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Partial cross tolerance to D-Ala2-D-Leu5-enkephalin after chronic spinal morphine infusion
Life Sciences, Vol. 32, pp, 2545-2550 Printed in the U.S.A.
Pergamon Press
PARTIAL CROSS TOLERANCE TO D-ALA2-D-LEU5-ENKEPHALIN AFTER CHRONIC SPINAL MORPHINE INFUSION Liang-Fu Tseng Toxicology Research Laboratory Veterans Administration Hospital Wood, Wisconsin 53193 and Department of Pharmacology and Toxicology The Medical College of Wisconsin Milwaukee, Wisconsin 53226 (Received in final form March 9, 1983)
Summary The development of tolerance to morphine and cross tolerance to D-Ala2-D-Leu5-enkephalin (DADL) at spinal cord level to the i n h i b i t i o n of t a i l f l i c k response was studied in rats t o l e r a n t to morphine. The long term intrathecal infusion of morphine sulfate was accomplished by means of an osmotic minipump. Intrathecal infusion of morphine sulfate (2 pg/hr) markedly elevated the t a i l f l i c k latency measured 24 hr a f t e r the start of infusion. The increased t a i l f l i c k latencies gradually decreased during 6 days of intrathecal infusion of morphine sulfate. Tolerance to morphine and DADL was determined by i n h i b i t i o n to the t a i l f l i c k response after intrathecal administration of cumulative doses of morphine sulfate and DADL. Chronic intrathecal infusion of morphine induced a marked tolerance to morphine but developed only a s l i g h t cross tolerance to DADL. The results indicate that there exists two separate types of opioid receptor, p- and 6-opioid receptor in the spinal cord of rats. Evidence has accumulated to support the contention that there exists at least two types of opioid receptors, p- and ~-receptors in the spinal cord of the r a t . This contention is based on the results obtained on comparat i v e potencies of opioid agonists on the production of analgesia and the d i f f e r e n t i a l s e n s i t i v i t y to the a n t a g o n i s t i c a c t i v i t y of naloxone ( 1 , 2 , 3 , ) . Intrathecal morphine and D-AlaL-D-Leu~-enkephalin (DADL) which stimulates p- and 6-opioid receptors respectively produced potent analgesia. The analgesia produced by morphine was r e a d i l y blocked by naloxone while DADL induced analgesia was r e s i s t a n t to naloxone. Another approach to support the presence of two separate types of opioid receptors involved tolerance studies. Previous studies demonstrated that rats given chronic intrathecal exposure of DADL led to development of tolerance to DADL and cross tolerance to morphine but the development of cross tolerance to morphine was blocked by concomitant administration of naloxone while the development of tolerance to DADL was not affected by naloxone (4). Extending these results f u r t h e r , the present investigation studies the tolerance to morphine and cross tolerance to DADL on the spinal t a i l f l i c k response in the rat after chronic spinal exposure to morphine. 0024-3205/83/222545-06503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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Noncross Tolerance to Intrathecal DADL
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Materials and Methods Male Sprague-Dawley rats (King Laboratory, Oregon, WI) weighing 360 gm to 380 gm were used. They were housed 2 per cage in a room maintained at 23°C ± 0.5°C having a 12 hr l i g h t - d a r k cycle. Food and water were a v a i l a b l e ad l i b i t u m . Tolerance to morphine was induced by chronic infusion of morphine s u l f a t e , 2 Ng/hr into the spinal subarachnoid space with an Alzet osmotic minipump (Alza Corp., Palo A l t o , CA). The surgical procedure f o r intrathecal cannulation and subcutaneous minipump implantation were performed as prev i o u s l y described (4,5). Analgesia was assessed with the t a i l f l i c k test (6). The i n t e n s i t y of the radiant heat was adjusted so that the control t a i l f l i c k latency was in the range of 3 to 5 sec. The t a i l f l i c k latency was tested once a day at i0 AM during 6 days of intrathecal drug infusion. To prevent tissue damage, the heat r a d i a t i o n was stopped at I0 sec i f the r a t did not f l i c k i t s t a i l . On day 6 of i n t r a t h e c a l infusion, the minipump was removed from rats under l i g h t ether anesthesia. The catheter was cleared of drug solution by i n j e c t i n g 6 ~I of drug vehicle solution. The degree of tolerance to morphine and cross tolerance to D-AIa2-D-Leu 5enkephalin (DADL) was then determined by i n h i b i t i o n to the t a i l f l i c k response a f t e r i n j e c t i o n of various doses of morphine s u l f a t e and DADL 4 hr a f t e r the removal of minipump. A cumulative dose schedule f o r DADL and morphine s u l f a t e was used. Drug solutions were injected i n t r a t h e c a l l y s t a r t i n g from 0.625 Ng and 0.0625 Ng of morphine s u l f a t e and DADL r e s p e c t i v e l y and doubling the dose every 10 min a f t e r each t a i l f l i c k measurement. The opioids were injected in a 4 ~I volume which was followed by a second i n j e c t i o n of 6 N1 of drug vehicle to clear the catheter. A control latency (T O) was obtained from the mean of two l a t e n c y d e t e r m i n a t i o n s measured before the s t a r t of the i n t r a t h e c a l injection. Latency (T I) was measured for each animal 10 min a f t e r each drug injection. The i n h i b i t i o n of t a i l f l i c k response was expressed as "percent analgesia" which was calculated as [(TI-To)/(T2-To) ] x i00 where the maximal response time (T2) was set at 12 sec. The maximal response time of 12 sec is about 3 times the t a i l f l i c k latency found in control animals. The doseresponse curves for the best f i t of the points were drawn v i s u a l l y . The test of p a r a l l e l i s m and dose r a t i o of the dose response curves were calculated according to the computer programs by T a l l a r i d a and Murray (7) using an Apple I I computer. Drugs used were D-Ala2-Leu5-enkephalin ( P e n i n s u l a Lab., Palo A l t o , CA) and morphine s u l f a t e (Mallinckrodt Chemical Works, St. Louis, MO). Sodium pentobarbital (Henry Schein Inc., Port Washington, NY) was used during the surgery for i n t r a t h e c a l cannulation and minipump implantation. The drug vehicle used to dissolve opioids consisted of NaCI (7.46 g) KCI (0.19 g), MgCI2 (0.19 g) and CaCI2 (0.14 g) per I000 ml of d i s t i l l e d water. Results The d a i l y t a i l f l i c k latencies during 6 days of intrathecal infusion with drug vehicle ( 1 N l / h r ) and morphine s u l f a t e (2 Ng/hr) are shown in Figure I . Infusion of drug vehicle did not a l t e r the t a i l f l i c k latencies to radiant heat during 6 days of infusion. Infusion of 2 Ng/hr of morphine s u l f a t e markedly elevated the t a i l f l i c k latency measured 24 hr a f t e r the s t a r t of infusion. The increased t a i l f l i c k latencies gradually decreased during 6 days of i n t r a t h e c a l morphine infusion i n d i c a t i n g that homeostatic tolerance developed (8). However, at day 6 of i n f u s i o n , the t a i l f l i c k latency was still s i g n i f i c a n t l y higher than that for the groups infused with the drug vehicle.
Vol. 32, No. 22, 1983
0
Noncross Tolerance to Intrathecal DADL
.10
~9 ~e ~7
2547
0 Vehicle, lJJl/hr, N:18 ~'----...~,~,
~ MorPhine Sulfate, 2ug/hr, N : 17
e-
•"J 6 U
"-5
--4
0.-.----0---.------~
0--------0------.~
~" 3
Days Of Intrathecal Infusion
FIG. i Tail f } i c k latencies during 6 days of i n t r a t h e c a l infusion of drug vehicle (I p l / h r ) and morphine s u l f a t e (2 Ng/hr) in r a t s . Vertical bars indicate the standard error of the mean. The standard error less than 0.2 sec is not shown in the f i g u r e . The t a i l f l i c k latencies of rats infused with morphine sulfate are s i g n i f i c a n t l y higher than vehicle controls (p < 0.01, student t t e s t , two t a i l e d ) , Four hours a f t e r removal of the minipump from rats c h r o n i c a l l y infused with morphine s u l f a t e , the t a i l f l i c k latency was not s i g n i f i c a n t l y d i f f e r e n t from rats c h r o n i c a l l y infused with drug vehicle (mean ± S.E., drug vehicle: 3.84 ± 0.27 sec, N:18; morphine s u l f a t e : 4.12 ± 0.21 sec, N=I7). Intrathecal i n j e c t i o n of morphine s u l f a t e at doses from 0.625 Ng to i0 ~g caused a dose related increase in the i n h i b i t i o n of t a i l f l i c k response in rats c h r o n i c a l l y infused with the drug vehicle (Figure 2). The ED50 value of morphine s u l fate was calculated to be 1.65 Ng. In contrast, morphine s u l f a t e at doses up to 40 Ng f a i l e d to i n h i b i t the t a i l f l i c k response and the dose-response curve of morphine s u l f a t e was completely f l a t in rats c h r o n i c a l l y infused with morphine s u l f a t e . Thus, more than 24 fold tolerance (40 Ng/l.65 Ng) developed. A dose of morphine s u l f a t e greater than 40 ~g e l i c i t e d stimulatory effects. Intrathecal i n j e c t i o n of DADL at doses from 0.0625 Ng to 2 Ng caused a dose dependent increase in the i n h i b i t i o n of t a i l f l i c k response in rats tested 4 hours a f t e r the termination of chronic i n t r a t h e c a l infusion with drug vehicle (Figure 3). The ED50 of DADL was calculated to be 0.064 pg. On molar basis, DADL was 44 fold more potent than morphine. In contrast to the r e s u l t s with morphine s u l f a t e , DADL at the same dose range used in vehicle infused rats caused a dose dependent elevation of the t a i l f l i c k latency in rats c h r o n i c a l l y infused with morphine s u l f a t e . The dose response curve, however, was shifted s l i g h t l y to the r i g h t by 4.7 f o l d . S t a t i s t i c a l analysis indicated that the two curves were p a r a l l e l . A high dose of DADL was able to e l i c i t maximum analgesia in most of chronic morphine infused rats (9 of 11 rats studied).
2548
Noncross Tolerance to Intrathecal DADL
~
lOO 90
Vol. 32, No. 22, 1983
j / 0
ve.,c,e JI
80
P_ 70~ ..-= 6o! <
50
~ 40 ~ ao 20 lO 1
lO
lOO
Morphine Sulfate,.ug
FIG. 2 I n t r a t h e c a l dose response curves of morphine s u l f a t e to i n h i b i t the t a i l f l i c k response in r a t s measured 4 hours a f t e r the t e r m i n a t i o n of the c h r o n i c i n t r a t h e c a l i n f u s i o n w i t h drug v e h i c l e ( I p l / h r ) or morphine s u l f a t e (MS, 2 pg/hr) f o r 6 days. N=6-7 animals per group. The v e r t i c a l bars i n d i c a t e the standard e r r o r of the mean.
100 V e h i ~
90
0
80 p_ 70
_~ 60 a
,~ 5o
~ 4o ~. 3o 2O
10 o.1
1.0
10.0
D-Ala=-D-Leu~-Enkephalin, .Ug
FIG. 3 Intrathecal dose r e s p o n s e c u r v e s of D - A l a 2 - L e u 5 - e n k e p h a l i n t o i n h i b i t the t a i l f l i c k response in r a t s measured 4 hours a f t e r t e r m i n a t i o n of the c h r o n i c i n t r a t h e c a l i n f u s i o n w i t h drug v e h i c l e ( i p l / h r ) or morphine s u l f a t e (2 ~ g / h r ) . N=II animals per group. The v e r t i c a l bars i n d i c a t e the standard e r r o r of the mean.
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Noncross Tolerance to Intrathecal DADL
2549
Discussion The development of tolerance to morphine by direct chronic administration of morphine into the spinal subarachnoid space has been reported previously (1,2,9,10). In most studies, a multiple injection technique was used. In the present study, an osmotic minipump was u t i l i z e d to chronically deliver the morphine sulfate into the spinal cord. The results showed that chronic i n t r a thecal infusion of morphine caused tolerance to intrathecal morphine. This is evidenced by the findings that elevated daily t a i l f l i c k latencies in morphine infused rats gradually decreased and approached the value of t a i l f l i c k latencies of rats infused with vehicle. In addition, 6 days after intrathecal morphine infusion, intrathecal morphine even at high dose failed to e l i c i t t a i l f l i c k inhibition. This loss of i n t r i n s i c a c t i v i t y of morphine in rats after chronic morphinization has been reported by others (11,12). The present study demonstrated that chronic intrathecal infusion of morphine developed only a slight cross tolerance to DADL. This is evidenced by the findings that DADL at the same dose range used in vehicle infused rats caused a dose dependent elevation of the t a i l f l i c k latency in rats chronic a l l y infused with morphine sulfate. The dose response curve of DADL was shifted s l i g h t l y to the right by 4.7 fold. This is in sharp contrast to that of morphine in which more than 24 fold tolerance developed. The results obtained from the present study are in agreement with the evidence that there exists at least two types of opioid receptors, p- and 6-opioid receptors, in the spinal cord of rats. B u t , the present finding for DADL indicates that morphine infusion produces some tolerance to DADL. This result is different from that of Yaksh (2) in that he found no cross tolerance. The results obtained from the present and previous studies (4) seem to indicate that DADL activates both 6- and p-opioid receptors to produce t a i l f l i c k inhibition and the induction of tolerance. Since chronic intrathecal exposure of morphine markedly reduced the p-opioid receptor a c t i v i t i e s , the 4.7 fold reduction of the potency of DADL in rats after chronic intrathecal morphine infusion may indicate that DADL partly acts on p-opioid receptors in addition to i t s major effect on 6-opioid receptors. Thus, i t appears that DADL has both p- and 6-agonist a c t i v i t y , and chronic morphine infusion which produces tolerance to p-agonist does produce some cross tolerance to DADL. This is in line with the finding in the previous report (8) that rats given chronic intrathecal exposure of DADL led to development of cross tolerance to morphine. Thus i t appears that morphine affects the p- more and the a-opioid receptors less, while DADL activates both 6- and N-opioid receptors on the production of spinal t a i l f l i c k inhibition and the development of tolerance. A further point to consider is the 4 hour interval between the termination of morphine sulfate infusion before the cross tolerance determinations were performed for DADL. Lange et al. (8,13) have described the concept of withrawal tolerance. Mice tested--at--3 days of morphine pellet implantation were not tolerant to intracerebroventricularly administered ( i . c . v . ) challenging doses of morphine. But, the mice manifested tolerance to i . c . v . morphine when tested after withdrawal of the morphine pellet: i . e . withdrawal tolerance. Furthermore, with the pellet in place, the mice were not tolerant to i . c . v , heroin or etorpine but did manifest cross tolerance to heroin and etorphine when tested after morphine pellet withdrawal. Thus, in the present study, I set the experiment such that i f cross tolerance were to occur, the situation (4 hours after termination of morphine sulfate infusion) favored e l i c i t i n g such a cross tolerance. In spite of this favored situation, the amount of cross tolerance seen to DADL (4.7 fold) was small in comparison to the large degree of tolerance seen to the morphine. These results again
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Noncross Tolerance to Intrathecal DADL
Vol. 32, No. 22, 1983
support the contention that d i f f e r e n t receptors are involved in the t a i l response and in addition DADL has some u- agonist a c t i v i t y .
flick
Acknowledgements These studies were supported in part by VA Grant 2960-003P. References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13.
L.F. Tseng. Life Sci. 29:1417-1424 (1981). T.L. Yaksh, In Advances--fn Pharmacology and Therapeutics I I , Volume 1, CNS Pharmacology-Neuropeptides, ed. by H. YOSHIDA, Y. HAGIHARA and S. EBASHI pp. 29-38, Pergammon Press, New York (1982). T.-C. Fu, S. Welch and W.L. Dewey. Pharmacologist 23:207 (1981). L.F. Tseng. Life Sci. 31:987-992 (1982). T.L. Yaksh and T.A. Rudy. Physiol. Behav. 17:1031-1036 (1976). W.L. Dewey and L.S. Harris, in Methods in Narcotic Research, ed. by S. EHRENPREIS and A. NEIDLE, pp. 101-109, Marcel Dekker, Inc. New York (1975). R.J. T a l l a r i d a and R.B. Murray, in Manual of Pharmacologic Calculations with Computer Programs, Springer-Verlag, New York, NY, Heidelberg, Berlin (1981). D.G. Lange, S.C. Roerig, J.M. Fujimoto and L.W. Busse. J. Pharmacol. Exp. Therap. 224:13-20 (1983). T.L. Yaksh, R.L. Kohl and T.A. Rudy. Europ. J. Pharmacol. 42:274-284 (1977). O.-G. Berge and K. Hole. Neuropharmacol. 20:653-657 (1981). J. Blasig, G. Meyer, V. H o l l t , J. Hengsten-b-e-rg, J. Dum and A. Herz. Neuropharmacol. 18:473-481 (1979). R.F. Mucha and H__Kalant. Psychopharmacol. 75:132-133 (1981). D.G. Lange, S.C. Roerig, J.M. Fujimoto and RT.H. Wang. Science 208: 72-74, 1980.
Pergamon Press
PARTIAL CROSS TOLERANCE TO D-ALA2-D-LEU5-ENKEPHALIN AFTER CHRONIC SPINAL MORPHINE INFUSION Liang-Fu Tseng Toxicology Research Laboratory Veterans Administration Hospital Wood, Wisconsin 53193 and Department of Pharmacology and Toxicology The Medical College of Wisconsin Milwaukee, Wisconsin 53226 (Received in final form March 9, 1983)
Summary The development of tolerance to morphine and cross tolerance to D-Ala2-D-Leu5-enkephalin (DADL) at spinal cord level to the i n h i b i t i o n of t a i l f l i c k response was studied in rats t o l e r a n t to morphine. The long term intrathecal infusion of morphine sulfate was accomplished by means of an osmotic minipump. Intrathecal infusion of morphine sulfate (2 pg/hr) markedly elevated the t a i l f l i c k latency measured 24 hr a f t e r the start of infusion. The increased t a i l f l i c k latencies gradually decreased during 6 days of intrathecal infusion of morphine sulfate. Tolerance to morphine and DADL was determined by i n h i b i t i o n to the t a i l f l i c k response after intrathecal administration of cumulative doses of morphine sulfate and DADL. Chronic intrathecal infusion of morphine induced a marked tolerance to morphine but developed only a s l i g h t cross tolerance to DADL. The results indicate that there exists two separate types of opioid receptor, p- and 6-opioid receptor in the spinal cord of rats. Evidence has accumulated to support the contention that there exists at least two types of opioid receptors, p- and ~-receptors in the spinal cord of the r a t . This contention is based on the results obtained on comparat i v e potencies of opioid agonists on the production of analgesia and the d i f f e r e n t i a l s e n s i t i v i t y to the a n t a g o n i s t i c a c t i v i t y of naloxone ( 1 , 2 , 3 , ) . Intrathecal morphine and D-AlaL-D-Leu~-enkephalin (DADL) which stimulates p- and 6-opioid receptors respectively produced potent analgesia. The analgesia produced by morphine was r e a d i l y blocked by naloxone while DADL induced analgesia was r e s i s t a n t to naloxone. Another approach to support the presence of two separate types of opioid receptors involved tolerance studies. Previous studies demonstrated that rats given chronic intrathecal exposure of DADL led to development of tolerance to DADL and cross tolerance to morphine but the development of cross tolerance to morphine was blocked by concomitant administration of naloxone while the development of tolerance to DADL was not affected by naloxone (4). Extending these results f u r t h e r , the present investigation studies the tolerance to morphine and cross tolerance to DADL on the spinal t a i l f l i c k response in the rat after chronic spinal exposure to morphine. 0024-3205/83/222545-06503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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Noncross Tolerance to Intrathecal DADL
Vol. 32, No. 22, 1983
Materials and Methods Male Sprague-Dawley rats (King Laboratory, Oregon, WI) weighing 360 gm to 380 gm were used. They were housed 2 per cage in a room maintained at 23°C ± 0.5°C having a 12 hr l i g h t - d a r k cycle. Food and water were a v a i l a b l e ad l i b i t u m . Tolerance to morphine was induced by chronic infusion of morphine s u l f a t e , 2 Ng/hr into the spinal subarachnoid space with an Alzet osmotic minipump (Alza Corp., Palo A l t o , CA). The surgical procedure f o r intrathecal cannulation and subcutaneous minipump implantation were performed as prev i o u s l y described (4,5). Analgesia was assessed with the t a i l f l i c k test (6). The i n t e n s i t y of the radiant heat was adjusted so that the control t a i l f l i c k latency was in the range of 3 to 5 sec. The t a i l f l i c k latency was tested once a day at i0 AM during 6 days of intrathecal drug infusion. To prevent tissue damage, the heat r a d i a t i o n was stopped at I0 sec i f the r a t did not f l i c k i t s t a i l . On day 6 of i n t r a t h e c a l infusion, the minipump was removed from rats under l i g h t ether anesthesia. The catheter was cleared of drug solution by i n j e c t i n g 6 ~I of drug vehicle solution. The degree of tolerance to morphine and cross tolerance to D-AIa2-D-Leu 5enkephalin (DADL) was then determined by i n h i b i t i o n to the t a i l f l i c k response a f t e r i n j e c t i o n of various doses of morphine s u l f a t e and DADL 4 hr a f t e r the removal of minipump. A cumulative dose schedule f o r DADL and morphine s u l f a t e was used. Drug solutions were injected i n t r a t h e c a l l y s t a r t i n g from 0.625 Ng and 0.0625 Ng of morphine s u l f a t e and DADL r e s p e c t i v e l y and doubling the dose every 10 min a f t e r each t a i l f l i c k measurement. The opioids were injected in a 4 ~I volume which was followed by a second i n j e c t i o n of 6 N1 of drug vehicle to clear the catheter. A control latency (T O) was obtained from the mean of two l a t e n c y d e t e r m i n a t i o n s measured before the s t a r t of the i n t r a t h e c a l injection. Latency (T I) was measured for each animal 10 min a f t e r each drug injection. The i n h i b i t i o n of t a i l f l i c k response was expressed as "percent analgesia" which was calculated as [(TI-To)/(T2-To) ] x i00 where the maximal response time (T2) was set at 12 sec. The maximal response time of 12 sec is about 3 times the t a i l f l i c k latency found in control animals. The doseresponse curves for the best f i t of the points were drawn v i s u a l l y . The test of p a r a l l e l i s m and dose r a t i o of the dose response curves were calculated according to the computer programs by T a l l a r i d a and Murray (7) using an Apple I I computer. Drugs used were D-Ala2-Leu5-enkephalin ( P e n i n s u l a Lab., Palo A l t o , CA) and morphine s u l f a t e (Mallinckrodt Chemical Works, St. Louis, MO). Sodium pentobarbital (Henry Schein Inc., Port Washington, NY) was used during the surgery for i n t r a t h e c a l cannulation and minipump implantation. The drug vehicle used to dissolve opioids consisted of NaCI (7.46 g) KCI (0.19 g), MgCI2 (0.19 g) and CaCI2 (0.14 g) per I000 ml of d i s t i l l e d water. Results The d a i l y t a i l f l i c k latencies during 6 days of intrathecal infusion with drug vehicle ( 1 N l / h r ) and morphine s u l f a t e (2 Ng/hr) are shown in Figure I . Infusion of drug vehicle did not a l t e r the t a i l f l i c k latencies to radiant heat during 6 days of infusion. Infusion of 2 Ng/hr of morphine s u l f a t e markedly elevated the t a i l f l i c k latency measured 24 hr a f t e r the s t a r t of infusion. The increased t a i l f l i c k latencies gradually decreased during 6 days of i n t r a t h e c a l morphine infusion i n d i c a t i n g that homeostatic tolerance developed (8). However, at day 6 of i n f u s i o n , the t a i l f l i c k latency was still s i g n i f i c a n t l y higher than that for the groups infused with the drug vehicle.
Vol. 32, No. 22, 1983
0
Noncross Tolerance to Intrathecal DADL
.10
~9 ~e ~7
2547
0 Vehicle, lJJl/hr, N:18 ~'----...~,~,
~ MorPhine Sulfate, 2ug/hr, N : 17
e-
•"J 6 U
"-5
--4
0.-.----0---.------~
0--------0------.~
~" 3
Days Of Intrathecal Infusion
FIG. i Tail f } i c k latencies during 6 days of i n t r a t h e c a l infusion of drug vehicle (I p l / h r ) and morphine s u l f a t e (2 Ng/hr) in r a t s . Vertical bars indicate the standard error of the mean. The standard error less than 0.2 sec is not shown in the f i g u r e . The t a i l f l i c k latencies of rats infused with morphine sulfate are s i g n i f i c a n t l y higher than vehicle controls (p < 0.01, student t t e s t , two t a i l e d ) , Four hours a f t e r removal of the minipump from rats c h r o n i c a l l y infused with morphine s u l f a t e , the t a i l f l i c k latency was not s i g n i f i c a n t l y d i f f e r e n t from rats c h r o n i c a l l y infused with drug vehicle (mean ± S.E., drug vehicle: 3.84 ± 0.27 sec, N:18; morphine s u l f a t e : 4.12 ± 0.21 sec, N=I7). Intrathecal i n j e c t i o n of morphine s u l f a t e at doses from 0.625 Ng to i0 ~g caused a dose related increase in the i n h i b i t i o n of t a i l f l i c k response in rats c h r o n i c a l l y infused with the drug vehicle (Figure 2). The ED50 value of morphine s u l fate was calculated to be 1.65 Ng. In contrast, morphine s u l f a t e at doses up to 40 Ng f a i l e d to i n h i b i t the t a i l f l i c k response and the dose-response curve of morphine s u l f a t e was completely f l a t in rats c h r o n i c a l l y infused with morphine s u l f a t e . Thus, more than 24 fold tolerance (40 Ng/l.65 Ng) developed. A dose of morphine s u l f a t e greater than 40 ~g e l i c i t e d stimulatory effects. Intrathecal i n j e c t i o n of DADL at doses from 0.0625 Ng to 2 Ng caused a dose dependent increase in the i n h i b i t i o n of t a i l f l i c k response in rats tested 4 hours a f t e r the termination of chronic i n t r a t h e c a l infusion with drug vehicle (Figure 3). The ED50 of DADL was calculated to be 0.064 pg. On molar basis, DADL was 44 fold more potent than morphine. In contrast to the r e s u l t s with morphine s u l f a t e , DADL at the same dose range used in vehicle infused rats caused a dose dependent elevation of the t a i l f l i c k latency in rats c h r o n i c a l l y infused with morphine s u l f a t e . The dose response curve, however, was shifted s l i g h t l y to the r i g h t by 4.7 f o l d . S t a t i s t i c a l analysis indicated that the two curves were p a r a l l e l . A high dose of DADL was able to e l i c i t maximum analgesia in most of chronic morphine infused rats (9 of 11 rats studied).
2548
Noncross Tolerance to Intrathecal DADL
~
lOO 90
Vol. 32, No. 22, 1983
j / 0
ve.,c,e JI
80
P_ 70~ ..-= 6o! <
50
~ 40 ~ ao 20 lO 1
lO
lOO
Morphine Sulfate,.ug
FIG. 2 I n t r a t h e c a l dose response curves of morphine s u l f a t e to i n h i b i t the t a i l f l i c k response in r a t s measured 4 hours a f t e r the t e r m i n a t i o n of the c h r o n i c i n t r a t h e c a l i n f u s i o n w i t h drug v e h i c l e ( I p l / h r ) or morphine s u l f a t e (MS, 2 pg/hr) f o r 6 days. N=6-7 animals per group. The v e r t i c a l bars i n d i c a t e the standard e r r o r of the mean.
100 V e h i ~
90
0
80 p_ 70
_~ 60 a
,~ 5o
~ 4o ~. 3o 2O
10 o.1
1.0
10.0
D-Ala=-D-Leu~-Enkephalin, .Ug
FIG. 3 Intrathecal dose r e s p o n s e c u r v e s of D - A l a 2 - L e u 5 - e n k e p h a l i n t o i n h i b i t the t a i l f l i c k response in r a t s measured 4 hours a f t e r t e r m i n a t i o n of the c h r o n i c i n t r a t h e c a l i n f u s i o n w i t h drug v e h i c l e ( i p l / h r ) or morphine s u l f a t e (2 ~ g / h r ) . N=II animals per group. The v e r t i c a l bars i n d i c a t e the standard e r r o r of the mean.
Vol. 32, No. 22, 1983
Noncross Tolerance to Intrathecal DADL
2549
Discussion The development of tolerance to morphine by direct chronic administration of morphine into the spinal subarachnoid space has been reported previously (1,2,9,10). In most studies, a multiple injection technique was used. In the present study, an osmotic minipump was u t i l i z e d to chronically deliver the morphine sulfate into the spinal cord. The results showed that chronic i n t r a thecal infusion of morphine caused tolerance to intrathecal morphine. This is evidenced by the findings that elevated daily t a i l f l i c k latencies in morphine infused rats gradually decreased and approached the value of t a i l f l i c k latencies of rats infused with vehicle. In addition, 6 days after intrathecal morphine infusion, intrathecal morphine even at high dose failed to e l i c i t t a i l f l i c k inhibition. This loss of i n t r i n s i c a c t i v i t y of morphine in rats after chronic morphinization has been reported by others (11,12). The present study demonstrated that chronic intrathecal infusion of morphine developed only a slight cross tolerance to DADL. This is evidenced by the findings that DADL at the same dose range used in vehicle infused rats caused a dose dependent elevation of the t a i l f l i c k latency in rats chronic a l l y infused with morphine sulfate. The dose response curve of DADL was shifted s l i g h t l y to the right by 4.7 fold. This is in sharp contrast to that of morphine in which more than 24 fold tolerance developed. The results obtained from the present study are in agreement with the evidence that there exists at least two types of opioid receptors, p- and 6-opioid receptors, in the spinal cord of rats. B u t , the present finding for DADL indicates that morphine infusion produces some tolerance to DADL. This result is different from that of Yaksh (2) in that he found no cross tolerance. The results obtained from the present and previous studies (4) seem to indicate that DADL activates both 6- and p-opioid receptors to produce t a i l f l i c k inhibition and the induction of tolerance. Since chronic intrathecal exposure of morphine markedly reduced the p-opioid receptor a c t i v i t i e s , the 4.7 fold reduction of the potency of DADL in rats after chronic intrathecal morphine infusion may indicate that DADL partly acts on p-opioid receptors in addition to i t s major effect on 6-opioid receptors. Thus, i t appears that DADL has both p- and 6-agonist a c t i v i t y , and chronic morphine infusion which produces tolerance to p-agonist does produce some cross tolerance to DADL. This is in line with the finding in the previous report (8) that rats given chronic intrathecal exposure of DADL led to development of cross tolerance to morphine. Thus i t appears that morphine affects the p- more and the a-opioid receptors less, while DADL activates both 6- and N-opioid receptors on the production of spinal t a i l f l i c k inhibition and the development of tolerance. A further point to consider is the 4 hour interval between the termination of morphine sulfate infusion before the cross tolerance determinations were performed for DADL. Lange et al. (8,13) have described the concept of withrawal tolerance. Mice tested--at--3 days of morphine pellet implantation were not tolerant to intracerebroventricularly administered ( i . c . v . ) challenging doses of morphine. But, the mice manifested tolerance to i . c . v . morphine when tested after withdrawal of the morphine pellet: i . e . withdrawal tolerance. Furthermore, with the pellet in place, the mice were not tolerant to i . c . v , heroin or etorpine but did manifest cross tolerance to heroin and etorphine when tested after morphine pellet withdrawal. Thus, in the present study, I set the experiment such that i f cross tolerance were to occur, the situation (4 hours after termination of morphine sulfate infusion) favored e l i c i t i n g such a cross tolerance. In spite of this favored situation, the amount of cross tolerance seen to DADL (4.7 fold) was small in comparison to the large degree of tolerance seen to the morphine. These results again
2550
Noncross Tolerance to Intrathecal DADL
Vol. 32, No. 22, 1983
support the contention that d i f f e r e n t receptors are involved in the t a i l response and in addition DADL has some u- agonist a c t i v i t y .
flick
Acknowledgements These studies were supported in part by VA Grant 2960-003P. References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13.
L.F. Tseng. Life Sci. 29:1417-1424 (1981). T.L. Yaksh, In Advances--fn Pharmacology and Therapeutics I I , Volume 1, CNS Pharmacology-Neuropeptides, ed. by H. YOSHIDA, Y. HAGIHARA and S. EBASHI pp. 29-38, Pergammon Press, New York (1982). T.-C. Fu, S. Welch and W.L. Dewey. Pharmacologist 23:207 (1981). L.F. Tseng. Life Sci. 31:987-992 (1982). T.L. Yaksh and T.A. Rudy. Physiol. Behav. 17:1031-1036 (1976). W.L. Dewey and L.S. Harris, in Methods in Narcotic Research, ed. by S. EHRENPREIS and A. NEIDLE, pp. 101-109, Marcel Dekker, Inc. New York (1975). R.J. T a l l a r i d a and R.B. Murray, in Manual of Pharmacologic Calculations with Computer Programs, Springer-Verlag, New York, NY, Heidelberg, Berlin (1981). D.G. Lange, S.C. Roerig, J.M. Fujimoto and L.W. Busse. J. Pharmacol. Exp. Therap. 224:13-20 (1983). T.L. Yaksh, R.L. Kohl and T.A. Rudy. Europ. J. Pharmacol. 42:274-284 (1977). O.-G. Berge and K. Hole. Neuropharmacol. 20:653-657 (1981). J. Blasig, G. Meyer, V. H o l l t , J. Hengsten-b-e-rg, J. Dum and A. Herz. Neuropharmacol. 18:473-481 (1979). R.F. Mucha and H__Kalant. Psychopharmacol. 75:132-133 (1981). D.G. Lange, S.C. Roerig, J.M. Fujimoto and RT.H. Wang. Science 208: 72-74, 1980.