As bone remodelling requires the immigration, proliferation and differentiation of osteoblasts at the fusion site, high dosages of intrawound vancomycin might interfere with regenerative processes and increase the risk of non-union. To allow an appropriate balance of infection risk and the risk of non-union, the minimal local concentration required should be determined by controlled in vivo studies.
Background The relatively membrane-impermeable lidocaine derivative QX-314 has been reported to permeate the ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential cation channel, subfamily A, member 1 (TRPA1) to induce a selective inhibition of sensory neurons. This approach is effective in rodents, but it also seems to be associated with neurotoxicity. The authors examined whether the human isoforms of TRPV1 and TRPA1 allow intracellular entry of QX-314 to mediate sodium channel inhibition and cytotoxicity. Methods Human embryonic kidney 293 (HEK-293) cells expressing wild-type or mutant human (h) TRPV1 or TRPA1 constructs as well as the sodium channel Nav1.7 were investigated by means of patch clamp and ratiometric calcium imaging. Cytotoxicity was examined by flow cytometry. Results Activation of hTRPA1 by carvacrol and hTRPV1 by capsaicin produced a QX-314–independent reduction of sodium current amplitudes. However, permeation of QX-314 through hTRPV1 or hTRPA1 was evident by a concentration-dependent, use-dependent inhibition of Nav1.7 activated at 10 Hz. Five and 30 mM QX-314 activated hTRPV1 via mechanisms involving the intracellular vanilloid-binding domain and hTRPA1 via unknown mechanisms independent of intracellular cysteins. Expression of hTRPV1, but not hTRPA1, was associated with a QX-314–induced cytotoxicity (viable cells 48 ± 5% after 30 mM QX-314) that was ameliorated by the TRPV1 antagonist 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (viable cells 81 ± 5%). Conclusions The study data demonstrate that QX-314 directly activates and permeates the human isoforms of TRPV1 and TRPA1 to induce inhibition of sodium channels, but also a TRPV1-dependent cytotoxicity. These results warrant further validation of this approach in more intact preparations and may be valuable for the development of this concept into clinical practice.
Introduction Surgical site infections represent a major complication of spinal surgery. The application of lyophilized vancomycin into the wound is reported to significantly decrease infection rates. As concentrations applied locally can exceed the minimal bacterial inhibitory concentration for more than a 1000-fold, toxic side effects on local tissue may be possible. Materials and Methods Primary osteoblast cell cultures were generated from bone tissue samples of 10 patients. Samples were incubated in absence or presence of 3, 6, or 12 mg/cm2 vancomycin according to a planned phase I clinical trial protocol. Changes in pH, osteoblast migration, proliferation, and viability were analyzed. Alkaline phosphatase and mineralization patterns were studied. Results The application of more than 3 mg/cm2 vancomycin induced a decline of pH toward the acidic range. The migration potential of osteoblasts was decreased from 100% (control samples) to zero (12 mg/cm2 vancomycin) in a dose-dependent manner. Cell proliferation was significantly inhibited at dosages above 3 mg/cm2. Significant cell death was observed if the dosage applied exceeded 6 mg/cm2. The synthesis of alkaline phosphatase was markedly reduced in all dosages applied and calcium deposition was significantly decreased in dosages above 3 mg/cm2. Conclusion As bone remodeling requires the immigration, proliferation, and differentiation of osteoblasts at the fusion site, high dosages of intrawound vancomycin might interfere with regenerative processes and increase the risk of nonunion. To allow an appropriate balance of infection risk and the risk of nonunion, the minimal local concentration required should be determined by controlled in vivo studies.
Introduction:The nonopioid analgesic and antipyretic dipyrone (metamizol) is frequently used worldwide. Dipyrone is a prodrug, and the metabolites 4-N-methylaminoantipyrine (MAA) and 4-aminoantipyrine (AA) seem to induce analgesia and antipyresia in part by inhibiting cyclooxygenase. In mice, however, the analgesic effect of dipyrone also seems to depend on the ion channel TRPA1. In this study, we explored the effects of dipyrone and its active metabolites on recombinant and native TRPA1 and TRPV1 channels.Methods:Constructs human (h) TRPA1 and TRPV1 were expressed in HEK293 cells, and dorsal root ganglion neurons were isolated from adult mice. Effects of dipyrone, MAA, and AA were explored by means of whole-cell patch clamp recordings and ratiometric calcium imaging.Results:Dipyrone failed to activate both hTRPA1 and hTRPV1. However, both MAA and AA evoked small outwardly rectifying membrane currents and an increase of intracellular calcium in cells expressing hTRPA1 or hTRPV1. MAA also sensitized both channels and thus potentiated inward currents induced by carvacrol (hTRPA1) and protons (hTRPV1). MAA-induced activation was inhibited by the antioxidant 10-mM glutathione included in the pipette, and the mutant constructs hTRPA1-C621/C641/C665S and hTRPV1-C158A/C391S/C767S were insensitive to both MAA and AA. Mouse dorsal root ganglion neurons exhibited a marginal calcium influx when challenged with MAA.Conclusion:The metabolites MAA and AA, but not dipyrone itself, activate and sensitize the nociceptive ion channels TRPA1 and TRPV1 in a redox-dependent manner. These effects may be relevant for dipyrone-induced analgesia and antipyresia.
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