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Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogs
Peptides 20 (1999) 1211–1217
Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogs P.P.-C. Tana, J.-C. Chenb, J.-Y. Lia, K.-W. Liangb, C.-H. Wonga, E.Y.-K. Huanga,* a
Department of Anesthesiology, Chang Gung Memorial Hospital, 5 Fu-Hsing Street, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China b Department of Pharmacology, Chang Gung University, 259 Wen Hua 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China Received 29 March 1999; accepted 7 May 1999
Abstract Neuropeptide FF (NPFF) has been reported to be an endogenous anti-opioid peptide that has significant effects on morphine tolerance and dependence. In the present study, we examined the chronic effects of NPFF and its synthetic analogs: the putative agonist, PFRFamide, and the putative antagonists, dansyl-PQRamide and PFR(Tic)amide on naloxone-precipitated morphine withdrawal syndromes in rats. After a 5-day co-administration with morphine [5 mg/kg, intraperitoneally (i.p.), twice per day (b.i.d.)] and the tested peptide [intracerebroventricularly (i.c.v.) or i.p., b.i.d.], naloxone (4 mg/kg, i.p.) was given systemically to evaluate the severity of the morphine withdrawal syndromes. Our results revealed that NPFF significantly potentiated the overall morphine withdrawal syndromes and, on the contrary, dansyl-PQRamide attenuated these syndromes. These results clearly indicate that modulation of the NPFF system in the mammalian central nervous system has significant effects on opiate dependence. In addition, morphine withdrawal syndromes could be practically applied as a valid parameter to functionally characterize the putative NPFF agonists and antagonists. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Withdrawal syndromes; Morphine; Neuropeptide FF; Behavior; Opiate dependence; Neuropeptides
1. Introduction In 1977, a cardioactive substance was purified from molluscs and identified as Phe-Met-Arg-Phe-NH2 (FMRFamide) [12]. Following the identification of FMRFamide, the mammalian counterpart of this neuropeptide was sought by many investigations. For instance, two mammalian FMRFamide-like peptides were identified from bovine brain, FLFQPQRFamide (NPFF) and AGEGLSSPFWSLAAPQRFamide (NPAF) [19]. These peptides were found to decrease the tail-flick latency in rats and attenuate the prolongation of the tail-flick latency induced by morphine [19]. Pretreatment with immunoglobulin G (IgG) from a NPFF antiserum prevented the naloxone-precipitated abstinence syndrome in morphine-dependent rats, while it restored the analgesic response to morphine in morphine-tolerant rats [5,7]. Moreover, the concentration of NPFF in the cerebrospinal fluid of morphine-dependent rats was higher than in naive rats [16]. Taken together, these reports suggest NPFF * Corresponding author. Tel.: ⫹886-3-3283016 ext. 5282; fax: ⫹8863-3283031. E-mail address: [email protected] (E.Y-K. Huang)
or its functionally related peptides play an important role in morphine tolerance and dependence [13]. The NPFF system could participate in the complexities of the pain-processing system that is closely related with the opioid system. The most noticeable property of NPFF is its unique binding site in the mammalian brain [1]. Although the receptor for NPFF has not yet been cloned, it has been proven to be different from all of the opioid receptors [1]. Therefore, it is generally believed that the study of NPFF and its receptor(s) could open a new avenue in the area of analgesia and drug addiction prevention. To investigate the significance of the NPFF system, the design of a specific antagonist for NPFF is essential. NPFF antagonists may possess an ability to reduce opiate dependence, which would be of great therapeutic benefit in developing a better treatment for opiate addicts. By using D-amino acids, N-methyl amino acids, or some other chemical modifications, many compounds have been designed and synthesized as NPFF agonists and antagonists [3,8]. However, untill now, no selective antagonist for NPFF has been fully characterized. The NPFF antagonist should possess a high affinity for NPFF receptors and functionally antagonize the bio-effects induced by NPFF. Because NPFF is considered to be a morphine withdrawal-
0196-9781/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 9 9 ) 0 0 1 2 5 - 4
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enhancing substance, the aim of the present study was to investigate the chronic effects of NPFF, PFRFamide (putative NPFF agonist), dansyl-PQRamide (putative NPFF antagonist), and PFR(Tic)amide (putative NPFF antagonist) on naloxone-precipitated morphine withdrawal syndromes. The current results indicate that NPFF agonists potentiate the naloxone-induced morphine withdrawal syndromes and, on the contrary, synthetic NPFF antagonists suppress the syndromes. Also, the application of morphine withdrawal model [5 mg/kg, intraperitoneally (i.p.), twice per day (b.i.d.) for 5 days; followed by naloxone 4 mg/kg, i.p. challenge] would be helpful for a structural and functional characterization of NPFF analogs.
2. Materials and methods 2.1. Peptide synthesis and purification NPFF, PFRFamide, dansyl-PQRamide, and PFR(Tic)amide were all synthesized manually on solid phase. In a stepwise manner at the 0.5-mmol scale, the Fmoc strategy was used (Atherton and Sheppard, 1989). Fmoc amino acids were coupled to Rink Amide MBHA resin (Novabiochem, Switzerland) by using 2-(1H-Benzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate as the coupling reagent to give C-terminally amidated peptides. For dansylPQRamide, the N-terminal dansylation was performed as the last coupling. Dansyl chloride (Sigma) in dimethylformamide was used for dansylation of proline on solid phase. The dansylation was repeated twice to assure that the N termini were completely dansylated. As for PFR(Tic)amide, N-1-Fmoc-L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (Fmoc-Tic) was used for the first coupling. Following the deprotection of Fmoc-Tic, the coupling with arginine was processed twice to assure the complete coupling. After the synthesis, the peptides were cleaved from the resin with a 97.5% trifluoroacetic acid solution for 1 h, and the crude peptides were desalted through a Sephadex G-10 column (Pharmacia, Uppsala, Sweden). Purification by analytical reverse-phase high-performance liquid chromatography (HPLC) was performed on a Beckman HPLC System (System Gold). The peptides were analysed by using the following conditions: column, Beckman Ultrasphere ODS C-18 (5 m, 0.46 ⫻ 25 cm; Beckman, Fullerton, CA, USA); eluent A, 0.1% (v/v) trifluoroacetic acid in H2O; eluent B, 0.1% (v/v) trifluoroacetic acid in acetonitrile; gradient B, 0% over 2 min, 0 – 80% over 32 min; flow rate 1 ml/min; absorbance 216 nm; fluorescence (Gilson 121, Middleton, WI, USA) detection for dansyl-PQRamide. Only peptides that showed a single peak on the HPLC profile were used for the following study. Fig. 1 shows the chemical structures of synthetic PFRFamide, PFR(Tic)amide, and dansyl-PQRamide.
2.2. Animals Male Sprague-Dawley rats, 200 –330 g (Academia Sinica, Taipei, Taiwan) were maintained five per cage with food and water available ad libitum and on a 12-h light/dark cycle. They were habituated to the animal room for at least 5 days before the experiments. Except for the experiment with dansyl-PQRamide, each rat was cannulated stereotaxically in the lateral ventricle under ketamine (50 mg/kg, i.p.) anesthesia and secured with stainless steel screws and dental cement. The i.c.v. implantation was carried out with a 23gauge guide cannula positioned in the lateral ventricle at the following coordinates: 0.8 mm posterior to bregma, 1.4 mm lateral from the midline, and 3.7 mm ventral from the dura [11]. Animals were allowed 7 days for recovery. Cannula placements subsequently were confirmed by methylene blue dye injection and histologic examination. 2.3. Chronic treatments with NPFF, PFRFamide, PFR(Tic)amide, and dansyl-PQRamide All rats were chronically treated with morphine (5 mg/ kg, b.i.d.) via i.p. injection for 5 consecutive days. For each experiment, rats were randomly divided into control and chronic peptide-treated groups. For the experiment with NPFF, the peptide was dissolved in saline at a concentration of 4 mg/ml. Following the systemic injection of morphine, 5 l (20 g of NPFF) was injected [intracerebroventricularly (i.c.v.), b.i.d.] over 1 min via a microdialysis pump (CMA 120, Stockholm, Sweden). The injection of NPFF also was performed for 5 consecutive days. Control rats received the same volume of saline injection (i.c.v., b.i.d.) in the same manner. For PFRFamide and PFR(Tic)amide, 5 l of 20 g of each peptide was individually injected. The corresponding controls were prepared by using the same method. A preliminary experiment indicated that the coadministration of a peptidase inhibitor, bestatin, did not prevent or induce any notable effect; thus, all of the peptides injected (i.c.v.) were in their free form. For the dansyl-PQRamide treated group, dansyl-PQRamide was dissolved in 20% dimethylsulfoxide at a concentration of 13 mg/ml. Following the injection of morphine, 13 mg/kg dansyl-PQRamide was injected (i.p., b.i.d.). The preparation was repeated for 5 consecutive days. The rats from the control group received a vehicle (0.3 ml of 20% dimethylsulfoxide) injection (i.p., b.i.d.). 2.4. Measurement of morphine withdrawal syndromes On the day of the behavioral test, all rats were injected with a high dose of morphine (20 mg/kg, i.p.). Four hours later, the rats received the naloxone (Sigma, St. Louis, MO, USA; 4 mg/kg, i.p.) injection to induce the morphine withdrawal syndromes. Twelve distinct behavioral signs (chewing, sniffing, grooming, wet dog shakes, stretching, yawning, rearing,
P. P.-C. Tan et al. / Peptides 20 (1999) 1211–1217
Fig. 1. The chemical structures of (1) PFRFamide, (2) PFR(Tic)amide, and (3) dansyl-PQRamide.
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jumping, teeth grounding, ptosis, diarrhea, and piloerection) were observed and scored during a 30-min period after the naloxone injection. The reactions of each animal were evaluated by an independent observer who did not have any prior knowledge of the nature of the treatment received. Chewing, sniffing, and grooming were all scored from 1.0 to 3.0 according to their frequency (1.0: occasional; 2.0: frequent; 3.0: continuous). The occurrences of wet dog shakes, stretching, yawning, rearing, and jumping were separately recorded. One to five occurrences of the above behavioral sign was scored as 1.0; 6 to 10 occurrences was scored as 2.0; and over 10 occurrences was scored as 3.0. For the other syndromes (teeth grounding, ptosis, diarrhea, and piloerection), 3.0 or 0 was given as the score to the rat for either showing or not showing the sign, respectively. Total scores were calculated by adding together the individual score for each withdrawal sign. 2.5. Statistics Multiple t-tests were used to examine the significance of the change of each syndrome. To compare with the control value, each total score for the peptide-treated group also was converted into the percentage of the control value with mean control value defined as 100%. The change for each peptide-treated group was compared with an unpaired Student’s t-test. A P value ⬍ 0.05 was considered significant.
3. Results 3.1. The effect of chronic NPFF administration on morphine withdrawal syndromes As illustrated in Table 1, the intensity of chewing, sniffing, grooming, wet dog shakes, rearing, and diarrhea displayed a higher score in the chronic NPFF-treated groups than the controls. Among these syndromes, sniffing, grooming, and diarrhea were statistically significant (Table 1). The total score for the withdrawal signs in the NPFF-treated groups was 17.20 ⫾ 0.97 (n ⫽ 5) vs. 12.40 ⫾ 1.44 (n ⫽ 5) in the control group, indicating a 38.71% increase (Fig. 2).
Table 1 Individual scores of the naloxone-precipitated morphine withdrawal syndromes in control (left column) and NPFF-treated (right column) groups Withdrawal sign
Control (score ⫾ SEM, n ⫽ 5)
NPFF-treated group (score ⫾ SEM, n ⫽ 5)
Chewing Sniffing Grooming Wet dog shakes Stretching Yawning Rearing Jumping Teeth grinding Ptosis Diarrhea Piloerection Total score
1.20 ⫾ 0.20 (5/5) 1.40 ⫾ 0.25 (5/5) 1.20 ⫾ 0.37 (4/5) 0.60 ⫾ 0.25 (3/5) 0 0.60 ⫾ 0.25 (3/5) 0.80 ⫾ 0.20 (4/5) 0 2.40 ⫾ 0.60 (4/5) 3.00 ⫾ 0.00 (5/5) 0 1.20 ⫾ 0.74 (2/5) 12.40 ⴞ 1.44
2.00 ⫾ 0.32 (5/5) 2.00 ⫾ 0.00 (5/5)* 2.60 ⫾ 0.40 (5/5)* 1.20 ⫾ 0.49 (4/5) 0 0 1.60 ⫾ 0.40 (5/5) 0 1.80 ⫾ 0.74 (3/5) 2.40 ⫾ 0.60 (4/5) 2.40 ⫾ 0.60 (4/5)** 1.20 ⫾ 0.74 (2/5) 17.20 ⴞ 0.97
The mean total scores are shown at the bottom of the table. The numbers within the bracket (a/b; a, the number of animals displaying the syndrome; b, the total number of animals) indicate the proportion of the animals showing the syndrome (* P ⬍ 0.05; ** P ⬍ 0.01).
3.3. The effect of chronic dansyl-PQRamide administration on morphine withdrawal syndromes Compared with the controls, the intensity of sniffing, grooming, wet dog shakes, stretching, yawning, rearing, teeth grounding, and ptosis was changed to a lower level in the chronic dansyl-PQRamide-treated groups (Table 3). Only the decrease of grooming has been found to be statistically significant (Table 3, P ⬍ 0.05). The total score for the withdrawal signs in the dansyl-PQRamide-treated groups was 7.00 ⫾ 1.23 (n ⫽ 7) vs. 11.00 ⫾ 1.48 (n ⫽ 7) in the control group, indicating a 36.36% decrease (Fig. 2, P ⫽ 0.0602).
3.2. The effect of chronic PFRFamide administration on morphine withdrawal syndromes As illustrated in Table 2, the intensity of chewing, sniffing, grooming, wet dog shakes, stretching, yawning, and rearing showed a higher score than those in the the control groups. Although none of these syndromes has reached to a statistical significance, the total score for the withdrawal signs in the PFRFamide-treated groups was 16.40 ⫾ 1.47 (n ⫽ 5) vs. 15.80 ⫾ 2.27 (n ⫽ 5) in the control group (Table 2).
Fig. 2. Chronic effects of NPFF, PFRFamide, dansyl-PQRamide, and PFR(Tic)amide on naloxone-precipitated morphine withdrawal syndromes. Each gray bar represents the mean total score of the peptide-treated group that was converted to the percentage of the corresponding control value with the mean control value defined as 100% (white bar). An unpaired Student’s t-test was used to examine the significance (* P ⬍ 0.05).
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Table 2 Individual scores of the naloxone-precipitated morphine withdrawal syndromes in control (left column) and PFRFamide-treated (right column) groups
Table 4 Individual scores of the naloxone-precipitated morphine withdrawal syndromes in control (left column) and PFR(Tic)amide-treated (right column) groups
Withdrawal sign
Control (score ⫾ SEM, n ⫽ 5)
PFRFa-treated group (score ⫾ SEM, n ⫽ 5)
Withdrawal sign
Control (score ⫾ SEM, n ⫽ 5)
Chewing Sniffing Grooming Wet dog shakes Stretching Yawning Rearing Jumping Teeth grinding Ptosis Diarrhea Piloerection Total score
1.60 ⫾ 0.40 (5/5) 1.00 ⫾ 0.00 (5/5) 1.60 ⫾ 0.25 (5/5) 1.20 ⫾ 0.58 (3/5) 0 0 2.40 ⫾ 0.40 (5/5) 0.20 ⫾ 0.20 (1/5) 1.20 ⫾ 0.74 (2/5) 1.80 ⫾ 0.74 (3/5) 1.80 ⫾ 0.74 (3/5) 3.00 ⫾ 0.00 (5/5) 15.80 ⴞ 2.27
2.60 ⫾ 0.25 (5/5) 1.40 ⫾ 0.60 (3/5) 2.40 ⫾ 0.40 (5/5) 2.20 ⫾ 0.49 (5/5) 0.40 ⫾ 0.25 (2/5) 0.40 ⫾ 0.25 (2/5) 2.80 ⫾ 0.20 (5/5) 0 1.20 ⫾ 0.74 (2/5) 1.20 ⫾ 0.74 (2/5) 1.80 ⫾ 0.74 (3/5) 0 16.40 ⴞ 1.47
PFR(Tic)a-treated group (score ⫾ SEM, n ⫽ 6)
Chewing Sniffing Grooming Wet dog shakes Stretching Yawning Rearing Jumping Teeth grinding Ptosis Diarrhea Piloerection Total score
1.60 ⫾ 0.25 (5/5) 1.60 ⫾ 0.25 (5/5) 1.80 ⫾ 0.37 (5/5) 1.80 ⫾ 0.49 (5/5) 0.20 ⫾ 0.20 (1/5) 0.20 ⫾ 0.20 (1/5) 2.40 ⫾ 0.40 (5/5) 0 1.20 ⫾ 0.74 (2/5) 1.20 ⫾ 0.74 (2/5) 1.80 ⫾ 0.74 (3/5) 2.40 ⫾ 0.60 (4/5) 16.20 ⴞ 1.28
1.17 ⫾ 0.17 (6/6) 0.33 ⫾ 0.21 (2/6)** 1.50 ⫾ 0.34 (6/6) 1.33 ⫾ 0.42 (5/6) 0.17 ⫾ 0.17 (1/6) 0.50 ⫾ 0.22 (3/6) 2.00 ⫾ 0.37 (6/6) 0 2.00 ⫾ 0.63 (4/6) 2.00 ⫾ 0.63 (4/6) 1.50 ⫾ 0.67 (3/6) 2.50 ⫾ 0.50 (5/6) 15.00 ⴞ 0.68
The mean total scores are shown at the bottom of the table. The numbers within the bracket (a/b; a, the number of animals displaying the syndrome; b, the total number of animals) indicate the proportion of the animals showing the syndrome.
3.4. The effect of chronic PFR(Tic)amide administration on morphine withdrawal syndromes Similar to dansyl-PQRamide, the intensity of chewing, sniffing, grooming, wet dog shakes, stretching, rearing, and diarrhea in the chronic PFR(Tic)amide-treated groups was lower when compared with the controls (Table 4). Only the decrease of sniffing has been found to be statistically significant (Table 4, P ⬍ 0.01). The total score for the withdrawal signs in the PFR(Tic)amide-treated groups was Table 3 Individual scores of the naloxone-precipitated morphine withdrawal syndromes in control (left column) and dansyl-PQRamide-treated (right column) groups Withdrawal sign
Control (score ⫾ SEM, n ⫽ 7)
Dansyl-PQRa-treated group (score ⫾ SEM, n ⫽ 7)
Chewing Sniffing Grooming Wet dog shakes Stretching Yawning Rearing Jumping Teeth grinding Ptosis Diarrhea Piloerection Total score
0.43 ⫾ 0.20 (3/7) 0.57 ⫾ 0.20 (4/7) 1.86 ⫾ 0.34 (7/7) 1.00 ⫾ 0.22 (6/7) 0.29 ⫾ 0.18 (2/7) 0.29 ⫾ 0.18 (2/7) 1.43 ⫾ 0.57 (4/7) 0 0.86 ⫾ 0.55 (2/7) 1.29 ⫾ 0.61 (3/7) 0.86 ⫾ 0.55 (2/7) 2.14 ⫾ 0.55 (5/7) 11.00 ⴞ 1.48
0.43 ⫾ 0.20 (3/7) 0.29 ⫾ 0.18 (2/7) 0.71 ⫾ 0.18 (5/7)* 0.29 ⫾ 0.29 (1/7) 0 0 0.43 ⫾ 0.20 (3/7) 0.14 ⫾ 0.14 (1/7) 0 0.43 ⫾ 0.43 (1/7) 1.29 ⫾ 0.61 (3/7) 3.00 ⫾ 0.00 (7/7) 7.00 ⴞ 1.23
The mean total scores are shown at the bottom of the table. The numbers within the bracket (a/b; a, the number of animals displaying the syndrome; b, the total number of animals) indicate the proportion of the animals showing the syndrome (* P ⬍ 0.05).
The mean total scores are shown at the bottom of the table. The numbers within the bracket (a/b; a, the number of animals displaying the syndrome; b, the total number of animals) indicate the proportion of the animals showing the syndrome (** P ⬍ 0.01).
15.00 ⫾ 0.68 (n ⫽ 6) vs. 16.20 ⫾ 1.28 (n ⫽ 5) in the control group, indicating a mild attenuation (7.41%) (Fig. 2).
4. Discussion The present study indicates that chronic administration of NPFF and its synthetic analogs significantly modulate the naloxone-precipitated morphine withdrawal syndromes. Given the evidence that acute NPFF injection was able to precipitate an opiate withdrawal syndrome in morphinedependent rats [6], our results clearly showed that continuous activation of NPFF receptors also would enhance the intensity of morphine withdrawal syndromes. Of interest, acute NPFF-induced withdrawal signs were reported to occur only when the drug was applied into the third ventricle but not into the lateral ventricle or injected intrathecally [14]. However, our results that chronic lateral ventricular injection of NPFF potentiated naloxone-precipitated morphine withdrawal syndromes suggest a time-dependent diffusion effect. Otherwise, it is possible that the chronic lateral ventricular injections enable NPFF to induce a more profound effect or indirectly modulate opiate dependence via other neurotransmitter systems. Although there was no immediate withdrawal sign(s) observed after each lateral ventricular injection, our results still highlight the importance of the NPFF system in the development of opiate dependence. The putative NPFF agonist PFRFamide first was predicted to possess a high affinity for NPFF receptors in a study of structure-activity relationship [10]. Despite the fact that the affinity of PFRFamide for NPFF receptors has not
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yet been determined in ligand-receptor binding assays, it has been reported to potentiate the spinal reflex in the isolated rat spinal cord preparation [4]. In that electrophysiological study, PFRFamide displayed a higher potency than that of NPFF for potentiation of spinal reflexes. If the increase in the magnitude of the reflex is through the activation of NPFF receptors, PFRFamide possibly would act as a full or partial NPFF agonist. In the present study, chronic treatment with PFRFamide appears to induce more stereotyped behaviors (chewing, sniffing, grooming, and rearing), and wet dog shakes, stretching, yawning in PFRFamide-treated rats than the saline controls. The results seem to implicate selective withdrawal signs that were turned on by PFRFamide. We also investigated the putative NPFF antagonist dansyl-PQRamide. Dansyl-PQRamide has been reported to be able to reduce the naloxone-precipitated morphine withdrawal syndromes when it was injected subcutaneously (s.c.) [9]. Its acute effect on morphine withdrawal syndromes has been well studied by Malin et al. [9]. In this study, we further characterized the chronic effect (i.p. injection) of dansyl-PQRamide and found that the result is compatible with the acute administration. Although dansylPQRamide might cross the blood– brain barrier and attenuate morphine withdrawal syndromes, the hydrophobicity makes it difficult to dissolve in water and produces a lot of problems for purification and for its high concentration in in vivo practical application [9]. However, this peptide still presents a typical structure for the design of NPFF analogs as antagonists. Another important property of dansyl-PQRamide is its high fluorescence due to its dansylated Nterminus. Thus, it could be used as a sensitive fluorescent probe in immunocytochemical or receptor binding studies. Therefore, PFR(Tic)amide, derived from PFRFamide, was designed and speculated to be an NPFF antagonist. L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic Because acid (Tic) has been successfully used to design and synthesize novel ␦-selective opioid antagonists and bradykinin antagonists [15,17,18], we attempted to convert the NPFF agonist PFRFamide into an antagonist by replacing Phe with Tic at its C terminus. It is expected the Tic residue in PFR(Tic)amide would be able to reduce the activation of NPFF receptors while the other three amino acids might maintain its high binding affinity. As we expected, the administration of PFR(Tic)amide to morphine-dependent rats produced an attenuation of several morphine withdrawal syndromes (chewing, sniffing, grooming, wet dog shakes, stretching, rearing, and diarrhea). Although the attenuation does not reach a statistical significance (92.59% of the control value), these results may still suggest that PFR(Tic)amide could be an effective NPFF antagonist. To gain the full scope of its pharmacological profile, this newly designed peptide requires further characterization. However, the possible ability of PFR(Tic)amide to reduce morphine withdrawal syndromes provides us a great therapeutic potential. In addition, the synthetic amino acid (Tic) could
make the peptide more hydrophobic, facilitating its crossing the blood– brain barrier and making it resistant to enzyme degradation. In summary, chronic treatment with NPFF significantly potentiated the naloxone-precipitated morphine withdrawal syndromes, whereas its putative antagonist, dansyl-PQRamide, showed an opposite effect. It is important to note that, to our knowledge, PFR(Tic)amide is the first design that was tested as an effective NPFF antagonist. Although its affinity for NPFF receptors has not yet been determined, PFR(Tic)amide also has displayed an apparent anti-NPFF cardiovascular effect in rats (in preparation). Our results suggested that PFR(Tic)amide or its related Tic-containing compounds may be useful for new drug development based on NPFF structure. In addition, our results should be helpful for further elucidation of the functional significance of the NPFF system in the mammalian central nervous system.
References [1] Allard M, Geoffre S, Legendre P, Vincent JD, Simonnet G. Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study. Brain Res 1989;500:169 –76. [2] Atherton E, Sheppard RC. Solid Phase Peptide Synthesis, a Practical Approach. Oxford, UK: IRL Press at Oxford University Press, 1989. [3] Gicquel S, Mazarguil H, Allard M, Simonnet G, Zajac JM. Analogues of F8Famide resistant to degradation, with high affinity and in vivo effects. Eur J Pharmacol 1992;222:61–7. [4] Huang EY-K, Bagust J, Sharma RP, Walker RJ. The effect of FMRFamide-like peptides on electrical activity in isolated mammalian spinal cord. Neurosci Res 1998;30:295–301. [5] Lake JR, Hammond MV, Shaddox RC, Hunsicker LM, Yang H.-YT, Malin DH. IgG from neuropeptide FF antiserum reverses morphine tolerance in the rat. Neurosci Lett 1991;132:29 –32. [6] Malin DH, Lake JR, Fowler DE, Hammond MV, Brown SL, Leyva JE, Prasco PE, Dougherty TM. FMRF-NH2-like mammalian peptide precipitates opiate-withdrawal syndrome in the rat. Peptides 1990;11: 277– 80. [7] Malin DH, Lake JR, Hammond MV, Fowler DE, Rogillio RB, Brown SL, Sims JL, Leecraft BM, Yang H-YT. FMRF-NH2-like mammalian octapeptide: possible role in opiate dependence and abstinence. Peptides 1990;11:969 –72. [8] Malin DH, Lake JR, Leyva JE, Hammond MV, Rogillio RB, Arcangeli KR, Ludgate K, Moore GM, Payza K. Analog of neuropeptide FF attenuates morphine abstinence syndrome. Peptides 1991;12:1011– 4. [9] Malin DH, Lake JR, Smith DA, Jones JA, Morel J, Claunch AE, Stevens PA, Payza K, Ho KK, Liu J, Ham I, Burgess K. Subcutaneous injection of an analog of neuropeptide FF prevents naloxoneprecipitated morphine abstinence syndrome. Drug Alcohol Depend 1995;40:37– 42. [10] Payza K, Akar CA, Yang H-YT. Neuropeptide FF receptors: structure-activity relationship and effect of morphine. J Pharmacol Exp Ther 1993;267:88 –94. [11] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. New York: Academic Press, 1986. [12] Price DA, Greenberg MJ. Structure of a molluscan cardioexcitatory neuropeptide. Science 1977;197:670 –1. [13] Rothman RB. A review of the role of anti-opioid peptides in morphine tolerance and dependence. Synapse 1992;12:129 –38. [14] Rothman RB, Brady LS, Xu H, Long JB. Chronic intracerebroventricular infusion of the antiopioid peptide, Phe-Leu-Phe-Gln-Pro-Gln-
P. P.-C. Tan et al. / Peptides 20 (1999) 1211–1217 Arg-Phe-NH2 (NPFF), downregulates mu opioid binding sites in rat brain. Peptides 1993;14:1271–7. [15] Schiller PW, Nguyen TM-D, Weltrowska G, Wilkes BC, Marsden BJ, Lemieux C, Chung NN. Differential stereochemical requirements of vs. ␦ opioid receptors for ligand binding and signal transduction: development of a class of potent and highly dpselective peptide antagonists. Proc Natl Acad Sci USA 1992;89: 11871–5. [16] Stinus L, Allard M, Gold L, Simonnet G. Change in CNS neuropeptide FF-like material, pain sensitivity, and opiate dependence following chronic morphine treatment. Peptides 1995;16:1235– 41.
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[17] Tancredi T, Salvadori S, Amodeo P, Picone D, Lazarus LH, Bryant SD, Guerrini R, Marzola G, Temussi PA. Conversion of enkephalin and dermorphin into ␦-selective opioid antagonists by single-residue substitution. Eur J Biochem 1994;224:241–7. [18] Thurieau C, Feletou M, Hennig P, Raimbaud E, Canet E, Fauchere J-L. Design and synthesis of new linear and cyclic bradykinin antagonists. J Med Chem 1996;39:2095–101. [19] Yang H-YT, Fratta W, Majane EA, Costa E. Isolation sequencing, synthesis and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 1985;82:7757– 61.
Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogs P.P.-C. Tana, J.-C. Chenb, J.-Y. Lia, K.-W. Liangb, C.-H. Wonga, E.Y.-K. Huanga,* a
Department of Anesthesiology, Chang Gung Memorial Hospital, 5 Fu-Hsing Street, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China b Department of Pharmacology, Chang Gung University, 259 Wen Hua 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China Received 29 March 1999; accepted 7 May 1999
Abstract Neuropeptide FF (NPFF) has been reported to be an endogenous anti-opioid peptide that has significant effects on morphine tolerance and dependence. In the present study, we examined the chronic effects of NPFF and its synthetic analogs: the putative agonist, PFRFamide, and the putative antagonists, dansyl-PQRamide and PFR(Tic)amide on naloxone-precipitated morphine withdrawal syndromes in rats. After a 5-day co-administration with morphine [5 mg/kg, intraperitoneally (i.p.), twice per day (b.i.d.)] and the tested peptide [intracerebroventricularly (i.c.v.) or i.p., b.i.d.], naloxone (4 mg/kg, i.p.) was given systemically to evaluate the severity of the morphine withdrawal syndromes. Our results revealed that NPFF significantly potentiated the overall morphine withdrawal syndromes and, on the contrary, dansyl-PQRamide attenuated these syndromes. These results clearly indicate that modulation of the NPFF system in the mammalian central nervous system has significant effects on opiate dependence. In addition, morphine withdrawal syndromes could be practically applied as a valid parameter to functionally characterize the putative NPFF agonists and antagonists. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Withdrawal syndromes; Morphine; Neuropeptide FF; Behavior; Opiate dependence; Neuropeptides
1. Introduction In 1977, a cardioactive substance was purified from molluscs and identified as Phe-Met-Arg-Phe-NH2 (FMRFamide) [12]. Following the identification of FMRFamide, the mammalian counterpart of this neuropeptide was sought by many investigations. For instance, two mammalian FMRFamide-like peptides were identified from bovine brain, FLFQPQRFamide (NPFF) and AGEGLSSPFWSLAAPQRFamide (NPAF) [19]. These peptides were found to decrease the tail-flick latency in rats and attenuate the prolongation of the tail-flick latency induced by morphine [19]. Pretreatment with immunoglobulin G (IgG) from a NPFF antiserum prevented the naloxone-precipitated abstinence syndrome in morphine-dependent rats, while it restored the analgesic response to morphine in morphine-tolerant rats [5,7]. Moreover, the concentration of NPFF in the cerebrospinal fluid of morphine-dependent rats was higher than in naive rats [16]. Taken together, these reports suggest NPFF * Corresponding author. Tel.: ⫹886-3-3283016 ext. 5282; fax: ⫹8863-3283031. E-mail address: [email protected] (E.Y-K. Huang)
or its functionally related peptides play an important role in morphine tolerance and dependence [13]. The NPFF system could participate in the complexities of the pain-processing system that is closely related with the opioid system. The most noticeable property of NPFF is its unique binding site in the mammalian brain [1]. Although the receptor for NPFF has not yet been cloned, it has been proven to be different from all of the opioid receptors [1]. Therefore, it is generally believed that the study of NPFF and its receptor(s) could open a new avenue in the area of analgesia and drug addiction prevention. To investigate the significance of the NPFF system, the design of a specific antagonist for NPFF is essential. NPFF antagonists may possess an ability to reduce opiate dependence, which would be of great therapeutic benefit in developing a better treatment for opiate addicts. By using D-amino acids, N-methyl amino acids, or some other chemical modifications, many compounds have been designed and synthesized as NPFF agonists and antagonists [3,8]. However, untill now, no selective antagonist for NPFF has been fully characterized. The NPFF antagonist should possess a high affinity for NPFF receptors and functionally antagonize the bio-effects induced by NPFF. Because NPFF is considered to be a morphine withdrawal-
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enhancing substance, the aim of the present study was to investigate the chronic effects of NPFF, PFRFamide (putative NPFF agonist), dansyl-PQRamide (putative NPFF antagonist), and PFR(Tic)amide (putative NPFF antagonist) on naloxone-precipitated morphine withdrawal syndromes. The current results indicate that NPFF agonists potentiate the naloxone-induced morphine withdrawal syndromes and, on the contrary, synthetic NPFF antagonists suppress the syndromes. Also, the application of morphine withdrawal model [5 mg/kg, intraperitoneally (i.p.), twice per day (b.i.d.) for 5 days; followed by naloxone 4 mg/kg, i.p. challenge] would be helpful for a structural and functional characterization of NPFF analogs.
2. Materials and methods 2.1. Peptide synthesis and purification NPFF, PFRFamide, dansyl-PQRamide, and PFR(Tic)amide were all synthesized manually on solid phase. In a stepwise manner at the 0.5-mmol scale, the Fmoc strategy was used (Atherton and Sheppard, 1989). Fmoc amino acids were coupled to Rink Amide MBHA resin (Novabiochem, Switzerland) by using 2-(1H-Benzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate as the coupling reagent to give C-terminally amidated peptides. For dansylPQRamide, the N-terminal dansylation was performed as the last coupling. Dansyl chloride (Sigma) in dimethylformamide was used for dansylation of proline on solid phase. The dansylation was repeated twice to assure that the N termini were completely dansylated. As for PFR(Tic)amide, N-1-Fmoc-L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (Fmoc-Tic) was used for the first coupling. Following the deprotection of Fmoc-Tic, the coupling with arginine was processed twice to assure the complete coupling. After the synthesis, the peptides were cleaved from the resin with a 97.5% trifluoroacetic acid solution for 1 h, and the crude peptides were desalted through a Sephadex G-10 column (Pharmacia, Uppsala, Sweden). Purification by analytical reverse-phase high-performance liquid chromatography (HPLC) was performed on a Beckman HPLC System (System Gold). The peptides were analysed by using the following conditions: column, Beckman Ultrasphere ODS C-18 (5 m, 0.46 ⫻ 25 cm; Beckman, Fullerton, CA, USA); eluent A, 0.1% (v/v) trifluoroacetic acid in H2O; eluent B, 0.1% (v/v) trifluoroacetic acid in acetonitrile; gradient B, 0% over 2 min, 0 – 80% over 32 min; flow rate 1 ml/min; absorbance 216 nm; fluorescence (Gilson 121, Middleton, WI, USA) detection for dansyl-PQRamide. Only peptides that showed a single peak on the HPLC profile were used for the following study. Fig. 1 shows the chemical structures of synthetic PFRFamide, PFR(Tic)amide, and dansyl-PQRamide.
2.2. Animals Male Sprague-Dawley rats, 200 –330 g (Academia Sinica, Taipei, Taiwan) were maintained five per cage with food and water available ad libitum and on a 12-h light/dark cycle. They were habituated to the animal room for at least 5 days before the experiments. Except for the experiment with dansyl-PQRamide, each rat was cannulated stereotaxically in the lateral ventricle under ketamine (50 mg/kg, i.p.) anesthesia and secured with stainless steel screws and dental cement. The i.c.v. implantation was carried out with a 23gauge guide cannula positioned in the lateral ventricle at the following coordinates: 0.8 mm posterior to bregma, 1.4 mm lateral from the midline, and 3.7 mm ventral from the dura [11]. Animals were allowed 7 days for recovery. Cannula placements subsequently were confirmed by methylene blue dye injection and histologic examination. 2.3. Chronic treatments with NPFF, PFRFamide, PFR(Tic)amide, and dansyl-PQRamide All rats were chronically treated with morphine (5 mg/ kg, b.i.d.) via i.p. injection for 5 consecutive days. For each experiment, rats were randomly divided into control and chronic peptide-treated groups. For the experiment with NPFF, the peptide was dissolved in saline at a concentration of 4 mg/ml. Following the systemic injection of morphine, 5 l (20 g of NPFF) was injected [intracerebroventricularly (i.c.v.), b.i.d.] over 1 min via a microdialysis pump (CMA 120, Stockholm, Sweden). The injection of NPFF also was performed for 5 consecutive days. Control rats received the same volume of saline injection (i.c.v., b.i.d.) in the same manner. For PFRFamide and PFR(Tic)amide, 5 l of 20 g of each peptide was individually injected. The corresponding controls were prepared by using the same method. A preliminary experiment indicated that the coadministration of a peptidase inhibitor, bestatin, did not prevent or induce any notable effect; thus, all of the peptides injected (i.c.v.) were in their free form. For the dansyl-PQRamide treated group, dansyl-PQRamide was dissolved in 20% dimethylsulfoxide at a concentration of 13 mg/ml. Following the injection of morphine, 13 mg/kg dansyl-PQRamide was injected (i.p., b.i.d.). The preparation was repeated for 5 consecutive days. The rats from the control group received a vehicle (0.3 ml of 20% dimethylsulfoxide) injection (i.p., b.i.d.). 2.4. Measurement of morphine withdrawal syndromes On the day of the behavioral test, all rats were injected with a high dose of morphine (20 mg/kg, i.p.). Four hours later, the rats received the naloxone (Sigma, St. Louis, MO, USA; 4 mg/kg, i.p.) injection to induce the morphine withdrawal syndromes. Twelve distinct behavioral signs (chewing, sniffing, grooming, wet dog shakes, stretching, yawning, rearing,
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Fig. 1. The chemical structures of (1) PFRFamide, (2) PFR(Tic)amide, and (3) dansyl-PQRamide.
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jumping, teeth grounding, ptosis, diarrhea, and piloerection) were observed and scored during a 30-min period after the naloxone injection. The reactions of each animal were evaluated by an independent observer who did not have any prior knowledge of the nature of the treatment received. Chewing, sniffing, and grooming were all scored from 1.0 to 3.0 according to their frequency (1.0: occasional; 2.0: frequent; 3.0: continuous). The occurrences of wet dog shakes, stretching, yawning, rearing, and jumping were separately recorded. One to five occurrences of the above behavioral sign was scored as 1.0; 6 to 10 occurrences was scored as 2.0; and over 10 occurrences was scored as 3.0. For the other syndromes (teeth grounding, ptosis, diarrhea, and piloerection), 3.0 or 0 was given as the score to the rat for either showing or not showing the sign, respectively. Total scores were calculated by adding together the individual score for each withdrawal sign. 2.5. Statistics Multiple t-tests were used to examine the significance of the change of each syndrome. To compare with the control value, each total score for the peptide-treated group also was converted into the percentage of the control value with mean control value defined as 100%. The change for each peptide-treated group was compared with an unpaired Student’s t-test. A P value ⬍ 0.05 was considered significant.
3. Results 3.1. The effect of chronic NPFF administration on morphine withdrawal syndromes As illustrated in Table 1, the intensity of chewing, sniffing, grooming, wet dog shakes, rearing, and diarrhea displayed a higher score in the chronic NPFF-treated groups than the controls. Among these syndromes, sniffing, grooming, and diarrhea were statistically significant (Table 1). The total score for the withdrawal signs in the NPFF-treated groups was 17.20 ⫾ 0.97 (n ⫽ 5) vs. 12.40 ⫾ 1.44 (n ⫽ 5) in the control group, indicating a 38.71% increase (Fig. 2).
Table 1 Individual scores of the naloxone-precipitated morphine withdrawal syndromes in control (left column) and NPFF-treated (right column) groups Withdrawal sign
Control (score ⫾ SEM, n ⫽ 5)
NPFF-treated group (score ⫾ SEM, n ⫽ 5)
Chewing Sniffing Grooming Wet dog shakes Stretching Yawning Rearing Jumping Teeth grinding Ptosis Diarrhea Piloerection Total score
1.20 ⫾ 0.20 (5/5) 1.40 ⫾ 0.25 (5/5) 1.20 ⫾ 0.37 (4/5) 0.60 ⫾ 0.25 (3/5) 0 0.60 ⫾ 0.25 (3/5) 0.80 ⫾ 0.20 (4/5) 0 2.40 ⫾ 0.60 (4/5) 3.00 ⫾ 0.00 (5/5) 0 1.20 ⫾ 0.74 (2/5) 12.40 ⴞ 1.44
2.00 ⫾ 0.32 (5/5) 2.00 ⫾ 0.00 (5/5)* 2.60 ⫾ 0.40 (5/5)* 1.20 ⫾ 0.49 (4/5) 0 0 1.60 ⫾ 0.40 (5/5) 0 1.80 ⫾ 0.74 (3/5) 2.40 ⫾ 0.60 (4/5) 2.40 ⫾ 0.60 (4/5)** 1.20 ⫾ 0.74 (2/5) 17.20 ⴞ 0.97
The mean total scores are shown at the bottom of the table. The numbers within the bracket (a/b; a, the number of animals displaying the syndrome; b, the total number of animals) indicate the proportion of the animals showing the syndrome (* P ⬍ 0.05; ** P ⬍ 0.01).
3.3. The effect of chronic dansyl-PQRamide administration on morphine withdrawal syndromes Compared with the controls, the intensity of sniffing, grooming, wet dog shakes, stretching, yawning, rearing, teeth grounding, and ptosis was changed to a lower level in the chronic dansyl-PQRamide-treated groups (Table 3). Only the decrease of grooming has been found to be statistically significant (Table 3, P ⬍ 0.05). The total score for the withdrawal signs in the dansyl-PQRamide-treated groups was 7.00 ⫾ 1.23 (n ⫽ 7) vs. 11.00 ⫾ 1.48 (n ⫽ 7) in the control group, indicating a 36.36% decrease (Fig. 2, P ⫽ 0.0602).
3.2. The effect of chronic PFRFamide administration on morphine withdrawal syndromes As illustrated in Table 2, the intensity of chewing, sniffing, grooming, wet dog shakes, stretching, yawning, and rearing showed a higher score than those in the the control groups. Although none of these syndromes has reached to a statistical significance, the total score for the withdrawal signs in the PFRFamide-treated groups was 16.40 ⫾ 1.47 (n ⫽ 5) vs. 15.80 ⫾ 2.27 (n ⫽ 5) in the control group (Table 2).
Fig. 2. Chronic effects of NPFF, PFRFamide, dansyl-PQRamide, and PFR(Tic)amide on naloxone-precipitated morphine withdrawal syndromes. Each gray bar represents the mean total score of the peptide-treated group that was converted to the percentage of the corresponding control value with the mean control value defined as 100% (white bar). An unpaired Student’s t-test was used to examine the significance (* P ⬍ 0.05).
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Table 2 Individual scores of the naloxone-precipitated morphine withdrawal syndromes in control (left column) and PFRFamide-treated (right column) groups
Table 4 Individual scores of the naloxone-precipitated morphine withdrawal syndromes in control (left column) and PFR(Tic)amide-treated (right column) groups
Withdrawal sign
Control (score ⫾ SEM, n ⫽ 5)
PFRFa-treated group (score ⫾ SEM, n ⫽ 5)
Withdrawal sign
Control (score ⫾ SEM, n ⫽ 5)
Chewing Sniffing Grooming Wet dog shakes Stretching Yawning Rearing Jumping Teeth grinding Ptosis Diarrhea Piloerection Total score
1.60 ⫾ 0.40 (5/5) 1.00 ⫾ 0.00 (5/5) 1.60 ⫾ 0.25 (5/5) 1.20 ⫾ 0.58 (3/5) 0 0 2.40 ⫾ 0.40 (5/5) 0.20 ⫾ 0.20 (1/5) 1.20 ⫾ 0.74 (2/5) 1.80 ⫾ 0.74 (3/5) 1.80 ⫾ 0.74 (3/5) 3.00 ⫾ 0.00 (5/5) 15.80 ⴞ 2.27
2.60 ⫾ 0.25 (5/5) 1.40 ⫾ 0.60 (3/5) 2.40 ⫾ 0.40 (5/5) 2.20 ⫾ 0.49 (5/5) 0.40 ⫾ 0.25 (2/5) 0.40 ⫾ 0.25 (2/5) 2.80 ⫾ 0.20 (5/5) 0 1.20 ⫾ 0.74 (2/5) 1.20 ⫾ 0.74 (2/5) 1.80 ⫾ 0.74 (3/5) 0 16.40 ⴞ 1.47
PFR(Tic)a-treated group (score ⫾ SEM, n ⫽ 6)
Chewing Sniffing Grooming Wet dog shakes Stretching Yawning Rearing Jumping Teeth grinding Ptosis Diarrhea Piloerection Total score
1.60 ⫾ 0.25 (5/5) 1.60 ⫾ 0.25 (5/5) 1.80 ⫾ 0.37 (5/5) 1.80 ⫾ 0.49 (5/5) 0.20 ⫾ 0.20 (1/5) 0.20 ⫾ 0.20 (1/5) 2.40 ⫾ 0.40 (5/5) 0 1.20 ⫾ 0.74 (2/5) 1.20 ⫾ 0.74 (2/5) 1.80 ⫾ 0.74 (3/5) 2.40 ⫾ 0.60 (4/5) 16.20 ⴞ 1.28
1.17 ⫾ 0.17 (6/6) 0.33 ⫾ 0.21 (2/6)** 1.50 ⫾ 0.34 (6/6) 1.33 ⫾ 0.42 (5/6) 0.17 ⫾ 0.17 (1/6) 0.50 ⫾ 0.22 (3/6) 2.00 ⫾ 0.37 (6/6) 0 2.00 ⫾ 0.63 (4/6) 2.00 ⫾ 0.63 (4/6) 1.50 ⫾ 0.67 (3/6) 2.50 ⫾ 0.50 (5/6) 15.00 ⴞ 0.68
The mean total scores are shown at the bottom of the table. The numbers within the bracket (a/b; a, the number of animals displaying the syndrome; b, the total number of animals) indicate the proportion of the animals showing the syndrome.
3.4. The effect of chronic PFR(Tic)amide administration on morphine withdrawal syndromes Similar to dansyl-PQRamide, the intensity of chewing, sniffing, grooming, wet dog shakes, stretching, rearing, and diarrhea in the chronic PFR(Tic)amide-treated groups was lower when compared with the controls (Table 4). Only the decrease of sniffing has been found to be statistically significant (Table 4, P ⬍ 0.01). The total score for the withdrawal signs in the PFR(Tic)amide-treated groups was Table 3 Individual scores of the naloxone-precipitated morphine withdrawal syndromes in control (left column) and dansyl-PQRamide-treated (right column) groups Withdrawal sign
Control (score ⫾ SEM, n ⫽ 7)
Dansyl-PQRa-treated group (score ⫾ SEM, n ⫽ 7)
Chewing Sniffing Grooming Wet dog shakes Stretching Yawning Rearing Jumping Teeth grinding Ptosis Diarrhea Piloerection Total score
0.43 ⫾ 0.20 (3/7) 0.57 ⫾ 0.20 (4/7) 1.86 ⫾ 0.34 (7/7) 1.00 ⫾ 0.22 (6/7) 0.29 ⫾ 0.18 (2/7) 0.29 ⫾ 0.18 (2/7) 1.43 ⫾ 0.57 (4/7) 0 0.86 ⫾ 0.55 (2/7) 1.29 ⫾ 0.61 (3/7) 0.86 ⫾ 0.55 (2/7) 2.14 ⫾ 0.55 (5/7) 11.00 ⴞ 1.48
0.43 ⫾ 0.20 (3/7) 0.29 ⫾ 0.18 (2/7) 0.71 ⫾ 0.18 (5/7)* 0.29 ⫾ 0.29 (1/7) 0 0 0.43 ⫾ 0.20 (3/7) 0.14 ⫾ 0.14 (1/7) 0 0.43 ⫾ 0.43 (1/7) 1.29 ⫾ 0.61 (3/7) 3.00 ⫾ 0.00 (7/7) 7.00 ⴞ 1.23
The mean total scores are shown at the bottom of the table. The numbers within the bracket (a/b; a, the number of animals displaying the syndrome; b, the total number of animals) indicate the proportion of the animals showing the syndrome (* P ⬍ 0.05).
The mean total scores are shown at the bottom of the table. The numbers within the bracket (a/b; a, the number of animals displaying the syndrome; b, the total number of animals) indicate the proportion of the animals showing the syndrome (** P ⬍ 0.01).
15.00 ⫾ 0.68 (n ⫽ 6) vs. 16.20 ⫾ 1.28 (n ⫽ 5) in the control group, indicating a mild attenuation (7.41%) (Fig. 2).
4. Discussion The present study indicates that chronic administration of NPFF and its synthetic analogs significantly modulate the naloxone-precipitated morphine withdrawal syndromes. Given the evidence that acute NPFF injection was able to precipitate an opiate withdrawal syndrome in morphinedependent rats [6], our results clearly showed that continuous activation of NPFF receptors also would enhance the intensity of morphine withdrawal syndromes. Of interest, acute NPFF-induced withdrawal signs were reported to occur only when the drug was applied into the third ventricle but not into the lateral ventricle or injected intrathecally [14]. However, our results that chronic lateral ventricular injection of NPFF potentiated naloxone-precipitated morphine withdrawal syndromes suggest a time-dependent diffusion effect. Otherwise, it is possible that the chronic lateral ventricular injections enable NPFF to induce a more profound effect or indirectly modulate opiate dependence via other neurotransmitter systems. Although there was no immediate withdrawal sign(s) observed after each lateral ventricular injection, our results still highlight the importance of the NPFF system in the development of opiate dependence. The putative NPFF agonist PFRFamide first was predicted to possess a high affinity for NPFF receptors in a study of structure-activity relationship [10]. Despite the fact that the affinity of PFRFamide for NPFF receptors has not
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yet been determined in ligand-receptor binding assays, it has been reported to potentiate the spinal reflex in the isolated rat spinal cord preparation [4]. In that electrophysiological study, PFRFamide displayed a higher potency than that of NPFF for potentiation of spinal reflexes. If the increase in the magnitude of the reflex is through the activation of NPFF receptors, PFRFamide possibly would act as a full or partial NPFF agonist. In the present study, chronic treatment with PFRFamide appears to induce more stereotyped behaviors (chewing, sniffing, grooming, and rearing), and wet dog shakes, stretching, yawning in PFRFamide-treated rats than the saline controls. The results seem to implicate selective withdrawal signs that were turned on by PFRFamide. We also investigated the putative NPFF antagonist dansyl-PQRamide. Dansyl-PQRamide has been reported to be able to reduce the naloxone-precipitated morphine withdrawal syndromes when it was injected subcutaneously (s.c.) [9]. Its acute effect on morphine withdrawal syndromes has been well studied by Malin et al. [9]. In this study, we further characterized the chronic effect (i.p. injection) of dansyl-PQRamide and found that the result is compatible with the acute administration. Although dansylPQRamide might cross the blood– brain barrier and attenuate morphine withdrawal syndromes, the hydrophobicity makes it difficult to dissolve in water and produces a lot of problems for purification and for its high concentration in in vivo practical application [9]. However, this peptide still presents a typical structure for the design of NPFF analogs as antagonists. Another important property of dansyl-PQRamide is its high fluorescence due to its dansylated Nterminus. Thus, it could be used as a sensitive fluorescent probe in immunocytochemical or receptor binding studies. Therefore, PFR(Tic)amide, derived from PFRFamide, was designed and speculated to be an NPFF antagonist. L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic Because acid (Tic) has been successfully used to design and synthesize novel ␦-selective opioid antagonists and bradykinin antagonists [15,17,18], we attempted to convert the NPFF agonist PFRFamide into an antagonist by replacing Phe with Tic at its C terminus. It is expected the Tic residue in PFR(Tic)amide would be able to reduce the activation of NPFF receptors while the other three amino acids might maintain its high binding affinity. As we expected, the administration of PFR(Tic)amide to morphine-dependent rats produced an attenuation of several morphine withdrawal syndromes (chewing, sniffing, grooming, wet dog shakes, stretching, rearing, and diarrhea). Although the attenuation does not reach a statistical significance (92.59% of the control value), these results may still suggest that PFR(Tic)amide could be an effective NPFF antagonist. To gain the full scope of its pharmacological profile, this newly designed peptide requires further characterization. However, the possible ability of PFR(Tic)amide to reduce morphine withdrawal syndromes provides us a great therapeutic potential. In addition, the synthetic amino acid (Tic) could
make the peptide more hydrophobic, facilitating its crossing the blood– brain barrier and making it resistant to enzyme degradation. In summary, chronic treatment with NPFF significantly potentiated the naloxone-precipitated morphine withdrawal syndromes, whereas its putative antagonist, dansyl-PQRamide, showed an opposite effect. It is important to note that, to our knowledge, PFR(Tic)amide is the first design that was tested as an effective NPFF antagonist. Although its affinity for NPFF receptors has not yet been determined, PFR(Tic)amide also has displayed an apparent anti-NPFF cardiovascular effect in rats (in preparation). Our results suggested that PFR(Tic)amide or its related Tic-containing compounds may be useful for new drug development based on NPFF structure. In addition, our results should be helpful for further elucidation of the functional significance of the NPFF system in the mammalian central nervous system.
References [1] Allard M, Geoffre S, Legendre P, Vincent JD, Simonnet G. Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study. Brain Res 1989;500:169 –76. [2] Atherton E, Sheppard RC. Solid Phase Peptide Synthesis, a Practical Approach. Oxford, UK: IRL Press at Oxford University Press, 1989. [3] Gicquel S, Mazarguil H, Allard M, Simonnet G, Zajac JM. Analogues of F8Famide resistant to degradation, with high affinity and in vivo effects. Eur J Pharmacol 1992;222:61–7. [4] Huang EY-K, Bagust J, Sharma RP, Walker RJ. The effect of FMRFamide-like peptides on electrical activity in isolated mammalian spinal cord. Neurosci Res 1998;30:295–301. [5] Lake JR, Hammond MV, Shaddox RC, Hunsicker LM, Yang H.-YT, Malin DH. IgG from neuropeptide FF antiserum reverses morphine tolerance in the rat. Neurosci Lett 1991;132:29 –32. [6] Malin DH, Lake JR, Fowler DE, Hammond MV, Brown SL, Leyva JE, Prasco PE, Dougherty TM. FMRF-NH2-like mammalian peptide precipitates opiate-withdrawal syndrome in the rat. Peptides 1990;11: 277– 80. [7] Malin DH, Lake JR, Hammond MV, Fowler DE, Rogillio RB, Brown SL, Sims JL, Leecraft BM, Yang H-YT. FMRF-NH2-like mammalian octapeptide: possible role in opiate dependence and abstinence. Peptides 1990;11:969 –72. [8] Malin DH, Lake JR, Leyva JE, Hammond MV, Rogillio RB, Arcangeli KR, Ludgate K, Moore GM, Payza K. Analog of neuropeptide FF attenuates morphine abstinence syndrome. Peptides 1991;12:1011– 4. [9] Malin DH, Lake JR, Smith DA, Jones JA, Morel J, Claunch AE, Stevens PA, Payza K, Ho KK, Liu J, Ham I, Burgess K. Subcutaneous injection of an analog of neuropeptide FF prevents naloxoneprecipitated morphine abstinence syndrome. Drug Alcohol Depend 1995;40:37– 42. [10] Payza K, Akar CA, Yang H-YT. Neuropeptide FF receptors: structure-activity relationship and effect of morphine. J Pharmacol Exp Ther 1993;267:88 –94. [11] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. New York: Academic Press, 1986. [12] Price DA, Greenberg MJ. Structure of a molluscan cardioexcitatory neuropeptide. Science 1977;197:670 –1. [13] Rothman RB. A review of the role of anti-opioid peptides in morphine tolerance and dependence. Synapse 1992;12:129 –38. [14] Rothman RB, Brady LS, Xu H, Long JB. Chronic intracerebroventricular infusion of the antiopioid peptide, Phe-Leu-Phe-Gln-Pro-Gln-
P. P.-C. Tan et al. / Peptides 20 (1999) 1211–1217 Arg-Phe-NH2 (NPFF), downregulates mu opioid binding sites in rat brain. Peptides 1993;14:1271–7. [15] Schiller PW, Nguyen TM-D, Weltrowska G, Wilkes BC, Marsden BJ, Lemieux C, Chung NN. Differential stereochemical requirements of vs. ␦ opioid receptors for ligand binding and signal transduction: development of a class of potent and highly dpselective peptide antagonists. Proc Natl Acad Sci USA 1992;89: 11871–5. [16] Stinus L, Allard M, Gold L, Simonnet G. Change in CNS neuropeptide FF-like material, pain sensitivity, and opiate dependence following chronic morphine treatment. Peptides 1995;16:1235– 41.
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[17] Tancredi T, Salvadori S, Amodeo P, Picone D, Lazarus LH, Bryant SD, Guerrini R, Marzola G, Temussi PA. Conversion of enkephalin and dermorphin into ␦-selective opioid antagonists by single-residue substitution. Eur J Biochem 1994;224:241–7. [18] Thurieau C, Feletou M, Hennig P, Raimbaud E, Canet E, Fauchere J-L. Design and synthesis of new linear and cyclic bradykinin antagonists. J Med Chem 1996;39:2095–101. [19] Yang H-YT, Fratta W, Majane EA, Costa E. Isolation sequencing, synthesis and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 1985;82:7757– 61.