Potentiating TRPA1 by Sea Anemone Peptide Ms 9a-1 Reduces Pain and Inflammation in a Model of Osteoarthritis.

Progressive articular surface degradation during arthritis causes ongoing pain and hyperalgesia that lead to the development of functional disability. TRPA1 channel significantly contributes to the activation of sensory neurons that initiate neurogenic inflammation and mediates pain signal transduction to the central nervous system. Peptide Ms 9a-1 from the sea anemone Metridium senile is a positive allosteric modulator of TRPA1 and shows significant anti-inflammatory and analgesic activity in different models of pain. We used a model of monosodium iodoacetate (MIA)-induced osteoarthritis to evaluate the anti-inflammatory properties of Ms 9a-1 in comparison with APHC3 (a polypeptide modulator of TRPV1 channel) and non-steroidal anti-inflammatory drugs (NSAIDs) such as meloxicam and ibuprofen. Administration of Ms 9a-1 (0.1 mg/kg, subcutaneously) significantly reversed joint swelling, disability, thermal and mechanical hypersensitivity, and grip strength impairment. The effect of Ms 9a-1 was equal to or better than that of reference drugs. Post-treatment histological analysis revealed that long-term administration of Ms9a-1 could reduce inflammatory changes in joints and prevent the progression of cartilage and bone destruction at the same level as meloxicam. Peptide Ms 9a-1 showed significant analgesic and anti-inflammatory effects in the model of MIA-induced OA, and therefore positive allosteric modulators could be considered for the alleviation of OA symptoms.

C-Terminal Region of Caveolin-3 Contains a Stretch of Amino Acid Residues Capable of Diminishing Symptoms of Experimental Autoimmune Encephalomyelitis but Not Rheumatoid Arthritis Modeled in Rats.

A short synthetic peptide from the C-terminal part of the caveolin-3 structure was tested for experimental autoimmune encephalomyelitis (EAE) treatment in rats. The structure-function similarity established between the novel synthetic peptide of pCav3 and the well-known immunomodulator immunocortin determined pCav3's ability to reduce EAE symptoms in Dark Agouti (DA) rats injected with pCav3 (500 µg/kg). pCav3 was found to interfere with the proliferation of lymphocytes extracted from the LNs of DA rats primed with homogenate injection, with IC50 = 0.42 µM (2.35 mcg/mL). pCav3 affected EAE in a very similar manner as immunocortin. The high degree of homology between the amino acid sequences of pCav3 and immunocortin corresponded well with the therapeutic activities of both peptides, as demonstrated on EAE. The latter peptide, possessing a homologous structure to pCav3, was also tested on EAE to explore whether there were structural restrictions between these peptides implied by the MHC-involved cell machinery. Consequently, immunocortin was further examined with a different autoimmune disease model, collagen-induced arthritis (CIA), established in Sprague-Dawley rats. CIA was established using an intentionally different genetic platform than EAE. Based on the results, it was concluded that the effectiveness of pCav3 and immunocortin peptides in EAE rat model was almost identical, but differed in the rat model of rheumatoid arthritis; thus, efficacy may be sensitive to the MHC type of animals used to establish the autoimmune disease model.

Analogs of 6-Bromohypaphorine with Increased Agonist Potency for α7 Nicotinic Receptor as Anti-Inflammatory Analgesic Agents.

Hypaphorines, tryptophan derivatives, have anti-inflammatory activity, but their mechanism of action was largely unknown. Marine alkaloid L-6-bromohypaphorine with EC50 of 80 µM acts as an agonist of α7 nicotinic acetylcholine receptor (nAChR) involved in anti-inflammatory regulation. We designed the 6-substituted hypaphorine analogs with increased potency using virtual screening of their binding to the α7 nAChR molecular model. Fourteen designed analogs were synthesized and tested in vitro by calcium fluorescence assay on the α7 nAChR expressed in neuro 2a cells, methoxy ester of D-6-iodohypaphorine (6ID) showing the highest potency (EC50 610 nM), being almost inactive toward α9α10 nAChR. The macrophages cytometry revealed an anti-inflammatory activity, decreasing the expression of TLR4 and increasing CD86, similarly to the action of PNU282987, a selective α7 nAChR agonist. 6ID administration in doses 0.1 and 0.5 mg/kg decreased carrageenan-induced allodynia and hyperalgesia in rodents, in accord with its anti-inflammatory action. Methoxy ester of D-6-nitrohypaphorine demonstrated anti-oedemic and analgesic effects in arthritis rat model at i.p. doses 0.05-0.26 mg/kg. Tested compounds showed excellent tolerability with no acute in vivo toxicity in dosages up to 100 mg/kg i.p. Thus, combining molecular modelling and natural product-inspired drug design improved the desired activity of the chosen nAChR ligand.

Nocistatin and Products of Its Proteolysis Are Dual Modulators of Type 3 Acid-Sensing Ion Channels (ASIC3) with Algesic and Analgesic Properties.

The neuropeptide nocistatin (NS) is expressed by the nervous system cells and neutrophils as a part of a precursor protein and can undergo stepwise limited proteolysis. Previously, it was shown that rat NS (rNS) is able to activate acid-sensing ion channels (ASICs) and that this effect correlates with the acidic nature of NS. Here, we investigated changes in the properties of rNS in the course of its proteolytic degradation by comparing the effects of the full-size rNS and its two cleavage fragments on the rat isoform 3 ASICs (ASIC3) expressed in X. laevis oocytes and pain perception in mice. The rNS acted as both positive and negative modulator by lowering the steady-state desensitization of ASIC3 at pH 6.8-7.0 and reducing the channel's response to stimuli at pH 6.0-6.9, respectively. The truncated rNSΔ21 peptide lacking 21 amino acid residues from the N-terminus retained the positive modulatory activity, while the C-terminal pentapeptide (rNSΔ30) acted only as a negative ASIC3 modulator. The effects of the studied peptides were confirmed in animal tests: rNS and rNSΔ21 induced a pain-related behavior, whereas rNSΔ30 showed the analgesic effect. Therefore, we have shown that the mode of rNS action changes during its stepwise degradation, from an algesic molecule through a pain enhancer to a pain reliever (rNSΔ30 pentapeptide), which can be considered as a promising drug candidate.

Efficacy of Ficus tikoua Bur. extract in ethylene glycol-induced urolithiasis model in SD rats.

The development of new herbal preparations for the treatment of urolithiasis is an urgent task of medical science. Ficus have attracted the attention of pharmacologists due to a wide range of biological properties, including antioxidant, anti-inflammatory, antibacterial and antifungal activity. We studied the effectiveness of Ficus tikoua Bur. in SD rats in which urolithiasis was induced by 6 weeks of oral administration of ethylene glycol 0.5% ad libitum instead of drinking water. Administration of the extract of Ficus tikoua Bur., as well as comparative drug Cystone® after modeling of urolithiasis lead to the restoration of diuresis and the concentration of inorganic phosphates starting from the 6th week of the experiment. The use of the Ficus tikoua Bur. extract for 6 weeks, both during the modeling of urolithiasis and during the recovery period, led to the restoration of the percentage of lymphocytes in the blood, content of sodium, chlorine and inorganic phosphates in the blood to the control level. Thus, the extract of Ficus tikoua Bur. seems to be a promising drug for effective treatment of the initial stages of the development of urolithiasis.

Analysis of Structural Determinants of Peptide MS 9a-1 Essential for Potentiating of TRPA1 Channel.

The TRPA1 channel is involved in a variety of physiological processes and its activation leads to pain perception and the development of inflammation. Peptide Ms 9a-1 from sea anemone Metridium senile is a positive modulator of TRPA1 and causes significant analgesic and anti-inflammatory effects by desensitization of TRPA1-expressing sensory neurons. For structural and functional analysis of Ms 9a-1, we produced four peptides-Ms 9a-1 without C-terminal domain (abbreviated as N-Ms), short C-terminal domain Ms 9a-1 alone (C-Ms), and two homologous peptides (Ms 9a-2 and Ms 9a-3). All tested peptides possessed a reduced potentiating effect on TRPA1 compared to Ms 9a-1 in vitro. None of the peptides reproduced analgesic and anti-inflammatory properties of Ms 9a-1 in vivo. Peptides N-Ms and C-Ms were able to reduce pain induced by AITC (selective TRPA1 agonist) but did not decrease AITC-induced paw edema development. Fragments of Ms 9a-1 did not effectively reverse CFA-induced thermal hyperalgesia and paw edema. Ms 9a-2 and Ms 9a-3 possessed significant effects and anti-inflammatory properties in some doses, but their unexpected efficacy and bell-shape dose-responses support the hypothesis of other targets involved in their effects in vivo. Therefore, activity comparison of Ms 9a-1 fragments and homologues peptides revealed structural determinants important for TRPA1 modulation, as well as analgesic and anti-inflammatory properties of Ms9a-1.

Derinat® has an immunomodulatory and anti-inflammatory effect on the model of acute lung injury in male SD rats.

To simulate acute lung injury (ALI) in SD male rats they we administered intratracheally with lipopolysaccharide (LPS) followed by hyperventilation of the lungs (HVL), which lead to functional changes in the respiratory system and an increase in the blood serum concentration of inflammatory cytokines. LPS + HVL after 4 h lead to pronounced histological signs of lung damage. We have studied the effectiveness of Derinat® when administered intramuscularly at dose of 7.5 mg/kg for 8 days in the ALI model. Derinat® administration lead to an increase in the concentration of most of the studied cytokines in a day. In the ALI model the administration of Derinat® returned the concentration of cytokines to its original values already 48 h after LPS + HVL, and also normalized the parameters of pulmonary respiration in comparison with animals without treatment. By the eighth day after LPS + HVL, respiratory parameters and cytokine levels, as well as biochemical and hematological parameters did not differ between groups, while histological signs of residual effects of lung damage were found in all animals, and were more pronounced in Derinat® group, which may indicate stimulation of the local immune response. Thus, the administration of Derinat® stimulates the immune response, has a pronounced protective effect against cytokinemia and respiratory failure caused by ALI, has immunomodulatory effect, and also stimulates a local immune response in lung tissues. Thus, Derinat® is a promising treatment for ALI.

TRPV1 Blocker HCRG21 Suppresses TNF-α Production and Prevents the Development of Edema and Hypersensitivity in Carrageenan-Induced Acute Local Inflammation.

Currently the TRPV1 (transient receptor potential vanilloid type 1) channel is considered to be one of the main targets for pro-inflammatory mediators including TNF-α. Similarly, the inhibition of TRPV1 activity in the peripheral nervous system affects pro-inflammatory mediator production and enhances analgesia in total. In this study, the analgesic and anti-inflammatory effects of HCRG21, the first peptide blocker of TRPV1, were demonstrated in a mice model of carrageenan-induced paw edema. HCRG21 in doses of 0.1 and 1 mg/kg inhibited edema formation compared to the control, demonstrated complete edema disappearance in 24 h in a dose of 1 mg/kg, and effectively reduced the productionof TNF-α in both doses examined. ELISA analysis of blood taken 24 h after carrageenan administration showed a dramatic cytokine value decrease to 25 pg/mL by HCRG21 versus 100 pg/mL in the negative control group, which was less than the TNF-α level in the intact group (40 pg/mL). The HCRG21 demonstrated potent analgesic effects on the models of mechanical and thermal hyperalgesia in carrageenan-induced paw edema. The HCRG21 relief effect was comparable to that of indomethacin taken orally in a dose of 5 mg/kg, but was superior to this nonsteroidal anti-inflammatory drug (NSAID) in duration (which lasted 24 h) in the mechanical sensitivity experiment. The results confirm the existence of a close relationship between TRPV1 activity and TNF-α production once again, and prove the superior pharmacological potential of TRPV1 blockers and the HCRG21 peptide in particular.

Anti-Inflammatory and Analgesic Effects of TRPV1 Polypeptide Modulator APHC3 in Models of Osteo- and Rheumatoid Arthritis.

Arthritis is a widespread inflammatory disease associated with progressive articular surface degradation, ongoing pain, and hyperalgesia causing the development of functional limitations and disability. TRPV1 channel is one of the high-potential targets for the treatment of inflammatory diseases. Polypeptide APHC3 from sea anemone Heteractis crispa is a mode-selective TRPV1 antagonist that causes mild hypothermia and shows significant anti-inflammatory and analgesic activity in different models of pain. We evaluated the anti-inflammatory properties of APHC3 in models of monosodium iodoacetate (MIA)-induced osteoarthritis and complete Freund's adjuvant (CFA)-induced rheumatoid monoarthritis in comparison with commonly used non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, and meloxicam. Subcutaneous administration of APHC3 (0.1 mg/kg) significantly reversed joint swelling, disability, grip strength impairment, and thermal and mechanical hypersensitivity. The effect of APHC3 was equal to or better than that of reference NSAIDs. Protracted treatment with APHC3 decreased IL-1b concentration in synovial fluid, reduced inflammatory changes in joints, and prevented the progression of cartilage degradation. Therefore, polypeptide APHC3 has the potential to be an analgesic and anti-inflammatory substance for the alleviation of arthritis symptoms.

Sevanol and Its Analogues: Chemical Synthesis, Biological Effects and Molecular Docking.

Among acid-sensing ion channels (ASICs), ASIC1a and ASIC3 subunits are the most widespread and prevalent in physiological and pathophysiological conditions. They participate in synaptic plasticity, learning and memory, as well as the perception of inflammatory and neurological pain, making these channels attractive pharmacological targets. Sevanol, a natural lignan isolated from Thymus armeniacus, inhibits the activity of ASIC1a and ASIC3 isoforms, and has a significant analgesic and anti-inflammatory effect. In this work, we described the efficient chemical synthesis scheme of sevanol and its analogues, which allows us to analyze the structure-activity relationships of the different parts of this molecule. We found that the inhibitory activity of sevanol and its analogues on ASIC1a and ASIC3 channels depends on the number and availability of the carboxyl groups of the molecule. At the structural level, we predicted the presence of a sevanol binding site based on the presence of molecular docking in the central vestibule of the ASIC1a channel. We predicted that this site could also be occupied in part by the FRRF-amide peptide, and the competition assay of sevanol with this peptide confirmed this prediction. The intravenous (i.v.), intranasal (i.n.) and, especially, oral (p.o.) administration of synthetic sevanol in animal models produced significant analgesic and anti-inflammatory effects. Both non-invasive methods of sevanol administration (i.n. and p.o.) showed greater efficacy than the invasive (i.v.) method, thus opening new horizons for medicinal uses of sevanol.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System.

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 µM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki = 16.7 ± 1.5 µM and αKi = 51.4 ± 4.1 µM for AChE and Ki = 70.5 ± 6.3 µM and αKi = 214 ± 17 µM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

Alkaloid Lindoldhamine Inhibits Acid-Sensing Ion Channel 1a and Reveals Anti-Inflammatory Properties.

Acid-sensing ion channels (ASICs), which are present in almost all types of neurons, play an important role in physiological and pathological processes. The ASIC1a subtype is the most sensitive channel to the medium's acidification, and it plays an important role in the excitation of neurons in the central nervous system. Ligands of the ASIC1a channel are of great interest, both fundamentally and pharmaceutically. Using a two-electrode voltage-clamp electrophysiological approach, we characterized lindoldhamine (a bisbenzylisoquinoline alkaloid extracted from the leaves of Laurus nobilis L.) as a novel inhibitor of the ASIC1a channel. Lindoldhamine significantly inhibited the ASIC1a channel's response to physiologically-relevant stimuli of pH 6.5-6.85 with IC50 range 150-9 µM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund's adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid (which correlated well with the function of ASIC1a in the peripheral nervous system). Thus, we describe lindoldhamine as a novel antagonist of the ASIC1a channel that could provide new approaches to drug design and structural studies regarding the determinants of ASIC1a activation.

Analgesic Activity of Acid-Sensing Ion Channel 3 (ASIС3) Inhibitors: Sea Anemones Peptides Ugr9-1 and APETx2 versus Low Molecular Weight Compounds.

Acid-sensing ion channel 3 (ASIC3) makes an important contribution to the development and maintenance of inflammatory and acid-induced pain. We compared different ASIC3 inhibitors (peptides from sea anemones (APETx2 and Ugr9-1) and nonpeptide molecules (sevanol and diclofenac)) in anti-inflammatory action and analgesic effects. All tested compounds had distinct effects on pH-induced ASIC3 current. APETx2 inhibited only transient current, whereas Ugr9-1 and sevanol decreased transient and sustained components of the current. The effect on mice was evaluated after administering an intramuscular injection in the acetic acid writhing pain model and the complete Freund's adjuvant-induced thermal hyperalgesia/inflammation test. The bell-shaped dependence of the analgesic effect was observed for APETx2 in the acetic acid-induced writhing test, as well as for sevanol and peptide Ugr9-1 in the thermal hyperalgesia test. This dependence could be evidence of the nonspecific action of compounds in high doses. Compounds reducing both components of ASIC3 current produced more significant pain relief than APETx2, which is an effective inhibitor of a transient current only. Therefore, the comparison of the efficacy of ASIC3 inhibitors revealed the importance of ASIC3-sustained currents' inhibition for promotion of acidosis-related pain relief.

New Disulfide-Stabilized Fold Provides Sea Anemone Peptide to Exhibit Both Antimicrobial and TRPA1 Potentiating Properties.

A novel bioactive peptide named τ-AnmTx Ueq 12-1 (short name Ueq 12-1) was isolated and characterized from the sea anemone Urticina eques. Ueq 12-1 is unique among the variety of known sea anemone peptides in terms of its primary and spatial structure. It consists of 45 amino acids including 10 cysteine residues with an unusual distribution and represents a new group of sea anemone peptides. The 3D structure of Ueq 12-1, determined by NMR spectroscopy, represents a new disulfide-stabilized fold partly similar to the defensin-like fold. Ueq 12-1 showed the dual activity of both a moderate antibacterial activity against Gram-positive bacteria and a potentiating activity on the transient receptor potential ankyrin 1 (TRPA1). Ueq 12-1 is a unique peptide potentiator of the TRPA1 receptor that produces analgesic and anti-inflammatory effects in vivo. The antinociceptive properties allow us to consider Ueq 12-1 as a potential analgesic drug lead with antibacterial properties.

Peptide from Sea Anemone Metridium senile Affects Transient Receptor Potential Ankyrin-repeat 1 (TRPA1) Function and Produces Analgesic Effect.

The transient receptor potential ankyrin-repeat 1 (TRPA1) is an important player in pain and inflammatory pathways. It is a promising target for novel drug development for the treatment of a number of pathological states. A novel peptide producing a significant potentiating effect on allyl isothiocyanate- and diclofenac-induced currents of TRPA1 was isolated from the venom of sea anemone Metridium senile. It is a 35-amino acid peptide cross-linked by two disulfide bridges named τ-AnmTX Ms 9a-1 (short name Ms 9a-1) according to a structure similar to other sea anemone peptides belonging to structural group 9a. The structures of the two genes encoding the different precursor proteins of Ms 9a-1 were determined. Peptide Ms 9a-1 acted as a positive modulator of TRPA1 in vitro but did not cause pain or thermal hyperalgesia when injected into the hind paw of mice. Intravenous injection of Ms 9a-1 (0.3 mg/kg) produced a significant decrease in the nociceptive and inflammatory response to allyl isothiocyanate (the agonist of TRPA1) and reversed CFA (Complete Freund's Adjuvant)-induced inflammation and thermal hyperalgesia. Taken together these data support the hypothesis that Ms 9a-1 potentiates the response of TRPA1 to endogenous agonists followed by persistent functional loss of TRPA1-expressing neurons. We can conclude that TRPA1 potentiating may be useful as a therapeutic approach as Ms 9a-1 produces significant analgesic and anti-inflammatory effects in mice models of pain.

A novel expression cassette delivers efficient production of exclusively tetrameric human butyrylcholinesterase with improved pharmacokinetics for protection against organophosphate poisoning.

Butyrylcholinesterase is a stoichiometric bioscavenger against poisoning by organophosphorus pesticides and nerve agents. The low level of expression and extremely rapid clearance of monomeric recombinant human butyrylcholinesterase (rhBChE) from bloodstream (t½≈2 min) limits its pharmaceutical application. Recently (Ilyushin at al., PNAS, 2013) we described a long-acting polysialylated recombinant butyrylcholinesterase (rhBChE-CAO), stable in the bloodstream, that protects mice against 4.2 LD50 of VR. Here we report a set of modifications of the initial rhBChE expression vector to improve stability of the enzyme in the bloodstream and increase its production in CHO cells by introducing in the expression cassette: (i) the sequence of the natural human PRAD-peptide in frame with rhBChE gene via "self-processing" viral F2A peptide under control of an hEF/HTLV promoter, and (ii) previously predicted in silico MAR 1-68 and MAR X-29 sequences. This provides fully tetrameric rhBChE (4rhBChE) at 70 mg/l, that displays improved pharmacokinetics (t½ = 32 ± 1.2 h, MRT = 43 ± 2 h). 3D Fluorescent visualization and distribution of (125)I-labeled enzyme reveals similar low level 4rhBChE and rhBChE-CAO accumulation in muscle, fat, and brain. Administered 4rhBChE was mainly catabolized in the liver and breakdown products were excreted in kidney. Injection of 1.2 LD50 and 1.1 LD50 of paraoxon to BALB/c and knockout BChE-/- mice pre-treated with 4rhBChE (50 mg/kg) resulted in 100% and 78% survival, respectively, without perturbation of long-term behavior. In contrast, 100% mortality of non-pre-treated mice was observed. The high expression level of 4rhBChE in CHO cells permits consideration of this new expression system for manufacturing BChE as a biopharmaceutical.