Development of Capsaicin-Containing Analgesic Silicone-Based Transdermal Patches.

Transdermal therapeutic systems (TTSs) enable convenient dosing in drug therapy. Modified silicone-polymer-based patches are well-controlled and cost-effective matrix diffusion systems. In the present study, we investigated the substance release properties, skin penetration, and analgesic effect of this type of TTS loaded with low-dose capsaicin. Release properties were measured in Franz diffusion cell and continuous flow-through cell approaches. Capsaicin was detected with HPLC-UV and UV spectrophotometry. Raman spectroscopy was conducted on human skin samples exposed to the TTS. A surgical incision or carrageenan injection was performed on one hind paw of male Wistar rats. TTSs were applied to the epilated dorsal skin. Patches were kept on the animals for 6 h. The thermal hyperalgesia and mechanical pain threshold of the hind paws were detected. Patches exhibited controlled, zero-order kinetic capsaicin release. According to the Raman mapping, capsaicin penetrated into the epidermis and dermis of human skin, where the target receptors are expressed. The thermal pain threshold drop of the operated rat paws was reversed by capsaicin treatment compared to that of animals treated with control patches. It was concluded that our modified silicone-polymer-based capsaicin-containing TTS is suitable for the relief of traumatic and inflammatory pain.

Investigation of the Role of the TRPA1 Ion Channel in Conveying the Effect of Dimethyl Trisulfide on Vascular and Histological Changes in Serum-Transfer Arthritis.

Rheumatoid arthritis (RA) is one of the most prevalent autoimmune diseases. Its therapy is often challenging, even in the era of biologicals. Previously, we observed the anti-inflammatory effects of garlic-derived organic polysulfide dimethyl trisulfide (DMTS). Some of these effects were mediated by activation of the TRPA1 ion channel. TRPA1 was mostly expressed in a subset of nociceptor neurons. We decided to investigate the action of DMTS in K/BxN serum-transfer arthritis, which is a relevant model of RA. TRPA1 gene knockout (KO) and wild-type (WT) mice were used. The interaction of DMTS and TRPA1 was examined using a patch clamp in CHO cells. Arthritis was characterized by mechanical hyperalgesia, paw swelling, movement range of the ankle joint, hanging performance, plasma extravasation rate, myeloperoxidase activity, and histological changes in the tibiotarsal joint. DMTS activated TRPA1 channels dose-dependently. DMTS treatment reduced paw swelling and plasma extravasation in both TRPA1 WT and KO animals. DMTS-treated TRPA1 KO animals developed milder collagen deposition in the inflamed joints than WT ones. TRPA1 WT mice did not exhibit significant cartilage damage compared to ones administered a vehicle. We concluded that DMTS and related substances might evolve into novel complementary therapeutic aids for RA patients.

Dimethyl Trisulfide Diminishes Traumatic Neuropathic Pain Acting on TRPA1 Receptors in Mice.

Pharmacotherapy of neuropathic pain is still challenging. Our earlier work indicated an analgesic effect of dimethyl trisulfide (DMTS), which was mediated by somatostatin released from nociceptor nerve endings acting on SST4 receptors. Somatostatin release occurred due to TRPA1 ion channel activation. In the present study, we investigated the effect of DMTS in neuropathic pain evoked by partial ligation of the sciatic nerve in mice. Expression of the mRNA of Trpa1 in murine dorsal-root-ganglion neurons was detected by RNAscope. Involvement of TRPA1 ion channels and SST4 receptors was tested with gene-deleted animals. Macrophage activity at the site of the nerve lesion was determined by lucigenin bioluminescence. Density and activation of microglia in the spinal cord dorsal horn was verified by immunohistochemistry and image analysis. Trpa1 mRNA is expressed in peptidergic and non-peptidergic neurons in the dorsal root ganglion. DMTS ameliorated neuropathic pain in Trpa1 and Sstr4 WT mice, but not in KO ones. DMTS had no effect on macrophage activity around the damaged nerve. Microglial density in the dorsal horn was reduced by DMTS independently from TRPA1. No effect on microglial activation was detected. DMTS might offer a novel therapeutic opportunity in the complementary treatment of neuropathic pain.

Antibacterial effect of local anaesthetics / Helyi érzéstelenítok antibakteriális hatása.

Összefoglaló. Gyógyszereink egy részének jelentos, az eredeti alkalmazástól eltéro hatása is van. Ezek felismerése fontos, hogy elkerüljük a nem várt mellékhatásokat, vagy kihasználjuk ezeket a kedvezo adottságokat. A helyi érzéstelenítok antibakteriális hatása 1909 óta ismert, de ennek több évtizeden keresztül nem tulajdonítottak jelentoséget. Az 1960-as években figyeltek fel eloször az álnegatív mikrobiológiai eredmények lehetoségére, helyi érzéstelenítoket használva a mintavételhez. Tanulmányok igazolták, hogy a bronchoszkópiás, seb-, bor- vagy fül-, orr-, gégészeti bakteriológiai eredmények is érintve lehetnek. A ma is használt gyógyszerek közül a 0,5%-os bupivakainnak és a 2%-os lidokainnak van jelentos antibakteriális hatása Gram-pozitív és Gram-negatív baktériumokkal szemben, ami kifejezettebb 37 °C-on, mint szobahomérsékleten. A legerosebb antibakteriális hatást a 0,5%-os bupivakain mutatta. A napi gyakorlatban alkalmazott koncentrációjuk magasabb, mint a különbözo klinikai izolátumokkal szemben meghatározott minimális gátló koncentráció. Fenti tulajdonságaik alapján felmerült szerepük a kórházi sebfertozések csökkentésében is. A hatásmechanizmus több pontja ismert, károsítják a sejthártya integritását, és több bakteriális enzim muködését gátolják. Orv Hetil. 2021; 162(5): 171-176. Summary. Medications may have important impacts other than the original effect. It is important to know about these to avoid side effects or use these beneficial capabilities. The antibacterial effect of local anaesthetics has been known since 1909. For decades, no attention has been payed to this fact. In the 1960s, the high number of negative microbiological results when local anaesthetics were used before sampling drew attention to the possible antibacterial effect. Studies suggested that cultures from bronchoscopy, wound, skin or nasal samples may be affected. Bupivacaine 0,5% and lidocaine 2% have the most noticeable effect against both Gram-positive and Gram-negative bacteria. This impact is more pronounced at 37 °C than at room temperature. Bupivacaine 0,5% has the most pronounced effect. The concentration of local anaesthetics in daily routine is higher than the minimal inhibitory concentration against various clinical isolates. In the view of these results, they may contribute to reduce surgical site infections. There are known details regarding the mechanism of action. Local anaesthetics have target sites on cellular membrane and inhibit bacterial enzymes. Orv Hetil. 2021; 162(5): 171-176.

Amiodarone that has antibacterial effect against human pathogens may represent a novel catheter lock.

Infection is one of the most feared hospital-acquired complications. Infusion therapy is frequently administered through a central line. Infusions facilitating bacterial growth may be a source of central line-associated bloodstream infections. On the other hand, medications that kill bacteria may protect against this kind of infection and may be used as a catheter lock.In this study, we examined the impact of amiodarone on bacterial growth. Amiodarone is used for controlling cardiac arrhythmias and can be administered as an infusion for weeks. Standard microbiological methods have been used to study the growth of laboratory strains and clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and multidrug-resistant Acinetobacter baumannii in amiodarone. The minimum inhibitory concentration (MIC) of amiodarone was determined. Bacterial growth from in use amiodarone syringes and giving sets was also investigated.Most examined strains were killed within 1 min in amiodarone. The other strains were killed within 1 h. The MICs of amiodarone were <0.5-32 µg/mL.Amiodarone infusion is unlikely to be responsible for bloodstream infections as contaminating bacteria are killed within 1 h. Amiodarone may also protect against central line infections if used as a catheter lock.

Nitrosopersulfide (SSNO-) Is a Unique Cysteine Polysulfidating Agent with Reduction-Resistant Bioactivity.

Aims: The aim of the present study was to investigate the biochemical properties of nitrosopersulfide (SSNO-), a key intermediate of the nitric oxide (NO)/sulfide cross talk. Results: We obtained corroborating evidence that SSNO- is indeed a major product of the reaction of S-nitrosothiols with hydrogen sulfide (H2S). It was found to be relatively stable (t1/2 ∼1 h at room temperature) in aqueous solution of physiological pH, stabilized by the presence of excess sulfide and resistant toward reduction by other thiols. Furthermore, we here show that SSNO- escapes the reducing power of the NADPH-driven biological reducing machineries, the thioredoxin and glutathione reductase systems. The slow decomposition of SSNO- produces inorganic polysulfide species, which effectively induce per/polysulfidation on glutathione or protein cysteine (Cys) residues. Our data also demonstrate that, in contrast to the transient activation by inorganic polysulfides, SSNO- induces long-term potentiation of TRPA1 (transient receptor potential ankyrin 1) channels, which may be due to its propensity to generate a slow flux of polysulfide in situ. Innovation: The characterized properties of SSNO- would seem to represent unique features in cell signaling by enabling sulfur and nitrogen trafficking within the reducing environment of the cytosol, with targeted release of both NO and polysulfide equivalents. Conclusion: SSNO- is a surprisingly stable bioactive product of the chemical interaction of S-nitrosothiol species and H2S that is resistant to reduction by the thioredoxin and glutathione systems. As well as generating NO, it releases inorganic polysulfides, enabling transfer of sulfane sulfur species to peptide/protein Cys residues. The sustained activation of TRPA1 channels by SSNO- is most likely linked to all these properties.

The fluorescent dye 3,3'-diethylthiatricarbocyanine iodide is unsuitable for in vivo imaging of myelination in the mouse.

There is a growing interest to use non-invasive optical imaging methods to study central nervous system diseases. The application of a myelin-binding fluorescent dye, 3,3-diethylthiatricarbocyanine iodide (DBT) was recently described for in vivo optical imaging of demyelination in the mouse. In the present study we aimed at adapting the method to our optical imaging systems, the IVIS Lumina II to measure epifluorescence and the fluorescent molecular tomograph (FMT) for 3-dimensional quantification of the fluorophore. Epifluorescent imaging was performed 5-30 min after DBT injection which was followed by FMT imaging at 40 min. Two mice also underwent micro-CT imaging in the FMT cassette for the purpose of FMT-CT co-registration. Ex vivo imaging of the brain and other tissues of the head and neck was carried out 1 h after injection. Both the FMT-CT co-registration and the ex vivo imaging of organs proved that DBT poorly crossed the blood-brain barrier. The dye did not accumulate in the myelin sheath of the sciatic nerve. In contrast, there was an intense accumulation in the pituitary and salivary glands. The FMT-CT co-registration unequivocally demonstrated that the signal localized to the head did not originate from beyond the blood-brain barrier. No myelin binding was demonstrated by the ex vivo imaging either. In conclusion, DBT is unsuitable for in vivo imaging of myelination due to its poor BBB penetration, accumulation in other structures of the head and neck region and lack of selective binding towards myelin in vivo.

TRPA1 Ion Channel Determines Beneficial and Detrimental Effects of GYY4137 in Murine Serum-Transfer Arthritis.

Modulation of nociception and inflammation by sulfide in rheumatoid arthritis and activation of transient receptor potential ankyrin 1 (TRPA1) ion channels by sulfide compounds are well documented. The present study aims to investigate TRPA1-mediated effects of sulfide donor GYY4137 in K/BxN serum-transfer arthritis, a rodent model of rheumatoid arthritis. TRPA1 and somatostatin sst4 receptor wild-type (WT) and knockout mice underwent K/BxN serum transfer and were treated daily with GYY4137. Functional and biochemical signs of inflammation were recorded, together with histological characterization. These included detection of hind paw mechanical hyperalgesia by dynamic plantar esthesiometry, hind paw volume by plethysmometry, and upside-down hanging time to failure. Hind paw erythema, edema, and passive movement range of tibiotarsal joints were scored. Somatostatin release from sensory nerve endings of TRPA1 wild-type and knockout mice in response to polysulfide was detected by radioimmunoassay. Polysulfide formation from GYY4137 was uncovered by cold cyanolysis. GYY4137 aggravated mechanical hyperalgesia in TRPA1 knockout mice but ameliorated it in wild-type ones. Arthritis score was lowered by GYY4137 in TRPA1 wild-type animals. Increased myeloperoxidase activity, plasma extravasation, and subcutaneous MIP-2 levels of hind paws were detected in TRPA1 knockout mice upon GYY4137 treatment. Genetic lack of sst4 receptors did not alter mechanical hyperalgesia, edema formation, hanging performance, arthritis score, plasma extravasation, or myeloperoxidase activity. TRPA1 WT animals exhibited smaller cartilage destruction upon GYY4137 administration. Sodium polysulfide caused TRPA1-dependent somatostatin release from murine nerve endings. Sulfide released from GYY4137 is readily converted into polysulfide by hypochlorite. Polysulfide potently activates human TRPA1 receptors expressed in Chinese hamster ovary (CHO) cells. According to our data, the protective effect of GYY4137 is mediated by TRPA1, while detrimental actions are independent of the ion channel in the K/BxN serum-transfer arthritis model in mice. At acidic pH in inflamed tissue sulfide is released from GYY4137 and reacts with neutrophil-derived hypochlorite. Resulting polysulfide might be responsible for TRPA1-mediated antinociceptive and anti-inflammatory as well as TRPA1-independent pro-inflammatory effects.

Effects of sulfide and polysulfides transmitted by direct or signal transduction-mediated activation of TRPA1 channels.

Hydrogen sulfide (H2 S) is a gaseous mediator in various physiological and pathological processes, including neuroimmune modulation, metabolic pathways, cardiovascular system, tumour growth, inflammation and pain. Now the hydrogen polysulfides (H2 Sn ) have been recognised as signalling molecules modulating ion channels, transcription factors and protein kinases. Transient receptor potential (TRP) cation channels can be activated by mechanical, thermal or chemical triggers. Here, we review the current literature regarding the biological actions of sulfide and polysulfide compounds mediated by TRP channels with special emphasis on the role of TRPA1, best known as ion channels in nociceptors. However, the non-neuronal TRPA1 channels should also be considered to play regulatory roles. Although sulfide and polysulfide effects in different pathological circumstances and TRPA1-mediated processes have been investigated intensively, our review attempts to present the first comprehensive overview of the potential crosstalk between TRPA1 channels and sulfide-activated signalling pathways. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Role of Transient Receptor Potential Ankyrin 1 Ion Channel and Somatostatin sst4 Receptor in the Antinociceptive and Anti-inflammatory Effects of Sodium Polysulfide and Dimethyl Trisulfide.

Transient receptor potential ankyrin 1 (TRPA1) non-selective ligand-gated cation channels are mostly expressed in primary sensory neurons. Polysulfides (POLYs) are Janus-faced substances interacting with numerous target proteins and associated with both protective and detrimental processes. Activation of TRPA1 in sensory neurons, consequent somatostatin (SOM) liberation and action on sst4 receptors have recently emerged as mediators of the antinociceptive effect of organic trisulfide dimethyl trisulfide (DMTS). In the frame of the present study, we set out to compare the participation of this mechanism in antinociceptive and anti-inflammatory effects of inorganic sodium POLY and DMTS in carrageenan-evoked hind-paw inflammation. Inflammation of murine hind paws was induced by intraplantar injection of carrageenan (3% in 30 µL saline). Animals were treated intraperitoneally with POLY (17 µmol/kg) or DMTS (250 µmol/kg) or their respective vehicles 30 min prior paw challenge and six times afterward every 60 min. Mechanical pain threshold and swelling of the paws were measured by dynamic plantar aesthesiometry and plethysmometry at 2, 4, and 6 h after initiation of inflammation. Myeloperoxidase (MPO) activity in the hind paws were detected 6 h after challenge by luminescent imaging. Mice genetically lacking TRPA1 ion channels, sst4 receptors and their wild-type counterparts were used to examine the participation of these proteins in POLY and DMTS effects. POLY counteracted carrageenan-evoked mechanical hyperalgesia in a TRPA1 and sst4 receptor-dependent manner. POLY did not influence paw swelling and MPO activity. DMTS ameliorated all examined inflammatory parameters. Mitigation of mechanical hyperalgesia and paw swelling by DMTS were mediated through sst4 receptors. These effects were present in TRPA1 knockout animals, too. DMTS inhibited MPO activity with no participation of the sensory neuron-SOM axis. While antinociceptive effects of POLY are transmitted by activation of peptidergic nerves via TRPA1, release of SOM and its effect on sst4 receptors, those of DMTS partially rely on SOM release triggered by other routes. SOM is responsible for the inhibition of paw swelling by DMTS, but TRPA1 does not contribute to its release. Modulation of MPO activity by DMTS is independent of TRPA1 and sst4.

Atropine and glycopyrrolate do not support bacterial growth-safety and economic considerations.

STUDY OBJECTIVE: Evaluation of bacterial growth in atropine and glycopyrrolate. DESIGN: Laboratory investigation. SUBJECTS AND MEASUREMENTS: Standard microbiological methods were used to evaluate the impact of atropine and glycopyrrolate on the growth of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Bacterial count was checked at 0, 1, 2, 3, 4, 6, and 24 hours. MAIN RESULTS: Atropine or glycopyrrolate did not support the growth of the above bacteria at any examined time at room temperature. Glycopyrrolate killed all of the examined strains (P < .05), whereas in atropine, only the clinical isolates of Staphylococcus and Acinetobacter were killed (P < .05). CONCLUSIONS: Drawing up atropine or glycopyrrolate at the beginning of the operating list and use within 24 hours if needed are a safe practice and do not pose infection hazard. We can also reduce hospital costs if we do not throw away these unused syringes following each case.

Capsaicin-Sensitive Sensory Nerves Mediate the Cellular and Microvascular Effects of H2S via TRPA1 Receptor Activation and Neuropeptide Release.

It is supposed that TRPA1 receptor can be activated by hydrogen sulphide (H2S). Here, we have investigated the role of TRPA1 receptor in H2S-induced [Ca(2+)]i increase in trigeminal ganglia (TRG) neurons, and the involvement of capsaicin-sensitive sensory nerves in H2S-evoked cutaneous vasodilatation. [Ca(2+)]i was measured with ratiometric technique on TRG neurons of TRPA1(+/+) and TRPA1(-/-) mice after NaHS, Na2S, allylisothiocyanate (AITC) or KCl treatment. Microcirculatory changes in the ear were detected by laser Doppler imaging in response to topical NaHS, AITC, NaOH, NaSO3 or NaCl. Mice were either treated with resiniferatoxin (RTX), or CGRP antagonist BIBN4096, or NK1 receptor antagonist CP99994, or K(+) ATP channel blocker glibenclamide. Alpha-CGRP(-/-) and NK1 (-/-) mice were also investigated. NaHS and Na2S increased [Ca(2+)]i in TRG neurons derived from TRPA(+/+) but not from TRPA1(-/-) mice. NaHS increased cutaneous blood flow, while NaOH, NaSO3 and NaCl did not cause significant changes. NaHS-induced vasodilatation was reduced in RTX-treated animals, as well as by pre-treatment with BIBN4096 or CP99994 alone or in combination. NaHS-induced vasodilatation was significantly smaller in alpha-CGRP(-/-) or NK1 (-/-) mice compared to wild-types. H2S activates capsaicin-sensitive sensory nerves through TRPA1 receptors and the resultant vasodilatation is mediated by the release of vasoactive sensory neuropeptides CGRP and substance P.

PACAP application improves functional outcome of chronic retinal ischemic injury in rats-evidence from electroretinographic measurements.

Retinoprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP) are well-known and have been demonstrated in various pathological conditions, such as diabetic retinopathy, excitotoxic retinal injury, UV light-induced degeneration, and ischemic retinal lesion. The neuronal degeneration observed in the different retinal layers under the above pathological conditions can be successfully decreased by PACAP; however, whether this morphological improvement is also reflected in functional amelioration remains unknown. Therefore, our purpose was to investigate the protective effect of PACAP on the rat retina after bilateral common carotid artery occlusion (BCCAO) with electroretinography (ERG) to parallel the functional data with the previous morphological and neurochemical observations. Control eyes received saline treatment while PACAP was injected into the vitreous space of the other eye immediately after the induction of ischemia. Retinal damage and protective effects of PACAP were quantified by the changes in the wave forms and amplitudes. On postoperative days 2 and 14, several parameters were assessed with special attention to the changes of b wave. The results confirm that the previously described morphological protection induced by PACAP treatment is reflected in functional improvement in ischemic retinal lesions.

Preconditioning with volatile anaesthetic sevoflurane in ischemic retinal lesion in rats.

Volatile anaesthetic agents have been recognized for their neuroprotective properties since the 1960s. However, little is known regarding the potential retinoprotective effects of preconditioning by anaesthetic drugs. Retinal ischemia can be modeled by permanent bilateral common carotid artery occlusion (BCCAO). Here we studied the degree of ischemic injury with preconditioning by sevoflurane in the rat retina. During the BCCAO operation and preconditioning Wistar rats were anaesthetized with 1 MAC of sevoflurane. The oxygen, carbon dioxide, and anaesthetic vapor concentration in the anaesthetizing box was monitored with a gas analyzer. We examined 4 groups: non- and preconditioning groups in control and BCCAO animals. The duration of preconditioning period was 1 h and it was performed 1 day before BCCAO. The retinas were processed for histological evaluation after 2 weeks survival to determine the cell number in the ganglion cell layer and the thickness of the whole retina and that of all retinal layers. BCCAO-induced retinal ischemic injury was ameliorated by sevoflurane preconditioning. Retinal thickness and the cell number in the ganglion cell layer were more retained in preconditioned animals after BCCAO compared to non-preconditioned group. These results suggest that preconditioning using sevoflurane could provide a new perspective in retinoprotective strategies.

A comparison of the antimicrobial property of lidocaine/prilocaine cream (EMLA) and an alcohol-based disinfectant on intact human skin flora.

BACKGROUND: The application of EMLA cream is indicated for topical anesthesia of the skin in connection with IV cannulation. Recently, we described that EMLA cream has an antibacterial effect in vitro. METHODS: The impact of the local anesthetic lidocaine/prilocaine cream (EMLA) on intact human skin flora was compared to that of an alcohol-based skin disinfectant (Skinsept Pur). Samples were taken from 0 to 12 h after treatment. RESULTS: The number of colony forming units (cfu) on the skin decreased significantly after both EMLA and Skinsept Pur treatment from 44.9 +/- 1.3 (42.4 +/- 7.0) to 0.9 +/- 0.17 (1.61 +/- 0.47) cfu/cm(2), respectively (mean +/- sem), at the first sampling time (1 h) and remained significantly below 0 h values for the study period. The cfu count was significantly lower with EMLA cream at 4, 6, and 12 h compared to Skinsept Pur. CONCLUSION: EMLA cream has a longer bacteriostatic effect after early bactericidal impact compared to skin disinfection with Skinsept Pur.

Occurrence of hlyA and sheA genes in extraintestinal Escherichia coli strains.

The association of a hemolytic phenotype with the carriage of the alpha-hemolysin gene (hlyA) and/or the silent hemolysin gene (sheA or clyA) among 540 extraintestinal clinical isolates of Escherichia coli and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal E. coli infection, the role of SheA is not completely clarified. Two hemolytic sheA+ E. coli strains that lacked hlyA and possessed no other hemolysin genes were identified. No hlyA+ sheA+ strains were identified, suggesting that there is possible incompatibility between hlyA and sheA in the chromosome of E. coli.

Bacterial growth in ropivacaine hydrochloride.

IMPLICATIONS: Drugs affecting bacterial growth may influence the occurrence of postoperative infections. Ropivacaine 10 mg/mL killed Staphylococcus aureus and Escherichia coli; ropivacaine 2 mg/mL supported the growth of E. coli.