Dermorphin was discovered in the early 1980s in the skin of South American 'monkey frogs' (Phyllomedusa). It locks onto the same mu-opioid receptors that morphine and fentanyl use, but grips them far more tightly, making it one of the strongest natural painkillers ever measured in a lab. Decades of animal research confirm it kills pain, but no one has ever run a human clinical trial on it, and there is no approved medicine made from it. In the real world, it is mostly known for turning up in horse-racing doping scandals, where it has been injected illegally to mask pain and boost performance.
How strong is the evidence?
Every study of actual dermorphin in this file is either an animal experiment (mostly rats and mice, plus one horse study) or a test-tube/cell-line experiment looking at how it grips opioid receptors. There is not one human clinical trial. A few newer 'peripherally acting' cousins of dermorphin (like DALDA) look promising for nerve and burn pain in rats without some of the classic opioid downsides, but that work is still animal-only. So the animal evidence that it's a powerful painkiller is strong and consistent across 40+ years of studies - the human evidence is simply nonexistent.
Uses
What people use it for
Pain research in animals
Animal / labUsed almost entirely as a research tool - injected into rats, mice, rabbits and other lab animals to study how opioid painkillers work and to test whether new pain-relief molecules are safer or more effective than morphine.
Illegal performance drug in horse racing
Animal / labDermorphin has been detected in postrace horse blood and urine samples. Scientists studied how quickly it clears from a horse's body specifically to help racing authorities catch this illegal doping use - it is not used therapeutically in horses.
Lab tool for mapping opioid receptors
Animal / labChemically tagged (fluorescent) versions of dermorphin let scientists see exactly where mu-opioid receptors sit on cells and how they behave, which helps in designing future painkillers.
Potential benefits
What it may help with
Blocks pain more powerfully than morphine - in animals
Animal / labIn rats, mice and rabbits, dermorphin produces deep, long-lasting pain relief. Injected directly into the brain it has been measured as up to several hundred times more potent than morphine; injected into the body (IV or under the skin) it is still roughly 25-30 times more potent. This is the most consistent and best-replicated finding across the entire research record.
May cause less tolerance and physical dependence than morphine
Animal / labIn rats and mice given repeated doses, dermorphin and its close relatives led to slower buildup of tolerance (needing more drug over time for the same effect) and a milder withdrawal reaction than morphine caused under the same conditions. This has never been tested in people.
Peripherally-restricted cousins may relieve nerve and burn pain without brain-related side effects
Animal / labA modified version called DALDA, engineered to mostly stay outside the brain, reduced pain from chemotherapy nerve damage, severe burn injury, and a chronic post-spine-surgery pain model in rats - without the sedation, addiction signs, or breathing problems typically seen with opioids that reach the brain. This is an early but genuinely promising research direction.
A dermorphin-based molecule showed antidepressant-like effects in mice
Animal / labA lab-made hybrid of dermorphin and another opioid peptide reduced signs of despair-like behavior in a standard mouse depression test, working through the same mu-opioid receptors involved in pain relief. This is a single, early, preclinical finding, not evidence dermorphin itself treats depression.
Studies:35908903
What to watch for
Side effects & risks
- Serious
Can suppress or destabilize breathing at higher doses
In rats, higher doses of dermorphin reduced breathing rate and volume, the same dangerous effect that makes opioid overdose deadly in people. Confusingly, very low doses sometimes briefly stimulated breathing instead, showing the effect is dose-dependent and unpredictable.
- Moderate
- Moderate
- Mild
- Mild
- Mild
Alters blood sugar hormones
In rats, an IV dose of dermorphin raised glucagon (a hormone that pushes blood sugar up) while blood glucose actually fell, suggesting it disrupts normal blood sugar regulation. This was a single small animal study.
- Moderate
Builds tolerance and physical dependence with repeated use
Animals dosed repeatedly needed escalating amounts for the same pain relief and showed withdrawal signs (like jumping and weight loss) when the drug was blocked - less severe than morphine withdrawal in the same studies, but still present.
- Serious
Extremely high potency makes overdose risk very high
Because dermorphin is active at doses hundreds of times smaller than morphine, even tiny dosing errors could be dangerous. This is a mechanistic/potency-based concern drawn directly from the potency data in these studies, not a reported overdose case.
Dosing
Dosing — what studies used
There is no established human dose for dermorphin - it has never been given to people in a clinical trial and has no approved medical use anywhere. Every dose in the literature comes from animal experiments (mostly rats and mice, plus one horse pharmacokinetics study tied to doping detection). Any 'dosing' information circulating outside of research is not based on human safety data and should not be treated as medical guidance.
Pain relief (analgesia) research in rodents
Animal study36-120 nmol by direct brain injection, or roughly 0.12-4.7 micromoles per kg body weight under the skin
Single dose per test · Effects measured over minutes to a few hours · Intracerebroventricular or subcutaneous
Used the analog [Lys7]dermorphin in rats and mice. This is a lab research dose, not applicable to humans.
Chronic pain after spine surgery, rat model, using the peripherally-acting analog DALDA
Animal study5 mg per kg body weight
Single injection · Tested acutely after model surgery · Intraperitoneal injection
DALDA is a modified, peripherally-restricted version of dermorphin, not dermorphin itself.
Burn-injury pain, rat model, using the analog DALDA
Animal study1, 3, or 10 mg per kg body weight (10 mg/kg worked best)
Dose-ranging test, repeated over the study period · Days following burn injury · Subcutaneous injection
Reduced ongoing pain without signs of addiction in this rat model.
Pharmacokinetics tied to horse-racing doping detection
Animal study9.3 ± 1.0 micrograms per kg body weight
Single dose · Tracked in blood for up to 12 hours and in urine for up to 72 hours · Intravenous or intramuscular injection
This dose reflects illegal doping use in racehorses, studied purely so regulators could detect it - it is not a therapeutic protocol.
Blood sugar hormone research in rats
Animal study0.1 to 10 micrograms per animal
Single intravenous dose · Blood measured over 30 minutes · Intravenous injection
Very small experimental dose used only to study effects on glucagon and glucose, not for pain relief.
All dosing data comes from animal research or a horse doping-detection study - none of it should be read as a safe or effective human dose. Dermorphin has no approved medical use in people.
These figures describe what researchers used in studies. They are not a recommendation or a prescription.
Mechanism
How it works
Dermorphin locks onto mu-opioid receptors - the same docking points in the brain and body that morphine, fentanyl, and your own natural painkilling chemicals (endorphins) use. It fits those receptors unusually tightly and selectively, which is why such tiny amounts produce such strong pain relief in animal studies. Turning on these receptors dulls pain signals, but the same receptors also control breathing, gut movement, body temperature, and heart rate - which is why dermorphin affects all of those things too, for better or worse. Newer lab-made versions try to keep the molecule out of the brain and spinal cord (working only on nerves in the body) to get the pain relief without the breathing and dependence risks that come from opioid receptors in the central nervous system.
Who should avoid it
- Should not be used outside of a licensed research setting - it is not an approved medicine
- Anyone with breathing problems, heart conditions, or low blood pressure, given the breathing, heart rate, and blood pressure effects seen in animal studies
- Pregnancy and breastfeeding - never studied and should be assumed unsafe like other potent opioids
- Athletes or competitors in any regulated or drug-tested sport, including horse racing - it is a banned doping substance
- Anyone combining it with other opioids, sedatives, or alcohol
Interactions to know
- Other opioid painkillers (morphine, fentanyl, oxycodone, etc.) - stacking opioids compounds breathing suppression and overdose risk
- Sedatives, benzodiazepines, and alcohol - all add to breathing suppression risk when combined with any strong opioid
- Naloxone (the opioid overdose antidote) blocks dermorphin's effects - used in research specifically to confirm it works through opioid receptors
The papers that matter most
Key studies
Established dermorphin as the most potent naturally occurring opioid painkiller known at the time, up to hundreds of times stronger than morphine when injected directly into the brain.
Characterisation and visualisation of [3H]dermorphin binding to mu opioid receptors in the rat brain
Comprehensive review showing dermorphin peptides are more potent painkillers than morphine in rodents and primates, with less tolerance and dependence in animal models.
The dermorphin peptide family
Showed that even injected into the body (not just the brain), a dermorphin analog gave strong, long-lasting pain relief with slower tolerance and milder withdrawal than morphine.
Production of antinociception by peripheral administration of [Lys7]dermorphin
Real-world relevance: this study exists because dermorphin was found in postrace horse samples, confirming its use as an illegal doping agent, and mapped how quickly it clears the body for detection purposes.
Pharmacokinetics and pharmacodynamics of dermorphin in the horse
A brain-sparing dermorphin analog (DALDA) relieved chemotherapy-induced nerve pain in rats without addiction or central nervous system side effects - a promising future direction, still preclinical.
Peripheral mu-opioid receptor activation by dermorphin [D-Arg2, Lys4] (1-4) amide alleviates behavioral and neurobiological aberrations in rat model of chemotherapy-induced neuropathic pain
The same peripherally-acting analog reduced burn-injury pain in rats at a 10 mg/kg dose without triggering addiction-like behavior, reinforcing the peripheral-opioid strategy.
Dermorphin [D-Arg2, Lys4] (1-4) Amide Attenuates Burn Pain by Inhibiting TRPV1/NR2B Mediated Neuroinflammatory Signalling
Bottom line
Dermorphin is a real, extraordinarily potent frog-derived opioid that reliably kills pain in animal studies - often far better than morphine - but it has never been tested in a single human clinical trial and has no approved medical use. In the real world it shows up mainly as an illegal doping agent in horse racing. This is a serious drug-safety story, not a wellness peptide, and it should be treated with the same caution as any unapproved, unregulated opioid.
Research papers
Studies we have on file for Dermorphin. Tap a title to open it on PubMed. Labels like “animal” or “human trial” are rough guides.
40 papers
The brain-gut-skin triangle: new peptides.
New data on tachykinins and bombesins are displayed and the present situation of research on the novel amphibian skin peptides sauvagine and dermorphin is illustrated. The potent stimulant effect of sauvagine on ACTH and beta-endorphin release has been confirmed both in vivo and on columns of isolated and dispersed rat pituitary cells, and similarly the potent inhibitory effect on PRL and GH release, both in the rat and man. Particular emphasis is laid on the occurrence of sauvagine-like immunoreactivity in fish urophysis and in amphibian nervous structures, including the retina. It is suggested that the long-searched corticotropin releasing factor and PRL release-inhibiting factor may be a sauvagine-like peptide. Dermorphin, in its turn, has been found to cause, by intracerebroventricular injection, not only analgesia and catalepsy, but also conspicuous EEG and behavioral changes in the rabbit and chick, as well as a sharp reduction in gastric emptying time and gastric acid output in the rat, together with marked stimulation of PRL release.
Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of μ-opioid peptide receptors.
The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.
The dermorphin peptide family.
1. In 1980, the skin of certain frogs belonging to the genus Phyllomedusinae was found to contain two new peptides that proved to be selective mu-opioid agonists. Given the name dermorphins, these were the first members of a peptide family that in the past 15 years has grown to reach a total of seven naturally occurring peptides and nearly 30 synthetic analogs. 2. Dermorphin peptides are potent analgesics in rodents and primates, including man. Some dermorphins can enter the blood-brain barrier and produce central antinociception after peripheral administration. 3. The dermorphin family also includes mu 1-opioid receptor selective agonists that produce intense opioid analgesia, but stimulate pulmonary ventilation. 4. Experiments in rats and mice chronically exposed to dermorphins have shown that not only do they have higher antinociceptive efficacy and potency than morphine, but they are also less likely than morphine to produce tolerance, dependence and opiate side effects.
Brain-gut-skin peptides: an update overview.
The authors present an overview of the main amphibian peptide families mainly derived from the skin and mostly discovered by Erspamer and his associates. The studies of the peptides do not only promote the understanding of their chemical, metabolical and physiological features of those molecules in amphibians, but also contribute to progress in our knowledge of the corresponding mammalian counterparts. Particular reference is made on sauvagine, tachykinin, bombesin and dermorphin families, offering new data mostly from personal contributions to this field.
Opioid peptides from frog skin.
The skin of the South American frogs Phyllomedusa secretes, in addition to numerous mammalian-like hormones and neuropeptides, several gene-encoded opioid peptides that contain a D-amino acid in position 2 of their sequence. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, dermenkephalin/deltorphin A, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 and the deltorphins, Tyr-D-Ala-Phe-Xaa-Val-Val-Gly-NH2 (where Xaa is either Asp or Glu) are highly potent at, and exquisitely selective, for the mu- and delta-opioid receptors. D-Ala and D-Met present in dermorphin and related peptides are coded for by the usual codons in the corresponding messenger RNAs. Prepro-dermorphin/dermenkephalin and prepro-deltorphins have considerable sequence identities to precursors encoding 10-46-residue-long antimicrobial peptides--dermaseptins, brevinins, temporins, esculentins and gaegurins--originating from various amphibian species. The similarity between the prepro-regions of precursors encoding end products with strikingly different structures and biological activities supports the suggestion that the genes encoding these peptides are all members of the same family.
Multiple ligands in opioid research.
The observation in 1979 that opioid receptors interact, led to the design of bivalent ligands in an attempt to improve selectivity and affinity towards the different subtypes( i.e. mu, delta, and kappa). Dimers of monovalent 'parent' opioid structures have been evaluated and include: (a) endogenous (e.g enkephalins) or exogenous (e.g dermorphin) peptide dimer analogues (b) mixed peptidic -non-peptidic bivalent ligands and (c) dual non-peptidic dimers. Chimeric structures, using an opioid pharmacophore in combination with a a non-opioid pharmacophore, have also been prepared. The common aim in all these studies is to improve the pharmacological profile of potential analgesics to minimize common opioid-induced side effects, such as physical dependence and tolerance. Here we present a brief overview efforts to develop bivalent opioid ligands for use in pain-related research.
Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine.
Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala(2) residue with d-Arg(2) makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg(2) have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg(2) may provide a new target molecule for developing novel analgesics that have fewer side effects.
Chemical space screening around Phe3 in opioid peptides: Modulating µ versus δ agonism by Suzuki-Miyaura cross-couplings.
In this study, affinities and activities of derivatized analogues of Dmt-dermorphin[1-4] (i.e. Dmt-d-Ala-Phe-GlyNH2, Dmt = 2',6'-dimethyl-(S)-tyrosine) for the µ opioid receptor (MOP) and δ opioid receptor (DOP) were evaluated using radioligand binding studies, functional cell-based assays and isolated organ bath experiments. By means of solid-phase or solution-phase Suzuki-Miyaura cross-couplings, various substituted regioisomers of the phenylalanine moiety in position 3 of the sequence were prepared. An 18-membered library of opioid tetrapeptides was generated via screening of the chemical space around the Phe3 side chain. These substitutions modulated bioactivity, receptor subtype selectivity and highly effective ligands with subnanomolar binding affinities, contributed to higher functional activities and potent analgesic actions. In search of selective peptidic ligands, we show here that the Suzuki-Miyaura reaction is a versatile and robust tool which could also be deployed elsewhere.
Production of antinociception by peripheral administration of [Lys7]dermorphin, a naturally occurring peptide with high affinity for mu-opioid receptors.
1. The opioid activity of the amphibian peptide, [Lys7]dermorphin, was studied in rats and mice. When administered intracerebroventricularly (i.c.v.), intravenously (i.v.) or subcutaneously (s.c.) it produced a long lasting analgesia. Its antinociceptive potency exceeded that of morphine 290 times by i.c.v. injection, and 25-30 times by peripheral administration. 2. The dose-response curves of [Lys7]dermorphin antinociception were shifted to the right by the pretreatment with naloxone (0.1 mg kg-1, s.c.) or with the mu 1-selective antagonist, naloxonazine (10 mg kg-1, i.v. 24 h before peptide injection). 3. The peptide also displayed potent antinociceptive effects in a chronic inflammatory pain model (rat Freund's adjuvant arthritis). In this pain model, systemic administration of the peptide raised the nociceptive threshold more in inflamed than in healthy paw. 4. High central and peripheral doses of [Lys7]dermorphin in rats produced catalepsy. The cataleptic response was antagonized by naloxone but left unchanged by naloxonazine pretreatment. 5. In rats and mice, central or peripheral administration of [Lys7]dermorphin induced a significantly slower development of tolerance to the antinociceptive effect than did morphine. 6. Upon naloxone precipitation of the withdrawal syndrome, [Lys7]dermorphin-dependent mice made fewer jumps and lost less weight than the morphine-dependent animals. Withdrawal hyperalgesia did not develop in [Lys7]dermorphin-dependent mice. 7. In conclusion, [Lys7]dermorphin seems to be a unique opioid peptide having a high penetration into the blood-brain barrier despite its low lipid solubility. This peptide causes fewer side-effects than other opioids and appears less likely than morphine to cause physical dependence in rats and mice.
Fluorescent opioid receptor ligands as tools to study opioid receptor function.
Opioid receptors are divided into the three classical types: MOP(μ:mu), DOP(δ:delta) and KOP(κ:kappa) that are naloxone-sensitive and an additional naloxone-insensitive nociceptin/orphanin FQ(N/OFQ) peptide receptor(NOP). Studies to determine opioid receptor location and turnover variably rely on; (i) measuring receptor mRNA, (ii) genetically tagging receptors, (iii) labelling receptors with radioligands, (iv) use of antibodies in immunohistochemistry/Western Blotting or (v) measuring receptor function coupled with the use of selective antagonists. All have their drawbacks with significant issues relating to mRNA not necessarily predicting protein, poor antibody selectivity and utility of radiolabels in low expression systems. In this minireview we discuss use of fluorescently labelled opioid receptor ligands. To maintain the pharmacological properties of the corresponding parent ligand fluorescently labelled ligands must take into account fluorophore (brightness and propensity to bleach), linker length and chemistry, and site to which the linker (and hence probe) will be attached. Use of donor and acceptor fluorophores with spectral overlap facilitates use in FRET type assays to determine proximity of ligand or tagged receptor pairs. There is a wide range of probes of agonist and antagonist nature for all four opioid receptor types; caution is needed with agonist probes due to the possibility for internalization. We have produced two novel ATTO based probes; DermorphinATTO488 (MOP) and N/OFQATTO594 (NOP). These probes label MOP and NOP in a range of preparations and using N/OFQATTO594 we demonstrate internalization and ligand-receptor interaction by FRET. Fluorescent opioid probes offer potential methodological advantages over more traditional use of antibodies and radiolabels.
Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo).
Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ. We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats. DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats. Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties.
Dermorphin: an opioid peptide from amphibian skin which affects endocrine pancreatic function.
We examined the effect of intravenous injection of the amphibian skin opioid heptapeptide dermorphin on plasma glucose, insulin, glucagon and somatostatin in the rat. Dermorphin in doses ranging from 0.1 to 10 micrograms per animal did not affect plasma insulin or somatostatin-like immunoreactivity (SLI). In contrast, the opiate peptide significantly stimulated plasma glucagon and decreased plasma glucose, both 10 and 30 min following injection. The results suggest an effect of dermorphin on the endocrine pancreas. Since dermorphin has been shown to be present in rat and porcine tissues, it may have a role in the regulation of glucose metabolism.
Characterizing a new rat model of chronic pain after spine surgery.
Chronic pain after spine surgery (CPSS) is a complex disorder characterized by multifactorial pathogenesis that occurs in 8%-40% of patients undergoing lumbar spine surgery. We aimed to develop a rat model that mimics clinical CPSS conditions by taking two sequential surgical procedures. Step 1: A plastic rod was inserted into the left L5 intervertebral foramen to produce a steady compression on the dorsal root ganglion (DRG) and the spinal nerve, a common cause of low back pain (LBP). Step 2: The rod was removed after 7 days when rats exhibited mechanical and heat hypersensitivity in the ipsilateral hindpaw, followed by a full L5 laminectomy to mimic spine decompression surgery in LBP patients. The retention of the rod induced a prolonged LBP-like behavior but was quickly resolved after rod removal without laminectomy. However, rats that received laminectomy after rod removal developed heightened mechanical and heat sensitivity in the hindpaw, impaired gait, and reduced spontaneous exploration activity, indicating CPSS. Patch clamp recording revealed a significant augmentation in the intrinsic excitability of small-diameter DRG neurons in CPSS rats. Administration of Dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA, 5 mg /kg, i.p.), a peripherally acting mu-opioid receptor (MOR)-preferred agonist, attenuated pain hypersensitivity, capsaicin-induced [Ca2+]i rising and the increased intrinsic excitability of DRG neurons from CPSS rats. Our findings suggest that this new model, which mirrors the nature of CPSS developed in patients, may be useful for future studies of the underlying mechanisms.
Pharmacokinetics and pharmacodynamics of dermorphin in the horse.
Dermorphin is a μ-opioid receptor-binding peptide that causes both central and peripheral effects following intravenous administration to rats, dogs, and humans and has been identified in postrace horse samples. Ten horses were intravenously and/or intramuscularly administered dermorphin (9.3 ± 1.0 μg/kg), and plasma concentration vs. time data were evaluated using compartmental and noncompartmental analyses. Data from intravenous administrations fit a 2-compartment model best with distribution and elimination half-lives (harmonic mean ± pseudo SD) of 0.09 ± 0.02 and 0.76 ± 0.22 h, respectively. Data from intramuscular administrations fit a noncompartmental model best with a terminal elimination half-life of 0.68 ± 0.24 (h). Bioavailability following intramuscular administration was variable (47-100%, n = 3). The percentage of dermorphin excreted in urine was 5.0 (3.7-10.6) %. Excitation accompanied by an increased heart rate followed intravenous administration only and subsided after 5 min. A plot of the mean change in heart rate vs. the plasma concentration of dermorphin fit a hyperbolic equation (simple Emax model), and an EC(50) of 21.1 ± 8.8 ng/mL was calculated. Dermorphin was detected in plasma for 12 h and in urine for 48 or 72 h following intravenous or intramuscular administration, respectively.
Peripheral mu-opioid receptor activation by dermorphin [D-Arg2, Lys4] (1-4) amide alleviates behavioral and neurobiological aberrations in rat model of chemotherapy-induced neuropathic pain.
Paclitaxel, a frequently utilized chemotherapeutic agent, often gives rise to severe and distressing sensory neuropathy in patients undergoing chemotherapy. Unfortunately, current therapeutics for chemotherapy-induced neuropathic pain (CINP) demonstrate limited effectiveness and are burdened with the potential for central side effects such as sedation, respiratory depression, cognitive impairment, and addiction, posing substantial clinical challenges. In light of these limitations, present study is designed to investigate the therapeutic potential of Dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a preferential peripherally acting mu-opioid receptor agonist, in rat model of CINP. The primary objective was to assess the analgesic properties of DALDA and elucidate the underlying mechanisms governing its therapeutic activity. Our findings revealed that DALDA treatment significantly ameliorated paclitaxel-induced evoked and spontaneous ongoing pain in rats without causing drug addiction and other central side effects. Molecular analyses further unveiled that paclitaxel administration resulted in increased expression of TRP channels, NR2B, voltage-gated sodium channels (VGSCs) and neuroinflammatory markers in both the dorsal root ganglion (DRG) and the spinal cord (L4-L5 region) of rats. DALDA treatment significantly downregulated ion channels (TRPs, VGSCs) and NR2B expressions, concomitant with the inhibition of microglial activation, resulting in the suppression of oxido-nitrosative stress and neuroinflammatory cascade. Findings from the current study suggests that peripheral mu-opioid receptors may offer a potential target for the treatment of patients suffering from CINP, offering new avenues for improved pain relief while minimizing central side effects.
χ-Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds.
Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these constricted scaffolds into the N-terminal tetrapeptide of dermorphin (i.e., H-Tyr-d-Ala-Phe-Gly-NH2) induced significant shifts in binding affinity, selectivity, and in vitro activity at the μ- and δ-opioid receptors (MOP and DOP, respectively). A reported constrained μ-/δ-opioid lead tetrapeptide H-Dmt-d-Arg-Aba-Gly-NH2 was modified through application of various constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when the aromatic moieties were turned toward the C-terminus of the peptide sequences, (partial) (ant)agonism at MOP and weak (ant)agonism at DOP were noticed, whereas the incorporation of the 1-Ana residue led toward balanced low nanomolar MOP/DOP binding and in vitro agonism.
Increased agonist affinity at the μ-opioid receptor induced by prolonged agonist exposure.
Prolonged exposure to high-efficacy agonists results in desensitization of the μ-opioid receptor (MOR). Desensitized receptors are thought to be unable to couple to G-proteins, preventing downstream signaling; however, the changes to the receptor itself are not well characterized. In the current study, confocal imaging was used to determine whether desensitizing conditions cause a change in agonist-receptor interactions. Using rapid solution exchange, the binding kinetics of fluorescently labeled opioid agonist, dermorphin Alexa594 (derm A594), to MORs was measured in live cells. The affinity of derm A594 binding increased after prolonged treatment of cells with multiple agonists that are known to cause receptor desensitization. In contrast, binding of a fluorescent antagonist, naltrexamine Alexa594, was unaffected by similar agonist pretreatment. The increased affinity of derm A594 for the receptor was long-lived and partially reversed after a 45 min wash. Treatment of the cells with pertussis toxin did not alter the increase in affinity of the derm A594 for MOR. Likewise, the affinity of derm A594 for MORs expressed in mouse embryonic fibroblasts derived from arrestin 1 and 2 knock-out animals increased after treatment of the cells with the desensitization protocol. Thus, opioid receptors were "imprinted" with a memory of prior agonist exposure that was independent of G-protein activation or arrestin binding that altered subsequent agonist-receptor interactions. The increased affinity suggests that acute desensitization results in a long-lasting but reversible conformational change in the receptor.
Structure-activity relationships of dermorphin synthetic analogues.
A number of dermorphin analogues have been reviewed for antinociceptive activity after systemic administration. Analgesic peptides as potent as or more potent than dermorphin are found among the hepta- and tetrapeptide analogues, probably because of enhanced stability towards carboxyldipeptidases imparted by the Pro6 and D-Ala2 residues lying on the right and left side, respectively, of the point of cleavage. Hexapeptide analogues are practically inactive. [D-Arg2]derivatives are very potent, particularly in the tetrapeptide series. In binding studies dermorphin behaves as a pure opioid agonist, with a marked affinity and selectivity for the mu-type opioid receptors. In the heptapeptide analogues a direct correlation seems to exist between mu-selectivity and "sodium shift," and between lipophilicity and MVD/GPI potency ratio.
Intracerebroventricular Administration of Dermorphin-Dynorphin Analogs Producing Antidepressant-Like Effects through Activation of μ1- and κ-Opioid Receptors in Mice.
The opioid system in the central nervous system regulates depressive-like behavior in animals. Opioid receptors and their endogenous ligands have been focused on as novel therapeutic targets for depression. We synthesized dermorphin (DRM)-dynorphin (DYN) analogs (DRM-DYN001-004) using the message-address concept concerning interactions with opioid receptors. It has previously been reported that DRM-DYN001, 003, and 004 have shown high affinities for μ- and κ-opioid receptors, whereas all analogs had a lower affinity for the δ-opioid receptor than for other receptors using a receptor binding assay. However, it remains unknown whether these analogs show antidepressant-like effects in mice. We examined the effects of DRM-DYN analogs on the duration of immobile behavior in a tail suspension test. Intracerebroventricular administration of DRM-DYN001 in mice shortened the duration of immobile behavior, but did not affect locomotion. The DRM-DYN001-induced antidepressant-like effect was inhibited by co-administration of naloxone (non-selective opioid receptor antagonist), naloxonazine (selective μ1-opioid receptor antagonist), or nor-BNI (κ-opioid receptor antagonist), but not naltrindole (δ-opioid receptor antagonist). These data suggest that DRM-DYN001 exerts an antidepressant-like effect via activation of the central μ1- and κ-opioid receptors in mice and may represent a new lead peptide for further investigation for the development of novel therapeutic approaches for depression.
Characterisation and visualisation of [3H]dermorphin binding to mu opioid receptors in the rat brain. Combined high selectivity and affinity in a natural peptide agonist for the morphine (mu) receptor.
Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1. Dermorphin was tested for its relative affinity to mu-, delta- and chi-opioid receptors by determining its potency in displacing the selective mu-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol = glycinol), the prototypic delta-receptor ligand [3H]Tyr-DPen-Gly-Phe-DPen (where DPen = beta, beta-dimethylcysteine) and the chi ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM, 62 nM and greater than 5000 nM respectively for mu, delta and chi sites, indicating a selectivity ratio Ki(delta)/Ki(mu) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity mu-agonist available, exhibited a selectivity ratio of 84. 2. Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd = 0.46 nM; Bmax = 92 fmol/mg membrane protein). 3. Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other mu-selective radiolabeled ligands. 4. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other mu probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than beta-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (mu) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
Differential involvement of mu 1-opioid receptors in dermorphin tetrapeptide analogues-induced antinociception.
The involvement of putative mu(1)-opioid receptors in the antinociception induced by the dermorphin tetrapeptide analogues Try-D-Arg-Phe-beta-Ala (TAPA) and Tyr-D-Arg-Phe-beta-Ala-NH(2) (TAPA-NH(2)) was determined in mice, using a tail-pressure test and a formalin test. TAPA and TAPA-NH(2) injected i.c.v. and i.t. produced dose-dependent antinociception in both assays. In the tail-pressure test, the antinociception induced by i.c.v. or i.t. injected TAPA, but not TAPA-NH(2), was significantly attenuated by pretreatment with naloxonazine, a selective antagonist for putative mu(1)-opioid receptors. Moreover, naloxonazine also significantly attenuated the antinociception induced by i.c.v. injected TAPA, but not TAPA-NH(2), in the formalin test. In contrast, the antinociception induced by both TAPA and TAPA-NH(2) given i.t. was significantly attenuated by pretreatment with naloxonazine in the formalin test. The present results suggest that TAPA and TAPA-NH(2) should be considered selective agonists for putative mu(1)- and mu(2)-opioid receptors, respectively. The C-terminal amidation of TAPA-NH(2) may be critical for distinguishing between putative mu(1)- and mu(2)-opioid receptors.
Differential opioid receptor expression and biochemical coupling profiles on glia.
The opioid receptor family comprises classical; MOP (mu/µ), KOP (kappa/k), DOP(delta/δ) receptors along with non-classical Nociceptin/Orphanin FQ (N/OFQ) peptide receptor (NOP). Expression on glial cells is controversial where there is a role for glia and opioids in pain processing and immunomodulation. Here we detail expression and function of opioid receptors in a wide range of established and primary glia. Namely, 1321N1 human astrocytoma, C6 rat astrocytoma, mouse primary astrocytes, human MO3.13 oligodendrocyte like cells, human HOG oligodendrocytoma, mouse EOC-20 microglia and mouse primary microglia. We used (i)-PCR for mRNA, (ii)-radioligand binding and (iii)-fluorescent probe binding for expression, (iv)-MAPK for activation and (v)-scratch assay for migration. MOP mRNA was detected in C6 and mouse primary astrocytes only. NOP mRNA was found in 1321N1, C6 and mouse primary astrocytes along with MO3.13 and HOG cells. There was variable expression of DOP and KOP; this was not probed further. Surprisingly, microglia (EOC-20 or primary mouse) did not express mRNA for opioid receptors unless treated with media from astrocytes. mRNA expression profile was generally matched by [3H]-DPN binding to classical and [3H]-N/OFQ binding to non-classical opioid receptors. In addition, C6, mouse primary and 1321N1 astrocytes along with MO3.13 and HOG cells bound the fluorescent NOP probe N/OFQATTO594. C6 and primary mouse astrocytes also bound the MOP fluorescent probe DermorphinATTO488. Where expressed both MOP and NOP supported Endomorphin-1 and N/OFQ-induced phosphorylation of ERK1/2 and reduced scratch migration. In microglia, astrocyte modulated opioid receptor expression could influence central opioid-immunomodulation and pain processing; further studies are required.
Dermorphin [D-Arg2, Lys4] (1-4) Amide Attenuates Burn Pain by Inhibiting TRPV1/NR2B Mediated Neuroinflammatory Signalling.
Burn injury-induced chronic pain is a highly debilitating condition that profoundly impacts the well-being of military veterans and the general population. Current pain management for burn injured patients mainly relies on central opioids, often causing sedation, addiction, and physical dependence. This study aims to investigate the effects of Dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a peripherally acting μ-opioid receptor (MOR) agonist in an animal model of burn injury-induced chronic pain while unravelling the underlying mechanisms. Soldering apparatus was used to induce burn pain in Sprague Dawley rats followed by testing for both evoked and ongoing pain behaviours. Molecular investigations were performed for TRPV1, NR2B, neuro-inflammatory and glia cell markers (TNF-α, IL-6, Iba-1 & ICAM-1), and neuropeptides (CGRP), using western blotting and RT-PCR analysis. Burn-injured rats exhibited significant hypersensitivity to mechanical, thermal, and cold stimuli, along with severe ongoing pain. Systemic administration of DALDA significantly reduced evoked pain behaviour in a dose-dependent manner (1, 3, and 10 mg/kg), with the 10 mg/kg s.c. dose effectively alleviating spontaneous pain without causing drug addiction. DALDA also restored antioxidant enzyme levels in the sciatic nerve and downregulated burn injury-induced molecular changes, including TRPV1, NR2B, and CGRP, as well as neuroinflammatory markers such as TNF-α and IL-6 in the DRG and spinal cord of rats. Activation of peripheral μ-opioid receptors efficiently mitigates both evoked and spontaneous pain in burn-injured rats, without causing central nervous system (CNS)-related side effects. Findings from the present study demonstrate a promising approach to mitigate burn pain, overcoming the limitations associated with centrally acting opioids.
Dermorphin-based potential affinity labels for mu-opioid receptors.
Dermorphin and [Lys7]dermorphin, selective micro -opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the 'message' or 'address' sequences as potential peptide-based affinity labels for micro -receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe3 and Phe5 was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine-containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing micro - and delta-opioid receptors. In dermorphin, introduction of the electrophilic groups in the 'message' domain lowered the binding affinity by > 1000-fold; only [Phe(p-NH2)3]dermorphin retained nanomolar affinity for micro -receptors. Modifications in the 'address' region of both dermorphin and [Lys7]dermorphin were relatively well tolerated. In particular, [Phe(p-NH2)5,Lys7]dermorphin showed similar affinity to dermorphin, with almost 2-fold higher selectivity for micro -receptors. [Phe(p-NHCOCH2Br)5]- and [Phe(p-NHCOCH2Br)5,Lys7]dermorphin exhibited relatively high affinity (IC50 = 27.7 and 15.1 nm, respectively) for micro -receptors. However, neither of these peptides inhibited [3H]DAMGO binding in a wash-resistant manner.
Innovative Opioid Peptides and Biased Agonism: Novel Avenues for More Effective and Safer Analgesics to Treat Chronic Pain.
Chronic pain states are clinically relevant and yet unsolved conditions impacting on quality of life and representing an important social and economic burden; these diseases are poorly treated with the currently available drugs, being urgent the need of innovative analgesics. In this frame, novel analogues of endomorphin-1 and dermorphin emerge as promising starting points to develop innovative, more effective analgesics to treat neuropathic pain. An extensive and structured search of bibliographic databases for peer-reviewed research literature was undertaken using focused review questions; all the retrieved papers were published on prestigious international journals by the experts of the field and were carefully analyzed to collect all the information and data necessary to the conceptual framework of this review. One hundred papers were included in this review; forty-one defined the up-to-date findings on neuropathic pain etiopathogenesis and its currently available treatment options. Thirty-five papers described all the advantages and drawbacks of using endomorphin-1 (23) or dermorphin (12) in the frame of neuropathic pain and outlined the most relevant advances in developing endomorphin-1 and dermorphin analogs useful as potential, innovative analgesics. Twenty-four papers provided the latest insights into exploiting biased agonism at opioid receptor as an innovative strategy to develop more effective and safer analgesics. This review reports that innovative opioid peptides will be of great help in better understanding the multifaceted scenario of neuropathic pain treatment, providing very interesting opportunities for the identification of novel and more effective opioid analgesics to be employed as medications.
Dermorphin gene sequence peptide with high affinity and selectivity for delta-opioid receptors.
Skin of the frog Phyllomedusa sauvagei contains a cDNA sequence that codes for the selective mu-receptor peptide dermorphin and a new heptapeptide we have designated as dermorphin gene-associated peptide (DGAP). Investigation of the opioid receptor binding characteristics of synthetic DGAP and [D-Met2]DGAP revealed that the latter peptide had high affinity and selectivity for delta-type opioid receptors in rat brain synaptosomes. The IC50 values for DGAP on mu- and delta-receptors were only 28 microM and 670 nM, respectively, while that for [D-Met2]DGAP was 0.80 nM for delta-receptors and greater than 1 microM for mu-receptors yielding a very high delta selectivity ratio (SR) of 1345. In comparison, the SR values for [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Pen2,5]enkephalin, ligands which are considered to be specific for delta-receptors, were 20, 42, and 301, respectively. Dermorphin, which contains a D-Ala2 residue and is a selective mu-receptor ligand (Lazarus, L.H., Guglietta, A., Wilson, W.E., Irons, B.J., and de Castiglione, R. (1989) J. Biol. Chem. 264, 354-362), exhibits a SR of 0.0055 similar to that for the conventional mu-agonist [D-Ala2,NMePhe4,Gly-ol]enkephalin (0.0040). This finding that frog skin cDNA contains the information to code for dermorphin and DGAP, or the presumed [D-Met2]DGAP molecule, which are among the most selective high affinity opioid ligands described for mu- and delta-receptors, may permit new insight into the design of future opioid receptor agonists and antagonists.
Dermorphin analog Tyr-D-Arg2-Phe-sarcosine-induced opioid analgesia and respiratory stimulation: the role of mu 1-receptors?
Tyr-D-Arg2-Phe-sarcosine4 (TAPS), a mu-selective tetrapeptide analog of dermorphin, induced sustained antinociception and stimulated ventilatory minute volume (MV) at the doses of 3 to 100 pmol i.c.v. The doses of 30 and 100 pmol i.c.v. induced catalepsy. The effect of TAPS on MV was in negative correlation with the dose and the maximal response was achieved by the lowest (3 pmol) dose (+63 +/- 23%, P < .05). Morphine, an agonist at both mu 1 and mu 2 sites, at a dose of 150 nmol i.c.v. (equianalgesic to 100 pmol of TAPS decreased the MV by 30%, due to a decrease in ventilatory tidal volume. The antinociceptive effect of TAPS was antagonized by naloxone and the mu 1 receptor antagonist, naloxonazine. Naloxonazine also attenuated the catalepsy produced by 100 pmol of TAPS i.c.v. and the respiratory stimulation produced by 3 pmol of TAPS i.c.v. Pretreatment with 30 pmol of TAPS antagonized the respiratory depression induced by the mu opioid agonist dermorphin (changes in MV after dermorphin alone at 1 or 3 nmol were -22 +/- 10% and -60 +/- 9% and, after pretreatment with TAPS, +44 +/- 11% and -18 +/- 5%, respectively). After combined pretreatment with naloxonazine and TAPS, 1 nmol of dermorphin had no significant effect on ventilation. In contrast, pretreatment with a low respiratory stimulant dose (10 pmol i.c.v.) of dermorphin did not modify the effect of 1 nmol of dermorphin. In conclusion, the antinociceptive, cataleptic and respiratory stimulant effects of TAPS appear to be a related to its agonist action at the mu 1 opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
2',6'-dimethylphenylalanine: a useful aromatic amino Acid surrogate for tyr or phe residue in opioid peptides.
Two aromatic amino acids, Tyr(1) and Phe(3) or Phe(4), are important structural elements in opioid peptides because they interact with opioid receptors. The usefulness of an artificial amino acid residue 2',6'-dimethylphenylalanine (Dmp) was investigated as an aromatic amino acid surrogate for several opioid peptides, including enkephalin, dermorphin, deltorphin, endomorphin, dynorphin A, and nociceptin peptides. In most peptides, substitutions of Phe(3) by a Dmp residue produced analogs with improved receptor-binding affinity and selectivity, while the same substitution of Phe(4) induced markedly reduced receptor affinity and selectivity. Interestingly, replacement of Tyr(1) by Dmp produced analogs with unexpectedly high affinity or produced only a slight drop in receptor affinity and bioactivity for most peptides. Thus, Dmp is also a useful surrogate for the N-terminal Tyr residue in opioid peptides despite the lack of a phenolic hydroxyl group, which is considered necessary for opioid activity. The Dmp(1)-substituted analogs are superior to 2',6'-dimethyltyrosine (Dmt)(1)-substituted analogs for high receptor selectivity since the latter generally have poor receptor selectivity. Thus, Dmp is very useful as an aromatic amino acid surrogate in opioid peptides and may be useful for developing other novel peptide mimetics with high receptor specificity.
Identification of a D-alanine-containing polypeptide precursor for the peptide opioid, dermorphin.
The naturally occurring amphibian skin peptides dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) are highly potent and selective agonists at the mu- and the delta-opioid receptors, respectively. For peptides synthesized by animal cells, they have a rather peculiar structural feature of containing a D-amino acid residue in their sequence which imparts biological activity on them. The cloned cDNA encoding the prodermorphin precursor contains the usual alanine and methionine codons at positions where D-alanine and D-methionine are present in the mature products. In this study, dermorphin precursor was characterized in extracts from amphibian skin by antisera recognizing distinct epitopes within the predicted structure of pro-dermorphin. Proteolytic digestion of purified endogenous pro-dermorphin generated a peptide containing a D-alanine in position 2, identified as prepro-dermorphin-(80-89), i.e. Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-Gly-Glu-Ala. In addition, analysis of skin extracts by enzyme immunoassays coupled to high performance liquid chromatography separations revealed the presence of, besides dermenkephalin, novel dermenkephalin-related peptides, i.e. [L-Met2]dermenkephalin, dermenkephalin-OH, and [Met(O)6]dermenkephalin. [L-Met2]dermenkephalin was present in frog skin in a concentration of about 100 times that of dermenkephalin. These observations confirm that, despite the presence of D-amino acid residues, dermorphin and dermenkephalin are genuine products of post-translational processing of a ribosomally made precursor. They suggest that D-Ala and D-Met develop from a dehydrogenation/hydrogenation stereoinversion of their corresponding L isomers incorporated into pro-dermorphin, a process that occurs with low efficiency at an early stage of biosynthesis.
Structural Characterization of Monomers and Oligomers of D-Amino Acid-Containing Peptides Using T-Wave Ion Mobility Mass Spectrometry.
The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer's disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers. Graphical Abstract ᅟ.
Pharmacological characterization of the dermorphin analog [Dmt(1)]DALDA, a highly potent and selective mu-opioid peptide.
The dermorphin-derived peptide [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2)), labels mu-opioid receptors with high affinity and selectivity in receptor binding assays. In mouse, radiant heat tail-flick assay [Dmt(1)]DALDA produced profound spinal and supraspinal analgesia, being approximately 5000- and 100-fold more potent than morphine on a molar basis, respectively. When administered systemically, [Dmt(1)]DALDA was over 200-fold more potent than morphine. Pharmacologically, [Dmt(1)]DALDA was distinct from morphine. [Dmt(1)]DALDA displayed no cross-tolerance to morphine in the model used and it retained supraspinal analgesic activity in morphine-insensitive CXBK mice. Supraspinally, it also differed from morphine in its lack of sensitivity towards naloxonazine. Finally, in antisense mapping studies, [Dmt(1)]DALDA was insensitive to MOR-1 exon probes that reduced morphine analgesia, implying a distinct receptor mechanism of action. Thus, [Dmt(1)]DALDA is an interesting and extraordinarily potent, systemically active peptide analgesic, raising the possibility of novel approaches in the design of clinically useful drugs.
Respiratory and cardiovascular effects of the mu-opioid receptor agonist [Lys7]dermorphin in awake rats.
1. Changes in respiratory variables, arterial blood pressure and heart rate were studied in awake rats after injection of the opioid peptide [Lys7]dermorphin and its main metabolites, [1-5]dermorphin and [1-4]dermorphin. 2. Fifteen minutes after injection, doses of [Lys7]dermorphin producing antinociception (i.c.v., 36-120 nmol; s.c., 0.12-4.7 micromol kg(-1)) significantly increased respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. This respiratory stimulation was reversed to depression by the 5-HT receptor antagonist ritanserin (2 mg kg(-1), s.c.), was blocked by naloxone (0.1 mg kg(-1), s.c.), significantly reduced by the mu1 opioid receptor antagonist naloxonazine (10 mg kg(-1), s.c., 24 h before) but unaffected by peripherally acting opioid antagonist naloxone methyl bromide (3 mg kg(-1), s.c.). Forty five minutes after injection, doses of the peptide producing catalepsy (s.c., 8.3-14.2 micromol kg(-1), i.c.v., 360 nmol) significantly reduced respiratory frequency and volume of rats breathing air and blocked the hypercapnic ventilator response of rats breathing from 4% to 10% CO2. I.c.v. administration of [1-5]dermorphin and [1-4]dermorphin (from 36 to 360 nmol) never stimulated respiration but significantly reduced basal and CO2-stimulated ventilation. Opioid respiratory depression was only antagonized by naloxone. 3. In awake rats, [Lys7]dermorphin (0.1-1 mg kg(-1), s.c.) decreased blood pressure. This hypotensive response was abolished by naloxone, reduced by naloxone methyl bromide and unaffected by naloxonazine. 4. In conclusion, the present study indicates that analgesic doses of [Lys7]dermorphin stimulate respiration by activating central mu1 opioid receptors and this respiratory stimulation involves a forebrain 5-hydroxytryptaminergic excitatory pathway.
DAMGO, a mu-opioid receptor selective agonist, distinguishes between mu- and delta-opioid receptors around their first extracellular loops.
The structural basis of mu-opioid receptor (OPR) for the specificity in its ligand binding was investigated using chimeric mu/delta-OPRs. Replacement of the region around the first extracellular loop of delta-OPR with the corresponding region of mu-OPR gave the resultant chimeric receptor the similar affinity to DAMGO compared with the native mu-OPR. The reciprocal replacement deprived the high affinity to DAMGO from mu-OPR. These results indicate that the difference(s) in the structure around the first extracellular loop is critical for DAMGO to distinguish between mu- and delta-OPRs. Furthermore, displacement studies revealed that this region is partly involved in the discrimination between mu- and delta-OPRs by other peptidic mu-selective ligands, such as dermorphin, morphiceptin and CTOP, but not by non-peptidic ligands, such as morphine and naloxone.
Caged naloxone reveals opioid signaling deactivation kinetics.
The spatiotemporal dynamics of opioid signaling in the brain remain poorly defined. Photoactivatable opioid ligands provide a means to quantitatively measure these dynamics and their underlying mechanisms in brain tissue. Although activation kinetics can be assessed using caged agonists, deactivation kinetics are obscured by slow clearance of agonist in tissue. To reveal deactivation kinetics of opioid signaling we developed a caged competitive antagonist that can be quickly photoreleased in sufficient concentrations to render agonist dissociation effectively irreversible. Carboxynitroveratryl-naloxone (CNV-NLX), a caged analog of the competitive opioid antagonist NLX, was readily synthesized from commercially available NLX in good yield and found to be devoid of antagonist activity at heterologously expressed opioid receptors. Photolysis in slices of rat locus coeruleus produced a rapid inhibition of the ionic currents evoked by multiple agonists of the μ-opioid receptor (MOR), but not of α-adrenergic receptors, which activate the same pool of ion channels. Using the high-affinity peptide agonist dermorphin, we established conditions under which light-driven deactivation rates are independent of agonist concentration and thus intrinsic to the agonist-receptor complex. Under these conditions, some MOR agonists yielded deactivation rates that are limited by G protein signaling, whereas others appeared limited by agonist dissociation. Therefore, the choice of agonist determines which feature of receptor signaling is unmasked by CNV-NLX photolysis.
Mu and delta opioid receptor regulation of pro-opiomelanocortin peptide secretion from the rat neurointermediate pituitary in vitro.
We investigated the ability of selective opioid agonists and antagonists to influence pro-opiomelanocortin peptide secretion from the rat neurointermediate lobe in vitro. The mu-opioid agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin) significantly stimulated beta-endorphin and alpha-melanocyte-stimulating hormone release relative to controls early (30 min) in the incubation period. Similar effects on beta-endorphin secretion were observed with the selective mu-opioid agonist dermorphin. The delta-opioid receptor agonist DPDPE ([D-Pen(2,5)]enkephalin) weakly inhibited beta-endorphin secretion relative to controls while the kappa-opioid receptor agonist U50488 had no effect. The mu-opioid selective antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)) inhibited basal beta-endorphin secretion while kappa- and delta-opioid receptor antagonists had no effect. Our data support a role for local mu-opioid receptor control of intermediate lobe pro-opiomelanocortin peptide secretion. Peptide secretion from melanotropes appears to be tonically stimulated by activation of mu-opioid receptors in the absence of intact neuronal innervation to the intermediate lobe.
In vitro and in vivo pharmaco-dynamic study of the novel fentanyl derivatives: Acrylfentanyl, Ocfentanyl and Furanylfentanyl.
Fentanyl derivatives (FENS) belongs to the class of Novel Synthetic Opioids that emerged in the illegal drug market of New Psychoactive Substances (NPS). These substances have been implicated in many cases of intoxication and death with overdose worldwide. Therefore, the aim of this study is to investigate the pharmaco-dynamic profiles of three fentanyl (FENT) analogues: Acrylfentanyl (ACRYLF), Ocfentanyl (OCF) and Furanylfentanyl (FUF). In vitro, we measured FENS opioid receptor efficacy, potency, and selectivity in calcium mobilization studies performed in cells coexpressing opioid receptors and chimeric G proteins and their capability to promote the interaction of the mu receptor with G protein and β-arrestin 2 in bioluminescence resonance energy transfer (BRET) studies. In vivo, we investigated the acute effects of the systemic administration of ACRYLF, OCF and FUF (0.01-15 mg/kg i.p.) on mechanical and thermal analgesia, motor impairment, grip strength and cardiorespiratory changes in CD-1 male mice. Opioid receptor specificity was investigated in vivo using naloxone (NLX; 6 mg/kg i.p) pre-treatment. In vitro, the three FENS were able to activate the mu opioid receptor in a concentration dependent manner with following rank order potency: FUF > FENT=OCF > ACRYLF. All compounds were able to elicit maximal effects similar to that of dermorphin, with the exception of FUF which displayed lower maximal effects thus behaving as a partial agonist. In the BRET G-protein assay, all compounds behaved as partial agonists for the β-arrestin 2 pathway in comparison with dermorphin, whereas FUF did not promote β-arrestin 2 recruitment, behaving as an antagonist. In vivo, all the compounds increased mechanical and thermal analgesia with following rank order potency ACRYLF = FENT > FUF > OCF and impaired motor and cardiorespiratory parameters. Among the substances tested, FUF showed lower potency for cardiorespiratory and motor effects. These findings reveal the risks associated with the use of FENS and the importance of studying the pharmaco-dynamic properties of these drugs to better understand possible therapeutic interventions in the case of toxicity.
mu-1 opioid receptor stimulation decreases body temperature in conscious, unrestrained neonatal rats.
The influence of mu-selective opioid agonists on neonatal thermoregulatory mechanisms has received little attention. Opioid treatment in adult subjects can cause either hyper- or hypothermia, depending on the experimental conditions, the strain of rat used, and the dose and route of administration of the drug. The present study assessed the effect of two mu opioid agonists on body temperature in neonatal Wistar rats aged 2 to 13 days. Rat pups were administered either saline or one of the two mu-selective opioid agonists, dermorphin (0.4 mg/kg) or fentanyl (0.06 mg/kg), by subcutaneous injection. Continuous rectal temperatures were measured both prior to and following drug or saline injection in freely moving, conscious animals. Ambient temperature in a plethysmograph chamber was maintained within or close to the thermoneutral zone for pups (32 degrees C). To distinguish between mu-1 and mu-2 effects, all animals received either saline or 10 mg/kg of the irreversible mu-1 antagonist naloxonazine (NALZ) 1 day prior to agonist administration. NALZ on its own had no effect on body temperature. Dermorphin and fentanyl both caused a fall in body temperature in pups of all age groups. The temperature decreases ranged from 0.8 degrees -2.2 degrees C. These opioid-induced changes were inhibited by NALZ pretreatment. Although there was no evidence for endogenous mu-1 opioid activity, this study indicated that stimulation of mu-1 opioid receptors causes a decrease in body temperature in conscious, unrestrained neonatal rats under or close to thermoneutral conditions.
Mu and kappa opioid system interactions in the expression of acute opioid dependence in isolated guinea-pig ileum.
In vivo studies have suggested that the kappa opioid system can partially inhibit the development of physical dependence to mu agonists. Vice versa, activation of mu receptors may inhibit the expression of physical dependence to kappa agonists. We studied mu-kappa interactions in the isolated guinea-pig ileum (GPI). In the isolated GPI briefly exposed to mu or kappa agonists the addition of the respective antagonists precipitated a withdrawal contracture. After a first withdrawal response, however, some tissues failed to exhibit subsequent mu or kappa withdrawal contractures. A withdrawal contracture to the selective mu antagonist, cyprodime, after repeated exposures to a selective mu agonist, dermorphin, was restored by nor-binaltorphimine (BNI), a selective kappa antagonist. Vice versa, after repeated exposures to the kappa agonist, U-50,488H, cyprodime restored tissue responsiveness to BNI. Tissues repeatedly exposed to dermorphin and washed after each exposure contracted to the addition of BNI. Tissues repeatedly exposed to U-50,488H contracted on the addition of cyprodime. These findings strongly suggest that exogenous agonist-elicited stimulation of the mu (or kappa) opioid system indirectly activates the endogenous kappa (or mu) system. The indirectly-activated endogenous system inhibits the withdrawal response to the exogenously-stimulated opioid system. In isolated GPI the mu and kappa opioid systems thus appear to interact, regulating each other.
Mu opioid receptor expressing neurons in the rostral ventromedial medulla are the source of mechanical hypersensitivity induced by repeated restraint stress.
Repeated exposure to psychophysical stress often causes an increase in sensitivity and response to pain. This phenomenon is commonly called stress-induced hyperalgesia (SIH). Although psychophysical stress is a well-known risk factor for numerous chronic pain syndromes, the neural mechanism underlying SIH has not yet been elucidated. The rostral ventromedial medulla (RVM) is a key output element of the descending pain modulation system. Descending signals from the RVM have a major impact on spinal nociceptive neurotransmission. In the present study, to clarify changes in the descending pain modulatory system in rats with SIH, we examined the expression of Mu opioid receptor (MOR) mRNA, MeCP2 and global DNA methylation in the RVM after repeated restraint stress for 3 weeks. Additionally, we microinjected neurotoxin dermorphin-SAP into the RVM. The repeated restraint stress for 3 weeks induced mechanical hypersensitivity in the hind paw, a significant increase in the expression of MOR mRNA and MeCP2, and a significant decrease in global DNA methylation in the RVM. The MeCP2 binding to MOR gene promoter in the RVM was significantly decreased in rats with repeated restraint stress. Furthermore, microinjection of dermorphin-SAP into the RVM prevented the mechanical hypersensitivity induced by repeated restraint stress. Although, because of the lack of specific antibody to MOR, we could not show a quantitative analysis in the number of MOR-expressing neurons after the microinjection, these results suggest that MOR-expressing neurons in the RVM induce SIH after repeated restraint stress.
In vitro and in vivo opioid activity of [DPro(6)]dermorphin, a new dermorphin analogue.
To study the effects of inducing stereo-chemical modifications in the structure of dermorphin (DM) so as to improve its mu-opioid receptor affinity and its resistance to C-terminal enzymatic degradation, in the Institute of Molecular Genetics of Moscow, we synthesized a new DM analogue ([DPro(6)]DM) and analyzed the changes induced in the biological activities of DM by substituting the Pro(6) residue with DPro(6). We compared the activity of the new DM analogue and DM in in vitro assays and in in vivo tests of analgesia, thermoregulation, heart rate recordings, and gastrointestinal motility in rats. In the in vitro tests, guinea pig ileum (GPI) and mouse vas deferens (MVD), although the opioid activities of [DPro(6)]DM indicated that the peptide was always less potent than DM, its lower IC(50) ratios (mu/delta) showed that it had higher mu-opioid receptor selectivity. In the in vivo analgesic test, [DPro(6)]DM, when injected intraperitoneally (i.p.) (0.5-5 and 10mg/kg) in rats, had the same antinociceptive efficacy as DM and when injected intranasally (i.n.) (0.005 and 0.02 mg/kg) it induced a more stable and long-lasting analgesia than DM (the AUC was about 91% higher for [DPro(6)]DM than for DM). Moreover, these data confirm that the intranasal route is advantageous for peripheral drug administration. In the heart rate study, [DPro(6)]DM and DM (0.5mg/kg, i.p.), induced a similar, weak bradycardia. The only difference was that [DPro(6)]DM induced a longer-lasting effect than DM. Conversely, in body temperature regulation [DPro(6)]DM induced weaker inhibitory activity than DM (56% of the DM-induced response); it did so only in a cold environment and at the maximal used dose (0.5mg/kg, i.p.) without inducing vasomotor effects. In the gastrointestinal study, [DPro(6)]DM and DM (0.005, 0.05, and 0.5mg/kg, i.p.) significantly slowed upper gastrointestinal transit of a charcoal meal and inhibited colonic propulsion. Comparison of the ED(50) values of [DPro(6)]DM (0.03 mg/kg) and DM (0.009 mg/kg) showed that the DM analogue was about three times less potent than DM in slowing gastrointestinal and colonic transit. In conclusion, all these data overall suggest that structural maneuvering in the Pro(6)-residue of the DM molecule changes its affinity for mu-opioid receptor subtypes and confirms the usefulness of experimental studies involving structural modifications in obtaining new therapeutic agents.
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