Analgesic Peptide Research Articles

Investigation of Mechanisms Underlying the Pharmacokinetics of Peptide Drugs and their Physiological Model Analysis.

In order to provide mechanistic insights into the pharmacokinetics of peptide drugs (including cytokines and growth factors), I investigated the mechanisms underlying the clearance and distribution of opioid peptides (β-endorphin, dynorphin, and dynorphin-like analgesic peptide), human insulin, and synthetic cyclopeptides (cyclosporine and PSC 833), by use of in vivo animals, perfused organs, and in vitro experimental systems. For opioid peptides, their tissue distribution was suggested to be governed by specific binding with K-type opioid receptors present in peripheral tissues including lung and liver, whereas for insulin the distribution and clearance were suggested to be governed by receptor binding and receptor-mediated endocytosis (RME), respectively, at the physiological concentration range in target organs. The “receptorrecycling” model, in which the internalized receptors are recycled back to the surface to be reutilized for subsequent binding, was developed to predict the hepatic handling of insulin in mice at low and high doses, and successfully incorporated in a physiologically-based pharmacokinetic model, together with transcapillary permeability and static receptor binding in extrahepatic tissues. For cyclopeptides, moreover, their brain penetration was shown to be modulated by P-glycoprotein-mediated efflux functioning at the blood-brain barrier. The kinetic RME analysis enables the prediction of not only the nonlinear target-mediated clearance and distribution of peptides, but also the down-regulation and subsequent recovery of surface receptors, which is useful for assessing the time-dependent changes of in vivo efficacy of peptide drugs. In conclusion, the therapeutic efficacy and protocols of peptide drugs should be assessed from its microscopic pharmacology based on the RME mechanisms, in conjunction with macroscopic pharmacokinetic modeling.

Indirect evidence that drug brain targeting using polysorbate 80-coated polybutylcyanoacrylate nanoparticles is related to toxicity.

To investigate the mechanism underlying the entry of the analgesic peptide dalargin into brain using biodegradable polybutylcyanoacrylate (PBCA) nanoparticles (NP) overcoated with polysorbate 80. The investigations were carried out with PBCA NP and with non biodegradable polystyrene (PS) NP (200 nm diameter). Dalargin adsorption was assessed by HPLC. Its entry into the CNS in mice was evaluated using the tail-flick procedure. Locomotor activity measurements were performed to compare NP toxicities. BBB permeabilization by PBCA NP was studied in vitro using a coculture of bovine brain capillary endothelial cells and rat astrocytes. Dalargin loading was 11.7 microg/mg on PBCA NP and 16.5 microg/ mg on PS NP. Adding polysorbate 80 to NP led to a complete desorption. Nevertheless, dalargin associated with PBCA NP and polysorbate 80 induced a potent and prolonged analgesia, which could not be obtained using PS NP in place of PBCA NP. Locomotor activity dramatically decreased in mice dosed with PBCA NP, but not with PS NP. PBCA NP also caused occasional mortality. In vitro, PBCA NP (10 microg/ml) induced a permeabilization of the BBB model. A non specific permeabilization of the BBB, probably related to the toxicity of the carrier, may account for the CNS penetration of dalargin associated with PBCA NP and polysorbate 80.

Intestinal absorption and analgesic activity of aminopeptidase-resistant cellobiose-coupled leucine enkephalinamide.

Leucine enkephalinamide (LEamide), aminopeptidase-degradable opioid peptide, was coupled with cellobiose (cellobiose-coupled LEamide, CcpLEamide). CcpLEamide was absorbed from the rat small intestine in vitro, whereas LEamide was not. CcpLEamide on the mucosal side was more stable than aminopeptidase-resistant cellobiose-coupled leucine enkephalin (CcpLE) in the presence of inhibitors of enkephalinase and angiotensin converting enzyme, and much more stable than LEamide. The absorption rate (clearance) of CcpLEamide was comparable with that of CcpLE in the presence of these peptidase inhibitors. Analgesic activities of these enkephalins were tested by the acetic acid writhing assay and hot-plate assay after subcutaneous administration to mice. Both assays indicated CcpLEamide-induced analgesia. On the other hand, the analgesic activity of LEamide was not observed, but pretreatment with amastatin (a peptidase inhibitor) produced LEamide-induced analgesia. These results indicate that CcpLEamide is stable in the body and has analgesic effects without pretreatment with peptidase inhibitors, and was stable enough to be absorbed from the small intestine. We propose CcpLEamide as an orally active analgesic peptide candidate.

An overview of current and investigational non-narcotic drugs for treatment of acute and chronic pain

A survey of newer analgesics in advanced clinical development suggests that cyclooxygenase-2 inhibitors for inflammatory pain, newer 5-hydroxytryptamine 1D (5-HT 1D) agonists for migraine pain, α-2 adrenergics, neuron-specific Ca++or NA+ channel antagonists, drugs that potentiate opioid analgesics or inhibit morphine tolerance development, or implantation of encapsulated living cell systems that release endorphins or other analgesic peptides, may improve analgesic responsiveness and reduce side effects for some patients with acute or chronic pain conditions. Among the more novel anti-inflammatory anti-arthritic drugs with different mechanisms of action than the dominant nonsteroidal anti-inflammatory drugs are the first new cyclooxygenase-2 inhibitors (celecoxib, vioxx) and two drugs that may have combined immunomodulatory and anti-inflammatory actions (pelubiprofen, enbrel). Other anti-inflammatory, anti-arthritic approaches in earlier clinical testing include two leukotriene antagonists, a C5a complement inhibitor, and matrix metalloproteinase inhibitor.This manuscript details the need for new pain relieving drugs, presents their status, and enumerates the drugs in development.The status of new nonsteroidal anti-inflammatory drugs is described with new mechanisms of action and efficacy for acute and chronic pain use.

The Metabolic Inhibition Model Which Predicts the Intestinal Absorbability and Metabolizability of Drug: Theory and Experiment.

The intestinal absorption of analgesic peptides (leucine enkephalin and kyotorphin) and modified peptides in rat were studied. Although these peptides were not absorbed, the absorbability (absorption clearance) of these peptides were increased in the presence of peptidase inhibitors. In order to kinetically analyze these phenomena, we proposed the metabolic inhibition model, which incorporated the metabolic clearance (metabolizability) with the absorption clearance. Metabolic activity was determined with intestinal homogenates. The higher the metabolic clearance was, the lower was the absorption clearance. The relationships between the absorption clearance and the metabolic clearance of the experimental data as well as of the theoretical values were hyperbolic. This model predicted the maximum absorption clearances of cellobiose-coupled leucine enkephalin (0.654 μl/min/cm) and kyotorphin (0.247 μl/min/cm). Details of the experimental methods are described.

Nociceptive stimulus induces release of endogenous β-endorphin in the rat brain

The hypothesis that the naturally occurring analgesic peptide, β-endorphin, is released in the brain in response to pain had never been directly validated. In this study, we applied a brain microdialysis method for monitoring β-endorphin release in vivo, to test this hypothesis in the brains of conscious, freely moving rats. Herein we first show that endogenous β-endorphin can be measured in vivo in the brain under physiological conditions. Upon induction of a nociceptive stimulus by injection of formalin into the hind-paws of rats, the extracellular levels of β-endorphin in their arcuate nucleus increased by 88%, corresponding to their nociceptive response. This direct evidence for the release of endogenous β-endorphin in the brain in response to nociceptive stimulus indicates a possible mechanism for organisms to cope with pain.

Isolation and characterization of anti morphine analgesic peptide from canine brain.

The anti-morphine peptide from canine brain, which is homologous to alpha subunit of hemoglobin of canine, has been isolated and characterized. The canine brain was homogenized and extracted in 0.1 M acetic acid. The supernatant was collected, and purified by chromatography of Sephadex G-50 and ion-exchange and RP-HPLC. A polypeptide was then obtained, which is termed as AMP5. It shows prominent antimorphine analgesic activity. AMP5 has an apparent molecular mass of 10.23kD. The isoelectric point was estimated to be around pH 6.7 by IEF. The amino acid composition of AMP5 and sequence of the N-terminal 50 amino acid residues was determined.

Differential expression of galanin immunoreactivities in the primary sensory neurons following partial and complete sciatic nerve injuries

Neuropeptide expression in primary sensory neurons is highly plastic in response to peripheral nerve axotomy. While neuropeptide changes following complete sciatic nerve injury have been extensively studied, much less is known about the effects of partial sciatic nerve injuries on neuropeptide plasticity. Galanin, a possible endogenous analgesic peptide, was up-regulated in primary sensory neurons following complete sciatic nerve injury. We investigated the effects of partial sciatic nerve injuries on galanin expression in primary sensory neurons, and compared this effect with that after complete sciatic nerve injury. Complete transection, partial transection and chronic constriction injury were made, respectively, on the sciatic nerves of three groups of rats at high thigh level. Animals were allowed to survive for four and 14 days before being killed. L4 and L5 dorsal root ganglia, L4–5 spinal cord and lower brainstem were processed for galanin immunocytochemical staining. After all three types of sciatic nerve injuries, galanin-immunoreactive neurons were significantly increased in the ipsilateral dorsal root ganglia, and galanin-immunoreactive axonal fibres were dramatically increased in the superficial laminae of the dorsal horn and the gracile nuclei, compared to the contralateral side. However, in partial injury models, the percentages of galanin-immunoreactive dorsal root ganglion neurons were significantly higher than in complete nerve transection. Size frequency distribution analysis detected that more medium- and large-size galanin-immunoreactive dorsal root ganglion neurons were present after partial nerve transection and constriction injury than after complete nerve transection. Using a combined approach of retrograde tracing of flurorescent dyes and galanin immunostaining, we found that a partial transection increased the proportions of galanin-immunoreactive neurons among both axotomized and non-axotomized neurons. Galanin-immunoreactive axonal fibres were not only detected in the superficial laminae, but also in the deeper laminae of the dorsal horn of partial injury animals. Furthermore, more galanin-immunoreactive axonal fibres were observed in the ipsilateral gracile nuclei of partially injured rats than in completely injured rats. We conclude that partial sciatic nerve injuries induced greater galanin up-regulation in medium- and large-size dorsal root ganglion neurons than complete sciatic nerve injury. Galanin expression in primary sensory neurons seems to be differentially regulated following partial and complete sciatic nerve injuries.

Synthesis and antinociceptive activity of peptides related to interleukin-1 beta 193-195 Lys-Pro-Thr.

To obtain information about the structure-activity relationships of analgesic peptides, we modified the previously reported tripeptide, H-Lys-Pro-Thr-OH (C). The proline part in C was replaced with various analogues of unconventional amino acids [(3aS, 7aS)-octahydroindole-2-carboxylic acid (Oic), (S,S,S,)-2-azabiciclo [3.3.0]octane-3-carboxylic acid (Aoc), D-Aoc, and (2S, 4R)-hydroxyproline (Hyp)] with varying lipophilic, steric, and conformational properties, and alternatively with Lys and Orn in the lysine part. Moreover, the threonine part was changed to various natural amino acids (Ser, Thr, Val, Leu). All the compound were screened in vivo for their analgesic effects in mouse writhing test. Compound 24 (H-Orn-Hyp-Val-OH), the most active compound within the series, showed an ED50 value of 10 mg/kg, which is comparable with the ED50 values exhibited by indometacin (4.1 mg/kg) and the dipeptide H-Lys-D-Pro-OH (6.9 mg/kg), both used as reference drugs.

A new potent analgesic peptide is a [formula omitted] selective agonist

A new potent analgesic peptide is a [formula omitted] selective agonist

Opioid peptides are substrates for the bifunctional enzyme LTA 4 hydrolase/aminopeptidase

We determined if any naturally occurring peptides could act as substrates or inhibitors of the bifunctional, Zn 2+ metalloenzyme LTA 4 hydrolase/aminopeptidase (E.C. 3.3.2.6). Several opioid peptides including met 5-enkephalin, leu 5-enkephalin, dynorphin 1–6, dynorphin 1–7, and dynorphin 1–8 competitively inhibited the hydrolysis of L-proline-p-nitroanilide by leukotriene A 4 hydrolase/ aminopeptidase, consistent with an interaction at its active site. The enzyme catalyzed the N-terminal hydrolysis of tyrosine from met 5-enkephalin with K m =450 ± 58 μM and V max =4.9 ± 0.6 nmol- hr −1- ug −1 and from leu 5-enkephalin with K m =387 ± 90 μM and V max =6.2 ± 2.5 nmol- hr −1- ug −1. Bestatin, captopril and carnosine inhibited the hydrolysis of the enkephalins. It is noteworthy that the bifunctional catalytic traits of this enzyme include generation of an hyperalgesic substance, LTB 4, and inactivation of analgesic opioid peptides.

In vivo transport of a dynorphin-like analgesic peptide, E-2078, through the blood-brain barrier: an application of brain microdialysis.

In vivo transport through the blood-brain barrier (BBB) has been demonstrated for a dynorphin-like analgesic peptide, CH3-[125I]Tyr-Gly-Gly-Phe-Leu-Arg-CH3Arg-D-Leu-NHC2H5 ( [125I]E-2078). A remarkable time-dependent increase in the distribution volume of [125I]E-2078 in the brain parenchyma separated from blood vessels and capillaries was observed during a brain perfusion. The distribution volume of [125I]E-2078 in the brain parenchyma after 20 min of perfusion was 2.18 +/- 0.09 microliters/g brain (mean +/- SE) and was significantly greater than the distribution volume of [3H]inulin (0.994 +/- 0.138 microliters/g brain), providing in vivo evidence for the penetration of [125I]E-2078 into the brain parenchyma. Brain microdialysis was carried out to collect directly the brain interstitial fluid (ISF) during the brain perfusion of [125I]E-2078. No metabolite of [125I]E-2078 in the brain ISF was found by high-performance liquid chromatographic analysis of the brain dialysate. The concentrations of [125I]E-2078 and [14C]sucrose in the brain ISF were estimated based on an in vitro evaluation of dialysis clearance. The concentration ratio of [125I]E-2078 between the brain ISF and the brain perfusate was determined to be 2.92 x 10(-1) +/- 0.50 x 10(-1) and was approximately 100 times higher than that of [14C]sucrose (2.71 x 10(-3) +/- 1.43 x 10(-3), demonstrating transport of [125I]E-2078 through the BBB in vivo. On the other hand, no remarkable difference in the cerebrospinal fluid (CSF)-to-perfusate concentration ratios of [125I]E-2078 and [14C]sucrose was observed, indicating little contribution of the blood-CSF barrier (BCSF barrier) transport to the penetration of [125I]E-2078 into the brain.

A parentrally active highly potent analgesic peptide with reduced abuse potential

A parentrally active highly potent analgesic peptide with reduced abuse potential

Synthesis and structure-activity relationships of (MeTyr1, MeArg7)-Dynorphin A(1-8)-OH analogues with substitution at position 8.

A series of [MeTyr1, MeArg7]-Dynorphin A (Dyn)(1-8)-OH analogues, modified at position 8 with various amino acids, is described. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after subcutaneous administration. None of the analogues tested displayed more potent kappa-opioid activity in the RVD than [MeTyr1, MeArg7, D-Leu8]-Dyn(1-8)-NHEt (1), which is a potent analgesic peptide with similar opioid receptor selectivity to that of Dyn. However, [MeTyr1, MeArg7, Melle8]-Dyn(1-8)-OH (11) showed about a twofold more potent analgesic effect than 1. Based on the obtained results it is conceivable that in the case of Dyn(1-8)-OH analogues both a lipophilic L-amino acid in position 8 and an unchanged 7-8 amide bond are essential to maintain potent kappa-opioid activity.

The opioid specificity of beta-endorphin enhancement of murine lymphocyte proliferation

Beta-endorphin (β-end) is a potent analgesic peptide which exhibits a variety of pharmacological activities in the central nervous system (CNS) following binding of its N-terminus to specific opioid receptors. Although C-terminal binding sites for this 31-amino-acid peptide have been characterized in CNS tissue, identification of their possible function has been facilitated by studies of β-end effects on lymphocyte activities. In this communication, we report a detailed analysis of the opioid specificity of the ability of β-end to enhance T cell mitogen-induced proliferation in unfractionated murine splenocytes. Intact 31-amino-acid β-end peptides from several species, including human, camel and rat, enhanced concanavalin A-stimulated [3H]thymidine uptake 50–640% in a dose-dependent, naloxone-irreversible fashion. The presence of the C-terminal amino acids was required for the enhancement activity, since met-enkephalin, α- and γ-endorphin, and human β-end 1–27 were ineffective. Accordingly, the truncated peptides, human β-end 6–31 and 18–31, were also able to enhance the Con A response. However, human β-end 18–31 was consistently not as effective as β-end 6–31 or the intact 31-residue peptide. These data suggest that although the C-terminus contains the primary active sequence, the N-terminus contributes to the overall potency of the effect. In support of this assertion, N-acetylation, which abolishes opioid binding activity, resulted in a reduced magnitude of enhancement. The data suggest that β-end interacts with a non-opioid receptor which has specificity characteristics strikingly similar to non-opioid receptors characterized in CNS tissue.

ChemInform Abstract: Conformational Properties of the Analgesic Peptide Dermorphin and Their Relationships to Biological Activity.

Abstract The conformational properties of the analgesic heptapetide dermorphin were investigated using potential energy calculations, by applying a variety of computer programs, especially aimed at detecting the low-energy minima of intramolecular potential energy functions. The most stable conformations are bent, due to various types of intramolecular hydrogen bonds, while the energy of extended structures is somewhat higher. This is in keeping with the conclusions drawn from experimental measurements in solution, that the pharmacological activity of dermorphin is likely to be due to a peculiar folded conformation, mainly resulting from the presence of Ala-2 in configuration D at the N -end and the Pro-6 — Ser-7 sequence, endowed with a high β-turn potential, at the C -end. A comparison was also made between the low-energy regions, as predicted by different force fields, and these predictions were found to be in substantial agreement.

Conformational properties of the analgesic peptide dermorphin and their relationships to biological activity

The conformational properties of the analgesic heptapetide dermorphin were investigated using potential energy calculations, by applying a variety of computer programs, especially aimed at detecting the low-energy minima of intramolecular potential energy functions. The most stable conformations are bent, due to various types of intramolecular hydrogen bonds, while the energy of extended structures is somewhat higher. This is in keeping with the conclusions drawn from experimental measurements in solution, that the pharmacological activity of dermorphin is likely to be due to a peculiar folded conformation, mainly resulting from the presence of Ala-2 in configuration D at the N-end and the Pro-6 — Ser-7 sequence, endowed with a high β-turn potential, at the C-end. A comparison was also made between the low-energy regions, as predicted by different force fields, and these predictions were found to be in substantial agreement.

OPIOIDS

From the Analgesic Peptide Reseatch Unit, Anesthesia Pain Unit, and the Department of Anesthesia, Massachusetts General Hospital and Shriners Burns Institute, Boston, MA.

Operation, anesthesia, and the endorphin system.

Endogenous opioid peptides regulate a multitude of neuronal pathways and extraneural targets (e.g., white blood cells). Acting in the periphery and at central sites of afferent integration within multiple compartments at multiple levels, they modify the secretion of other hormones yet are themselves secreted during stress to produce important biological effects. Usually quiescent, the opioid system becomes active during a host of stresses to optimize, integrate, and buffer bodily responses. During stress, endorphins act to limit sympathetically driven responses such as tachycardia and vasoconstriction; however, if given to resting subjects, opiates may evoke the very responses they inhibit during stress. As the roles of individual opioid peptides and opiate receptors are studied in increasing depth, an amazing degree of functional heterogeneity and detail has emerged. Such knowledge has barely begun to be exploited in creating the next generation of therapeutics.

Neo-kyotorphin, an analgesic peptide isolated from human lung carcinoma

Neo-kyotorphin, an analgesic peptide isolated from human lung carcinoma