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Inhibition of inflammatory pain by naloxone and its N methyl quaternary analogue
Life Sciences, Vol. 31, pp. 1209-1212 Printed in the U.S.A.
Pergamon Press
INHIBITION OF INFLAMMATORY PAIN BY NALOXONE AND ITS N METHYL QUATERNARY ANALOGUE L. RIOS and J.J.C. JACOB Laboratory of Pharmacology, Pasteur Institute, F75724 Paris C~dex 15 (Received in final form June 14, 1982) Sun~nary Naloxone hydrochloride (Nx) and methylnaloxone methylsulfate (MeNx) antagonized the inflammatory pain, but not oedema, produced by intraplantar carrageenan in rats. The liminar effective dose (3 pg/kg) was the same for the two drugs after intraplantar or subcutaneous injection. The s.c. dose effects curves were, as a whole, similar for the two drugs. The effect was long lasting and stereospecific. The low "analgesic" s.c. doses of Nx were able to antagonize morphine analgesia, those of MeNx were not. The interpretation is difficult; a peripheral action or better the local production of morphinomimetic metabolite(s) might account for most facts. It is well known that naloxone, a specific opiate antagonist, produces hyperalgesia in experimental animal and in man with a variety of nociceptive methods, a property indicating the regulatory function of endogenous opioids (i). It is however ineffective in some tests and more recently an appearantly paradoxical, opposite effect of this drug has been described: low doses of naloxone were found to inhibit inflammatory pain induced either acutely with intraplantar prostaglandin (2) or chronically with Freund's adjuvant (3). Ferreira and Nakamura (2) obtained the analgesic effect with intraplantar injection of naloxone and suggested a peripheral local site of action, an hypothesis which was questioned by Kayser and Guilbaud (2) who used the i.v. route. To gain some precision about the site and mechanism of this action, we have compared the efficacy of naloxone (Nx) and of its N methyl-quaternary analogue (MeNx) after intraplantar (i.pl.) and subcutaneous (s.c.) administrations. MeNx is assumed to act mainly at peripheral levels; to verify that it had no relevant central action we have studied comparatively its ability to antagonize morphine (Mo) analgesia, MR 2266 and MR 2267 [respectively (-) and (+) 5,9 e diethyl-2 (3 furyl-methyl) 2' hydroxy 6,7 benzomorphan (4)] were studied to assess the stereospecificity of the action. Methods Male Sprague-Dawley rats (200-250 g) were used; inflammation was produced by i.pl. injection of carrageenan (500 pg in 0.i ml); the opiate antagonist dissolved in saline was injected i.pl. (0.01 ml/100 g in the inflamed hindpaw) or s.c. (0.I ml/100 g) .5 hr after carrageenan. Pain thresholds were measured according to Randall and Sellito (6) with a Basile analgesimeter on both inflamed and controlateral hindpaws just before carrageenan and .5 - 1.5 - 2.5 - 3.5 and 4.5 hrs after the opiate antagonist. Controls received saline instead of the opiate antagonist. The antagonism of Mo analgesia was studied in distinct groups of rats with the hot plate test (55 °C). Morphine sulfate (15 mg/kg s.c.) was injected 30 min before the test ~nd the antagonist at times corresponding to those of the Randall and Sellito test. To avoid learning, rats were exposed only once (cut off-time: 60 sec) and distinct groups of rats were used for each time interval. Groups of 6 rats were used in each experiment and most experiments were dupllca0024-3205/82/121209-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1210
Analgesia by Naloxone and Methylnaloxone
Vol. 31, No.s 12 & 13, 1982
ted. Nx was used as the hydrochloride and MeNx as the methylsulfate; the doses are expressed in Nx base for the two drugs. FIG. 1 Nx 3 IJg/k 9 MeNx Thr,~sh Inhibition of inflammaI PI tory pain by a low dose of naloxone and methyl N naloxone. Ordinates: variations in pain thres-1 holds of the inflamed paws. T: treated. C: controls injected with carrageenan alone (one unit = 30 g pressure; base line thresholds wio ~ " [,, thout or before inflammation were 4 to 5 units) Abscissae: Time in hr _1 a2.____. ::Significant for P = 0.05 (t-test)
Results Nx and MeNx prevented pain produced by intraplantar carrageenan. The liminar effective dose was 3 Ug/kg for the two drugs and the two routes, i.pl. and s.c. (Fig. i), I ~g being ineffective; for MeNx, the effect of 3 Ug was greater by the i.pl. than by the s.c. route (not reproducible). A higher dose (i0 ug/kg s.c.) of Nx or MeNx had an unexpected long duration of action (2.5 hr) which was delayed for MeNx (Fig. 2); this delay was still observed after 30 Ug s.c. (Fig. 4) but no more after higher doses of MeNx (not illustrated). As a whole the analgesic effects by the s.c. route increased up to 300 ug/kg being almost equal for the two drugs (Fig. 3); with 3 mg/kg, MeNx was still analgesic but Nx was no more; on the contrary Nx increased the carrageenan hyperalgesia in one out of two experiments; this latter observation was made also by Kaiser and Guilbaud (3). The analgesic effect of opiate antagonists is stereospecific; 30 Ug of MR 2266 [the (-) isomer] were definltely analgesic whereas MR 2267 had no significant effect at the same dosage (Fig. 2). i0 ~g of the-two drugs were inactive (not illustrated). At several occasions, in treated animals, the pain thresholds of the inflamed paw became somewhat higher than before the injection of carrageenan, indicating an analgesic effect greater than the mere antagonism of inflaTmnatory pain. On the contrary, the pain thresholds of the controlateral, non inflamed paw, were hardly modified; slight hyperalgesla was sometimes observed which was significant occasionally, at variable and erratic time intervals. Oedema measured in all experiments was not diminished; it was also not increased in these and other experiments where a liminar dose of i.pl. carrageenan (i0 Ug) was used. In distinct experiments (Fig. 4), the low analgesic doses of Nx (3-10 and 30 ~g/kg s.c.) were found to antagonize the morphine analgesia; the time curve of this effect was nearly the same as for the antagonism of carrageenan hyperalgesia. On the contrary 3 and i0 ~g/kg s.c. of MeNx had no significant antimorphine action and 30 Ug had a transient one, being ineffective after 2.5 hr when the "analgesic" action on the inflamed paw reached a maximum. With i00 ~g/kg s.c. of MeNx, the two activities were no more dissociated (not illustrated). Discussion Our results confirm the observations of Ferreira and Nakamura (2) and of Kaiser and Guilbaud (4) that low doses of Nx inhibit inflammatory pain. Further-
Vgl. 31, No.s 12 & 13, 1982
Analgesia by Naloxone and Methylnaloxone
Nx
A
FIG. 2
MeNx
Thresh
Duration of the "analgesic" effect of i0 p$/k$ s.c. of Nx or MeNx - Stereospecificity of the effect of MR 2266 Ordinates, abscissae and symbols as in Fig. I - - treated --- controls. Note that for all figures the variations of the thresholds from the base line values of the inflamed paws are corrected for the slight variations occuring in the controlateral non inflamed paw.
10 ,AJg
~--~"-l--)+~-~:
--" --
~ 2
MR 2266
MR 2267
30
o
pg
\: CA
"'-.T~ ,
J"
.5
t.5
2.5
3.5
,
CA ~'~""~'~~"~+' i" J i I
F'T--[
: :. __,,.5 0
\
4.5 h
- . 5 0 .5
1.5
2.5
1211
I
3.5
I
4.5 h
FIG. 3 Dose effect curves for Nx (e) and MeNx (+) "analgesic" action. Ordinates: analgesic action in units, calculated by adding the differences, for each time interval, between controls and treated. Abscissae: s.c. doses, log. scale. t
$
10
30
t00
300
3o00 ~a;)/l~
FIG. 4
anal u~lrs
ant, Ha Y.
Nx
MeNx
i ~00
~ - 60
Comparisons of the "analgesic" and antimorphine effects of low doses of Nx and MeNx. Ordinates: + analgesic action in units (for each time interval, difference between the thresholds of inflamed paws of control and treated animals) • Antagonism of Mo analgesia in % calculated according to the formula T - C I00- Mo----~C x i00 where Mo,
3 ~g %
i 20
~
~] T
i
J
•.
T~i
-100 2-
\\
T
T and C are the latencies (in sec) of the licking reaction for animals treated respectively:saline and morphine, the antagonist and morphine and saline alone. MeNx was not more effective in another series of experiments where the dose of morphine was reduced to I0 mg/kg s.c. Abscissae: interval in hr between the injection of the antagonist and the test.
,,- \ l ÷
r~ r \ ',
.°oJ '
,o
t .5
1.5
2.5
3.5
,~.5
.5
1.5
2.5
3.5
4.5 h
1212
Analgesia by Naloxone and Methylnaloxone
Vol. 31, No.s 12 & 13, 1982
more the liminar effective dose (3 ~g/kg) was the same for the two routes, intraplantar and subcutaneous; this result does not allow to know if the site of action is local or central; it indicates only that the distribution of Nx or of active metabolite(s) is similar for the two routes of administration at the time of the measurement (shortest: .5 hr). Our results differ from those of the cited authors by the unexpected long duration of action of low doses of Nx. As a whole, the analgesic effectiveness of MeNx was almost the same as that of Nx for most of the studied dosages. This observation argues in favour of a peripheral site of action as MeNx is not expected to permeate the blood brain harrier (B.B.B.); this is strongly supported by the fact that low "analgesic" doses of MeNx - unlike Nx - were unable to antagonize the (central) Mo analgesia. A higher dose of MeNx (I00 ~g/kg s.c.) on the contrary antagonized Mo analgesia and probably permeate the B.B.B. to an appreciable extend. This analgesic action does not reflect a general antagonism of mediators of acute inflammation e.g. prostaglandins as the carrageenan oedema was not influenced. Stereospecific opioid receptors are involved as MR 2266 was effective and its (+) isomer, MR 2267 almost not. A troublesome point is that MeNx appeared equiactive with Nx whereas it has been reported to have much less affinity for opiate receptors (6). Among other possibilities might be relevant a) the use of another salt (the methylsulfate instead of the bromide) which dissociates less readily b) the production of active metabolite(s); such metabolite(s) might be Nx itself (by N demethylation) and also morphinomimetic opiates: oxymorphone (by N deallylation) or noroxymorphone; this latter "agonist" is also the demethylation product of Nx. As analgesia by Nx and MeNx have been observed at the level on the inflamed paw but not at the controlateral paw, it might be suggested that N deallylation of both antagonist, yelding morphlnomimetic metabolite(s) occurs more readily in inflamed tissue. This would account for the contrast between the "analgesic" and antimorphine action of low doses of naloxone. This and other possible hypotheses deserve further work. Whatsoever, this action might be of therapeutical interest due to the long duration of action of minute doses of substances not liable to produce addiction. Acknowledgments The authors wish to thank Dr. Ferster (Endo-Laboratory) for naloxone hydrochloride, Dr. Moulineau (Clin-Midy) for methylnaloxone methylsulfate, Ms Prudhomme for technical assistance. This work was supported by grants of the INSERM (contracts C.R.L. n ° 79.4.525.3 et A.T.P. n ° 80.79.112), and of the DRET (contract 81-524). References 1. 2. 3. 4.
J.J.C. JACOB and K. RAMABADRAN, Pharmac. Ther. 14 177-196 (1981). S.H. FERREIRA and M. NAKAMURA, Prostaglandins 18 191-208 (1979). V. KAISER and G. GUILBAUD, Brain Research 226 344-348 (1981). H. MERZ, A. LANGBEIN, K. STOKHAUS, G. WALTER and H. WICK, Narcotic antagonist Adv. Biochem. Psychopharmac. pp. 91-107, Raven Press, New York (1974). 5. L.O. RANDALL and J.J. SELLITO, Arch. Int. Pharmacodyn. iii 409-419 (1957). 6. A.K. KILLIAN, C.R. SCHUSTER, B.H. WAINER and H. MERZ, Life Sciences 28 12391243 (1981).
Pergamon Press
INHIBITION OF INFLAMMATORY PAIN BY NALOXONE AND ITS N METHYL QUATERNARY ANALOGUE L. RIOS and J.J.C. JACOB Laboratory of Pharmacology, Pasteur Institute, F75724 Paris C~dex 15 (Received in final form June 14, 1982) Sun~nary Naloxone hydrochloride (Nx) and methylnaloxone methylsulfate (MeNx) antagonized the inflammatory pain, but not oedema, produced by intraplantar carrageenan in rats. The liminar effective dose (3 pg/kg) was the same for the two drugs after intraplantar or subcutaneous injection. The s.c. dose effects curves were, as a whole, similar for the two drugs. The effect was long lasting and stereospecific. The low "analgesic" s.c. doses of Nx were able to antagonize morphine analgesia, those of MeNx were not. The interpretation is difficult; a peripheral action or better the local production of morphinomimetic metabolite(s) might account for most facts. It is well known that naloxone, a specific opiate antagonist, produces hyperalgesia in experimental animal and in man with a variety of nociceptive methods, a property indicating the regulatory function of endogenous opioids (i). It is however ineffective in some tests and more recently an appearantly paradoxical, opposite effect of this drug has been described: low doses of naloxone were found to inhibit inflammatory pain induced either acutely with intraplantar prostaglandin (2) or chronically with Freund's adjuvant (3). Ferreira and Nakamura (2) obtained the analgesic effect with intraplantar injection of naloxone and suggested a peripheral local site of action, an hypothesis which was questioned by Kayser and Guilbaud (2) who used the i.v. route. To gain some precision about the site and mechanism of this action, we have compared the efficacy of naloxone (Nx) and of its N methyl-quaternary analogue (MeNx) after intraplantar (i.pl.) and subcutaneous (s.c.) administrations. MeNx is assumed to act mainly at peripheral levels; to verify that it had no relevant central action we have studied comparatively its ability to antagonize morphine (Mo) analgesia, MR 2266 and MR 2267 [respectively (-) and (+) 5,9 e diethyl-2 (3 furyl-methyl) 2' hydroxy 6,7 benzomorphan (4)] were studied to assess the stereospecificity of the action. Methods Male Sprague-Dawley rats (200-250 g) were used; inflammation was produced by i.pl. injection of carrageenan (500 pg in 0.i ml); the opiate antagonist dissolved in saline was injected i.pl. (0.01 ml/100 g in the inflamed hindpaw) or s.c. (0.I ml/100 g) .5 hr after carrageenan. Pain thresholds were measured according to Randall and Sellito (6) with a Basile analgesimeter on both inflamed and controlateral hindpaws just before carrageenan and .5 - 1.5 - 2.5 - 3.5 and 4.5 hrs after the opiate antagonist. Controls received saline instead of the opiate antagonist. The antagonism of Mo analgesia was studied in distinct groups of rats with the hot plate test (55 °C). Morphine sulfate (15 mg/kg s.c.) was injected 30 min before the test ~nd the antagonist at times corresponding to those of the Randall and Sellito test. To avoid learning, rats were exposed only once (cut off-time: 60 sec) and distinct groups of rats were used for each time interval. Groups of 6 rats were used in each experiment and most experiments were dupllca0024-3205/82/121209-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1210
Analgesia by Naloxone and Methylnaloxone
Vol. 31, No.s 12 & 13, 1982
ted. Nx was used as the hydrochloride and MeNx as the methylsulfate; the doses are expressed in Nx base for the two drugs. FIG. 1 Nx 3 IJg/k 9 MeNx Thr,~sh Inhibition of inflammaI PI tory pain by a low dose of naloxone and methyl N naloxone. Ordinates: variations in pain thres-1 holds of the inflamed paws. T: treated. C: controls injected with carrageenan alone (one unit = 30 g pressure; base line thresholds wio ~ " [,, thout or before inflammation were 4 to 5 units) Abscissae: Time in hr _1 a2.____. ::Significant for P = 0.05 (t-test)
Results Nx and MeNx prevented pain produced by intraplantar carrageenan. The liminar effective dose was 3 Ug/kg for the two drugs and the two routes, i.pl. and s.c. (Fig. i), I ~g being ineffective; for MeNx, the effect of 3 Ug was greater by the i.pl. than by the s.c. route (not reproducible). A higher dose (i0 ug/kg s.c.) of Nx or MeNx had an unexpected long duration of action (2.5 hr) which was delayed for MeNx (Fig. 2); this delay was still observed after 30 Ug s.c. (Fig. 4) but no more after higher doses of MeNx (not illustrated). As a whole the analgesic effects by the s.c. route increased up to 300 ug/kg being almost equal for the two drugs (Fig. 3); with 3 mg/kg, MeNx was still analgesic but Nx was no more; on the contrary Nx increased the carrageenan hyperalgesia in one out of two experiments; this latter observation was made also by Kaiser and Guilbaud (3). The analgesic effect of opiate antagonists is stereospecific; 30 Ug of MR 2266 [the (-) isomer] were definltely analgesic whereas MR 2267 had no significant effect at the same dosage (Fig. 2). i0 ~g of the-two drugs were inactive (not illustrated). At several occasions, in treated animals, the pain thresholds of the inflamed paw became somewhat higher than before the injection of carrageenan, indicating an analgesic effect greater than the mere antagonism of inflaTmnatory pain. On the contrary, the pain thresholds of the controlateral, non inflamed paw, were hardly modified; slight hyperalgesla was sometimes observed which was significant occasionally, at variable and erratic time intervals. Oedema measured in all experiments was not diminished; it was also not increased in these and other experiments where a liminar dose of i.pl. carrageenan (i0 Ug) was used. In distinct experiments (Fig. 4), the low analgesic doses of Nx (3-10 and 30 ~g/kg s.c.) were found to antagonize the morphine analgesia; the time curve of this effect was nearly the same as for the antagonism of carrageenan hyperalgesia. On the contrary 3 and i0 ~g/kg s.c. of MeNx had no significant antimorphine action and 30 Ug had a transient one, being ineffective after 2.5 hr when the "analgesic" action on the inflamed paw reached a maximum. With i00 ~g/kg s.c. of MeNx, the two activities were no more dissociated (not illustrated). Discussion Our results confirm the observations of Ferreira and Nakamura (2) and of Kaiser and Guilbaud (4) that low doses of Nx inhibit inflammatory pain. Further-
Vgl. 31, No.s 12 & 13, 1982
Analgesia by Naloxone and Methylnaloxone
Nx
A
FIG. 2
MeNx
Thresh
Duration of the "analgesic" effect of i0 p$/k$ s.c. of Nx or MeNx - Stereospecificity of the effect of MR 2266 Ordinates, abscissae and symbols as in Fig. I - - treated --- controls. Note that for all figures the variations of the thresholds from the base line values of the inflamed paws are corrected for the slight variations occuring in the controlateral non inflamed paw.
10 ,AJg
~--~"-l--)+~-~:
--" --
~ 2
MR 2266
MR 2267
30
o
pg
\: CA
"'-.T~ ,
J"
.5
t.5
2.5
3.5
,
CA ~'~""~'~~"~+' i" J i I
F'T--[
: :. __,,.5 0
\
4.5 h
- . 5 0 .5
1.5
2.5
1211
I
3.5
I
4.5 h
FIG. 3 Dose effect curves for Nx (e) and MeNx (+) "analgesic" action. Ordinates: analgesic action in units, calculated by adding the differences, for each time interval, between controls and treated. Abscissae: s.c. doses, log. scale. t
$
10
30
t00
300
3o00 ~a;)/l~
FIG. 4
anal u~lrs
ant, Ha Y.
Nx
MeNx
i ~00
~ - 60
Comparisons of the "analgesic" and antimorphine effects of low doses of Nx and MeNx. Ordinates: + analgesic action in units (for each time interval, difference between the thresholds of inflamed paws of control and treated animals) • Antagonism of Mo analgesia in % calculated according to the formula T - C I00- Mo----~C x i00 where Mo,
3 ~g %
i 20
~
~] T
i
J
•.
T~i
-100 2-
\\
T
T and C are the latencies (in sec) of the licking reaction for animals treated respectively:saline and morphine, the antagonist and morphine and saline alone. MeNx was not more effective in another series of experiments where the dose of morphine was reduced to I0 mg/kg s.c. Abscissae: interval in hr between the injection of the antagonist and the test.
,,- \ l ÷
r~ r \ ',
.°oJ '
,o
t .5
1.5
2.5
3.5
,~.5
.5
1.5
2.5
3.5
4.5 h
1212
Analgesia by Naloxone and Methylnaloxone
Vol. 31, No.s 12 & 13, 1982
more the liminar effective dose (3 ~g/kg) was the same for the two routes, intraplantar and subcutaneous; this result does not allow to know if the site of action is local or central; it indicates only that the distribution of Nx or of active metabolite(s) is similar for the two routes of administration at the time of the measurement (shortest: .5 hr). Our results differ from those of the cited authors by the unexpected long duration of action of low doses of Nx. As a whole, the analgesic effectiveness of MeNx was almost the same as that of Nx for most of the studied dosages. This observation argues in favour of a peripheral site of action as MeNx is not expected to permeate the blood brain harrier (B.B.B.); this is strongly supported by the fact that low "analgesic" doses of MeNx - unlike Nx - were unable to antagonize the (central) Mo analgesia. A higher dose of MeNx (I00 ~g/kg s.c.) on the contrary antagonized Mo analgesia and probably permeate the B.B.B. to an appreciable extend. This analgesic action does not reflect a general antagonism of mediators of acute inflammation e.g. prostaglandins as the carrageenan oedema was not influenced. Stereospecific opioid receptors are involved as MR 2266 was effective and its (+) isomer, MR 2267 almost not. A troublesome point is that MeNx appeared equiactive with Nx whereas it has been reported to have much less affinity for opiate receptors (6). Among other possibilities might be relevant a) the use of another salt (the methylsulfate instead of the bromide) which dissociates less readily b) the production of active metabolite(s); such metabolite(s) might be Nx itself (by N demethylation) and also morphinomimetic opiates: oxymorphone (by N deallylation) or noroxymorphone; this latter "agonist" is also the demethylation product of Nx. As analgesia by Nx and MeNx have been observed at the level on the inflamed paw but not at the controlateral paw, it might be suggested that N deallylation of both antagonist, yelding morphlnomimetic metabolite(s) occurs more readily in inflamed tissue. This would account for the contrast between the "analgesic" and antimorphine action of low doses of naloxone. This and other possible hypotheses deserve further work. Whatsoever, this action might be of therapeutical interest due to the long duration of action of minute doses of substances not liable to produce addiction. Acknowledgments The authors wish to thank Dr. Ferster (Endo-Laboratory) for naloxone hydrochloride, Dr. Moulineau (Clin-Midy) for methylnaloxone methylsulfate, Ms Prudhomme for technical assistance. This work was supported by grants of the INSERM (contracts C.R.L. n ° 79.4.525.3 et A.T.P. n ° 80.79.112), and of the DRET (contract 81-524). References 1. 2. 3. 4.
J.J.C. JACOB and K. RAMABADRAN, Pharmac. Ther. 14 177-196 (1981). S.H. FERREIRA and M. NAKAMURA, Prostaglandins 18 191-208 (1979). V. KAISER and G. GUILBAUD, Brain Research 226 344-348 (1981). H. MERZ, A. LANGBEIN, K. STOKHAUS, G. WALTER and H. WICK, Narcotic antagonist Adv. Biochem. Psychopharmac. pp. 91-107, Raven Press, New York (1974). 5. L.O. RANDALL and J.J. SELLITO, Arch. Int. Pharmacodyn. iii 409-419 (1957). 6. A.K. KILLIAN, C.R. SCHUSTER, B.H. WAINER and H. MERZ, Life Sciences 28 12391243 (1981).