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Further Characterization of a Novel Tetrapeptide with an Analgesic Action in the Central and Peripheral Nervous System in Rats

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Abstract

The novel tetrapeptide FLPS has been previously shown to induce antinociception in a model of post-incisional pain in the rat. It has been demonstrated in membrane preparations of rat brain that 10 μM tetrapeptide did not compete with [3H]naloxone for its binding site on the opioid receptors which differentiates it from typical opioids. In the first set of experiments, the stimulation of [35S]GTPγS binding by the tetrapeptide to membrane preparations of recombinant cell lines expressing human μ, δ, and κ receptors has been investigated. The specific [35S]GTPγS binding did not change in the presence of 100 μM tetrapeptide and DAMGO, DPDPE, or U-69593 at submaximal and maximal concentrations, indicating that the tetrapeptide was not an agonist or antagonist of the opioid receptors and it did not stimulate or blocked the stimulation of G-proteins coupled to these receptors. Studies with naloxone and naloxone methiodide pretreatment suggested that the tetrapeptide was either directly or indirectly affected by a naloxone-binding site located primarily within the central nervous system and to a lesser extent in the periphery. Naloxone pretreatment had a dual effect on morphine antinociception based on the concentration used. At 2 mg/kg, naloxone competed reversibly with 25 mg/kg morphine on its binding site on the opioid receptors, while at 5.4 mg/kg it blocked antinociception induced by 10 mg/kg morphine, 2.7 mg/kg aspirin, or 75 mg/kg tetrapeptide. These data suggest that a new naloxone-binding site is involved in alleviation of pain by at least three classes of analsegics (opioids, cycloogenase 2 inhibitors, and the tetrapeptide). The biological activity of the peptide was discovered first and the mechanism of action is now being studied. In an attempt to identify the therapeutic target of the tetrapeptide, a total of 23 radioligand binding assays to targets within the CNS were conducted. The results of these assays were negative and they reinforce the notion that the tetrapeptide activates an unknown mechanism involved in pain perception after surgery.

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Abbreviations

ANOVA:

One-way analysis of variance

CGP 54,626:

[S-(R*,R*)]-[3-[[1-(3,4-Dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl) phosphinic acid

COX-2:

Cyclooxygenase 2

CNS:

Central nervous system

DMSO:

Dimethyl sulfoxide

DPCPX:

8-Cyclopentyl-1,3-dipropylxanthine

GABA:

γ-Aminobutyric acid

5-HT:

5-Hydroxytryptamine

NSAID:

Non-steroidal anti-inflammatory drug

SCH 23,390:

(R)-(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine

R-PIA:

N6-[(R)-1-methyl-2-phenyl–ethyl]adenosine

Tyr-S36057:

Dodecapeptide melanin-concentrating hormone (MCH 6–17)

WIN 55,212-2:

(R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone

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Acknowledgments

We thank Dr. Michelle D. Brot for helpful comments and Ricerca Biosciences for the screening assays.

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Authors and Affiliations

  1. NeoPro Pain, 1124 Columbia St., Seattle, WA, 98104, USA

    Hanna Skubatz

  2. Calvert Laboratories, Inc., Scott Technology Park, 130 Discovery Drive, Scott Township, PA, 18447, USA

    Brian Klatt

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  1. Hanna Skubatz
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  2. Brian Klatt
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Correspondence to Hanna Skubatz.

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Skubatz, H., Klatt, B. Further Characterization of a Novel Tetrapeptide with an Analgesic Action in the Central and Peripheral Nervous System in Rats. Int J Pept Res Ther 18, 41–52 (2012). https://doi.org/10.1007/s10989-011-9277-5

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