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Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit 5 6
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Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions
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Daniele Corti, Valentina Galbiati ⇑, Nicolò Gatti, Marina Marinovich, Corrado L. Galli, Emanuela Corsini
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Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Università degli Studi di Milano, Milan, Italy
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a r t i c l e
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Article history: Received 28 October 2014 Revised 19 April 2015 Accepted 20 April 2015 Available online xxxx Keywords: Drug allergy Cytokines Surface markers p38 MAPK PKC
a b s t r a c t Despite important impacts of systemic hypersensitivity induced by pharmaceuticals, for such endpoint no reliable preclinical approaches are available. We previously established an in vitro test to identify contact and respiratory allergens based on interleukin-8 (IL-8) production in THP-1 cells. Here, we challenged it for identification of pharmaceuticals associated with systemic hypersensitivity reactions, with the idea that drug sensitizers share common mechanisms of cell activation. Cells were exposed to drugs associated with systemic hypersensitivity reactions (streptozotocin, sulfamethoxazole, neomycin, probenecid, clonidine, procainamide, ofloxacin, methyl salicylate), while metformin was used as negative drug. Differently to chemicals, drugs tested were well tolerated, except clonidine and probenecid, with no signs of cytotoxicity up to 1–2 mg/ml. THP-1 activation assay was adjusted, and conditions, that allow identification of all sensitizing drugs tested, were established. Next, using streptozotocin and selective inhibitors of PKC-b and p38 MAPK, two pathways involved in chemical allergen-induced cell activation, we tested the hypothesis that similar pathways were also involved in drug-induced IL-8 production and CD86 upregulation. Results indicated that drugs and chemical allergens share similar activation pathways. Finally, we made a structure–activity hypothesis related to hypersensitivity reactions, trying to individuate structural requisite that can be involved in immune mediated adverse reactions. Ó 2015 Elsevier Ltd. All rights reserved.
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1. Introduction
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Chemical hypersensitivity is described as adverse health effects resulting from the stimulation of specific immune response by chemicals. Hypersensitivity reactions are the result of normally beneficial immune responses acting inappropriately against benign antigens, causing inflammatory reactions and tissue damage (Corsini and Kimber, 2007). Hypersensitivity drug reactions (HDRs) are the adverse effect of pharmaceutical formulations (including active drugs and excipients) that clinically resemble allergy (Demoly et al., 2014). HDRs are the most frequent immunotoxic effects of drugs. HDRs account for approximately 1/6 of drug-induced adverse effects (Pichler, 2001). They include immune-mediated (‘allergic’) and non immune-mediated (‘pseudoallergic’) reactions, while drug-induced autoimmune reactions, either systemic or organ-specific, are seemingly rare (Descotes, 2005; D’Cruz, 2000). HDRs belong to type B adverse drug reactions, which are defined
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⇑ Corresponding author at: Laboratory of Toxicology, Dipartimento di Scienze Farmacologiche e Biomolecolari, Via G. Balzaretti 9, 20133 Milan, Italy. E-mail address: [email protected] (V. Galbiati).
by the World Health Organization as the dose-independent, unpredictable, noxious, and unintended response to a drug taken at a dose normally used in humans. HDRs are unavoidable and linked to the intrinsic proprieties of the drug molecule and the genetic predisposition of the patient (Adkinson et al., 2002). As severe and unpredicted adverse events dramatically showed in the recent years that the immune system is a critical aspect of drug safety, enhanced prediction of nonclinical immune safety evaluation is crucial (Descotes, 2012). The process of developing a new chemical entity (NCE) into a marketed pharmaceutical has many routes to failure and is very expensive. Overall, approximately 80% of NCEs that enter clinical trials do not become approved drugs, and 20% of all failures are related to safety issues (DiMasi, 2001). Despite the important adverse reactions linked to immune-mediated hypersensitivity (the classical four types of hypersensitivity reactions described by Gell and Coombs (1963)) and autoimmunity, nowadays there are no validated standard in vitro or in vivo methods to perform a screening test suitable for assessing the sensitizing potential of a drug during the pre-clinical phase. Animals are generally good predictors of human responses to drugs, however, for some categories of toxicity,
http://dx.doi.org/10.1016/j.tiv.2015.04.012 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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including systemic hypersensitivity reactions (Kimber, 2001; Pieters, 2007), animal models are not currently considered adequate to predict potential human toxicity. With the exception of contact sensitisation, very few animal models and assays can reliably predict the potential for (unspecific) immunostimulation, systemic hypersensitivity reactions or autoimmunity (Descotes, 2006). With systemic hypersensitivity reactions, we refer to the immune-mediated reactions that involve the entire body, to distinguish them from local (i.e. contact dermatitis) reactions. At present, the evaluation of the contact sensitization potential of chemicals is done using the local lymph node assay (LLNA), as described in the OECD guideline 429, as an alternative method to the traditional Guinea Pig assays (Angers-Loustau et al., 2011; Gerberick et al., 2007). Over the last years, many in vitro models have been proposed to identify the potential of chemicals to induce skin sensitization to meet current animal welfare, public opinions and legislation constrains (Corsini et al., 2014). On December 12th 2013 the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM, part of the European Commission Joint Research Centre) published its Recommendation on the Direct Peptide Reactivity Assay (DPRA) for skin sensitisation, capable to distinguish sensitizer from non-sensitizers, and on February 17th 2014 its recommendation on the KeratinoSens™ assay. Other assays (i.e. human Cell Line Activation Test, h-CLAT) will follow shortly. It is expected that a predictive method to totally replace animal testing will be in the form of a test battery comprising molecular, cell-based, and/or computational methods, the so-called ‘‘Integrated Approaches to Testing and Assessment’’ (Basketter et al., 2013). The development of alternative in vitro assays to detect the sensitizing potential during the development phase of a drug would reduce the use of animals but also of cost and time, possibly increasing safety and specificity of the results. Most drugs are small molecules (PM < 1000 Da) and as such are not able to activate a specific immune reaction. They must bind to carrier proteins to form a complete immunogenic compound. Some of these are not inherently immunogenic, but they must undergo metabolic transformations in order to become haptens and trigger the allergic response (Chang and Gershwin, 2010). The progress of allergic reaction then requires the activation of dendritic cells (DC), which cause the activation of specific T cells (Martin, 2012). DC are antigen-presenting cells that play a central role in the initiation and regulation of adaptive immune responses. Following the contact with antigens they undergo a process of maturation associated with the expression on the membrane of several co-stimulatory molecules such as CD80, CD86 and CD40 and various adhesion molecules including CD2, CD11a, CD54, CD58 (Quah and O’Neill, 2005). During the maturation process DC secrete various cytokines, such as IL-1b and IL-8. Activated DC move out of tissues where encounter with the antigen took place and migrate into the regional lymphatic system or the blood. In the lymph node or in the spleen, DC present antigen to specific T lymphocyte, using MHC class II molecules (Ryan et al., 2007). Adhesion molecules on both the DC (i.e. CD86) and the T-cell (i.e. CD28) ensure appropriate contact and co-stimulation. Following the stimulus, a clone of T cells is produced with the ability to react to the antigen, which caused their expansion. We previously demonstrated in the human promyelocytic cell line THP-1 that all chemical allergens tested, with the exception of the prohapten isoeugenol, induced a dose-related release of interleukin-8 (IL-8). In contrast to IL-8 release, CD54 and CD86 expression did not provide a sensitive method failing to correctly identify approximately 30% of the tested compounds. Although CD86 appears to be a more sensitive marker than CD54 when discriminating allergens from irritants neither of these markers provided robust methodology in our hands (Mitjans et al., 2008).
The purpose of this study was to assess the possibility to use or in case adapt the THP-1 assay for the in vitro identification of drugs, which may be associated with in vivo immediated and nonimmediated drug hypersensitivity reactions, and to understand their molecular mechanism of action. The drugs used in this work were selected on the basis of definite immune-adverse reactions reported in literature, postmarketing data or labeling information, use in in vivo animal or in vitro studies and on the commercial availability as pure drugs (Speer, 1979; D’Cruz, 2000; Weaver et al., 2005; You et al., 2014). Clonidine, ofloxacin, procainamide, streptozotocin, sulfamethoxazole have been associated with a relatively high incidence of immune-mediated hypersensitivity reactions (Weaver et al., 2005). Methyl salicylate in topical analgesic preparations as well al probenecid has been reported to cause irritant or allergic contact dermatitis and anaphylactic reactions (Chan, 1996; Myers et al., 1998). The proposed screening assay has the potential to identify adverse effects on the immune system (i.e. immune-mediated hypersensitivity reactions, including both immediate and nonimmediate HDRs), and can be easily incorporated into drug development prior to phase III clinical trials.
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2. Materials and methods
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2.1. Chemicals
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Streptozotocin, sulfamethoxazole, procainamide hydrochloride, ofloxacin, neomycin sulfate, probenecid, clonidine and methyl salicylate were selected as sensitizing molecules according to their clinical episodes of hypersensitivity reactions. Metformin was selected as non-sensitizing compound as there are not evidences of hypersensitivity reactions in the history of its clinical use. All reagents and chemicals were purchased from Sigma–Aldrich Co. (St. Louis, Mo, USA) at the highest purity available. Lipopolysaccharide (LPS) from Escherichia coli serotype 0127:B8 was obtained from Sigma and stock solution dissolved in PBS. Streptozotocin and methyl salicylate were dissolved in DMSO (final concentration of DMSO in culture medium 2000 >1000 >2000 >1000 >2000 750 >1000 >600 >2000
106/ml cells were treated for 24–48 h with increasing concentration of the selected chemicals or DMSO as vehicle control (0.2%). Cell viability was then assessed by PI staining. Sulfamethoxazole, ofloxacin, clonidine, probenecid and metformin were dissolved directly at the maximum concentration in medium culture (RPMI) due to their low solubility both in PBS and DMSO. CV75 (the concentration resulting in 75% of cells viability compared to vehicle treated cells) was calculated by linear regression analysis of data.
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Fig. 1. Effects of the selected drugs on IL-8 release. THP-1 cells were treated for 24 h, and in specific cases (sulfamethoxazole, probenecid, metformin) also for 48 h, with increasing concentrations of the selected drug sensitizers (streptozotocin, sulfamethoxazole, procainamide hydrochloride, ofloxacin, probenecid, neomycin, clonidine, methyl salicylate) and with metformin as negative drug. Vehicle treated cells were included as a vehicle control. IL-8 release was measured by ELISA in culture supernatants, results are expressed as pg/ml. Each value represents the mean ± SD, n = 3. Data are representative of one experiment out of three. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄p < 0.05 and ⁄⁄p < 0.01 vs vehicle treated cells (control).
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3.2. Effects of the selected chemicals on the release of IL-8 and CD86 expression In Figs. 1 and 2 the effects of the selected drugs on IL-8 release (Fig. 1) and CD86 expression (Fig. 2) following 24–48 h of treatment are reported. As shown in Fig. 1, a statistically significant increase in IL-8 release after 24 h of treatment was observed for streptozotocin, procainamide, methylsalicylate, ofloxacin, clonidine, neomycin and
probenecid, while sulfamethoxazole failed to induce significant release of IL-8 after 24 h treatment. The experiment was repeated exposing cells to sulfamethoxazole, probenecid and metformin for 48 h. Under this condition a modest but statistically significant release of IL-8 was detected following sulfamethoxazole treatment. Probenecid induced a dose-related release of IL-8, confirming the data obtained at 24 h. It was tested at 48 h as at 24 h it failed to up-regulate CD86 (see below). As expected metformin did not induce IL-8 release at either times.
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Fig. 2. Effects of the selected drugs on the expression of cell surface marker CD86. THP-1 cells were treated for 24 h and in specific cases for 48 h (neomycin, sulfamethoxazole, probenecid, metformin), with increasing concentrations of the selected drugs. Results are expressed as stimulation index (SI). The dotted line on the y-axis at 1.5 of SI represents the threshold above which the drug is considered as sensitizer. Each value represents the mean ± SD, n = 3. Data are representative of one experiment out of three. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄p < 0.05 and ⁄⁄p < 0.01 vs vehicle treated cells (control).
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Neomicyn 5
Based on these results, in order to correctly identify all drug sensitizers tested the combination of IL-8 production (mRNA expression at 3 h or IL-8 release at 24 h), CD86 and CD54 expression at 24 h will allow the proper identification. In alternative, the assessment of only IL-8 release at 24 and 48 h will be sufficient for the identification. Thus, depending on the laboratory’s equipments and skills, the proposed protocol will allow a certain degree of flexibility.
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3.3. Effect on IL-8 mRNA expression
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Another flexibility may be provided by the previously observation we made on the fact that chemical allergens, which failed to induced the release of IL-8, were able to up-regulate IL-8 mRNA expression at 3 h (Galbiati et al., 2012). Therefore, we investigated the effects of sulfamethoxazole and metformin, both negative at 24 h, on IL-8 mRNA expression. LPS (10 ng/ml) was used as positive control. As shown in Fig. 4, only sulfamethoxazole was able to induce a dose-related IL-8 mRNA expression as assessed by Real Time PCR, metformin as expected was negative. LPS was, as expected, positive in both sets of experiments. Based on these results, we propose a tier approach for the in vitro identification of drug sensitizers. We propose initially to investigate the effect on IL-8 release and CD86 expression following 24 h of exposure, if positive (statistically significant release of IL-8 at any concentration and/or a SI P 1.5 for CD86) the drug will be considered as sensitizer. If negative, in order to exclude any activation, IL-8 release and CD86 expression at 48 h (statistically
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As in the h-CLAT, one of methods recently validated at EURL-ECVAM, chemical allergens are identified based on CD86 and/or CD54 expression (Ashikaga et al., 2008). In parallel to IL-8 release, the effect of the selected drugs on CD86 expression was investigated. As shown in Fig. 2, very similar results as those of IL-8 release were observed, with the exception of neomycin, which failed to induce CD86 also at 48 h. Streptozotocin, procainamide, methylsalicylate, ofloxacin, clonidine induced a statistical significant increase in the expression of CD86, while sulfamethoxazole and probenecid failed to induce significant up-regulation of CD86 expression after 24 h treatment. The experiment was repeated exposing cells for 48 h, under this condition a dose-related increase in CD86 expression was observed both for sulfamethoxazole and probenecid. As expected, metformin failed to induce CD86 at either times. In supplementary Fig. 1 the biological variability in drug-induced CD86 upregulation is reported. Neomycin failed to induce CD86 expression at both 24 and 48 h. As in the h-CLAT, alternatively to CD86, some chemical allergens induce only the upregulation of CD54 (Ashikaga et al., 2008), we investigated the effect of neomycin on CD54 expression. As shown in Fig. 3A, neomycin induced a statistically significant up-regulation of CD54 expression. We also investigated the ability of the negative drug metformin to induced CD54 expression, as shown in Fig. 3B metformin was negative in the CD54 expression.
CD 54 SI
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Fig. 3. Effects of the selected drugs on the expression of cell surface marker CD54. THP-1 cells were treated for 24 h with increasing concentrations of neomycin and metformin. Results are expressed as stimulation index (SI). The dotted line on the yaxis at 2 of SI represents the threshold above which the drug is considered as sensitizer. Each value represents the mean ± SD, n = 3. Data are representative of one experiment out of three. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄p < 0.05 and ⁄⁄p < 0.01 vs vehicle treated cells (control).
Fig. 4. Effects of the selected drugs on IL-8 mRNA expression. THP-1 cells were treated for 3 h with the selected drug (sulfamethoxazole, metformin) or with LPS 10 ng/mL as positive control. IL-8 mRNA expression was evaluated by Real TimePCR as described in Section 2. Results are expressed as 2-DDCT. Each value represents the mean ± SD, n = 3 independent experiments. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄⁄p < 0.01 vs vehicle treated cells (control).
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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significant release of IL-8 at any concentration and/or a SI P 1.5 for CD86), or CD54 expression (SI P 2.0) at 24 h or alternatively IL-8 mRNA expression (2-CT > 3.0) at 3 h should be assessed. Only if negative results were obtained in all parameters the drug will be considered as non-sensitizer. 3.4. Role of PKC-b and p38 MAPK in drug-induced CD86 expression and IL-8 release
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4. Discussion
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Despite the important adverse reactions linked to hypersensitivity and autoimmunity and our improved mechanistic understanding, nowadays there are no validated standard in vitro or in vivo methods to perform a screening test suitable for assessing the sensitizing potential of a drug during the pre-clinical phase. Here, we describe a strategy based on IL-8 production, CD86 and/or CD54 expression in THP-1 cells for the in vitro identification of drug sensitizers. The proposed test allowed the correct identification of all the selected drug sensitizers tested, including
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We have previously shown a key role of p38 MAPK and PKC-b activation in chemical allergen-induced CD86 expression and IL-8 release in both THP-1 cells and primary DC (Mitjans et al., 2008; Corsini et al., 2014). We, therefore, wanted to investigate if similar pathways are also involved in drug-induced CD86 expression and IL-8 release. Using specific inhibitors, we found that both pathways are involved in streptozotocin-induced IL-8 release and CD86 expression. Streptozotocin was chosen as reference drug as it induced both markers. THP-1 cells were cultured for 2 h in presence or absence of PKC-b pseudosubstrate (Pseudo, 5 lM) in RPMI without FCS. Then, FCS (10% final concentration) and streptozotocin (Strepto, 1500 lg/ml) were added for 24 h. By blocking PKC-b activation, a significant reduction in both CD86 expression (Fig. 5A) and IL-8 release (Fig. 5B) was obtained. Similarly, the selective p38 MAPK inhibitor SB 202190 (50 nM), significantly reduced streptozotocin-induced CD86 expression (Fig. 5C), and completely blocked IL-8 release (Fig. 5D), confirming previously results obtained with chemical allergens.
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sulfamethoxazole, probenecid and procainamide for which metabolism is needed. The proposed in vitro method, for the hazard identification of immune-mediated hypersensitivity reactions induced by pharmaceuticals, benefits from a rationalistic approach with the idea that allergenic drugs share with chemical allergens common mechanisms of cell activation. A straightforward test method based on immunological knowledge and read out parameters was developed. Within the European project SENS-IT-IV, we have previously established an in vitro test able to identify contact and respiratory allergens based on the use of the human THP-1 cell line and IL-8 release (Mitjans et al., 2008, 2010). Among the recently proposed in vitro tests, the release of IL-8 is one of the most promising biomarkers to distinguish sensitizers from non-sensitizers (Toebak et al., 2006; Nukada et al., 2008; dos Santos et al., 2011; Takahashi et al., 2011). IL-8 is a well-established potent chemotactic peptide for neutrophils as well as for T lymphocytes, basophils (Leonard et al., 1990), and NK cells (Sebok et al., 1993). Although it is impossible to demonstrate the exact role of IL-8 in contact hypersensitivity due to the lack of the mouse counter part of IL-8, recently, MCP-1 (CCL2), that is coordinately regulated with IL-8, has been shown to play a crucial role of dendritic cell maturation and skin sensitization in vivo, indicating the importance of IL-8 in DC activation step (Ishimoto et al., 2008). In parallel to IL-8 production, several of the proposed in vitro methods based on DC use CD86 alone or in combination with CD54 expression for the identification of chemical allergens (see review by dos Santos et al., 2011). Both markers are physiologically involved in antigen presentation and T cells activation (Nuriya et al., 1996; Banchereau et al., 2000; Yoshida et al., 2003). As the THP-1 activation assay detected chemical sensitizers associated with both type I and IV hypersensitivity reactions, we hypothesized that the test could also identify substances associated with type II and III hypersensitivity reactions. We aimed, therefore, to assess the possibility to use or in case adapt the THP-1 assay for the in vitro identification of drugs, which may be associated in vivo with hypersensitivity reactions, and to understand their molecular mechanism of action. The drugs selected, namely streptozotocin, sulfamethoxazole, neomycin, clonidine, probenecid, procainamide, ofloxacin, methyl salicylate, had various
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Fig. 5. Role of PKC-b and p38 MAPK in streptozotocin-induced CD86 expression and IL-8 release. PKC-b pseudosubstrate (5 lM) and SB202190 (50 nM) were used as selective inhibitors of PKC-b and p38 MAPK, respectively, as described in Section 2. (A) Role of PKC-b on streptozotocin-induced CD86 expression. (B) Role of PKC-b on streptozotocininduced IL-8 release. (C) Role of p38 MAPK on streptozotocin-induced CD86 expression. (D) Role of p38 MAPK on streptozotocin-induced IL-8 release. Results are expressed as mean ± SD, n = 3. Data are representative of one experiment out of three. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄p < 0.05 and ⁄⁄ p < 0.01 vs relative controls and §p < 0.05 and §§p < 0.01 vs streptozotocin (Strepto) alone.
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Fig. 6. Structures of the selected drugs and relative CAS number.
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origins, different mechanism of action and different therapeutic application. They were selected on the basis of immune-adverse reactions reported in literature, postmarketing data or labeling information and on the commercial availability as pure drugs (D’Cruz, 2000; Weaver et al., 2005; You et al., 2014). Penicillin G, another drug frequently associated with hypersensitivity reactions, was previously tested, and found to be able to induced a
dose-related release of IL-8 following 48 h of exposure (Mitjans et al., 2008). The THP-1 assay originally developed, based on the release of IL-8, needed to be adapted in order to identify all the drug sensitizers tested. We found that exposure of THP-1 cells to sensitizing drugs results in most cases in dose related release of IL-8 and increase of CD86 expression, with some differences among drugs,
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Fig. 7. Chemical allergens structurally related to the selected pharmaceuticals.
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markers and times of exposure. Using only the release of IL-8 as a marker, 48 h will be required to identify all positive drugs, as sulfamethoxazole was negative at 24 h. Using only CD86 expression, neomycin will be negative, failing to induced CD86 both at 24 and 48 h, while 48 h will also be required for sulfamethoxazole and probenecid, as it was for sulfamethoxazole for IL-8. The combination of both IL-8 and CD86 expression at 24 h will allow the
identification of all drugs tested, except sulfamethoxazole. The use of IL-8 mRNA expression at 3 h or CD54 expression at 24 h may offer an alternative to the 48 h exposure and increase our confidence in the negativity of a drug. The expression of IL-8 mRNA at 3 h is based on previous observation we made on chemical allergens failing to induce the release of IL-8: all chemical sensitizers tested including pro-hapten induced IL-8 mRNA at 3 h (Galbiati
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et al., 2012). Sulfamethoxazole induced a dose-related increase of IL-8 mRNA at 3 h. The use of CD54 expression is based on the h-CLAT, in which chemicals are classified as sensitizers if they induce CD86 (MFI > 150) and/or CD54 (MFI > 200) (Ashikaga et al., 2008). Using CD54 expression at 24 h a dose-related increase was observed following exposure to neomycin. There are, however, some important differences with the h-CLAT. First, the method we use to calculate CD86 and CD54 expression is different from the one described in the h-CLAT protocol. In the h-CLAT only the MFI is considered, while we also include in the calculation the percentage of positive cells. In the case of the drugs used in the current study, the use of MFI only will lead to negative judgment for almost all the drug tested as many of the selected compounds induced a MFI below 150. As drug sensitizers also increase the percentage of positive cells, its inclusion into the calculation allow to reach a SI of 1.5, the threshold for positivity. Second problem following the h-CLAT protocol, which was optimized for chemical sensitizers, it is the concentration tested. With the exception of clonidine, no CV75 could be calculated for the other drugs. Drugs compared to chemicals are less cytotoxic, which necessitated increasing the concentration to 2000 lg/ml. In the h-CLAT protocol for DMSO soluble compounds 1000 lg/ml is set as the highest concentration. In order to detect drugs using CD86 and/or CD54 expression, the h-CLAT protocol should be therefore adapted. The negative drug metformin was negative in all conditions and for all parameters. Concerning the mechanisms of action, using streptozotocin as reference allergenic drug, we found, similarly to chemical allergens, a key role for p38 MAPK and PKC-b activation in streptozotocin-induced IL-8 release and CD86 expression, confirming previous results obtained with chemical allergens (Mitjans et al., 2008; Corsini et al., 2013; Lin et al., 2011). Using other allergenic drugs, we are currently obtaining results similar to those of streptozotocin, indicating that the cellular activation follows the same pathway induced by chemical sensitizers. Finally, observing results obtained and the common structural elements of the different drug tested, we made a structure–activity hypothesis related to hypersensitivity reactions, starting from the definition of pharmacophore and making then a ‘‘Structure– Hypersensitivity-Relationship’’. Paul Ehrlich defined the word pharmacophore for the first time in 1909 as ‘‘the drug molecule’s smallest part responsible for the biological activity’’. A pharmacophore is then a set of structural elements, suitably arranged in the three dimension of space, which takes over directly or indirectly in the specific interaction with a receptor or a target, initiating the biological response. Starting from this rational, observing the structures of the drugs used in this study (Fig. 6), we compared them with the structures of chemical compounds known to be allergens (Fig. 7), making a first hypothesis to search for possible common structural features that may act as a hapten, and correlate the action of sensitizer in the body. First, we observed that in the structure of all the sensitizing drugs analyzed (Fig. 6), with the exception of neomycin and streptozotocin, is present an aromatic ring with electronegative substituents in ortho or para position, and a side chain capable to generate H bonds (clonidine, methylsalicylate, procainamide, probenecid, sulfamethoxazole). The side chain on the aromatic ring of all these drugs is metabolically unstable, in particular for procainamide. The hydrolysis that can occur in vivo, generates more simple anilinic compounds, which can be assimilated to para-phenylenediamine, an aromatic di-amine used in the textile processing and dye hair known to be a potent allergen. This may suggest that the degradation products of the drugs may undergo further metabolic processes before the elimination from the body, activated to hapten, and then recognized as antigen when bound to the plasma proteins. The greater chemical stability of probenecid’s and sulfamethoxazole’s sulfonamidic group may
explain why it was necessary to perform a 48 h treatment to observe a sensitizing effect. THP-1 cells are known to have a limited metabolic activity, and may require longer time to generate an active compound. Further analysis revealed that some of the drugs tested have structural characteristics similar to other compounds known to be allergens, i.e. eugenol, isoeugenol, p-benzylbromide, or with other drugs with aromatic groups which have been associated with allergic reactions, i.e. acetylsalicylic acid, paracetamol. Although more difficult to detect, these features can also be observed within the ofloxacin structure. Different appears the situation of streptozotocin and neomycin, where this assumption is not applicable. It is, however, possible to considerate two other important factors. First, both drugs are of bacterial origin, and as such they should be considered toxins or defense elements developed by microorganism. It is therefore plausible to assume that they can be recognized by the immune system as non-self molecules. A possible confirmation of this hypothesis is the observation of similarities with the complex structure of LPS, the bacterial compound used as classical activator of innate immunity. The second point to consider is that these compounds are characterized by a high number of polar and hydrophilic groups (–OH, –NH2): in vivo they can stably bind to plasma proteins, suggesting the possibility to easily make complete antigens able to be recognized as non-self by the immune system. On the other side, observing the structure of metformin (the drug selected as non-sensitizing) clearly the characteristics outlined above are absent. It is possible to affirm that the presence of aromatic rings, in particular anilines with electronegative substituents in ortho/para position, may be a necessary requirement to generate in vivo derivatives that can result in the activation of the immune system. In conclusion, we establish experimental conditions and markers to correctly identify drug sensitizers. The proposed screening assay can be easily incorporated into drug development to identify drugs, which may have the potential to cause allergic or systemic hypersensitivity reactions, an important clinical problem.
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Conflict of interest
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Authors declare of not having any financial, personal, or association with any of individuals or organizations that could have inappropriately influenced the submitted work.
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Uncited references
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Banchereau and Steinman (1998), Burchiel et al. (1997), Coombs and Gell (1975), Corsini and Roggen (2009), Ehlrich (1909), Fernandez et al. (2014), Gell and Coombs (1963), Jerschow et al. (2001), Kimber et al. (2002), Vandebriel et al. (2005).
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tiv.2015.04.012.
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Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit 5 6
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Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions
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Daniele Corti, Valentina Galbiati ⇑, Nicolò Gatti, Marina Marinovich, Corrado L. Galli, Emanuela Corsini
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Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Università degli Studi di Milano, Milan, Italy
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a r t i c l e
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Article history: Received 28 October 2014 Revised 19 April 2015 Accepted 20 April 2015 Available online xxxx Keywords: Drug allergy Cytokines Surface markers p38 MAPK PKC
a b s t r a c t Despite important impacts of systemic hypersensitivity induced by pharmaceuticals, for such endpoint no reliable preclinical approaches are available. We previously established an in vitro test to identify contact and respiratory allergens based on interleukin-8 (IL-8) production in THP-1 cells. Here, we challenged it for identification of pharmaceuticals associated with systemic hypersensitivity reactions, with the idea that drug sensitizers share common mechanisms of cell activation. Cells were exposed to drugs associated with systemic hypersensitivity reactions (streptozotocin, sulfamethoxazole, neomycin, probenecid, clonidine, procainamide, ofloxacin, methyl salicylate), while metformin was used as negative drug. Differently to chemicals, drugs tested were well tolerated, except clonidine and probenecid, with no signs of cytotoxicity up to 1–2 mg/ml. THP-1 activation assay was adjusted, and conditions, that allow identification of all sensitizing drugs tested, were established. Next, using streptozotocin and selective inhibitors of PKC-b and p38 MAPK, two pathways involved in chemical allergen-induced cell activation, we tested the hypothesis that similar pathways were also involved in drug-induced IL-8 production and CD86 upregulation. Results indicated that drugs and chemical allergens share similar activation pathways. Finally, we made a structure–activity hypothesis related to hypersensitivity reactions, trying to individuate structural requisite that can be involved in immune mediated adverse reactions. Ó 2015 Elsevier Ltd. All rights reserved.
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1. Introduction
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Chemical hypersensitivity is described as adverse health effects resulting from the stimulation of specific immune response by chemicals. Hypersensitivity reactions are the result of normally beneficial immune responses acting inappropriately against benign antigens, causing inflammatory reactions and tissue damage (Corsini and Kimber, 2007). Hypersensitivity drug reactions (HDRs) are the adverse effect of pharmaceutical formulations (including active drugs and excipients) that clinically resemble allergy (Demoly et al., 2014). HDRs are the most frequent immunotoxic effects of drugs. HDRs account for approximately 1/6 of drug-induced adverse effects (Pichler, 2001). They include immune-mediated (‘allergic’) and non immune-mediated (‘pseudoallergic’) reactions, while drug-induced autoimmune reactions, either systemic or organ-specific, are seemingly rare (Descotes, 2005; D’Cruz, 2000). HDRs belong to type B adverse drug reactions, which are defined
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⇑ Corresponding author at: Laboratory of Toxicology, Dipartimento di Scienze Farmacologiche e Biomolecolari, Via G. Balzaretti 9, 20133 Milan, Italy. E-mail address: [email protected] (V. Galbiati).
by the World Health Organization as the dose-independent, unpredictable, noxious, and unintended response to a drug taken at a dose normally used in humans. HDRs are unavoidable and linked to the intrinsic proprieties of the drug molecule and the genetic predisposition of the patient (Adkinson et al., 2002). As severe and unpredicted adverse events dramatically showed in the recent years that the immune system is a critical aspect of drug safety, enhanced prediction of nonclinical immune safety evaluation is crucial (Descotes, 2012). The process of developing a new chemical entity (NCE) into a marketed pharmaceutical has many routes to failure and is very expensive. Overall, approximately 80% of NCEs that enter clinical trials do not become approved drugs, and 20% of all failures are related to safety issues (DiMasi, 2001). Despite the important adverse reactions linked to immune-mediated hypersensitivity (the classical four types of hypersensitivity reactions described by Gell and Coombs (1963)) and autoimmunity, nowadays there are no validated standard in vitro or in vivo methods to perform a screening test suitable for assessing the sensitizing potential of a drug during the pre-clinical phase. Animals are generally good predictors of human responses to drugs, however, for some categories of toxicity,
http://dx.doi.org/10.1016/j.tiv.2015.04.012 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.
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including systemic hypersensitivity reactions (Kimber, 2001; Pieters, 2007), animal models are not currently considered adequate to predict potential human toxicity. With the exception of contact sensitisation, very few animal models and assays can reliably predict the potential for (unspecific) immunostimulation, systemic hypersensitivity reactions or autoimmunity (Descotes, 2006). With systemic hypersensitivity reactions, we refer to the immune-mediated reactions that involve the entire body, to distinguish them from local (i.e. contact dermatitis) reactions. At present, the evaluation of the contact sensitization potential of chemicals is done using the local lymph node assay (LLNA), as described in the OECD guideline 429, as an alternative method to the traditional Guinea Pig assays (Angers-Loustau et al., 2011; Gerberick et al., 2007). Over the last years, many in vitro models have been proposed to identify the potential of chemicals to induce skin sensitization to meet current animal welfare, public opinions and legislation constrains (Corsini et al., 2014). On December 12th 2013 the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM, part of the European Commission Joint Research Centre) published its Recommendation on the Direct Peptide Reactivity Assay (DPRA) for skin sensitisation, capable to distinguish sensitizer from non-sensitizers, and on February 17th 2014 its recommendation on the KeratinoSens™ assay. Other assays (i.e. human Cell Line Activation Test, h-CLAT) will follow shortly. It is expected that a predictive method to totally replace animal testing will be in the form of a test battery comprising molecular, cell-based, and/or computational methods, the so-called ‘‘Integrated Approaches to Testing and Assessment’’ (Basketter et al., 2013). The development of alternative in vitro assays to detect the sensitizing potential during the development phase of a drug would reduce the use of animals but also of cost and time, possibly increasing safety and specificity of the results. Most drugs are small molecules (PM < 1000 Da) and as such are not able to activate a specific immune reaction. They must bind to carrier proteins to form a complete immunogenic compound. Some of these are not inherently immunogenic, but they must undergo metabolic transformations in order to become haptens and trigger the allergic response (Chang and Gershwin, 2010). The progress of allergic reaction then requires the activation of dendritic cells (DC), which cause the activation of specific T cells (Martin, 2012). DC are antigen-presenting cells that play a central role in the initiation and regulation of adaptive immune responses. Following the contact with antigens they undergo a process of maturation associated with the expression on the membrane of several co-stimulatory molecules such as CD80, CD86 and CD40 and various adhesion molecules including CD2, CD11a, CD54, CD58 (Quah and O’Neill, 2005). During the maturation process DC secrete various cytokines, such as IL-1b and IL-8. Activated DC move out of tissues where encounter with the antigen took place and migrate into the regional lymphatic system or the blood. In the lymph node or in the spleen, DC present antigen to specific T lymphocyte, using MHC class II molecules (Ryan et al., 2007). Adhesion molecules on both the DC (i.e. CD86) and the T-cell (i.e. CD28) ensure appropriate contact and co-stimulation. Following the stimulus, a clone of T cells is produced with the ability to react to the antigen, which caused their expansion. We previously demonstrated in the human promyelocytic cell line THP-1 that all chemical allergens tested, with the exception of the prohapten isoeugenol, induced a dose-related release of interleukin-8 (IL-8). In contrast to IL-8 release, CD54 and CD86 expression did not provide a sensitive method failing to correctly identify approximately 30% of the tested compounds. Although CD86 appears to be a more sensitive marker than CD54 when discriminating allergens from irritants neither of these markers provided robust methodology in our hands (Mitjans et al., 2008).
The purpose of this study was to assess the possibility to use or in case adapt the THP-1 assay for the in vitro identification of drugs, which may be associated with in vivo immediated and nonimmediated drug hypersensitivity reactions, and to understand their molecular mechanism of action. The drugs used in this work were selected on the basis of definite immune-adverse reactions reported in literature, postmarketing data or labeling information, use in in vivo animal or in vitro studies and on the commercial availability as pure drugs (Speer, 1979; D’Cruz, 2000; Weaver et al., 2005; You et al., 2014). Clonidine, ofloxacin, procainamide, streptozotocin, sulfamethoxazole have been associated with a relatively high incidence of immune-mediated hypersensitivity reactions (Weaver et al., 2005). Methyl salicylate in topical analgesic preparations as well al probenecid has been reported to cause irritant or allergic contact dermatitis and anaphylactic reactions (Chan, 1996; Myers et al., 1998). The proposed screening assay has the potential to identify adverse effects on the immune system (i.e. immune-mediated hypersensitivity reactions, including both immediate and nonimmediate HDRs), and can be easily incorporated into drug development prior to phase III clinical trials.
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2. Materials and methods
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2.1. Chemicals
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Streptozotocin, sulfamethoxazole, procainamide hydrochloride, ofloxacin, neomycin sulfate, probenecid, clonidine and methyl salicylate were selected as sensitizing molecules according to their clinical episodes of hypersensitivity reactions. Metformin was selected as non-sensitizing compound as there are not evidences of hypersensitivity reactions in the history of its clinical use. All reagents and chemicals were purchased from Sigma–Aldrich Co. (St. Louis, Mo, USA) at the highest purity available. Lipopolysaccharide (LPS) from Escherichia coli serotype 0127:B8 was obtained from Sigma and stock solution dissolved in PBS. Streptozotocin and methyl salicylate were dissolved in DMSO (final concentration of DMSO in culture medium 2000 >1000 >2000 >1000 >2000 750 >1000 >600 >2000
106/ml cells were treated for 24–48 h with increasing concentration of the selected chemicals or DMSO as vehicle control (0.2%). Cell viability was then assessed by PI staining. Sulfamethoxazole, ofloxacin, clonidine, probenecid and metformin were dissolved directly at the maximum concentration in medium culture (RPMI) due to their low solubility both in PBS and DMSO. CV75 (the concentration resulting in 75% of cells viability compared to vehicle treated cells) was calculated by linear regression analysis of data.
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Fig. 1. Effects of the selected drugs on IL-8 release. THP-1 cells were treated for 24 h, and in specific cases (sulfamethoxazole, probenecid, metformin) also for 48 h, with increasing concentrations of the selected drug sensitizers (streptozotocin, sulfamethoxazole, procainamide hydrochloride, ofloxacin, probenecid, neomycin, clonidine, methyl salicylate) and with metformin as negative drug. Vehicle treated cells were included as a vehicle control. IL-8 release was measured by ELISA in culture supernatants, results are expressed as pg/ml. Each value represents the mean ± SD, n = 3. Data are representative of one experiment out of three. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄p < 0.05 and ⁄⁄p < 0.01 vs vehicle treated cells (control).
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3.2. Effects of the selected chemicals on the release of IL-8 and CD86 expression In Figs. 1 and 2 the effects of the selected drugs on IL-8 release (Fig. 1) and CD86 expression (Fig. 2) following 24–48 h of treatment are reported. As shown in Fig. 1, a statistically significant increase in IL-8 release after 24 h of treatment was observed for streptozotocin, procainamide, methylsalicylate, ofloxacin, clonidine, neomycin and
probenecid, while sulfamethoxazole failed to induce significant release of IL-8 after 24 h treatment. The experiment was repeated exposing cells to sulfamethoxazole, probenecid and metformin for 48 h. Under this condition a modest but statistically significant release of IL-8 was detected following sulfamethoxazole treatment. Probenecid induced a dose-related release of IL-8, confirming the data obtained at 24 h. It was tested at 48 h as at 24 h it failed to up-regulate CD86 (see below). As expected metformin did not induce IL-8 release at either times.
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Fig. 2. Effects of the selected drugs on the expression of cell surface marker CD86. THP-1 cells were treated for 24 h and in specific cases for 48 h (neomycin, sulfamethoxazole, probenecid, metformin), with increasing concentrations of the selected drugs. Results are expressed as stimulation index (SI). The dotted line on the y-axis at 1.5 of SI represents the threshold above which the drug is considered as sensitizer. Each value represents the mean ± SD, n = 3. Data are representative of one experiment out of three. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄p < 0.05 and ⁄⁄p < 0.01 vs vehicle treated cells (control).
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Based on these results, in order to correctly identify all drug sensitizers tested the combination of IL-8 production (mRNA expression at 3 h or IL-8 release at 24 h), CD86 and CD54 expression at 24 h will allow the proper identification. In alternative, the assessment of only IL-8 release at 24 and 48 h will be sufficient for the identification. Thus, depending on the laboratory’s equipments and skills, the proposed protocol will allow a certain degree of flexibility.
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3.3. Effect on IL-8 mRNA expression
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Another flexibility may be provided by the previously observation we made on the fact that chemical allergens, which failed to induced the release of IL-8, were able to up-regulate IL-8 mRNA expression at 3 h (Galbiati et al., 2012). Therefore, we investigated the effects of sulfamethoxazole and metformin, both negative at 24 h, on IL-8 mRNA expression. LPS (10 ng/ml) was used as positive control. As shown in Fig. 4, only sulfamethoxazole was able to induce a dose-related IL-8 mRNA expression as assessed by Real Time PCR, metformin as expected was negative. LPS was, as expected, positive in both sets of experiments. Based on these results, we propose a tier approach for the in vitro identification of drug sensitizers. We propose initially to investigate the effect on IL-8 release and CD86 expression following 24 h of exposure, if positive (statistically significant release of IL-8 at any concentration and/or a SI P 1.5 for CD86) the drug will be considered as sensitizer. If negative, in order to exclude any activation, IL-8 release and CD86 expression at 48 h (statistically
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As in the h-CLAT, one of methods recently validated at EURL-ECVAM, chemical allergens are identified based on CD86 and/or CD54 expression (Ashikaga et al., 2008). In parallel to IL-8 release, the effect of the selected drugs on CD86 expression was investigated. As shown in Fig. 2, very similar results as those of IL-8 release were observed, with the exception of neomycin, which failed to induce CD86 also at 48 h. Streptozotocin, procainamide, methylsalicylate, ofloxacin, clonidine induced a statistical significant increase in the expression of CD86, while sulfamethoxazole and probenecid failed to induce significant up-regulation of CD86 expression after 24 h treatment. The experiment was repeated exposing cells for 48 h, under this condition a dose-related increase in CD86 expression was observed both for sulfamethoxazole and probenecid. As expected, metformin failed to induce CD86 at either times. In supplementary Fig. 1 the biological variability in drug-induced CD86 upregulation is reported. Neomycin failed to induce CD86 expression at both 24 and 48 h. As in the h-CLAT, alternatively to CD86, some chemical allergens induce only the upregulation of CD54 (Ashikaga et al., 2008), we investigated the effect of neomycin on CD54 expression. As shown in Fig. 3A, neomycin induced a statistically significant up-regulation of CD54 expression. We also investigated the ability of the negative drug metformin to induced CD54 expression, as shown in Fig. 3B metformin was negative in the CD54 expression.
CD 54 SI
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CD 54 SI
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Fig. 3. Effects of the selected drugs on the expression of cell surface marker CD54. THP-1 cells were treated for 24 h with increasing concentrations of neomycin and metformin. Results are expressed as stimulation index (SI). The dotted line on the yaxis at 2 of SI represents the threshold above which the drug is considered as sensitizer. Each value represents the mean ± SD, n = 3. Data are representative of one experiment out of three. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄p < 0.05 and ⁄⁄p < 0.01 vs vehicle treated cells (control).
Fig. 4. Effects of the selected drugs on IL-8 mRNA expression. THP-1 cells were treated for 3 h with the selected drug (sulfamethoxazole, metformin) or with LPS 10 ng/mL as positive control. IL-8 mRNA expression was evaluated by Real TimePCR as described in Section 2. Results are expressed as 2-DDCT. Each value represents the mean ± SD, n = 3 independent experiments. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄⁄p < 0.01 vs vehicle treated cells (control).
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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significant release of IL-8 at any concentration and/or a SI P 1.5 for CD86), or CD54 expression (SI P 2.0) at 24 h or alternatively IL-8 mRNA expression (2-CT > 3.0) at 3 h should be assessed. Only if negative results were obtained in all parameters the drug will be considered as non-sensitizer. 3.4. Role of PKC-b and p38 MAPK in drug-induced CD86 expression and IL-8 release
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4. Discussion
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Despite the important adverse reactions linked to hypersensitivity and autoimmunity and our improved mechanistic understanding, nowadays there are no validated standard in vitro or in vivo methods to perform a screening test suitable for assessing the sensitizing potential of a drug during the pre-clinical phase. Here, we describe a strategy based on IL-8 production, CD86 and/or CD54 expression in THP-1 cells for the in vitro identification of drug sensitizers. The proposed test allowed the correct identification of all the selected drug sensitizers tested, including
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3
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We have previously shown a key role of p38 MAPK and PKC-b activation in chemical allergen-induced CD86 expression and IL-8 release in both THP-1 cells and primary DC (Mitjans et al., 2008; Corsini et al., 2014). We, therefore, wanted to investigate if similar pathways are also involved in drug-induced CD86 expression and IL-8 release. Using specific inhibitors, we found that both pathways are involved in streptozotocin-induced IL-8 release and CD86 expression. Streptozotocin was chosen as reference drug as it induced both markers. THP-1 cells were cultured for 2 h in presence or absence of PKC-b pseudosubstrate (Pseudo, 5 lM) in RPMI without FCS. Then, FCS (10% final concentration) and streptozotocin (Strepto, 1500 lg/ml) were added for 24 h. By blocking PKC-b activation, a significant reduction in both CD86 expression (Fig. 5A) and IL-8 release (Fig. 5B) was obtained. Similarly, the selective p38 MAPK inhibitor SB 202190 (50 nM), significantly reduced streptozotocin-induced CD86 expression (Fig. 5C), and completely blocked IL-8 release (Fig. 5D), confirming previously results obtained with chemical allergens.
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sulfamethoxazole, probenecid and procainamide for which metabolism is needed. The proposed in vitro method, for the hazard identification of immune-mediated hypersensitivity reactions induced by pharmaceuticals, benefits from a rationalistic approach with the idea that allergenic drugs share with chemical allergens common mechanisms of cell activation. A straightforward test method based on immunological knowledge and read out parameters was developed. Within the European project SENS-IT-IV, we have previously established an in vitro test able to identify contact and respiratory allergens based on the use of the human THP-1 cell line and IL-8 release (Mitjans et al., 2008, 2010). Among the recently proposed in vitro tests, the release of IL-8 is one of the most promising biomarkers to distinguish sensitizers from non-sensitizers (Toebak et al., 2006; Nukada et al., 2008; dos Santos et al., 2011; Takahashi et al., 2011). IL-8 is a well-established potent chemotactic peptide for neutrophils as well as for T lymphocytes, basophils (Leonard et al., 1990), and NK cells (Sebok et al., 1993). Although it is impossible to demonstrate the exact role of IL-8 in contact hypersensitivity due to the lack of the mouse counter part of IL-8, recently, MCP-1 (CCL2), that is coordinately regulated with IL-8, has been shown to play a crucial role of dendritic cell maturation and skin sensitization in vivo, indicating the importance of IL-8 in DC activation step (Ishimoto et al., 2008). In parallel to IL-8 production, several of the proposed in vitro methods based on DC use CD86 alone or in combination with CD54 expression for the identification of chemical allergens (see review by dos Santos et al., 2011). Both markers are physiologically involved in antigen presentation and T cells activation (Nuriya et al., 1996; Banchereau et al., 2000; Yoshida et al., 2003). As the THP-1 activation assay detected chemical sensitizers associated with both type I and IV hypersensitivity reactions, we hypothesized that the test could also identify substances associated with type II and III hypersensitivity reactions. We aimed, therefore, to assess the possibility to use or in case adapt the THP-1 assay for the in vitro identification of drugs, which may be associated in vivo with hypersensitivity reactions, and to understand their molecular mechanism of action. The drugs selected, namely streptozotocin, sulfamethoxazole, neomycin, clonidine, probenecid, procainamide, ofloxacin, methyl salicylate, had various
§§ 1
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Fig. 5. Role of PKC-b and p38 MAPK in streptozotocin-induced CD86 expression and IL-8 release. PKC-b pseudosubstrate (5 lM) and SB202190 (50 nM) were used as selective inhibitors of PKC-b and p38 MAPK, respectively, as described in Section 2. (A) Role of PKC-b on streptozotocin-induced CD86 expression. (B) Role of PKC-b on streptozotocininduced IL-8 release. (C) Role of p38 MAPK on streptozotocin-induced CD86 expression. (D) Role of p38 MAPK on streptozotocin-induced IL-8 release. Results are expressed as mean ± SD, n = 3. Data are representative of one experiment out of three. Statistical analysis was performed with Dunnett’s multiple comparison test, with ⁄p < 0.05 and ⁄⁄ p < 0.01 vs relative controls and §p < 0.05 and §§p < 0.01 vs streptozotocin (Strepto) alone.
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Fig. 6. Structures of the selected drugs and relative CAS number.
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origins, different mechanism of action and different therapeutic application. They were selected on the basis of immune-adverse reactions reported in literature, postmarketing data or labeling information and on the commercial availability as pure drugs (D’Cruz, 2000; Weaver et al., 2005; You et al., 2014). Penicillin G, another drug frequently associated with hypersensitivity reactions, was previously tested, and found to be able to induced a
dose-related release of IL-8 following 48 h of exposure (Mitjans et al., 2008). The THP-1 assay originally developed, based on the release of IL-8, needed to be adapted in order to identify all the drug sensitizers tested. We found that exposure of THP-1 cells to sensitizing drugs results in most cases in dose related release of IL-8 and increase of CD86 expression, with some differences among drugs,
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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Fig. 7. Chemical allergens structurally related to the selected pharmaceuticals.
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markers and times of exposure. Using only the release of IL-8 as a marker, 48 h will be required to identify all positive drugs, as sulfamethoxazole was negative at 24 h. Using only CD86 expression, neomycin will be negative, failing to induced CD86 both at 24 and 48 h, while 48 h will also be required for sulfamethoxazole and probenecid, as it was for sulfamethoxazole for IL-8. The combination of both IL-8 and CD86 expression at 24 h will allow the
identification of all drugs tested, except sulfamethoxazole. The use of IL-8 mRNA expression at 3 h or CD54 expression at 24 h may offer an alternative to the 48 h exposure and increase our confidence in the negativity of a drug. The expression of IL-8 mRNA at 3 h is based on previous observation we made on chemical allergens failing to induce the release of IL-8: all chemical sensitizers tested including pro-hapten induced IL-8 mRNA at 3 h (Galbiati
Please cite this article in press as: Corti, D., et al. Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.04.012
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et al., 2012). Sulfamethoxazole induced a dose-related increase of IL-8 mRNA at 3 h. The use of CD54 expression is based on the h-CLAT, in which chemicals are classified as sensitizers if they induce CD86 (MFI > 150) and/or CD54 (MFI > 200) (Ashikaga et al., 2008). Using CD54 expression at 24 h a dose-related increase was observed following exposure to neomycin. There are, however, some important differences with the h-CLAT. First, the method we use to calculate CD86 and CD54 expression is different from the one described in the h-CLAT protocol. In the h-CLAT only the MFI is considered, while we also include in the calculation the percentage of positive cells. In the case of the drugs used in the current study, the use of MFI only will lead to negative judgment for almost all the drug tested as many of the selected compounds induced a MFI below 150. As drug sensitizers also increase the percentage of positive cells, its inclusion into the calculation allow to reach a SI of 1.5, the threshold for positivity. Second problem following the h-CLAT protocol, which was optimized for chemical sensitizers, it is the concentration tested. With the exception of clonidine, no CV75 could be calculated for the other drugs. Drugs compared to chemicals are less cytotoxic, which necessitated increasing the concentration to 2000 lg/ml. In the h-CLAT protocol for DMSO soluble compounds 1000 lg/ml is set as the highest concentration. In order to detect drugs using CD86 and/or CD54 expression, the h-CLAT protocol should be therefore adapted. The negative drug metformin was negative in all conditions and for all parameters. Concerning the mechanisms of action, using streptozotocin as reference allergenic drug, we found, similarly to chemical allergens, a key role for p38 MAPK and PKC-b activation in streptozotocin-induced IL-8 release and CD86 expression, confirming previous results obtained with chemical allergens (Mitjans et al., 2008; Corsini et al., 2013; Lin et al., 2011). Using other allergenic drugs, we are currently obtaining results similar to those of streptozotocin, indicating that the cellular activation follows the same pathway induced by chemical sensitizers. Finally, observing results obtained and the common structural elements of the different drug tested, we made a structure–activity hypothesis related to hypersensitivity reactions, starting from the definition of pharmacophore and making then a ‘‘Structure– Hypersensitivity-Relationship’’. Paul Ehrlich defined the word pharmacophore for the first time in 1909 as ‘‘the drug molecule’s smallest part responsible for the biological activity’’. A pharmacophore is then a set of structural elements, suitably arranged in the three dimension of space, which takes over directly or indirectly in the specific interaction with a receptor or a target, initiating the biological response. Starting from this rational, observing the structures of the drugs used in this study (Fig. 6), we compared them with the structures of chemical compounds known to be allergens (Fig. 7), making a first hypothesis to search for possible common structural features that may act as a hapten, and correlate the action of sensitizer in the body. First, we observed that in the structure of all the sensitizing drugs analyzed (Fig. 6), with the exception of neomycin and streptozotocin, is present an aromatic ring with electronegative substituents in ortho or para position, and a side chain capable to generate H bonds (clonidine, methylsalicylate, procainamide, probenecid, sulfamethoxazole). The side chain on the aromatic ring of all these drugs is metabolically unstable, in particular for procainamide. The hydrolysis that can occur in vivo, generates more simple anilinic compounds, which can be assimilated to para-phenylenediamine, an aromatic di-amine used in the textile processing and dye hair known to be a potent allergen. This may suggest that the degradation products of the drugs may undergo further metabolic processes before the elimination from the body, activated to hapten, and then recognized as antigen when bound to the plasma proteins. The greater chemical stability of probenecid’s and sulfamethoxazole’s sulfonamidic group may
explain why it was necessary to perform a 48 h treatment to observe a sensitizing effect. THP-1 cells are known to have a limited metabolic activity, and may require longer time to generate an active compound. Further analysis revealed that some of the drugs tested have structural characteristics similar to other compounds known to be allergens, i.e. eugenol, isoeugenol, p-benzylbromide, or with other drugs with aromatic groups which have been associated with allergic reactions, i.e. acetylsalicylic acid, paracetamol. Although more difficult to detect, these features can also be observed within the ofloxacin structure. Different appears the situation of streptozotocin and neomycin, where this assumption is not applicable. It is, however, possible to considerate two other important factors. First, both drugs are of bacterial origin, and as such they should be considered toxins or defense elements developed by microorganism. It is therefore plausible to assume that they can be recognized by the immune system as non-self molecules. A possible confirmation of this hypothesis is the observation of similarities with the complex structure of LPS, the bacterial compound used as classical activator of innate immunity. The second point to consider is that these compounds are characterized by a high number of polar and hydrophilic groups (–OH, –NH2): in vivo they can stably bind to plasma proteins, suggesting the possibility to easily make complete antigens able to be recognized as non-self by the immune system. On the other side, observing the structure of metformin (the drug selected as non-sensitizing) clearly the characteristics outlined above are absent. It is possible to affirm that the presence of aromatic rings, in particular anilines with electronegative substituents in ortho/para position, may be a necessary requirement to generate in vivo derivatives that can result in the activation of the immune system. In conclusion, we establish experimental conditions and markers to correctly identify drug sensitizers. The proposed screening assay can be easily incorporated into drug development to identify drugs, which may have the potential to cause allergic or systemic hypersensitivity reactions, an important clinical problem.
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Conflict of interest
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Authors declare of not having any financial, personal, or association with any of individuals or organizations that could have inappropriately influenced the submitted work.
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Uncited references
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Banchereau and Steinman (1998), Burchiel et al. (1997), Coombs and Gell (1975), Corsini and Roggen (2009), Ehlrich (1909), Fernandez et al. (2014), Gell and Coombs (1963), Jerschow et al. (2001), Kimber et al. (2002), Vandebriel et al. (2005).
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tiv.2015.04.012.
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