Modeling skin sensitization potential of mechanistically hard-to-be-classified aniline and phenol compounds with quantum mechanistic properties

Skin sensitization related dermatitis and rash represents the most common manifestation of chemical immunotoxicity in humans, which results in a cost estimated $1 billion annually due to lost work, reduced productivity, medical care, and disability payments in USA [1, 2]. In addition, as part of the regulatory review process, an increase in the incidence of skin allergies and hypersensitivity-related adverse events associated with the use of FDA regulated products or approved drugs has been observed, suggesting a safety gap between premarket review and the post market surveillance [2].

Common testing methods to assess skin sensitization potential of materials include: (1) guinea pig maximization test (GPMT); (2) murine-based local lymph node assay (LLNA). In GPMT tests, hazard identification is done by visual observations of erythema and edema reactions, which are subjective, are difficult to differentiate between contact allergens and strong irritants, and is time consuming [3]. The LLNA is recommended by international regulatory agencies; however, inconsistencies between LLNA and clinical observations have been documented [4]. Considering the existence of vast compounds around today, developing rapid and effective methods for chemical sensitizer identification/risk assessment is still a challenge [2].

In silico approaches are an attractive alternative to animal testing through analyzing the structural features of sensitizers/non-sensitizers to derive predictive rules or models [5]. The risks of thousands of commercially available chemicals could be assessed in a cost effective manner. Among these approaches, mechanism based rules, which investigate the structural characteristics of sensitizers, are promising [6].

Historically, the first study of chemical reactivity and skin sensitization was reported in 1936 [7]. A mechanism of small organic molecules to form an immunogenic complex by reacting with macromolecules (proteins or others) in the skin to cause sensitization was postulated. Currently, a more plausible mechanism reported involves a formation of covalent bonding between electrophilic allergens and nucleophilic moieties of amino acids from skin proteins (usually side chains) [8]. Such amino acids include cysteine thiol (mainly) and lysine (amino), and to a lesser extent arginine, histidine, methionine and tyrosine [9]. Based on the well-established principles of mechanistic organic chemistry, the skin sensitization potential of a chemical in many cases was predicted by its reactivity with these residues [9, 10]. However, some compounds need to be activated via either autoxidation outside the skin (prehaptens) or bioactivation inside the skin (prohaptens) to be able to form immunogenic complexes with skin proteins [11].

Structure-activity relationship (SAR) studies of skin sensitization potential have been successfully carried out for epoxyaldehydes [12], enone [13], halogenated aromatics [14], benzaldehydes [15], dienes [16], oximes [17], aldehydes [18] and epoxides [19]. Aniline/aromatic amine and/or phenol derivatives are two large classes of frequently sensitizing chemicals. Quite a few pilot studies have been conducted [20–23]. Roberts et al. specifically investigated the sensitization mechanisms of diaminobenzenes or dihydroxylbenzenes [24]. However, the predictability of the skin sensitization potential for these two classes of chemicals is unsatisfactory [6, 25]. Further exploration of novel sensitization mechanisms will be informative for constructing better SAR models/rules. In addition, aniline and phenol moieties that are often present in approved drugs can also cause skin sensitization. For example, contact dermatitis occurs in one individual following prolonged subcutaneous infusion of hydromorphone [26], a cancer pain treatment agent which contains one phenol moiety.

Drug-induced skin reactions may be associated with several biological mechanisms, but in many cases the precise mechanism is unclear [27]. It is well-known that Type IV allergic reaction induced by many chemicals and drugs is a T-cell mediated delay type hypersensitivity which can cause skin sensitization/dermatitis [27].

In this study, we intended to establish an in silico model for a class of mechanistically hard-to-classify anilines and phenols to study the relationship between their chemical reactivity and biological allergic response. We then investigated sensitization mechanisms of action associated with these compounds based on the features of this model. The model was further utilized to analyze a subset of FDA approved drugs containing aniline and/or phenol groups in skin sensitization potential. The predicted skin sensitization potential for these drugs was validated according to relevant literatures and adverse event reports.

The compounds with aniline and/or phenol moieties can be classified into a single subclass for consideration of skin sensitizers. However, not all of the compounds possessing aniline or phenol groups are sensitizers, suggesting some compounds can form covalent bonds with skin proteins whereas others cannot. In this study, the sensitization potential of anilines and phenols were modeled using quantum mechanical descriptors.

Modeling the skin sensitization potential by quantum properties of anilines and phenols

The coefficient constant of ϵ _HOMO was determined as the highest weighting factor based on the results of linear regression analysis. The skin sensitization potential of anilines and phenols can be formulated as:

$\mathrm{Predicted}\mathrm{Value}\left(P\right)=15.30*{\mathit{\in }}_{\mathrm{HOMO}}\left(\mathrm{Hartree}\right)+5.08\left(1\right)$

(1)

The median of the symbolized skin sensitization potency, 0.50, was considered as the threshold for prediction of sensitizers and non-sensitizers. An aniline or phenol is predicted as a sensitizer if P is greater than 0.50, and as a non-sensitizer if P is below 0.5. With a threshold of P =0.50, Equation 1 implies that a chemical within the applicability domain is predicted to be a skin sensitizer if the HOMO energy is greater than -0.30 Hartree ((0.5-5.08)/15.30 = -0.299 ≈ -0.30). The experimental allergenicity categories (sensitizer or non-sensitizer) and predicted results of the training set are shown in Figure 1, where red-open circle dots indicate well-known sensitizers at 1 and non-sensitizers at 0, respectively. The blue-solid-diamond dots indicate the predicted values. All of the training compounds were correctly predicted by Formula 1. The same model was applied to the test set. Interestingly, all test compounds were correctly predicted (Figure 2). The total prediction accuracy of chemicals in training and test sets was 100% (30/30). The model shows very high accuracy and only depends on the value of ϵ _HOMO, suggesting that ϵ _HOMO is a key factor for the assessment of skin sensitization potential of those aniline and phenol containing compounds.

The linear relationship between the predicted values and ϵ _HOMO also suggests that a chemical with higher predicted value implies a higher reactivity for oxidation consequently resulting in higher skin sensitization potential. The LLNA data as a quantitative endpoint, posed a semi-dose-dependent manner, allows for prediction of potency. The EC3 values (effective concentration for a three-fold proliferation of lymph node cells) from the reported LLNA experiments of most allergen phenols were also collected as shown in Table 1. Weak sensitizers with higher EC3 values (meaning lower sensitization potential) have smaller P values. For example, P values of five weak sensitizers with100 > EC3 > 10 i.e., eugenol (CAS: 97-53-0, EC3 = 13.95), Dihydroeugenol (CAS: 2785-87-7, EC3 = 12.45), 2,2’-azodiphenol (CAS: 2050-14-8, EC3 = 27.90), 3-methyleugenol (CAS: 186743-26-0,EC3 = 32), and aniline (CAS: 62-53-3, EC3 = 89) were 0.549, 0.672, 0.580, 0.580, and 0.702, respectively. On the other hand, two moderate sensitizers, 2-Methoxy-4-methyl-phenol (CAS: 93-51-6, EC3 = 5.8), 4-Chloroaniline (CAS: 106-47-8, EC3 = 6.5), have a slightly higher P value 0.672, 0.656, respectively. Another two moderate sensitizers with smaller EC3 value, isoeugenol (CAS: 97-54-1, EC3 = 3.5) and 1-naphthol (CAS: 90-15-3, EC3 = 1.3) have greater P values, 0.840 and 0.886, respectively. For most chemicals, their -logEC3 values correlate with P values quite well, but for aniline, its –logEC3 value is much less potent than its P value predicted. This may indicate that the initial oxidation of aniline, which is quite fast, is not in this case the rate-determining step for protein haptenation. The analysis of the relationship between EC3 and ϵ_HOMO for these nine chemicals was reported in the Additional file 1.

Possible reaction mechanisms of aromatic anilines and phenols

Occurrence of electrophilic–nucleophilic reactions between chemical and skin proteins is a primary reason of chemical induced skin sensitization [8]. Most chemicals with high skin sensitization potential can be classified as Michael acceptors (MA), SN₂ electrophiles, S_NAr electrophiles, Schiff base formers, or acylation agents. The reaction mechanisms of anilines and phenols, however, are poorly understood and very few of them can be classified into the aforementioned five categories. One proposed mechanism is that sensitization occurs via oxygen attack ortho to an amino group or via oxidative quinone-methide formation [25, 41]. For example, Roberts et al. reported the mechanistic chemistry of aromatic diamino-, dihydroxy-, and amino-hydroxy compounds [24] where two parallel chemical mechanisms were described as the most possible processes: oxidation to electrophilic (protein reactive) quinones, quinone imines, or quinone di-imines or formation of protein reactive free radicals. These mechanisms, unfortunately, are not applicable to the all single NH/OH substituted anilines and phenols. For instance, aniline and 4-butylaniline are sensitizers whereas 4-aminobenzoic acid, 4-aminobenzenesulfonamide, and 4-aminobenzenesulfonic acid are non-sensitizers. Beside the solubility effects and the formation of ions/zwitterions, the reactivity variety of chemical entities by substituent effects play an important role in reducing dermal penetration and immunogenicity of protein conjugates.

By analyzing the relationship between quantum properties and chemical reactivity, we successfully modeled the skin sensitization potential of two groups of chemicals (aromatic anilines and phenols) with a single coefficient of ϵ _HOMO, while the energy of the lowest unoccupied molecular orbital (ϵ _LUMO), considered as the critical factor for most electrophilic reactions [8, 11], was poorly correlated with sensitization potential. These results suggest the skin sensitization mechanism of those compounds may result from several steps but not a directly electrophilic reaction.

The ϵ _HOMO dependent results implied that a process of losing electron may be involved in the activation of those sensitizers. Those compounds may be activated via an autoxidation mechanism to further interact with skin proteins as prehaptens. In addition, the mechanisms where these chemicals directly react with free radical of skin proteins also should be considered [42]. In the present study, we proposed that two potential pathways could lead these compounds to cause skin sensitization as shown in Scheme 1[42]. In the first pathway (Scheme 1a), an aniline (or a phenol) is readily oxidized to a radical cation through loss of an electron at the aromatic ring [43] and forms two possible reactive intermediates. A protein-associated sulfhydryl radical then attacks the aromatic of the radical cation to form a covalent bond at the orth- or para-position. Or, the reactive intermediates bind to nucleophilic moieties on proteins through the Michael addition. The second pathway corresponds to what Lepoittevin defined as a direct haptenation route [44], whereby attack of a protein associated sulfhydryl radical on the ring gives an intermediate radical (Scheme 1b).

A compound with lower energy of HOMO appears either more stable or less reactive when reacting with a protein associated sulfhydryl radical [24]. Therefore, as shown in Figure 3, the sensitizers e.g., the aniline (CAS: 62-53-3) and the eugenol (CAS: 97-53-0) equipped with higher ϵ _HOMO values can lose electron(s) more easily to form radical cation intermediates than non-sensitizers (e.g., 2-fluoro-5-nitroaniline (CAS: 369-36-8), 4-aminobenzoic acid (CAS: 150-13-0), salicylic acid (CAS: 69-72-7), and 3-hydroxy-4-nitrobenzoic acid (CAS: 619-14-7)). We noted that the non-sensitizers of anilines and phenols are those that have the electron withdrawing groups attached to the aromatic ring, such as –F, -NO₂, -COOH. This implies that introduction of electron withdrawing groups to the aromatic ring of anilines or phenols may be one of the effective ways to reduce the potency of skin sensitizers.

This study has demonstrated how quantum chemical calculations can be utilized to predict skin sensitization potential and to infer the reaction mechanism for a class of mechanistically hard-to-be-classified chemicals containing aniline and phenol moieties. The outcomes emphasized that the energy of highest occupied molecular orbital plays an important role for predicting skin sensitization potential of these compounds, indicating the activation process occurred via either autoxidation or direct reaction with free radical. Our model was further applied to predict the allergenic potential of the approved drugs containing aniline and/or phenol moieties. Several of these drugs were identified as sensitizers and the prediction agreed well with their “allergic dermatitis” side effect. Thus, the data indicate that our newly developed in silico algorithm shows promise as a preclinical risk assessment tool for screening allergenic potential.

Again, we should point out that skin allergic reactions are not commonly seen for drugs given via the oral route. Though they may share similar mechanisms, caution should be taken when extrapolating our model from skin sensitization potential for topically applied chemicals to predict “allergic potential” of drugs.