- Open Access
PK20, a new opioid-neurotensin hybrid peptide that exhibits central and peripheral antinociceptive effects
© Kleczkowska et al; licensee BioMed Central Ltd. 2010
Received: 5 August 2010
Accepted: 6 December 2010
Published: 6 December 2010
The clinical treatment of various types of pain relies upon the use of opioid analgesics. However most of them produce, in addition to the analgesic effect, several side effects such as the development of dependence and addiction as well as sedation, dysphoria, and constipation. One solution to these problems are chimeric compounds in which the opioid pharmacophore is hybridized with another type of compound to incease antinociceptive effects. Neurotensin-induced antinociception is not mediated through the opioid system. Therefore, hybridizing neurotensin with opioid elements may result in a potent synergistic antinociceptor.
Using the known structure-activity relationships of neurotensin we have synthesized a new chimeric opioid-neurotensin compound PK20 which is characterized by a very strong antinociceptive potency. The observation that the opioid antagonist naltrexone did not completely reverse the antinociceptive effect, indicates the partial involvement of the nonopioid component in PK20 in the produced analgesia.
The opioid-neurotensin hybrid analogue PK20, in which opioid and neurotensin pharmacophores overlap partially, expresses high antinociceptive tail-flick effects after central as well as peripheral applications.
The opioids system is the major endogenous pathway that modulates pain signal transmission and perception. Therefore most pain medicines, available for the treatment of severe pain, express affinity for the opioid receptors. The search for selective opioid compounds is still a main avenue in the development of new analgesics. However, pain is mediated by various complementary and/or alternative pathways that participate in the creation of a final level of pain perception. Therefore, we have proposed a new, multitarget approach in searching for new analgesics . According to this concept, the new type of analgesics should interact with a broad spectrum of pathways involved in pain transmission and modulation. The use of a peptidomimetic in the chemical structure of the drug allows to modulate the permeability of the active substance and, in consequence, creates "site specificity of action". This concept has been positively proven with the creation of multitarget molecules interacting with broad spectrum of opioid receptors  or opioid and NK1-, [3, 4] CCK-, or neurotensin receptors .
The tridecapeptide neurotensin, (NT, p-Glu1-Leu2-Tyr3-Glu4-Asn5-Lys6-Pro7-Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) [7–10] exerts antinociceptive activity, and is therefore considered as a pain modulating factor . Microinjection experiments have provided evidence that NT can modulate pain transmission in several brain regions and pathways that are involved in the central integration of pain responses, including the central amygdale, the hypothalamic medial preoptic nucleus (MPO), certain thalamic nuclei, the periaqueductal gray (PAG), and the rostroventral medulla (RVM) [11, 12]. Interestingly, neurotensin has bipolar (facilitatory and inhibitory) effects on pain modulation, which depend on the injected doses. Facilitation predominates at low (picomolar) doses of NT injected into the RVM, whereas higher doses (nanomolar) produce antinociception . Until now, the NTS1 and NTS2 receptor subtypes, which belong to the class of G protein-coupled receptors, appear to be required for different aspects of neurotensin-induced analgesia [11, 14, 15].
Structure-activity relationship studies with a number of neurotensin analogs and partial sequences have established that the C-terminal hexapeptide of NT contains all the structural requirements for receptor binding and activation.
Measurement of antinociception by intrathecally administered hybrid PK20 into rats
PK20, the new opioid-neurotensin hybrid peptide was injected intrathecally into male Wistar rats (weighing 225-250 g) by way of implanted cannulae. Animals were housed separately and given full access to food and water. The technique of intrathecal drug administration, originally described by Yaksh & Rudy , was used to test the spinal action of the investigated opioid-neurotensin hybrid peptide, PK20. The analgesic activity of PK20 was evaluated by using the tail-flick test (Model 33 Tail Flick Analgesia Meter, USA), in which the role of the nociceptor agent was fulfilled by a light beam . The measurement parameters (beam temperature, the time of rat's tail exposition on the irrigation of laser beam) were set properly in order to avoid the burn of tails.
The effect of action of PK20 was measured at different doses per rat and within a time frame of 120 minutes (at 5, 15, 30, 60 and 120 minutes after injection). For each time period three measurements were carried out.
Control responses for each rat in the tail-flick test, and for each mouse in hot water tail-flick test, were determined before the injection. Following an intrathecal injection of saline, morphine, and naltrexone, the response to PK20 was determined. Regarding naltrexone, PK20 was i.t. administered after 10 min interval after the injection of the opioid blocker.
where pdr - post drug response, br - baseline response, co - cut-off value. The cut-off was 7 s for all experiments (n equals 6).
Measurement of antinociception by intravenously administered hybrid PK20 into mice
Measurements of antinociception were also carried out by intravenous administration of PK20 into SWISS WEBSTER male white mice (weighing 35 - 40 g) and using the hot water tail-flick test. The hybrid compound was injected at a dose of 10 and 4 mg/kg. The effect of PK20 was measured within a period of 120 minutes (at 5, 15, 30, 60 and 120 minutes after injection). For each time period three measurements were carried out.
Hot water tail-flick measurements were taken in water warmed to a temperature of 55.5°C ± 1°C. Latency was measured as the time that the mice needed to remove its tail after it was placed into hot water.
The hot water tail-flick test was scored as % MPE, as was mentioned above. However in this case the applied cut-off was 10 s.
All reported data represent the mean % MPE ± SEM. Significant difference at individual time points between two groups was determined by Student's T-test with use of Statistica® 7.1 software (StatSoft, Tulsa, USA) with *p < 0.05 and **p < 0.001 being considered significant.
Antinociceptive effects of intrathecally administered PK20 hybrid peptide in tail-flick test
To evaluate the possible analgesic effect of the neurotensin part of PK20, a μ-opioid receptor blocker, naltrexone, which antagonizes the antinociceptive effect, induced by the opioid subunit in PK20, was used.
It was observed that, although naltrexone significantly reduced the analgesic effect induced by PK20, the growing profile of PK20's antinociceptive action is still preserved.
Effects of PK20 hybrid peptide in hot water tail-flick test after intravenous application in mice
To evaluate PK20's ability to act after peripheral administration, and thus to cross the blood-brain barrier (BBB), we examined the analgesic effect induced by intravenous application of the peptide at two doses of 4 and 10 mg/kg, respectively.
Our in vivo studies have shown that PK20 treatment results in long-standing time-dependent antinociception when administered centrally as well as peripherally. This novel opioid-neurotensin hybrid peptide has a significantly intensified analgesic effect, when compared to saline and morphine. The improved analgesia mediated by this peptide suggests a possible plasma stability and implies a delayed enzymatic degradation (data not published). Intrathecal injection of PK20 at a dose of 0.02 nmol/rat exerts a similar analgesic action to that observed for morphine at 3 nmol/rat, indicating a very high antinociceptive potency of the investigated peptide. Interestingly, by increasing the concentration of the administered compound (range between 0.1-0.5 nmol/rat) the antinociceptive effect at 0.5 nmol/rat starts from a lower value than at 0.1 nmol/rat during the first 15 minutes after injection, which might be interpreted as a short delay in response to nociceptive stimuli.
Naltrexone, injected 10 min before the compound, effectively attenuated the antinociceptive action produced by the hybrid peptide. However, a slight and still time-dependent growing profile of analgesic effect of PK20 is still preserved. These findings indicate that naltrexone only partially inhibited the antinociceptive action of PK20, suggesting that also the neurotensin fragment is involved in analgesia. The analgesic response induced by PK20 is mediated not only through activation of the opioid pathway, but also through action at neurotensin receptors.
Our study also indicates the ability of PK20 to cross the highly selective blood-brain barrier, which was examined by its intravenous administration into mice (hot water tail-flick test). Since only a few neurotensin analogs were reported to cross the BBB, like a compound from the Eisai group  or NT66L and NT69L [19, 28], PK20 seems to be a very interesting novel pain relieving drug.
Neurotensin as well as opioids exert analgesia. Opioids drugs block pain signals by interacting especially with mu-opioid receptors, whereas NT or its analogs act independently of the opioid pathways. Therefore, by combining these two elements, the antinociceptive effect might be obtained either by the opioid or neurotensin part alone, or by synergy of two interacting parts, thus acting more efficiently than in case of separate administration of each of them.
Having in mind the fact that chronic administration of opiates generally produce tolerance [29, 30] and dependence, which are highly undesirable effects, the creation of novel compounds such as this hybrid may have an influence on the reduction of these side-effects and gives a hope to obtain new drugs with the ability to sufficiently relief pain states.
The comparative studies on tolerance development after multiple application of PK20 and morphine are in progress.
The opioid-neurotensin hybrid analogue PK20, in which opioid and neurotensin pharmacophores partially overlap, expresses high antinociceptive tail-flick effects after central as well as peripheral application.
Supported by European Grant "Normolife", LSHC-CT-2006-037733 and by Grant G.000.08 of the Fund for Scientific Research-Flanders(FWO-Vlaanderen)
- Lipkowski AW, Misicka A, Hruby VJ, Carr DB: Opioid peptide analogues: Reconsideration as a potentially new generation of analgesics. Polish J Chem 1994, 68: 907–912.Google Scholar
- Kosson D, Maszczynska Bonney I, Carr DB, Mayzner-Zawadzka E, Lipkowski AW: Antinociceptive properties of biphalin after intrathecal application in rats: a reevaluation. Pharmacol Rep 2005, 57: 545–549.PubMedGoogle Scholar
- Maszczynska Bonney I, Foran SE, Marchand JE, Lipkowski AW, Carr DB: Spinal antinociceptive effects of AA501, a novel chimeric peptide with opioid receptor agonist and tachykinin receptor antagonist moieties. Eur J Pharmacol 2004, 488: 91–99. 10.1016/j.ejphar.2004.02.023View ArticleGoogle Scholar
- Foran SE, Carr DB, Lipkowski AW, Maszczyńska I, Marchand JE, Misicka A, Beinborn M, Kopin A, Kream RM: Substance P - opioid chimeric peptide as a novel non-tolerance forming analgesic. Proc Natl Acad Sci USA 2000, 97: 7621–7626. 10.1073/pnas.130181897PubMed CentralPubMedView ArticleGoogle Scholar
- Lee YS, Agnes RS, Davis P, Ma SW, Badghisi H, Lai J, Porreca F, Hruby VJ: Partial retro-inverso, retro, and inverso modifications of hydrazide linked bifunctional peptides for opioid and cholecystokinin (CCK) receptors. J Med Chem 2007, 50: 165–168. 10.1021/jm061268pPubMed CentralPubMedView ArticleGoogle Scholar
- Yano K, Kimura S, Imanishi Y: Simultaneous activation of two different receptor systems by enkephalin/neurotensin conjugates having spacer chins of various lengths. Eur J Pharm Sci 1998, 7: 41–48. 10.1016/S0928-0987(98)00002-5PubMedView ArticleGoogle Scholar
- Binder EB, Kinkead B, Owens MJ, Nemeroff CB: Neurotensin and dopamine interactions. Pharmacol Rev 2001, 53: 453–486.PubMedGoogle Scholar
- Carraway R, Leeman SE: The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem 1973, 248: 6854–6861.PubMedGoogle Scholar
- Carraway R, Leeman SE: The amino acid sequence of a hypothalamic peptide, neurotensin. J Biol Chem 1975, 250: 1907–1911.PubMedGoogle Scholar
- Hermans E, Malateaux JM: Mechanism of regulation of neurotensin receptors. Pharmacol Ther 1998, 79: 89–104. 10.1016/S0163-7258(98)00009-6PubMedView ArticleGoogle Scholar
- Dobner PR: Neurotensin and pain modulation. Peptides 2006, 27: 2405–2414. 10.1016/j.peptides.2006.04.025PubMedView ArticleGoogle Scholar
- Gui X, Carraway RE, Dobner PR: Endogenous neurotensin facilitates visceral nociception and is required for stress-induced antinociception in mice and rats. Neurosci 2004, 126: 1023–1032. 10.1016/j.neuroscience.2004.04.034View ArticleGoogle Scholar
- Smith DJ, Hawranko AA, Monroe PJ, Gully D, Urban MO, Craig CR, Smith JP, Smith DL: Dose-dependent pain-facilitatory and -inhibitory actions of neurotensin are revealed by SR 48692, a nonpeptide neurotensin antagonist: influence on the antinociceptive effect of morphine. J Pharmacol Exper Ther 1997, 282: 899–908.Google Scholar
- Buhler AV, Proudfit HK, Gebhart GF: Neurotensin-produced antinociception in the rostral ventromedial medulla is partially mediated by spinal cord norepinephrine. Pain 2008, 135: 280–290. 10.1016/j.pain.2007.06.010PubMed CentralPubMedView ArticleGoogle Scholar
- Horvath G, Kekesi G: Interaction of endogenous ligands mediating antinociception. Br Res Rev 2006, 52: 69–92. 10.1016/j.brainresrev.2006.01.001View ArticleGoogle Scholar
- Clineschmidt BV, McGuffin JC, Bunting PB: Neurotensin: antinocisponsive action in rodents. Eur J Pharmacol 1979, 54: 129–139. 10.1016/0014-2999(79)90415-1PubMedView ArticleGoogle Scholar
- Holmes BB, Rady JJ, Smith DJ, Fujimoto JM: Supraspinal neurotensin-induced antianalgesia in mice is mediated by spinal cholecystokinin. Jpn J Pharmacol 1999, 79: 141–149. 10.1254/jjp.79.141PubMedView ArticleGoogle Scholar
- Tyler-McMahon B, Boules M, Richelson E: Neurotensin: peptide for the next millennium. Reg Peptides 2000, 93: 125–136. 10.1016/S0167-0115(00)00183-XView ArticleGoogle Scholar
- Boules M, Frederickson P, Richelson E: Bioactive analogs of neurotensin: focus on CNS effects. Peptides 2006, 27: 2523–2533. 10.1016/j.peptides.2005.12.018PubMedView ArticleGoogle Scholar
- Bruehlmeier M, Garcia-Garayoa E, Blanc A, Holzer B, Gergely S, Tourwé D, Schubiger PA, Bläuenstein P: Stabilization of neurotensin analogues: effect on peptide catabolism, biodistribution and tumor binding. Nucl Med Biol 2002, 29: 321–327. 10.1016/S0969-8051(01)00304-3PubMedView ArticleGoogle Scholar
- Kokko KP , Hadden MK, Price KL, Orwig KS, See RE, Dix TA: In vivo behavioural effects of stable, receptor-selective neurotensin[8–13] analogues that cross the blood-brain barrier. Neuropharmacol 2005, 48: 417–425. 10.1016/j.neuropharm.2004.10.008View ArticleGoogle Scholar
- Granier C, van Rietschoten J, Kitabgi P, Poustis C, Freychet P: Synthesis and characterization of neurotensin analogue for structure-activity relationship studies. Eur J Biochem 1982, 124: 117–125. 10.1111/j.1432-1033.1982.tb05913.xPubMedView ArticleGoogle Scholar
- Tyler BM, Douglas CL, Fauq A, Ping-Pang Y, Stewart JA, Cusack B, McCormick DJ, Richelson E: In vitro binding and CNS effects of novel neurotensin agonists that cross the blood-brain barrier. Neuropharmacol 1999, 38: 1027–1034. 10.1016/S0028-3908(99)00011-8View ArticleGoogle Scholar
- Kleczkowska P, Kaczorowska E, Ruszczyńska-Bartnik K, Ejchart A, Tourwé D, Lipkowski AW: Chimeric opioid-neurotensin ligands as new prospective analgesics in chronic pain. In Peptides 2008 Chemistry of Peptides in Life Science Technology and Medicine (Proceeding of the 30th European Peptide Symposium). Edited by: Lankinen H, Vallivirta J, Strandin T, Hepojoli J. FIPS, Helsinki; 2008:538–539.Google Scholar
- Garcia-Garayoa E, Bläuenstein P, Bruehlmeier M, Blanc A, Iterbeke K, Conrath P, Tourwé D, Schubiger PA: Preclinical evaluation of a new stabilized neurotensin(8–13) pseudopeptide radiolabeled with 99m Tc. J Nucl Med 2002, 43: 374–383.PubMedGoogle Scholar
- Yaksh TL, Rudy TA: Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976, 17: 1031–1036. 10.1016/0031-9384(76)90029-9PubMedView ArticleGoogle Scholar
- Yoburn BC, Morales R, Kelly DD, Inturrisi CE: Constrain of the tail-flick assay: morphine analgesia and tolerance are dependent upon locus of tail stimulation. Life Sci 1984, 34: 1755–1762. 10.1016/0024-3205(84)90575-7PubMedView ArticleGoogle Scholar
- Mazella J, Vincent JP: Functional roles of the NTS2 and NTS3 receptors. Peptides 2006, 27: 2469–2475. 10.1016/j.peptides.2006.04.026PubMedView ArticleGoogle Scholar
- Gallagher RM, Rosenthal LJ: Chronic pain and opiates: balancing pain control and risks in long-term opioid treatment. Arch Phys Med Rehabil 2008,89(Suppl 1):77–82. 10.1016/j.apmr.2007.12.003View ArticleGoogle Scholar
- Haghparast A, Semnanian S, Fathollahi Y: Morphine tolerance and dependence in the nucleus paragigantocellularis: single unit recording study in vivo. Brain Res 1998, 814: 71–77. 10.1016/S0006-8993(98)01029-4PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<url>http://creativecommons.org/licenses/by/2.0</url>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.