of low Km cyclic AMP-phosphodiesterase of adenylate

mechanisms

with a 19-adrenergic agonist,

findings

of the

metabolism

of the hypertension

the common

Their findings suggest

which increase

Although

for the effectiveness

of the cyclic nucleotide

of the causes

found similar metabolic

If similar biochemical methods

aberration

tests

level of cyclic GMP.

to be due to the increased

also due to the decreased

already

reported

Regardless

screening

constants

the effectiveness

in various

organs

of these drugs

which could be used to select the most effective compound

using

will yield a

for clinical use.

Cyclic 3H-AMP was purchased from New England Nuclear, Boston, Mass. and cyclic 3H-GMP from Amersham1Searle , Arlington Heights, 111. Their specific activities were 40 and 15 Ci/mmole, checked

by thin-layer

(Analtech, with 95 GMP

respectively.

The radiochemical

chromatography

purity of the compounds

on cellulose

Newark, Delaware and Brinkman, Westbury, ethanol-IM ammonium acetate (70:35, v/v).

were obtained

from

Sigma Chem.

grade or the highest grade commercially

terase

inhibitors,

available.

Roche.

Other new inhibitors,

467 and EG 626) which were a gift from Professors of Japan

Arteriosclerosis

Research,

Tokyo,

12 week old C57 mice immediately removed

from brain

proximal

portion

of quadriceps

auditory

meatus

(free of cartilage).

prepared

rats and platelets

product

(cerebrum),

before

The enzyme

after

Appropriate

each enzyme

assay.

fresh from human

or guanosine

for 10-15 min, the reaction

ponding

mixture

with 95 % ethanol:

III (Beckman, tration

tration

and the products

0.4 ,, Omnifiuor

muscle-3.4,

was measured

0.1 mm in thickness

acetate

C9H,O,N

were

from albino

by Scott and Solomon AMP or GMP,

(4).

was further

The con

The labelled substrate Assay mixture

by the method

on cellulose

contained

Nuclear,

spectrophotometry.

aorta-7.2

thin-layer

plates and

Spots corres

vials to which 200 ,ol present was detected

Boston,

The final protein

0.8.

of mostly

by a motor driven keratome

epidermis

with little dermal

C„H,,;O,N_

concen

heart-1.1,

The protein

of Lowry et al. (5). The effects of inhibitors obtained

in

Mass.) and 2 % BBS

mixture was: brain-0.08,

and platelets

5 mm and floated on Hank"s medium

and EG 626

at 37 °C, generally

(75:35, v/v) for 2 hr.

The radioactivity

(New England

The skin samples and consisted

After incubation

were scraped into counting

in ;gig/,'d of the reaction

skin-0.6,

The skin slices were cut 5 EG 467

of homogenate

in ice water and 5 ,el of 2N HCl was added to each

Calif.) by liquid scintillation

tested on skin slices.

leaf of liver,

pH 8.0; 5 mM MgCI2 i 0.1 tlg/ld of snake

(1 PI) was spotted

1M ammonium

of the tissue homognate

liver=0.75,

of tissue were

was obtained

of 5'-nucleotidase.

of 1 M LiCl2 was added to elute the nucleotides. a medium containing

pieces

donors.

as enzyme source.

was arrested

The reaction

to the substrate

Institute

of cyclic AMP (or cyclic GMP) with the labelled cyclic nucleotides

(105 cpm) ; and the tissue homogenate

developed

however,

by thin layer chromatography.

a final volume of 20 /d: 100 mM glycylglycine,

of the tubes.

Small

a

(EG

fresh from 8 to

edge of the largest

reaction,

by the addition

were separated

amounts

and S. Ishikawa,

concentrations

Aorta,

of cyclic nucleotide-phosphodiesterase

venom; variable

frontal

was

compounds+

muscle, and the skin from the inner surface of the external

were prepared

verted to adenosine

(R020-1724)

Tissues were removed

cervical fracture.

apex of heart,

were

One of the new phosphodies

T. Shimamoto

Japan.

plates

All other chemicals

are phthalazinol

assay system is similar to that described

and final product

Mo.

4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone

gift from Hoffmann-La

thin layer

N.Y.). The plates were developed Unlabelled cyclic AMP and cyclic

Co., St. Louis,

reagent

freshly

or DEAE-cellulose

was regularly

concen were also

were approx. contamination.

with the keratin layer up

for 5 min at 37'C. to the Hank's addition

The addition

or 5'-adenosine

medium causes cyclic AMP to accumulate

of the phosphodiesterase

lation.

The cyclic AMP

protein

of epinephrine

method

inhibitors

contents

further

monophosphate

in the skin slices (6). potentiates

Simultaneous

the cyclic AMP accumu

in the skin slices were measured

(7) with minor modifications

(5'-AMP)

by Gilman's

binding

(6).

RESULTS Cyclic AMP phosphodiesterase Fig. I shows

double reciprocal

from all tissues showed (Fig.

1).

Fig. 1. inhibition

The double

reciprocal

by theophylline constants

of the reciprocal

plots in the presence the inhibitor

was competitive were calculated

The typical inhibitors

marized

in Table

effective)

double

are shown 1.

Inhibitor

for papaverine

of decreasing

effectiveness

in Fig. 2.

(Y-axis):

in

thus the

with abscissa (X-axis)

i.e. 1/Km and 1/Kp respectively,

of various

of this graphical

inhibitors

The inhibitor

constants

and the largest

(the least effective)

on phosphodiesterase

inhibitors

in separate

in the presence

of

thus computed

are sum

were the smallest

(the most

for theophylline.

was papaverine,

This order was not changed

where

determination,

were plotted

plots for the low Km enzyme

of these inhibitors

there is no clear-cut

are also shown

for both high and low Km phosphodiesterase.

constants

1724, EG 467 and theophylline. Although

of theophylline

To increase the precision

reciprocal

phosphodiesterase

intersect on the ordinate

plots with and without the inhibitor;

Kp is written for Km (I -! i/Ki).

various

The phosphodiesterase

from the points of intersection

the high Kms and low Kms in the presence graphs.

enzyme.

two Kms, high and low, similar to the platelet

The lines with and without

The inhibition

plots for the platelet

The order

EG 626, R020

in all tissues thus far tested.

tissue specificity of the inhibitors,

it appears

that both pa

FIG. 1 Cyclic AMP hydrolysis by phosphodiesterase from platelets. A kinetic analysis with double reciprocal plots (Lineweaver-Burk plot) shows two Kms. Assay conditions are as described in the text. Double reciprocal plots in the presence of theophylline is also shown (the broken line). The unit of cyclic AMP concentration was in mM shown in abscissa.

FIG. 2 Double reciprocal plots of "low Km" muscle phosphodiesterase in the presence of various inhibitors. Concentration of the inhibitors used were 250 uM each of EG 626 EG 467=M, RO20-1724 (not shown); 62.5 uM of papaverine = 0; and 500 uM of theophylline=. . EG 626, EG 467, and R020-1724 were dis solved in ethanol and then added to the reagent mixture. The final ethanol con centration did not exceed 2 The effect of 2 % ethanol is also shown in this figure ([]) and the plots are the same as those of the control (x. ).

TABLE

I

Inhibitor

Constants

for

Cyclic

AMP-Phosphodiesterase

All figures are the average of two experiments. Ki= x 10-s M ( )*=Only mation is listed, since a clear-cut linearity was not always obtainable.

approxi

paverine

and EG 626 are extremely

quite effective in arterial

inhibitory

to platelet

phosphodiesterase

and also are

phosphodiesterase.

Cyclic GMP phosphodiesterase When cyclic GMP concentration

was varied from 300 /cM to 0.6 /eM, we again obtained

two Km values for each tissue tested. cases,

thus,

inhibitors terase.

the inhibition

constants

tested, R020-1724 Particularly,

but is a relatively

sistent with findings of Prasad AMP-phosphodiesterase was otherwise theophylline, TABLE 2

did not inhibit

inhibitor

Inhibitor

manner.

neuroblastorna

Effect

Among

the

inhibition

This is con

was specific for cyclic

cells.

The effectiveness

order

of papaverine,

of the inhibitors EG

626, EG 467,

(Table 2).

Constants

Accumulation

all

for cyclic GMP-phosphodies

for

Cyclic

GMP-Phosphodiesterase

All figures are the average of two experiments. Ki= x 10-s M; ( )*=Only mation is listed, since a clear-cut linearity was not always obtainable.

TABLE 3

in nearly

the brain cyclic GMP-phosphodiesterase;

R020-1724

i.e. in the decreasing

and R020-1724

in the same

inhibitor

for brain cyclic AMP-phosphodiesterase.

et al. (8) that

in mouse

unchanged:

was fully competitive

were calculated

was the least effective

this agent potent

The inhibition

of

Phosphodiesterase in Skin

Slices

Inhibitors

on

Cyclic

AMP

approxi

Effects of phosphodiesterase inhibitors on skin slices In Table 3, a preliminary experiment using skin slices is shown.

When skin slices were

incubated with epinephrine, the intracellular cyclic AMP levels were markedly increased. The addition of phosphodiesterase

inhibitors further potentiated this increase (Table 3).

When the skin slices were incubated with 5'-AMP, the addition of theophylline blocked this activation, but EG 626 or EG 467 further potentiated the activation.

Theophylline appears

to block the AMP-receptor site on a cell membrane, hence it cannot be used as a phosphodies terase inhibitor, should AMP be one of natural activators of cyclic AMP in skin in vivo. DISCUSSION The clinical effectiveness of the phosphodiesterase factors:

inhibitors is influenced by several

1. their potency as an inhibitor, 2. tissue specificity, 3. toxicity, 4. permeability

and local retention at the site of action etc.

The in vitro system used in the present study

gave the first two parameters and should be a useful screening method prior to clinical testing. The in vitro effectiveness is represented by the inhibition constant, i.e. the smaller the inhibitor constant, the better the clinical effectiveness.

In this regard, both papaverine and

EG 626 appear to be the most effective, but the former drug cannot be used clinically for any long-term therapy. side effects (9).

EG 626, on the other hand, can be used without the problem of

Shimamoto (10, 11) has treated certain atherosclerotic diseases with these

inhibitors and observed that EG 626 gave much faster and better clinical response than did EG 467.

These findings parallel their in vitro effectiveness.

Our data suggest that there is no clear-cut tissue specificity for the inhibitors. relative degree of in vitro effectiveness (Ki) is paralleled in different organs.

For example,

EG 467 is approx. twice as effective as theophylline in brain, heart, liver, etc. Kis of papaverine and EG 626 for platelets and arterial phosphodiesterase tremely small figures.

Their

However,

exhibited ex

Their Km/Ki ratio is 1-2 in platelets as compared with 5-1 in skin.

These drugs, therefore, can be effectively used as anti-platelet phosphodiesterase inhibitors. Among the inhibitors tested, the in vitro effectiveness of theophylline, EG 467 and papaverine is the same for both cyclic AMP-and cyclic GMP-phosphodiesterase. Papaverine has the smallest Ki for both cyclic nucleotides. The relative effectiveness of EG 626 and R020-1724 toward cyclic AMP-phosphodiesterase GMP-specific enzyme.

is far better than that toward the cyclic

This is particularly so in platelets and brain in the case of EG 626

and specifically so in brain in the case of RO20-1724.

The administration of these drugs

should theoretically cause an increase in cyclic AMP level either in platelets and brain or in brain respectively, without greatly effecting the cyclic GMP level, and such may be of future clinical significance. The solubility and permeability of an inhibitor are also important factors in determining their possible clinical usefulness.

The in vitro data do not yield this information.

in vitro system to test these factors is possible.

A "semi"

A tissue-slice model might be used to deter

mine the increase in intracellular cyclic AMP potentiated by the addition of the inhibitor.

A preliminary

experiment

that the inhibitors

using skin slices is shown in Table 3.

are permeable

The results clearly indicate

into skin slices and increase the intracellular

cyclic AMP

levels.

REFERENCES 1) AMER, M.S., GOMOLL,A.W., PERHACH,JR., J.L., FERGUSON,H.C. AND MCKINNEY,G.R.: Proc. natn. Acad. Sci. U.S.A. 71, 4930 (1974) 2) AMER, M.S., DOBA, N. AND REIs, D.J.: Proc. natn. Acad. Sci. U.S.A. 72, 2135 (1975) 3) NUMANO,F., MAEZAWA,H., SHIMAMOTO, T. AND ADACHI, K.: Ann. N. Y. Acad. Sci. 275, 311 (1976) 4) SCOTT,W.A. AND SOLOMON,B.: Biochem. biophys. Res. Commun. 53, 1024 (1973) 5) LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. AND RANDALL,R.J.: J. biol. Chem. 193, 265 (1951) 6) YOSHIKAWA,K., ADACHI,K., HALPRIN, K.M. AND LEVINE,V.: Brit. J. Derm. 92,249 (1975) 7) GILMAN,A.G.: Proc. natn. Acad. Sci. U.S.A. 67, 305 (1970) 8) PRASAD, K.N., BECKER,G. AND TRIPATHY,K.: Proc. Soc. exp. Biol. Med. 149, 757 (1975) 9) SHIMAMOTO, T.: Cardiovascular Disease, Edited by RUSSEK,H.I., Univ. Park Press, Baltimore (1974) 10) SHIMAMOTO, T.: Japan. Heart J. 16, 76 (1975) 11) SHIMAMOTO, T. MURASE,H. and NUMANO,F.: Mechanisms of Ageing and Development 5, 241 (1976)