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.     

Polypeptide Modulators of TRPV1 Produce Analgesia without Hyperthermia

Transient receptor potential vanilloid 1 receptors (TRPV1) play a significant physiological role. The study of novel TRPV1 agonists and antagonists is essential. Here, we report on the characterization of polypeptide antagonists of TRPV1 based on in vitro and in vivo experiments. We evaluated the ability of APHC1 and APHC3 to inhibit TRPV1 using the whole-cell patch clamp approach and single cell Ca²⁺ imaging. In vivo tests were performed to assess the biological effects of APHC1 and APHC3 on temperature sensation, inflammation and core body temperature. In the electrophysiological study, both polypeptides partially blocked the capsaicin-induced response of TRPV1, but only APHC3 inhibited acid-induced (pH 5.5) activation of the receptor. APHC1 and APHC3 showed significant antinociceptive and analgesic activity in vivo at reasonable doses (0.01–0.1 mg/kg) and did not cause hyperthermia. Intravenous administration of these polypeptides prolonged hot-plate latency, blocked capsaicin- and formalin-induced behavior, reversed CFA-induced hyperalgesia and produced hypothermia. Notably, APHC3’s ability to inhibit the low pH-induced activation of TRPV1 resulted in a reduced behavioural response in the acetic acid-induced writhing test, whereas APHC1 was much less effective. The polypeptides APHC1 and APHC3 could be referred to as a new class of TRPV1 modulators that produce a significant analgesic effect without hyperthermia.     

Cyclisation Increases the Stability of the Sea Anemone Peptide APETx2 but Decreases Its Activity at Acid-Sensing Ion Channel 3

APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3.     

Lyophilization Serves as an Effective Strategy for Drug Development of the α9α10 Nicotinic Acetylcholine Receptor Antagonist α-Conotoxin GeXIVA[1,2]

α-Conotoxin GeXIVA[1,2] is a highly potent and selective antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype. It has the advantages of strong efficacy, no tolerance, and no effect on motor function, which has been expected help patients with neuropathic pain. However, drug development for clinical use is severely limited owing to its instability. Lyophilization is applied as the most preferred method to solve this problem. The prepared lyophilized powder is characterized by differential scanning calorimetry (DSC), powder X-ray diffractometry (PXRD), and Fourier transform infrared spectroscopy (FTIR). Molecular simulation is also used to explore the internal distribution and forces formed in the system. The analgesic effect on paclitaxel-induced neuropathic pain following single and 14-day repeated administrations are evaluated by the von Frey test and the tail-flick test. Trehalose combined with mannitol in a ratio of 1:1 is employed as the excipients in the determined formulation, where trehalose acts as the stabilizer and mannitol acts as the bulking agent, according to the results of DSC, PXRD, and FTIR. Both GeXIVA[1,2] (API) and GeXIVA[1,2] lyophilized powder (formulation) could produce stable analgesic effect. These results indicated that GeXIVA[1,2] lyophilized powder could improve the stability and provide an effective strategy to push it into clinical use as a new analgesic drug.     

小麦硝酸盐转运蛋白基因 TaNRT1.1的鉴定及其等位变异分析

为解析小麦硝酸盐转运蛋白基因 TaNRT1.1的生物学功能,本研究通过同源克隆的方法从普通小麦中克隆了小麦硝酸盐转运蛋白基因 TaNRT1.1(TaNRT1.1-1A、 TaNRT1.1-1B和 TaNRT1.1-1D)。生物信息学分析表明,这三个同源基因编码的蛋白均为疏水蛋白,含有丰富的α-螺旋和无未见则卷曲,主要定位于质膜上。小麦不同组织qRT-PCR分析表明, TaNRT1.1-1A和 TaNRT1.1-1B基因在根中表达量最高,其次是叶和茎, TaNRT1.1-1D基因在茎中表达量最高,其次是叶和根。因此,推测 TaNRT1.1-1A和 TaNRT1.1-1B基因在硝酸盐吸收过程中发挥了重要作用, TaNRT1.1-1D基因在硝酸盐转运过程中发挥了重要作用。通过对小麦 TaNRT1.1基因多态性筛选发现,在 TaNRT1.1-1A基因启动子上游1 120 bp的位置有一个8 bp(TGCATGCA)的插入位点,该位点可能与小麦氮利用效率相关。不同氮利用效率小麦品种qRT-PCR分析结果表明,氮高效小麦品种（基因型为 TaNRT1.1-1A-b）苗期根中 TaNRT1.1-1A基因的相对表达量显著高于氮低效小麦品种（基因型为 TaNRT1.1-1A-a）。