TRPA1-dependent and -independent activation by commonly used preservatives

Abstract

Background and purpose: Addition of preservatives ensures microbial stability, especially in multidose containers of parenterally administered pharmaceuticals. These compounds can cause side effects, and particularly at the site of application, might elicit or facilitate pain. TRPA1 is a cation channel expressed in peripheral neurons which contributes to pain and inflammation and is sensitive to many irritants. The most commonly used preservatives, in particular with a focus on parenteral formulations, were investigated for their potential to activate TRPA1. Experimental approach: Sixteen preservatives were screened for mediating calcium influx in human TRPA1-transfected HEK293t cells. Untransfected cells served as control, results were further validated in mouse sensory neurons. In addition, proinflammatory mediators serotonin, histamine and prostaglandin E2 were co-administered to probe a potential sensitisation of preservative-induced TRPA1 activation. Key results: Butylparaben, propylparaben, ethylparaben, bronopol, methylparaben, phenylethyl alcohol and phenol induced a TRPA1-dependent calcium influx in transfected HEK293t cells at concentrations used for preservation. Other preservatives increased calcium within the used concentration ranges, but to a similar degree in untransfected controls. Serotonin, histamine, and prostaglandin enhanced TRPA1 activation of phenylethyl alcohol, bronopol, ethylparaben, propylparaben and butylparaben. Conclusion and implications: Systematic screening of common preservatives applied for parenterally administered drugs resulted in identifying several preservatives with substantial TRPA1 channel activation. This activation was enhanced by the addition of proinflammatory meditators. This allows selecting a preservative without TRPA1 activation, particularly in case of pharmaceuticals that could act proinflammatory.

Keywords: TRP channel; TRPA1; inflammation; pain; parenteral; preservative; sensitisation.

FIGURE 1

Preservatives for parenteral formulation activate TRPA1. (A) Experimental protocol on the fluorescence plate reader. Substances were automatically added after 30 s. The shaded area indicates the area under the curve (AUC) analysed in all further experiments. In this experiment TRPA1 agonist allylisothiocyanate 25 µM (AITC) is used as positive control. This causes a response in hTRPA1 transfected (red trace), but not in untransfected HEK293t cells (black trace). Traces are the mean of five repetitions, normalized to the 30 s before application. (B) Identical experiment with application of a preservative, in this panel bronopol 50 µM. Traces are the mean of five repetitions. (C-R) Concentration-response of the indicated preservative in hTRPA1-transfected and untransfected HEK293t cells. Shaded area reflects the typical range in clinical use. The panels are sorted from high to no TRPA1 activation at the lower end of the commonly used preservatives concentration. *indicates a significantly larger response in hTRPA1-transfected compared to untransfected cells. (S) Activation by the preservatives at the left border of the shaded concentration range. In contrast to the focus on TRPA1 in the other panels, the bar chart was sorted by total activation. The stacked histogram visualizes the TRPA1-independent (grey) activation, and the additional TRPA1-dependent (red) activation on top.

FIGURE 2

Preservatives with TRPA1-independent action release intracellular calcium. (A) m-Cresol 9.52 mM was applied (black bar) in the in presence of extracellular calcium (solid black line) or in absence of extracellular calcium and presence of EGTA 6.66 mM (dashed grey line). (B) Benzyl alcohol 48.1 mM, (C) benzethonium 0.09 mM and (D) chlorobutanol 15.5 mM were tested with the same approach. Traces represent the mean, shaded areas the standard error of the mean.

FIGURE 3

Preservatives for parenteral formulation activate sensory neurons. (A) Phenylethyl alcohol 30 mM induced a substantial increase in intracellular calcium (red trace, n = 954), compared with controls (black trace, n = 1,190), or sodium benzoate 10 mM (blue trace, n = 942). Sensory neurons of TRPA1^−/− mice (green trace, n = 773) showed a complete reduction in phenylethyl alcohol induced activation. Black bars indicate substance application. After a preservative, TRPA1 agonist AITC 100 µM and capsaicin 1 µM was applied. Traces represent the mean, shaded areas of the same colour the standard error of the mean. (B) Area under the curve calculated over a 60 s interval starting with the preservative application. Dots represent the means of the dishes. Horizontal bar and variance indicates mean ± SEM (395–1701 cells), tested on independent experimental days. Chlorhexidine 300 µM served as an additional preservative without neuronal activation. In contrast, calcium-elevation by phenylethyl alcohol is fully TRPA1-dependent, as it is absent in sensory neurons lacking the ion channel.

FIGURE 4

Inflammatory mediators sensitise TRPA1 activation of phenylethyl alcohol. (A) TRPA1-activating phenylethyl alcohol 30 mM applied alone (black) or co-applied with histamine 1 μM, serotonin 1 µM or prostaglandin E2 1 µM (PGE₂). Traces are calcium-dependent fluorescence, normalised to the last 10 s before application, data are mean ± standard error of the mean. (B–D) Areas under the curve for phenylethyl alcohol around the typical range were higher in the presence of inflammatory mediators compared to control. Data are geometric least squares means with 95% confidence intervals estimated from a linear model. * indicates p < 0.05. Typical concentrations of four further preservatives for subcutaneous formulation tested for sensitisation of the TRPA1 response by inflammatory mediators (Infl. med.). (E) Bronopol 1.8 µM, (F) ethylparaben 100 μM, (G) propylparaben 100 μM, (H) butylparaben 100 µM were tested alone or coapplied with inflammatory mediators. Each trace represents the mean of three experiments, normalised to the last 10 s before application. (I–L) Area under the curve for experiments in panels (E–H). Area under the curves of the 30 s application period. Data are mean with standard error of the mean. * indicates p < 0.05 vs. control.