Effects of narcotic analgesics on lipase activity in vitro

Pharmacologica/ Research Communications, Vol. 14, No. 4, 1982

327

EFFECTS OF NARCOTIC ANALGESICS ON LIPASE ACTIVITY IN VITRO

J. R. Traynor and P. D. Kelly Biochemical Pharmacology Laboratory, Department of Chemistry, University of Technology, Loughborough, Leics. ReceivedmfinalformO0

LEII 3TU, U.K.

1982

S~qARY

The effects of a number of narcotic analgesic drugs on the hydrolytic

activity of a partially purified lipase from the mold Rhizopus arrhizus have been studied.

The narcotics stimulate activity in the order

etorphine>diprenorphine>buprenorphine>levorphanol>morphine=dextrorphan> codeine.

The stimulatory effect is stereospecific, levorphanol being

i00 times more potent than dextrorphan.

At high doses all drugs inhibit

enzymic activity in a non-stereospecific manner.

It is suggested that a

direct interference with lipase enzyme activity may contributo to the previously reported lipolytic effects of morphine.

INTRODUCTION In vivo acute and chronic morphinisation alters lipase activity (Fresia et al, 1967; Sabl4-Amplis et al, 1975; |Vong et al, 1977).

In

vitro morphine causes a dose-dependent increase in fatty acid release

from the rat epididymal fat pad, reaching a maximum effect at 2.5 mm CWong et al, 1977).

It has been proposed that morphine may exert its

lipolytic effect via a low affinity interaction with the plasma membrane adrenaline receptor CWong et al, 1977).

Recently the morphine-like

peptide 8-endorphin has been reported to have lipolytic activity in isolated fat cells obtained from rabbits at nearly physiological

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© 1982 The Italian Pharmacological Society

328

Pharmacological Research Communications, VoL I4, No. 4, I 9 8 2

c o n c e n t r a t i o n (Schwandt e t a l ,

1979).

This e f f e c . t i s n o t m e d i a t e d

by an o p i a t e r e c e p t o r .

N a r c o t i c drugs have l o c a l a n a e s t h e t i c p r o p e r t i e s (Simon and g o s e n b e r g , 1970).

We have p r e v i o u s l y e s t a b l i s h e d

drugs with l o c a l a n a e s t h e t i c p r o p e r t i e s triglyceride

lipase

a t high doses

affect the activity

1970) i n v i t r o .

o r d e r to d e t e r m i n e w h e t h e r d i r e c t e n z y m a t i c i n t e r a c t i o n s r e s p o n s i b l e for the observed l i p o l y t i c

the lipase

of a

( T r a y n o r and Kunze, 1976), and o t h e r i i p o l y t i c

enzymes ( T r a y n o r and Kunze, 1975; Kunze e t a l ,

studied the e f f e c t s

that

effects

might be

o f morphine we have

o f s e v e r a l o p i a t e compounds on t h e a c t i v i t y

(EC 3 . 1 , 1 . 3 )

In

of

d e r i v e d from t h e mold Rhizopus a r r h i z u s .

This i s a w e l t c h a r a c t e r i s e d enz>~e which we have p r e v i o u s l y used as a model ( T r a y n o r and Kunze, 1976).

8~TERIALS AND METHODS

Chemicals

All reagents and solvents used were o f analytical grade.

Tributyroylglycerol was purchased from B.D.H. (Poole, U.K.) and its purity checked by thin-layer chromatography.

Partially purified

lipase (EC 3.1.1.3) from the mold R. arrhizus was purchased from Boehringer (Nannheim G.F,R.).

Samples of opiates were gifts from

HacFarlan Smith, Edinburgh (Codeine and morphine), Reck~tt and Colman, llull (etorphine, buprenorphine and diprenorphine) andRoche Products Ltd., Welwyn Garden City (dextrophan and levorphanol).

Enzyme assays

The activity of the mold lipase towards triacyl-

glycerols was measured using emulsified tributyroylglycerol as substrate, This substrate is recommended over longer chain substrates for kinetic investigations of lipase enz~:.es (Semeriva et al, 1971) since it

329

Pharmaco/ogica/ Research Communications, VoL 14, No. 4, 1982 emulsifies spontaneously in dilute NaCL solutions, thus simplifying the system, whilst the released butyric acid has a low pka, The a s s a y c o n s i s t e d added vi_~a a g a s - t i g h t the r e q u i r e d stirring

syringe

amounts o f t r i b u t y r o l y g l y c e r o l ,

i n 30 ml, o f 100 mH NaC1 c o n t a i n i n g

amount o f o p i a t e .

f o r 5 min. a t

incubation

of the desired

The s u b s t r a t e

1000 r e v .

m i x t u r e was a d j u s t e d

was e m u l s i f i e d

min. -1 a t a t e m p e r a t u r e o f 25 ° . t o pH 8 . 0 by t h e a d d i t i o n

The enzyme (10 ~g) was added and t h e l i b e r a t i o n a stream of nitrogen, a pH s t a t least

of butyric

was f o l l o w e d a t 25 ° as a f u n c t i o n

c h a r g e d w i t h 20 n~l NaOH.

3 min.

All determinations

by

Kinetic

o f NaOH. acid,

under

o f time u s i n g

c u r v e s were l i n e a r

were made a t l e a s t

The

for at

in duplicate

and

reproducibility was found within ± 5%.

RESULTS Under the experimental conditions employed all of the narcotic analgesics stimulated the hydrolysis of emulsified tributryoylglycerol by the mold lipase (Fig. i).

Potency decreased in the order, etorphine,

which was active at nH concentrations, >diprenorphine>buprenorphine> levorphanol>morphine=dextrorphan>codeine, although the drugs do show varying degrees of maximal obtainable stimulation.

It is of note that

levorphanol is iO0 times more active than its isomer dextrorphan. At higher concentrations all of the opiates had a powerful inhibitory effect on the activity of the lipase towards the emulsified substrate (Table i).

Indeed if high enough doses were employed maximal

inhibition of activity could be observed, for exa)~le morphine at iO mM caused a 96 % inhibition.

The very active oripavine alkaloids etorphine

Pharmacological Research Communications, Vol. 14, No. 4, 1982

330 40

c

Q

~ o

0

@

lb-'

lo

153

-

Opiate (r,,,9

Fig. 1

S t i m u l a t i o n o f t h e h y d r o l y s i s o f t r i b u t y r i n by the mold l i p a s e in the p r e s e n c e o f 0 e t o r p h i n e ; • b u p r e n o r p h i n e ; @ diprenorphine; A levorphanol; A dextrorphan; [[]morphine; D c o d e i n e . Experimental d e t a i l s as in the text.

and diprenorphine would also appear to require mN levels to cause effective inhibition.

Thus unlike, stimulation, where a large

variation in drug effect was observed, the opiates are almost equipotent as lipase inhibitors.

Table i

Inhibition of lipase activity by opiates

Opiate ( c o n c e n t r a t i o n )

% Inhibition

Dextrorphan (5 mN)

21

Levorphanol (5 ~1)

50

Morphine (5 raM)

S1

Diprenorphine ( 1 . 0 raM)

36

Etorphine (O.i n~4)

14

DISCUSSION All of the drugs with narcotic properties increase the activity of the triglyceride lipase from Rhizopus arrhizus, whilst at high doses

Pharmacologica/ Research Communications, Vol. 14~ No. 4, 1982

causing inhibition, properties of this

331

ltowever, major d i f f e r e n c e s a r e s e e n i n the inhibition

when compared with s t i m u l a t i o n .

Tile drugs v a r y g r e a t l y i n t h e i r a b i l i t y

t o cause s t i m u l a t i o n .

E t o r p h i n e i s a p p r o x i m a t e l y 10,000 times as p o t e n t as c o d e i n e , and l e v o r p h a n o l i s 100 times more p o t e n t than morphine, range f o r i n h i b i t i o n i n t h e n~,! r a n g e .

i s much l e s s e x t e n s i v e and a l l compounds a r e a c t i v e

Of t h e i s o m e r i c compounds d e x t r o r p h a n and l e v o r p h a n o l

only levorphanol is a n a l g e s i c a l l y

active since it,

e n a n t i o m e r , has t h e c o r r e c t s t e r e o c h e m i s t r y t o f i t receptor.

tlowever, t h e dose

but n o t i t s the a n a l g e s i c

In tile p r e s e n t work l e v o r p h a n o l i s markedly more p o t e n t

as a " l i p a s e - s t i m u l a t o r "

than d e x t r o r p h a n , w h i l s t t h e compounds have

similar inhibitory effects.

This would s u g g e s t t h a t s t i m u l a t i o n i s

stereospecific, but inhibition is non-specific. In previous work we have demonstrated that drugs with local anaesthetic properties affect the activity of lipolytic enzymes, causing both stimulation and inhibition.

Two hypotheses have been

put forward to explain these effects, namely an interference with substrate, thus changing its physico-chemical properties and susceptibility to hydrolysis (Scherphof et al, 1972) and a binding to the enzyme, possibly displacing calcium from a binding site (Waite and Sisson, 1972).

Although no calcium ions are involved in

the present system the observed stereospecificity and the wide range of drug activitSes, suggest that the stimulatory effects of the drugs may well be due to an interaction with enzymic sites.

Certainly we

have previously demonstrated that the major tranquilliser chloropromazine, which "is also a potent local anaesthetic, is able to bind to phospholipase A 2 (Kunze et al, 1976).

On the other hand the

observed lack of stereospecificity and the equipotency of a range of drugs, suggests that inhibition of lipase-catalysed hydrolysis is due

332

Pharmacologica/ Research Communications, Vol. 14, No. 4. 1982

to a non-specific interaction, possibly with tile lipid substrate. Certainly this inhibition occurs at doses where non-specific local anaesthetic-type

actions of narcotics are seen (Kosterlitz et al, 1967).

Finally the results of this study suggest that the observed in vivo and in vitro lipolytic effects of opiates (ffong et al, 1977) might, in part, be caused by a direct action on lipolysis.

We thank the S.R.C. and Reckitt and Colman Ltd. for support.

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