Endopeptidase 24.15 inhibition and opioid antinociception

Psychopharmacology (1992) 106:408-416 Psychopharmacology © Springer-Verlag 1992

Endopeptidase 24.15 inhibition and opioid antinociception Benjamin Kest 1, Marion Orlowski ~, and Richard J. Bodnar ~

1 Department of Psychology, Neuropsychology Doctoral Sub-Program, Queens College, CUNY, 65-30 Kissena Boulevard, Flushing, NY 11367, USA 2 Department of Pharmacology, Mount Sinai School of Medicine, New York, NY 10029, USA

Received May 6, 1991 / Final version July 10, 199I

Abstract. Whereas endopeptidase 24.11 cleaves the Gly- Phe bond in both Met- and Leu-enkephalin, endopeptidase 24.15 rapidly converts dynorphin A1 - s, alpha and beta-neoendorphin into Leu-enkephalin, and Met-enkephalin-Arg6-GlyV-Leu 8 (MERGL) into Met-enkephalin. Inhibitors of both endopeptidase 24.11 and endopeptidase 24.15 each produce antinociception, and inhibitors of endopeptidase 24.11 increase the magnitude of enkephalin antinociception. The present study com- pared the central antinociceptive effect of an inhibitor of endopeptidase 24.15, N-[1-(R-S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB) with one of endopeptidase 24.11 N-[1-(RS)-carboxy-3- phenylpropyl]-Phe-p-aminobenzoate (cFP-F-pAB) upon central opioid antinociception induced by MERGL, met- enkephalin and dynorphin Ai-8. cFP-AAF-pAB, but not cFP-F-pAB increased MERGL antinociception on the tail-flick and jump tests, In contrast, cFPoFopAB, but not cFP-AAF-pAB increased met-enkephalin antinociception. Whereas central dynorphin A1-8 failed to induce antinociception itself, co-administration of cFP- AAF-pAB and dynorphin Al - s increased nociceptive thresholds. This effect was not accompanied by motor dysfunction, but was blocked by systemic pretreatment with naloxone or central pretreatment with naltrexone or nor-binaltorphamine, but not beta-funaltrexamine. These data indicate that endopeptidase 24.15 may be re- sponsible for the degradation of specific opioid peptides (e.g., MERGL, dynorphin), and that this process may prevent the futl expression of their antinociceptive properties.

Key words: Endopeptidase 24.15 - Endopeptidase 24.11 - MERGL - Dynorphin - A,_ 8 - Nor-binaltorphamine

Antinociception

Evidence indicates that several peptidases are involved in the metabolism of opioid peptides. Endopeptidase 24.11

Offprint requests to: R.J. Bodnar

("enkephalinase') cleaves the Gly-Phe bond in both Met- and Leu-enkephalin (Malfroy et al. 1978; Sullivan et al. 1978; Almenoff et al. 1981; Fulcher et al. 1982). In- hibitors of this enzyme such as thiorphan (Roques et al. !980), acetorphan (Lecomte et al. 1986), SCH 34826 (Chipkin et al. 1988) and cFP-F-pAB (Pozsgay et al. 1986) slow the degradation of enkephalins in vivo and possess antinociceptive properties (Roques et al. 1980; Chipkin et al. 1982, 1988; DeLaBaume et al. 1983; Lecomte et al. 1986; Kest et al. 1991). Other studies using specific enzyme inhibitors indicate that aminopeptidases and angiotensin converting enzyme (ACE) also contrib- ute to the degradation of opioid peptides (Turner et al. 1985). Thus, administration of the aminopeptidase inhibitors, bestatin and amastatin, alone or in combination with the ACE inhibitor captopril, increase basal nociceptive thresholds and potentiate opioid antinociception (Chaillet et al. 1983; Chou et al. 1984; Fournie-Zaluski et al. t984).

Unlike the opioid degrading enzymes described above, endopeptidase 24.15 (EC3.4.24.15) converts larger prodynorphin and proenkephalin-derived peptides into the corresponding enkephalins which are resistant to further degradation by this enzyme (Orlowski et al. 1983, 1989; Chu and Orlowski 1984, 1985; Acker et al. 1987). Thus, endopeptidase 24.15 rapidly converts dynorphin A1-8, alpha and beta-neoendorphin into Leu-enkephalin, and MERGL and metorphamide into Met-enkephalin. Two active site-directed inhibitors of this enzyme, cFP-AAF-pAB and N-[t-(RS)-carboxy-3-phenylpropyl]-Ala-D-Ala-Phe-p-aminobenzoate (cFP- A(D)AF-pAB; Chu and Orlowski 1984; Orlowski et at. 1988), produce a dose-dependent antinociception on the tail-flick and jump tests which is reversed by naloxone (Kest et al. 1991). It was therefore of interest to compare the antinociceptive effect of inhibitors of endopeptidase 24.15 with that of endopeptidase 24.11 upon opioid antinociception. The present study examined the central antinociceptive effects of cFP-AAF-pAB and cFP-F- pAB following co-administration with MERGL, Met- enkephalin and dynorphin A1-8 on the tail-flick (D'Amour and Smith 1941) and jump (Evans 196I) tests.

W h e r e a s m e t - e n k e p h a l i n and M E R G L have each been shown to elicit a sho r t - ac t ing an t inoc i cep t ion (e.g., Belluzzi et al. 1976; I a d o r o l a et al. 1986; M e l l s t r o m et al. 1987), d y n o r p h i n pep t ides exer t p o o r sup ra sp ina l an t ino - c icep t ion (e.g., F r i e d m a n et al. 1981; C h a v k i n et al. 1982). O n the bas is o f thei r i nh ib i t o ry ac t iv i ty o f o p i o i d pep t ide d e g r a d a t i o n , it was expec ted t ha t c F P - A A F - p A B w o u l d po t en t i a t e M E R G L a n d d y n o r p h i n A 1 - s, b u t no t m e t - e n k e p h a l i n an t inoc icep t ion , whereas c F P - F - p A B w o u l d po t en t i a t e m e t - e n k e p h a t i n an t inoc icep t ion , b u t no t the others . This s tudy also eva lua t ed whe ther the an t inoc i cep t ion el ici ted by c F P - A A F - p A B and d y n o r - ph in A I _ a c o - a d m i n i s t r a t i o n was a l te red by the o p i o i d r ecep to r an t agon i s t s n a l o x o n e a n d na l t r exone , the k a p p a o p i o i d r ecep to r an tagon i s t , n o r - b i n a l t o r p h a m i n e (Nor - B N I : T a k e m o r i et al. 1988) o r the m u op io id r ecep to r an tagon i s t , b e t a - f u n a l t r e x a m i n e ( B - F N A : T a k e m o r i et al. 1981). F ina l ly , the effects o f c F P - A A F - p A B and dy- n o r p h i n A 1 - 8 c o - a d m i n i s t r a t i o n were eva lua t ed in different tests o f m o t o r abil i t ies.

General methods

Subjects, surgery and histology. Male albino Sprague-Dawtey rats (350-550 g, Charles River Laboratories, Wilmington, MA) were housed individually in the Queens College Vivarium Facility ac- cording to an approved protocol by the Queens College Institution- al Animal Care and Use Committee. Rats were maintained on a 12 h light: 12 h dark cycle at ambient temperatures between 22 ° and 25 ° C with ad libitum access to food and water. Following anes- thesia with chlorpromazine (3 mg/kg, IP) 20 rain prior to ketamine HCL (100 mg/kg, IM), a stainless steel quide cannula (Plastic Products, 22 gauge) was stereotaxically (Kopf Instruments) aimed so that its tip was positioned 0.3 mm above the left lateral ventricle. With the incisor bar set at + 5 mm, coordinates were 0.5 mm anterior to the bregma suture, t.3 mm lateral to the sagittal suture and 3.6 mm from the top of the skull. The cannula was secured to three stainless steel anchor screws with dental acrylic. All animals were allowed 10 days to recover from surgery before behavioral testing began. Following experimental testing, each animal was anesthetized with a barbiturate mixture (Euthanasia # 5, H. Schein Co.), sacrificed, and its brain removed. The brains were cut through the lateral ventricle, and examined under light microscopy to deter- mine cannula placement. Only animals with a cannula in the lateral ventricle were included in the statistical analysis.

Nociceptive tests. All rats were tested on the tail-flick and jump tests in that order to minimize carry-over effects between tests. On the tail-flick test, the beam (IITC Co.) was positioned 8 cm above the dorsum and 3 8 cm proximal to the tip of the rat's tail with the thermal intensity set to produce baseline tail-flick latencies between 2 and 3.5 s. Each session consisted of three latency determinations separated by 10-s intertrial intervals. To avoid tissue damage, a trial was automatically terminated if a response did not occur within t0 s. On the jump test, electric shock was delivered to the feet of the rat by a shock generator (BRS/LVE) and shock scrambler (Camden Instruments). The jump threshold was defined in mA as the lowest of two consecutive ascending intensities in which the animal simul- taneously removed both hindpaws from the grids. Each of six trials began with the animal receiving a 300 ms footshock at a current intensity of 0.10 mA with subsequent shocks increased in 0.05 mA increments at 10 s intervals until the jump threshold was deter- mined. Baseline tail-flick latencies and jump thresholds were deter- mined for at least 4 days before experimental testing began.

409

Motor tests. The locomotor activity of individual rats was measured (Omicron Animal Activity Meter, Omnitech Co.) by counting the number of vertical or horizontal movements. Righting reflexes were evaluated by placing each rat upside down and recording whether the criterion latency to righting (< 1 s) was achieved. Finally, rats placed in the middle of a wire-mesh inclined plane at either 45 ° or 90 ° were assessed for their ability to maintain their position.

Drugs. cFP-F-pAB and cFP-AAF-pAB were synthesized as described previously (Pozsgay et al. 1986; Ortowski et al. 1988) with chosen dose and time course based upon basal nociceptive effects (Kest et al. 1991). MERGL, met-enkephatin and dynorphin A I_8 (Peninsula Laboratories, Belmont, CA) doses were chosen based upon their antinociceptive effects (Belluzzi et al. 1976; Friedman et al. 1981 ; Chavkin et al. 1982; Herman and Goldstein 1985; Iadoro- la et al. 1985). Naloxone and naltrexone (Sigma, St. Louis, MO) doses were based upon their effects to block opioid actions (see review: Sawynok et al. 1979). B-FNA and Nor-BNI (Research Biochemicals, Natick, MA) doses were based upon their resepective effects to block mu and kappa receptors (e.g., Takemori et al. 1981, 1988). All ICV injections were infused in a 5 pl volume by a Hamilton syringe and polyethylene tubing at a rate of 1 ~tl every 10 s through a stainless steel internal cannuta (Plastic Products, 28 gauge) which protruded 0.5 mm beyond the tip of the guide cannula. The vehicle solute for all compounds was 0.9% normal saline, except cFP-F-pAB which was 0.9% normal saline and 0.2% acetic acid to adjust the pH to 7.0. All injections were administered between 1 and 8 h into the light cycle to avoid circadian differences in nociceptive thresholds (Frederickson et al. 1977).

Statistical analyses. Split-plot analyses of variance corrected for repeated measures on each dependent variable (tail-flick latencies and jump thresholds) were used to assess significant differences among treatment conditions and across the time course. Whereas significant agonist effects from corresponding vehicle control conditions were assessed with Dunnett comparisons (P < 0.05), significant agonist effects as a function of endopeptidase inhibitor coadministration were made using Dunn comparisons (P < 0.05) of difference scores which were derived by subtracting each postdrug effect from its corresponding vehicle value~

Experiment 1: Endopeptidase inhibitors and MERGL antinociception

Protocol

Subgroups o f ra t s received a m a x i m u m of four pa i rs o f mic ro in jec t ion cond i t ions at weekly intervals in counte r - b a l a n c e d o rde r (Table 1A). These cond i t ions inc luded a vehicle-vehicle cont ro l , a dose range o f M E R G L (25-200 gg), a dose range o f c F P - A A F - p A B (2.5-25 nmol ) p a i r e d wi th M E R G L (5-100 gg), c F P - F - p A B pa i r ed wi th M E R G L and c F P - A A F - p A B alone. A 10-min in terval s epa ra t ed each pa i r o f mic ro in jec t ions wi th la tencies and th resholds assessed at 5, 10, 15, 30, 45 and 60 min fo l lowing the second inject ion.

Results

Signif icant effects were obse rved a m o n g cond i t ions [tail- f l ick: F ( 8 , 6 0 ) = 1 2 . 2 1 , P < 0 . 0 0 1 ; j u m p F = 6 . 0 7 , P < 0.001], across the t ime course [tail-fl ick: F ( 5 , 3 0 0 ) = 18.66, P < 0 . 0 0 1 ; j u m p : F = 14.68, P < 0 . 0 0 1 ] and for the in t e rac t ion be tween cond i t ions and t imes

410

Table 1. Injection groups used to evaluate endopeptidase 24.15 and 24.11 inhibition effects upon MERGL, met-enkephalin and dynorphin A~-s antinociception

First injection (nmol) Second injection (gg) n

A. MERGL paradigm

Vehicle Vehicle 24 Vehicle MERGL (25) 10 Vehicle MERGL (100) 10 Vehicle MERGL (200) 9 cFP-AAF- pAB (25) Vehicle 6 cFP-AAF-pAB (25) MERGL (5) 5 cFP-AAF-pAB (25) MERGL (25) 7 cFP-AAF-pAB (25) MERGL (100) 7 cFP-AAF-pAB (2.5) MERGL (100) 9 cFP-F-pAB (25) MERGL (100) 9

B. Met-enkephalin paradigm

Vehicle Vehicle 24 Vehicle Met-Enk (25) 7 Vehicle Met-Enk (50) 8 Vehicle Met-Enk (100) 8 Vehicle Met-Enk (200) 11 cFP-F-pAB (25) Vehicle 7 cFP-F-pAB (25) Met-Enk (25) 7 cFP-F-pAB (25) Met-Enk (50) 8 cFP-F-pAB (25) Met-Enk (100) 9 cFP-AAF--pAB (25) Met-Enk (100) 8

C. Dynorphin paradigm

Vehicle Vehicle 60 Vehicle Dyn (5) 8 Vehicle Dyn (20) 5 cFP-AAF-pAB (25) Vehicle 5 cFP-AAF-pAB (25) Dyn (1) 10 cFP-AAF-pAB (25) Dyn (5) 1t cFP-AAF-pAB (2=5) Dyn (5) 8 cFP-F-pAB (25) Vehicle 5 cFP-F-pAB (25) Dyn (5) 8

[tail-flick: F(40,300)=2.98, P<0 .001 ; jump: F=3.05, P < 0.001]. Figure 1 illustrates the alterations in tail-flick latencies (left panel) and jump thresholds (right panel) induced by the central co-administration of M E R G L (100 gg) with vehicle, cFP-AAF-pAB (25 nmol) or cFP- F-pAB (25 nmol). The vehicle-MERGL conditions significantly increased latencies (100 gg: 14~17%, 5-10 rain; 200 gg: 16-30%, 5-30 min) and thresholds (100 gg: 13-19%, 5-10 min; 200 gg: 18-21%, 5-10 min), except at the 25 gg dose (Table 2). Pairing cFP-AAF-pAB (25nmol) with M E R G L (100 gg) significantly increased latencies (20-27%) and thresholds (11-30%) across the time course relative to vehicle-vehicle treatment, and significantly increased latencies (10-26%, 10-60 rain) and thresholds (13-15%, 15-60 min) relative to vehicle- M E R G L treatment. The increase in M E R G L antinociception by cFP-AAF-pAB was not due to the latter's intrinsic antinociceptive properties since cFP-AAF-pAB failed to significantly alter latencies or thresholds across this time course. In contrast, pairing cFP-F-pAB (25 nmol) with M E R G L (100 gg) significantly increased only latencies (11 14%, 5-10 min) relative to vehicle-vehicle treatment, but not relative to vehicle-MERGL treatment. A lower 2.5 nmol dose of cFP-AAF-pAB also potentiated M E R G L (100 gg) antinociception on the tail-flick (15%; 5-10 rain) and jump (11%; 5 min) tests. Further, if cFP-AAF-pAB (25 nmol) was paired with M E R G L (25 gg), latencies (12-17%, 5-45 min) and thresholds (10-13%, 5-30 min) were significantly increased relative to vehicle-vehicle treatment. Thus, an endopeptidase 24.15 inhibitor, cFP-AAF-pAB, but not an endopeptidase 24.11 inhibitor, cFP-F-pAB, increased M E R G L antinociception on the tail-flick and jump tests.

Experiment 2: Endopeptidase inhibitors and met-enkephalin antinociception

Protocol

New subgroups of rats received a maximum of four injection conditions (Table 1 B) as described previously,

VEHICLEJMERGL ~- 2.0- ~ cFP-AAF~pAB/MERGL E E 0,15

J I == 1 I -o- g .

g l sj ~ o.~o ,~ ~ *

, , , ~ ~ ,~. "a" ,~ 1.0 I ~ ~.._ ~ A " L "~" ¢c ~ ~ 7, ¢~ *

0.05

o.o ' 9 o.oo

z ~ -o o5, . . . . -0.5 ' ' ' r,- • 5 1 0 1 5 3 0 4'5 6 0 ~ ~'o ~5 3'0 45 60

TIME (min)

Fig. 1. Alterations in central MERGL (100 gg, tCV) antinociception on the tail-flick (Ieftpanel) and jump (right panel) tests following central pretreatment 10 min earlier with vehicle, the endopeptidase 24.15 inhibitor, cFP-AAF-pAB (25 nmol) or the endopeptidase 24.11 inhibitor, cFP-F-pAB (25 nmol). Thefilled stars denote

TIME (rain)

significant increases in latencies and thresholds relative to the vehicle/vehicle condition (Dunnett comparisons, P<0.05) while the open stars denote significant increases in latencies and thresholds relative to the vehicle/MERGL condition (Dunn comparisons, P < 0.05)

Table 2. Summary of endopeptidase 24.15 and 24.11 inhibition upon MERGL antinociception

411

Group 5 10 15 30 45 60

A. Tailfliek test

Veh/Veh 3.41 3,42 3.44 3.36 3.38 3.27 cFP-AAF-pAB 25/Veh 3.45 3,42 3.45 3.64 3.57 3.41 Veh/MERGL 25 3.17 3,37 3.17 3.23 3.56 3.19 Veh/MERGL I00 3.98* 3.91" 3.75 3.39 3.40 3,27 Veh/MERGL 200 4,23* 4.34* 4.34* 3.89* 3.68 3.34 cFP-AAF-pAB/MERGL 25 5 3.71 3.64 3.48 3.60 3.73 3.4t 25 25 4.00* 3.82 3.91" 3.92* 3.96* 3.61 2.5 100 3.93* 3.95* 3.53 3.38 3.30 3.19 25 100 4.34* 4.35* 4.13" 4.28* 4.09* 3,97* cFP-F-pAB/MERGL 25 100 3,88* 3.86* 3.60 3.53 3.38 3,16

B. Jump test

Veh/Veh 0.338 0.349 0.352 0.338 0.351 0,340 cFP-AAF-pAB 25/Veh 0.361 0.353 0.344 0.358 0.353 0,356 Veh/MERGL 25 0.331 0.339 0.333 0.344 0.354 0.353 Veh/MERGL 100 0,402* 0.393* 0.355 0,346 0.341 0.349 Veh/MERGL 200 0,413" 0.403* 0.360 0,353 0.350 0.336 cFP-AAF-pAB/MERGL 25 5 0.340 0.350 0.347 0.340 0.333 0.330 25 25 0.381" 0.374 0.360 0.367 0.372 0.367 2.5 100 0.374* 0.364 0.339 0.331 0.326 0.320 25 100 0.438* 0.427* 0.410" 0.390* 0.388* 0.395* cFP-F-pAB/MERGL 25 100 0.361 0.354 0.343 0.326 0.321 0.330

* Significant difference relative to Veh/Veh treatment (Dunnett comparison, P<0.05)

including a vehicle-vehicle control, a dose range of met- enkephalin (25-200 gg), cFP-F-pAB paired with met- enkephalin (25 100 lag), cFP-AAF-pAB paired with met- enkephalin and cFP-F-pAB alone. A 10-rain interval separated each pair of microinjections with latencies and thresholds assessed at 5, 10, 15, 30, 45 and 60 min following the second injection.

Results

Significant effects were observed among conditions [tail- flick: F(9,86)=24.96, P<0 .001 ; jump F=7.93 , P<0.001) , across the time course [tail-flick: F(5,430)=77.90, P<0 .001 ; jump: F=52.60, P<0.001] and for the interaction between conditions and times [tail-flick: F(45,430)= 5.79, P < 0.001; jump: F = 6.69, P < 0.001]. Figure 2 illustrates the alterations in latencies and thresholds induced by the central co-administration of met-enkephalin (I00 lag) with vehicle, cFP-AAF-pAB (25 nmol) or cFP-F-pAB (25 nmol). The vehicle-met- enkephalin conditions at doses of 25, 100 and 200 lag, but not at 50 lag, significantly increased latencies relative to vehicle-vehicle treatment (Table 3). Pairing cFP-F-pAB (25 nmol) with met-enkephalin (100 lag) significantly increased latencies (13-34%, 5-60 rain) and thresholds (12-33 %, 5-30 rain) relative to vehicle-vehicle treatment, and significantly increased latencies (12-27 %, 10-45 min) and thresholds (20-24%, 5 ~ 0 min) relative to vehicle-

met-enkephalin treatment. The increased met-enkephalin antinociception by cFP-F-pAB was not due to the latter's intrinsic antinociceptive properties since cFP-F- pAB failed to significantly alter latencies or thresholds across this time course. In contrast, pairing cFP-AAF- pAB (25 nmol) with met-enkephalin (100 lag) significantly increased latencies (14%, 5-15 min) and thresholds (13%, 10 rain) relative to vehicle-vehicle treatment, but not relative to vehicle-met-enkephalin treatment. When cFP-F-pAB (25 nmol) was paired with met-enkephalin (50 lag), latencies (5-30 min, 34%) and thresholds (5-15 min, 21%) were significantly increased relative to vehicle-vehiqle treatment. Thus, an endopeptidase 24.11 inhibitor, cFP-F-pAB, but not an endopeptidase 24.15 inhibitor, cFP-AAF-pAB, increased met-enkephalin antinociception on the tail-flick and jump tests.

Experiment 3: Endopeptidase inhibitors and dynorphin A1-s antinociception

Protocol 3A

New subgroups of rats received a maximum of four injection conditions (Table 1C) as previously described, including a vehicle-vehicle control, a dose range of dynorphin A1-8 (5-20 lag), cFP-AAF-pAB (2.5-25 nmol)

412

paired with dynorphin A1-8 (1-5 gg), and cFP-F-pAB paired with dynorphin AI -8 . A 10-min interval separated each of the microinjections with latencies and thresholds assessed at 15, 30, 45, 60 and 90 min following the second injection.

Protocol 3B

Six rats received the following four triads of injection conditions at weekly intervals in counterbalanced order: a) vehicle (SC)-vehMe (ICV)-vehicle (ICV); b) vehicle ( (SC)-cFP-AAF-pAB (25 nmol, ICV)-dynorphin A1-8 (5 gg, tCV); c) naloxone (1 mg/kg, SC)-vehicle (ICV)- vehicle (ICV) and d) naloxone (1 mg/kg, SC)-cFP-AAF-

pAB (25 nmol, ICV)-dynorphin A1 ~8 (5 gg, ICV). The systemic injection preceded the first microinjection by 30 min, while the two microinjections were separated by a 10-min interval. Latencies and thresholds were assessed at 15, 30, 45, 60 and 90 min following the second microinjection.

Protocol 3C

Rats received the following four microinjection pretreatments at weekly intervals in counterbalanced order: a) vehicle (n = 9); b) Nor -BNI (20 gg, n = 9); c) naltrexone (20 pg, n = 8) and b) B-FNA (20 gg, n = 9). The first three pretreatments were administered 1 h prior to co-

VEHICLF-JM ET-ENKEPHALIN ~, ~ cFp=AAF-pA B/MET-EN KIEPHALIN E

2.0" I ~ cFP-F-pAB/MET-ENKEPHALIN ~ 0,151 I ~ cFP-F-pAB/VEHICLE ~ I ~i"

000 1 == o.s

....= 5 10 15 30 45 60 ~ " " 5 1'0 1'5 30 4'5 6'0 p- TiME (MIN) TIME (rain)

Fig. 2. Alterations in central met-enkephalin (100 gg, ICV) antinociception on the tail-flick (left panel) and jump (right panel) tests following central pretreatment 10 min earlier with vehicle, cFP-AAF-pAB (25 nmol) or cFP-F-pAB (25 nmol). The filled and open stars, respectively, denote significant increases in latencies and thresholds relative to the vehicle/vehicle and the vehicle/met-enkephalin conditions

Table 3. Summary of endopeptidase 24.I5 and 24.11 inhibition upon met-enkephalin antinociception

Group 5 10 15 30 45 60

A, Tail-flick test Veh/Veh 3.40 3.43 3.29 3.31 3.30 3.18 cFP-F-pAB 25/Veh 3.35 3.48 3.36 3.41 3.34 3.20 Veh/Met-Enk 25 3.40 3.29 3.24 3.37 3.40 3.34 Veh/Met-Enk 50 3.60 3.81" 3.54 3,25 3.10 3.33 Veh/Met-Enk 100 4.34* 3.87* 3.46 3,47 3.34 3.37 Veh/Met-Enk 200 4.27* 4.05* 3.58 3.26 3.20 3.12

cFP--F-pAB/Met-Enk 25 25 3.62 3.44 3.37 3.07 3.15 3.12 25 50 4.56* 4.19" 4.17" 3.84* 3.47 3.41 25 100 4.50* 4.50* 4.41" 3.99* 3.73* 3.62* cFP-AAF-pAB/Met-Enk 25 100 3.97* 4.10" 3.97* 3.49 3.27 3.26

B. Jump test Veh/Veh 0.384 0.379 0.375 0.378 0.381 0.377 cFP-F-pAB 25/Veh 0.345 0.376 0.367 0.369 0.364 0.350 Veh/Met-Enk 25 0.421" 0.417" 0.426* 0.417" 0.400 0.393 Veh/Met-Enk 50 0.390 0.387 0.377 0.383 0.388 0.367 Veh/Met-Enk 100 0.425* 0.377 0.356 0.344 0.346 0.350 Veh/Met-Enk 200 0.486* 0.476* 0.421" 0.417" 0 . 4 0 8 0.408* cFP-F-pAB/Met-Enk 25 25 0.421" 0 . 4 0 0 0 . 4 1 6 " 0 .412" 0 .417" 0.402 25 50 0.463* 0.458 0.423* 0,396 0.356 0.371 25 100 0.509* 0.452* 0.443* 0.422* 0.400 0.383 cFP-~AAF pAB/Met-Enk 25 100 0.406 0.425* 0.396 0.372 0.373 0.354

* Significant difference relative to Veh/Veh treatment (Dunnett comparison, P< 0.05)

CENTRAL PRETREATMENT

VEHICLE 2.01 g 0.15 I "'Q)'-- cFP-AAF-pAB (2.5 nmol)

• ~ cFP-AAF*pAB (25 nmol) ¢ m 1.5 t ~ cFP-F-pAB (25 nmol) O O O m 0.10

~, 0.00 >.. 0.0 , . , o

-o.5 ,,=, -o.o~ 5 ~ s 3 0 4 5 8 0 9 0

I- TIME (min)

1 '5 3'0

administration of cFP-AAF-pAB (25 nmol)-dynorphin A1_8 (5 gg) since this interval maximizes these opioid antagonist effects (e.g., Sawynok et al. 1979; Takemori et al. 1988). The last pretreatment was administered 24 h prior to co-administration of cFP-AAF-pAB (25 nmol)-dynorphin A1 -8 (5 gg), since this interval maximizes mu opioid receptor blockade (e.g., Takemori et al. 1981). Nociceptive thresholds were assessed at 15, 30, 45, 60 and 90 min following administration of dynorphin Al - s .

Protocol 3D

Six rats received microinjections of either vehicle-vehicle or cFP-AAF-pAB (25 nmol)-dynorphin A1-8 (5 gg) which were counterbalanced and tested at weekly intervals. Immediately after the last injection, animals were

Table 4. Summary of endopeptidase 24.15 and 24.11 inhibition upon dynorphin A l_s antinociception

Group 15 30 45 60 90

A. Tail-flick test Veh/Veh 3.44 3.50 3.47 3.54 Veh/Dyn 5 3.01 3.12 2.92 3.00 Veh/Dyn 20 3.93 3.87 3.80 3.75 cFP-AAF-pAB/Dyn 25 1 3.45 3.29 3.31 3.09 2.5 5 4.16" 4.08* 4.14" 4.19" 25 5 3.76* 3.45 3.82* 3.32 cFP-F-pAB/Dyn 25 5 3.99* 3.67 3.67 3.47

B. Jump test

Veh/Veh 0.331 0.328 0.334 0.340 Veh/Dyn 5 0.316 0.311 0.304 0.307 Veh/Dyn 20 0.325 0.335 0.332 0.332 cFP-AAF-pAB/Dyn 25 1 0.310 0.325 0.338 0.325 2.5 5 0,374* 0.377* 0.392* 0.385* 25 5 0.353* 0.352* 0.378* 0.377* cFP-F-pAB/Dyn 25 5 0.349 0.346 0.375* 0.381"

3.50 2.95 3.83

3.27 4.04* 3.73*

3.53

0.336 0.301 0.328

0.329 0.377* 0.351"

0.372*

* Significant difference relative to Veh/Veh treatment (Dunnett comparison, P< 0.05)

4'5 6'0 9'0 TIME (rain)

413

Fig. 3. Alterations in nociception on the tail- ~ck (left panel) and jump (right panel) tests in rats receiving central dynorphin A1-8 (5 gg, ICV) 10 rnin following central pretreatment with vehicle, cFP-AAF-pAB (2.5 or 25 nmol) or cFP-F-pAB (25 nmol). The filled stars denote significant increases in latencies and thresholds relative to the vehicle/vehicle condition

assessed for righting reflexes and performance on an inclined plane. Locomotor activity was assessed at 10-60 min after the last injection by monitoring counts at 10-rain intervals. Righting reflexes and performance on an inclined plane were then reassessed.

Results

Protocol 3A. Significant effects were observed for both nociceptive measures among agonist and/or endopeptidase inhibitor conditions [tail-flick: F(7,52) = 9.27, P < 0.001 ; jump: F = 4.43, P < 0.001] and between vehicle and different drug treatments [tail-flick: F(1,52)= 8.72, P<0.005; jump: F = 17.10, P<0.001]. Significant differences were observed across the time course for the jump [F(4,208)=4.89, P<0.001], but not the tail-flick (F=0.58) tests. Figure 3 illustrates the alterations in latencies and thresholds induced by the central co- administration of dynorphin AI - s (5 gg) with vehicle, cFP-AAF-pAB (25 and 2.5 nmol) or cFP-F-pAB (25 nmol). Vehicle paired with dynorphin A1-8 at doses of 5 and 20 gg failed to alter latencies or thresholds relative to vehicle-vehicle treatment (Table 4). Pairing cFP-AAF-pAB (25 nmol) with dynorphin A~-a (5 gg) significantly increased latencies (9-15%, 15 and 90 min) and thresholds (10-22%, 15-90 min) relative to vehicle- vehicle treatment, and significantly increased latencies (16-18%, 15 and 90 min) and thresholds (9-24%, 15-90 min) relative to vehicle-dynorphin AI - s treatment. The lower dose of cFP-AAF-pAB (2.5 nmol) paired with dynorphin A~_e (5 gg) significantly increased latencies (7-12%; 15-90 rain) and thresholds (10-14%, 15-90 min) relative to vehicle-vehicle treatment. The induction of dynorphin A1-s antinociception by cFP-AAF-pAB was not due to the latter's intrinsic antinociceptive properties, since cFP-AAF-pAB failed to significantly alter latencies or thresholds across this time course. In contrast, pairing cFP-F-pAB (25 nmol) with dynorphin A1-8 (5 pg) significantly increased latencies (8 %, 15 min) and thresholds (6-7%, 45-90 min) relative to vehicle-vehicle treatment, but not relative to vehicle-dynorphin A1_8 treatment. Thus, cFP-AAF-pAB, but not cFP-F-pAB, induced mild, though significant dynorphin A~ _ s antinociception on the tail-flick and jump tests.

Protocol 3B. Significant differences were observed among conditions [tail-flick: / '(2,10)=7.59, P<0.01; jump:

414

O u)

==

Ci

<,

SYSTEMIC PRETREATMENT

2 .0 ~ VEHICLE JL NALOXONE

1,5

1.0

0.5"

-0.5 ; 1'5 3'0 4'5

TIME (rain)

6 "0 9 '0

g 0.15

n- O

0.10 ffl

t~ 0.05 ,,=,

o 0.00

o

-0.05 B: ;z I,-

i

15 3'0 4 5 6 0 9 0

TIME (min)

Fig. 4. Alterations in antinociception induced by ¢oadministration of cFP-AAF-pAB (25 nmol, ICV) and dynorphin AI_ 8 (5 pg, ICV) following systemic pretreatment with either vehicle or natoxone (t mg/kg, SC). The filled stars denote significant increases in latencies and thresholds relative to the vehicle/ vehicle condition

2.0"

¢ 1~5- 0

1.0~

0.5

o 0.0

z -0.5 g

CENTRAL PRETREATMENT

VEHICLE NOR-BNI B-FNA

NALTREXONE

1 '5 3 '0 4 '5

TIME (min)

g

0 0

o

0

6'0 9 0 ~: I--.

0.15

0,10

0.05 *

O,OG

-0.05 1 '5 3'0 45

TIME (min)

6'5 9'0

Fig. 5. Alterations in antinociception induced by coadministration of cFP-AAF-pAB (25 nmol, ICV) and dynorphin A1 8 (5 pg, ICV) following central pretreatment with either vehicle, the kappa opioid antagonist, nor- binaltorphamine (NOR-BNI, 20 gg, ICV), the mu opioid antagonist, beta-funaltrexamine (B-FNA, 20 pg, ICV) or nattrexone (20 pg, [CV). Thejqlled stars' denote significant increases in latencies and thresholds relative to the vehicle/vehicle condition

F=39.69, P<0.001], across the time course [tail-flick: F(4,20) = 3.10, P < 0.05; jump: F - 3.58, P < 0.05] and for the interaction between conditions and times [tail-flick: F(8,40)=2.27, P<0.05; jump: F=5.55, P<0.001]. Co- administration of cFP-AAF-pAB and dynorphin A1_ 8 significantly increased latencies (9-18 %, 15-60 min) and thresholds (6-19%, 15---60 min) relative to vehicle-vehicle treatment. Pretreatment with naloxone eliminated these antinociceptive effects on the tail-flick and jump tests without affecting basal latencies or thresholds (Fig. 4).

Protocol3C. Significant differences were observed among conditions [tail-flick: F(4,32) = 23.90, P < 0.001 ; jump: F=21.59, P<0.001], across the time course [tail-flick: F(4,32)-24.35, P<0.001; jump: F = 10.04, P<0.001] and for the interaction between conditions and times [tail-flick: F(16,128)=4.84, P<0.001; jump: F=5.49; P<0.001]. Co-administration of cFP-AAF-pAB and dynorphin A1 -8 significantly increased latencies (9-24 %, 15-45 min) and thresholds (7-19%, 15 60 min) relative to vehicle-vehicle treatment. Central pretreatment with either naltrexone or Nor-BNI completely eliminated these antinociceptive effects on both tests (Fig. 5). In contrast, pretreatment with B-FNA failed to alter antinociception induced by co-administration of cFP- AAF-pAB and dynorphin A1-8.

Protocol 3D. Significant differences failed to occur in either locomotor activity, righting reflexes or performance on an inclined plane following vehicle-vehicle or cFP-AAF-pAB/dynorphin AI_ 8 treatment (data not shown).

Discussion

Central pretreatment with the inhibitor of endopeptidase 24.15, cFP-AAF-pAB produced small, but significant increases in MERGL antinociception on the tail-flick and jump tests without significantly altering basal latencies or thresholds during this time course. Further, the duration of MERGL antinociception was significantly lengthened from 10 to 60 min by endopeptidase 24.15 inhibitor co-administration. Since cFP-AAF-pAB is an effective inhibitor of endopeptidase 24.15 (Chu and Or- lowski 1984; Ortowski et al. 1988), and since endopeptidase 24.15 is implicated in the degradation of MERGL (Chu and Orlowski 1985; Acker et al. 1987; Orlowski et al. 1989), these data suggest that the small magnitude and brief duration of MERGL antinociception are due to its rapid degradation by endopeptidase 24.15. These data complement the previous observation that bestatin also potentiates MERGL antinociception (Mellstrom et al. 1987). The degradation of MERGL by endopeptidase 24.15 would yield met-enkephalin, a peptide sensitive to degradation by endopeptidase 24.11. It is therefore surprising that cFP-F-pAB failed to consistently alter MERGL antinociception.

As expected, the endopeptidase 24.11 inhibitor, cFP- F-pAB produced small, but significant increases in met- enkephalin antinociception on both tests. These data support previous findings of increased antinociception induced by enkephalins or their analogues following pretreatment with other endopeptidasc 24.11 inhibitors such as thiorphan, acetorphan and SCH 34826 (Roques et al. 1980; Chipkin et al. 1982, 1988; Lecomte et al. 1986).

415

Since cFP-AAF-pAB possesses no inhibitory activity upon endopeptidase 24.11, and since endopeptidase 24.15 does not participate in the degradation of pen- tapeptide enkephalins, it is not surprising that cFP-AAF- pAB failed to alter met-enkephalin antinociception, thereby confirming the specificity of this inhibitor.

The small, but significant increases in tail-flick latencies and jump thresholds following central co-administration of cFP-AAF-pAB and dynorphin A1-s is some- what controversial given the previous failure to observe antinociception following central dynorphin fragments (e.g., Friedman et al. 1981 ; Chavkin et al. 1982), and the observations of weak spinal antinociception accompanied by motor dysfunction (Herman and Goldstein 1985; Faden and Jacobs 1987; Long et al. 1988). However, an explanation for dynorphin's equivocal ability to induce antinociception relates to its rapid enzymatic degradation (Young et al. 1986). Endopeptidase 24.15 appears to play a major role in this degradation process (Chu and Orlowski 1985; Acker et al. 1987; Orlowski et al. 1989) such that exogenous administration of dynorphin Al_s into the lateral ventricle of behaving rats is minutely recovered from the fourth ventricle in perfusate studies (Molineaux and Ayala 1990). Co-administration of cFP- AAF-pAB significantly increased the levels and the duration of recoverable dynorphin A~-s. Interestingly, the time course of antinociception by co-administration of cFP-AAF-pAB and dynorphin Al -s was similar to the time course of recoverable dynorphin in the perfusate studies. These data support other findings of dynorph]n antinociception utilizing either synthetic analogues (Wal- ker et al. 1987) or aminopeptidase inhibition (Nakazawa et al. 1989). Given the lack of participation of endopeptidase 24.11 in the degradation of dynorphin-related peptides, it is not surprising that cFP-F-pAB was relatively ineffective in eliciting antinociception when paired with dynorphin A~ -s-

The antinociception induced by cFP-AAF-pAB and dynorphin A~-s coadministration appeared to be mediated through opioid receptors given its sensitivity to blockade by systemic naloxone and central naltrexone. The effectiveness of Nor-BNI, but not B-FNA to significantly reduce antinociception induced by cFP-AAF-pAB and dynorphin A~_ s coadministration suggests that the kappa receptor may be participating in this small antinociceptive effect. This supports the contention that dynorphin acts primarily through kappa sites (e.g., Chavkin et al. 1982), and that the kappa receptor is involved in a minor way in supraspinal antinociception (Millan 1989, 1990). Intrathecal dynorphin produces antinociception and motor dysfunction (Faden and-Jacobs 1983; Her- man and Goldstein 1985); the latter effect appears to be mediated by degradation of dynorphin into nonopioid peptides (Long et al. 1988). The failure to observe motor dysfunction following central coadministration of cFP- AAF-pAB and dynorphin A~-8 suggests that the increases in tail-flick latencies and jump thresholds are due to activation of intrinsic pain-inhibitory processes.

The present observation that cFP-AAF-pAB produces small increases in MERGL and dynorphin antinociception, together with the previously-described

small and time-dependent antinociceptive actions of this inhibitor (Kest et al. 1991), indicates that endopeptidase 24.15, like endopeptidase 24.t 1, aminopeptidase, and ACE, is involved in the modulation of opioid antinociception. The specificity of each endopeptidase inhibitor to affect some, but not all forms of opioid antinociception suggests their possible utility as a specific tool to treat specific subsets of pain states while reducing side effects.

Acknowledgements. This research was supported in part by PSC/ CUNY Grant 669213 and NIH BRSG PRO7064 (RJB) and NIH Grant DK25377 (MO). Whe thank Dr. Christopher Molineaux for his advice in the formulation of the cFP-AAF-pAB-dynorphin A1-8 study. This paper is dedicated to the memory of Dr. Dennis Kelly, a friend, colleague and mentor.

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Endopeptidase 24.15 inhibition and opioid antinociception

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