Mechanistic Studies on the Photoallergy Mediated by Fenofibric Acid

Article pubs.acs.org/crt

Mechanistic Studies on the Photoallergy Mediated by Fenofibric Acid: Photoreactivity with Serum Albumins Ignacio Vayá,† Inmaculada Andreu,‡ Vicente T. Monje,† M. Consuelo Jiménez,*,† and Miguel A. Miranda*,† †

Departamento de Química/Instituto de Tecnología Química UPV-CSIC, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain ‡ Unidad Mixta de Investigación IIS La Fe-UPV, Hospital Universitari i Politècnic La Fe, Avenida de Fernando Abril Martorell 106, 46026 Valencia, Spain S Supporting Information *

ABSTRACT: The photoreactivity of fenofibric acid (FA) in the presence of human and bovine serum albumins (HSA and BSA, respectively) has been investigated by steady-state irradiation, fluorescence, and laser flash photolysis (LFP). Spectroscopic measurements allowed for the determination of a 1:1 stoichiometry for the FA/SA complexes and pointed to a moderate binding of FA to the proteins; by contrast, the FA photoproducts were complexed more efficiently with SAs. Covalent photobinding to the protein, which is directly related to the photoallergic properties of the drug, was detected after long irradiation times and was found to be significantly higher in the case of BSA. Intermolecular FA-amino acid and FA-albumin irradiations resulted in the formation of photoproducts arising from coupling between both moieties, as indicated by mass spectrometric analysis. Mechanistic studies using model drug−amino acid linked systems indicated that the key photochemical step involved in photoallergy is formal hydrogen atom transfer from an amino acid residue to the excited benzophenone chromophore of FA or (more likely) its photoproducts. This results in the formation of caged radical pairs followed by C−C coupling to give covalent photoaducts.

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also photosensitize damage to DNA,7 peroxidation of fatty acids,8 and red blood cell lysis.9 The photochemical reactivity and photophysical properties of FA, attributed mainly to its first triplet excited state, have been well characterized in organic and aqueous buffered solutions; the intersystem crossing quantum yield is higher in organic solvents than in aqueous medium.9−12 The free acid displays the typical benzophenone photoreactivity; however, the sodium salt undergoes photodecarboxylation. Its main photoproducts8−11,13 exhibit a higher hydrophobicity and maintain the benzophenone chromophore; hence, they can interact to a greater extent with biomolecules, resulting in an enhanced photosensitized damage.14,15 Serum albumins (SAs) are abundant proteins in blood and plasma and have a well-known structure.16 For these reasons, they have been widely used as model proteins in diverse studies.17−19 Their main physiological function is to transport exogenous and endogenous agents such as drugs, metabolites, fatty acids, etc., through the bloodstream.20,21 Hence, complexation of drugs with proteins is a key process, which modulates a number of properties such as drug solubility in plasma, transport, toxicity, susceptibility to oxidation, in vivo half-life, etc.22,23

INTRODUCTION

Drug-induced photosensitivity refers to adverse cutaneous sideeffects that result from exposure to certain drugs and light; these include phototoxic and photoallergic reactions. Phototoxicity, which normally appears at high drug and light doses, is the result of direct cellular damage produced by photochemical reactions mediated by drugs. In contrast, photoallergic reactions do not show strong dependence on drug concentration and may operate at low doses. From a mechanistic point of view, photoallergy normally involves covalent drug−protein photobinding leading to the formation of a complete photoantigen, which may trigger a hypersensitivity reaction due to a cell-mediated immune response.1 Fenofibrate (FB) is a third-generation fibric acid derivative commonly used to treat hypertriglyceridemia. It reduces the concentration of triglycerides by inhibiting hepatic synthesis and increasing their catabolism.2 It is a pharmacologically inactive pro-drug, and after administration, it undergoes rapid hydrolysis of the ester bond to form the active metabolite fenofibric acid (FA). The latter is known to generate photosensitivity side effects associated with photoallergic responses and characterized by erythematovesicular or eczematous eruptions.3−5 Both FB and FA are able to absorb UVA photons inducing in vitro prostaglandin synthesis,6 the mediators of the inflammatory response. Moreover, they can © 2015 American Chemical Society

Received: August 27, 2015 Published: December 3, 2015 40

DOI: 10.1021/acs.chemrestox.5b00357 Chem. Res. Toxicol. 2016, 29, 40−46

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Chemical Research in Toxicology

HPLC analysis was performed by means of a Waters HPLC system connected to a PDA Waters 2996 detector, using an isocratic flux (0.8 mL/min) of EtOAc/Hexane/HOAc (30/70/0.15%, v/v/v) as eluent, and a direct phase Teknokroma Kromasil 100 Si column, 5 μm (25 × 0.46 cm). Preparative HPLC was carried out with a JASCO HPLC equipment, composed of a DG-2080-54 degasification system, LG-2080-04 mixer, and a PU-2080 pump connected to a UV-1575 detector. An isocratic flux of 2 mL/min of EtOAc/Hexane/HOAc (30/70/0.15%, v/v/v) as eluent and a direct phase Teknokroma Tracer Excel 120 Si, 5 μm (25 × 1.0 cm) was employed. Gas chromatography was run with an HP-6890 Series and a HP 19091S-433 column, with an automatic Agilent Technologies 7683 Series injector coupled to an Agilent 5973 Network mass selective detector. Synthesis and Characterization of the New Compounds. To a solution of FA (100 mg, 0.31 mmol) in methylene chloride (20 mL), 0.33 mmol of EDC and 0.33 mmol of BtOH were added. The mixture was maintained under stirring, and then 0.31 mmol of (S)-Tyr or (S)Trp in 2 mL of methylene chloride was added dropwise. After 3 h, the crude was washed consecutively with diluted NaHCO3, 1 M HCl, and brine, and then dried over MgSO4. Final purification was performed by preparative layer chromatography (methylene chloride/ethyl acetate, 90/10, v/v), followed by recrystallization. The yields were 72% and 70% for FA-Tyr and FA-Trp, respectively. All new compounds were characterized by 1H and 13C NMR spectroscopy, as well as by high resolution mass spectrometry (HRMS). Their purity was confirmed by gas chromatography (GC) and high performance liquid chromatography (HPLC). A summary of the most relevant data follows. Compound 4: 4-(4-Chlorobenzoyl)phenyl acetate. 1H NMR (CDCl3) (δ, ppm) 2.35 (s, 3H), 7.23 (d, J = 8 Hz, 2H, ArH), 7.47 (d, J = 8 Hz, 2H, ArH), 7.75 (d, J = 8 Hz, 2H, ArH), 7.82 (d, J = 8 Hz, 2H, ArH). 13C NMR (CDCl3) (δ, ppm) 194.3, 168.9, 154.1, 139.0, 135.8, 134.7, 131.5 (×2), 131.4 (×2), 128.7 (×2), 121.7 (×2), 21.2. Exact Mass (ESI) [MH+] Calcd for C15H12ClO3: 275.0475. Found: 275.0471. FA-Tyr: Methyl 2-(2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanamido)-3-(4-hydroxyphenyl)-(2S)-propanoate. 1 H NMR (CDCl3) (δ, ppm) 1.50 (s, 3H), 1.58 (s, 3H), 2.84 (dd, J1 = 16.0 Hz, J2 = 8.0 Hz, 1H), 3.12 (dd, J1 = 16.0 Hz, J2 = 4.0 Hz, 1H), 3.74 (s, 3H), 4.74−4.79 (m, 1H), 6.12 (NH, 1H), 6.56−7.75 (m, 13H, ArH). 13 C NMR (CDCl3) (δ, ppm) 195.3, 173.9, 171.8, 158.4, 155.1, 139.0, 135.8, 131.8 (×2), 131.5 (×2), 131.2, 129.8 (×2), 128.7 (×2), 127.1, 118.8 (×2), 116.0 (×2), 81.4, 53.2, 52.4, 36.7, 26.5, 23.7. Exact Mass (ESI) [MH+] Calcd for C27H26ClNO6: 496.1512. Found: 496.1527. FA-Trp: Methyl 2-(2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanamido)-3-(1H-indol-3-yl)-(2S)-propanoate. 1H NMR (CDCl3) (δ, ppm) 1.50 (s, 3H), 1.58 (s, 3H), 3.22 (dd, J1 = 16.0 Hz, J2 = 8.0 Hz, 1H), 3.31 (dd, J1 = 16.0 Hz, J2 = 4.0 Hz, 1H), 3.71 (s, 3H), 4.88− 4.93 (m, 1H), 6.69−7.72 (14H, ArH), 8.12 (NH, 1H). 13C NMR (CDCl3) (δ, ppm) 194.6, 173.9, 172.1, 158.4, 138.8, 136.1, 136.0, 131.6 (×2), 131.4, 131.3 (×2), 128.6 (×2), 127.2, 122.5, 122.3, 119.7, 119.2 (×2), 118.4, 111.3, 109.7, 81.6, 52.6, 52.4, 27.4, 26.1, 24.0. Exact Mass (ESI) [MH+] Calcd for C29H27ClN2O5: 519.1664. Found: 519.1687. Laser Flash Photolysis Experiments. A XeCl excimer laser with an excitation wavelength of 308 nm was employed. The equipment consists of a pulsed laser, a 77250 Oriel monochromator, and an oscilloscope DP04054 Tektronix with connection to a personal computer for the transference of the output signal. The single pulses were of ca. 17 ns in duration, and the energy was maintained at 20 mJ/ pulse. Besides, a Q-switched Nd:YAG laser (Quantel Brilliant, 266 or 355 nm, 15 mJ per pulse, 5 ns fwhm) was coupled to a mLFP-111 Luzchem miniaturized equipment. This transient absorption spectrometer includes a ceramic xenon light source, 125 mm monochromator, Tektronix 9-bit digitizer TDS-3000 series with 300 MHz bandwidth, compact photomultiplier and power supply, cell holder and fiber optic connectors, fiber optic sensor for laser-sensing pretrigger signal, computer interfaces, and a software package developed in the LabVIEW environment from National Instruments.

The photobehavior of drugs in the presence of proteins is an important issue. This is because, as mentioned above, covalent drug−protein photobinding is in the origin of photoallergy but also because the complexation process may provide protection from photooxidation or other undesired reactions.24,25 In addition, the photosensitized modification of proteins may result in the loss of their biological functions. Fibric acid derivatives are generally well absorbed from the gastrointestinal tract and display a high degree of binding to SAs. In this context, it is known that about 99% of FA binds to serum proteins under physiological conditions.26,27 Furthermore, the FA glucuronide derivative has been studied in the presence of HSA and human plasma; hydrolysis of the glucuronide and acyl group rearrangement, as well as the formation of covalent adducts with albumin and plasma proteins, have been detected.28 The photoreactivity of FA has been investigated in different biomolecular environments (DNA, lipids, etc.).7,8 However, the direct photochemical interaction between FA and serum albumins has not been addressed in detail. In this work, we have investigated the photoreactivity of FA in the presence of human and bovine serum albumins (HSA and BSA, respectively) as well as in model linked systems (Chart 1). Chart 1. Chemical Structures of FA, FA-Tyr, and FA-Trp

Our results are in agreement with a moderate noncovalent binding of FA to the protein in the dark. Besides, photobinding has been found to occur both with FA and its photoproducts as a significant photoreaction pathway. Laser flash photolysis (LFP) experiments with FA/SA mixtures have shed some light on the mechanistic pathways leading to the undesired phototoxicology of the drug.

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MATERIALS AND METHODS

General. C o m m e r c i a l FA , (S ) -T r p , (S )- T yr, 1 -( 3(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (BtOH), dithiothreitol (DTT), iodoacetamide (IAM), and trifluoroacetic acid (TFA) were purchased from SigmaAldrich. Spectrophotometric HPLC or reagent grade solvents were obtained from Scharlab and used without further purification. Solutions of phosphate-buffered saline (PBS) (0.01 M, pH 7.4) were prepared by dissolving phosphate-buffered saline tablets (from Sigma) in Milli-Q water. Human and bovine serum albumins were purchased from Sigma-Aldrich. The 1H NMR and 13C NMR spectra were recorded in CDCl3 at 400 and 100 MHz, respectively, using a Bruker AVANCE III instrument; chemical shifts are reported in ppm. Steady state absorption spectra were recorded in a JASCO V-630 spectrophotometer. Emission spectra were recorded using a JASCO spectrofluorometer system provided with a monochromator in the wavelength range 200−900 nm. The solutions were placed into 10 × 10 mm2 quartz cells, and measurements were performed at room temperature. 41


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Chemical Research in Toxicology Scheme 1. Photoproducts of FA in PBS and MeCN Solutions

operated in positive ionization mode at 100 °C with the capillary voltage at 3.0 kV and a temperature of desolvation of 300 °C. The cone and desolvation gas flows were 40 and 800 L/h, respectively. The collision gas flow applied was 0.2 mL/min. All data collected in Centroid mode were acquired using Masslynx software (Waters Corp., Milford, MA, USA). Leucine−enkephalin was used at a concentration of 250 pg/mL as the lock mass generating an [M + H]+ ion (m/z 556.2771) and fragment at m/z 120.0813 with a flow rate of 50 mL/ min to ensure accuracy during the MS analysis. The photoadducts were identified by means of exact mass, and the tolerance of the measurements was ≤5 ppm. Protein Digestion and LC-ESI-MS/MS Analysis. HSA was enzymatically digested into smaller peptides using trypsin. Subsequently, these peptides were analyzed using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LCMS/MS). Briefly, 20 μg of sample was taken (according to Qubit quantitation), and the volume was set to 20 μL. Digestion was achieved with sequencing grade trypsin (Promega) according to the following steps: (i) 2 mM DTT in 50 mM NH4HCO3, V = 25 μL, 20 min (60 °C); (ii) 5.5 mM IAM in 50 mM NH4HCO3, V = 30 μL, 30 min (dark); (iii) 10 mM DTT in 50 mM NH4HCO3, V = 60 μL, 30 min; and (iv) trypsin (trypsin/protein ratio 1:20 w/w), V = 64 μL, overnight 37 °C. Digestion was stopped with 7 μL of 10% TFA (Cf protein ca 0.28 μg/μL). Next, 5 μL of sample (except the main bands) was loaded onto a trap column (NanoLC Column, 3 μ C18−CL, 350 μm × 0.5 mm; Eksigent) and desalted with 0.1% TFA at 3 μL/min during 5 min. The peptides were then loaded onto an analytical column (LC Column, 3 μm C18−CL, 75 μm × 12 cm, Nikkyo) equilibrated in 5% acetonitrile 0.1% formic acid. Elution was carried out with a linear gradient of 5 to 45% B in A for 30 min (A, 0.1% formic acid; B, acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min. Peptides were analyzed in a mass spectrometer nanoESI qQTOF (5600 TripleTOF, ABSCIEX). The tripleTOF was operated in information-dependent acquisition mode, in which a 0.25-s TOF MS scan from 350−1250 m/z was performed, followed by 0.05-s product ion scans from 100 to 1500 m/ z on the 50 most intense 2−5 charged ions. ProteinPilot v4.5. (ABSciex) search engine default parameters were used to generate a peak list directly from 5600 TripleTOF wiff files. The obtained mgf was used for identification with MASCOT (v 4.0, Matrix- Science). Database searches were performed on SwissProt database. Searches were done with tryptic specificity allowing one missed cleavage and a tolerance on the mass measurement of 100 ppm in MS mode and 0.6 Da in MS/MS mode. Carbamidomethylation of Cys was used as a fixed modification and oxidation of Met and deamidation of Asn and Gln as variable modifications. Two different modifications were defined in Y, W for FA.

The LFP equipment supplies 5 V trigger pulses with programmable frequency and delay. The rise time of the detector/digitizer is ∼3 ns up to 300 MHz (2.5 GHz sampling). The monitoring beam is provided by a ceramic xenon lamp and delivered through fiber optic cables. The laser pulse is probed by a fiber that synchronizes the LFP system with the digitizer operating in the pretrigger mode. Transient spectra were recorded employing 10 × 10 mm2 quartz cells with 4 mL capacity and were bubbled for 15 min with N2 before acquisition, when necessary. The concentration of FA in MeCN or PBS was 2.5 × 10−5 M, and for the protein complexes, a FA/SA 1:1 molar ratio was prepared. All of the experiments were carried out at room temperature. Steady-State Photolysis. Steady-state photolysis was performed by using a multilamp Luzchem photoreactor emitting at λmax = 300 nm (14 × 8 W lamps). Solutions of FA (10−4 M) were irradiated at different times in acetonitrile or PBS under N2 or air and in the absence or in the presence of protein (FA/SA 1:1 molar ratio) through Pyrex. First, calibration curves of FA and photoproducts were performed with the aim of quantifying the concentration of photoproduct formation after irradiation of the drug in aqueous solution and in the presence of SAs. Then, solutions of FA and FA/SA mixtures (4 mL) in PBS were irradiated in aerated conditions at different times. In the case of FA, the aqueous solution was washed with DCM (×3), dried with Na2SO4, and filtered. The organic layer was concentrated and dissolved in EtOAc. The crude was then analyzed by HPLC. In the case of samples containing albumins, protein was precipitated with 20 mL of acetone at −20 °C (5× dilution), stored at least 1.5 h at −20 °C, and centrifuged during 30 min (4 °C) at 6000 rpm. The supernatant was filtered with PTFE filters of 0.45 μm. The acetone was evaporated, and the resultant aqueous phase was washed with DCM (×3) to extract the organic compounds. Finally, DCM was evaporated, and the crude was dissolved in EtOAc and analyzed by HPLC. Products were eluted using a direct phase Teknokroma Kromasil 100 Si column, 5 μm (25 × 0.46 cm) with an isocratic flux of 0.8 mL/min and using EtOAc/ hexane/HOAc (30/70/0.15%, v/v/v) as eluent. Quantification of photoproduct formation was performed determining the area of each peak in the chromatogram and interpolating the obtained value to the calibration curves previously prepared. Detection of FA/Tyr and FA/Trp Photoadducts by UPLC-MS/ MS. Photoadducts formed between FA and Tyr or Trp were resolved using an Ultra Performance Liquid Chromatography (UPLC) system (ACQUITY, Waters Corp. Milford, MA, USA) with a conditioned autosampler at 4 °C and an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7 mm) maintained at 40 °C. The analysis was performed using acetonitrile and water (80:20, v/v, containing 0.01% formic acid) as the mobile phase with a flow rate of 0.5 mL/min and an injection volume of 5 μL. The UPLC system was connected to a Waters ACQUITY XevoQToF Spectrometer (Waters Corp., Milford, MA, USA) via an electrospray ionization interface. This source was 42


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RESULTS AND DISCUSSION The stoichiometry of FA/SA complexes was determined by means of Job plot analysis,29,30 where the absorbance of FA/ protein mixtures was represented as a function of the protein molar fraction. In all cases, a maximum at χprotein = 0.5 was observed, indicating the formation of 1:1 complexes (see Figure S1). Steady-state photolysis of FA (10−4 M) was performed in aerated phosphate buffer solution (PBS) and in the presence of equimolar amounts of HSA or BSA. Irradiations were done in a multilamp photoreactor with λmax = 300 nm through Pyrex; under these conditions, FA was irradiated selectively (see Figure S2). The course of the photoreaction was followed by HPLC (detection at λ = 254 nm). As anticipated from the literature, three main photoproducts (1, 2, and 3) resulting from decarboxylation of FA were obtained. Photolysis of FA in aerated MeCN evidenced the formation of an additional photoproduct 4 that was not previously identified; its formation can be explained through trapping of the intermediate radical species by oxygen (Scheme 1). Drug photodegradation was found to be faster in the bulk solution than in the presence of protein, the reactivity being lower in HSA than in BSA (see Figure 1); this points to a slowdown in the photodecarboxylation process within the protein cavities, which is more remarkable in the case of HSA.

Having demonstrated the photoreactivity of FA with isolated amino acids, the posible formation of covalent FA-protein photoadducts was investigated by digestion of an irradiated FA/ HSA mixture with trypsin, followed by LC-MS/MS analysis. Only one peptide sequence was covalenty modified by FA, namely, 469RMPCADEY#LSVVLNQLCVLHEK490, in which the Tyr-476 residue incorporated FA and FA-H2O as variable modifications. No modification was observed in the Trp residue probably because FA does not bind to site I of HSA, where the only Trp of the protein is located. With the aim of understanding the photoreaction mechanism of FA in the presence of proteins, LFP experiments were performed at λexc = 308 nm since at this excitation wavelength, the drug is the only absorbing species. Figure 2 shows the

Figure 2. Laser flash photolysis of FA (red), FA/HSA (black), and FA/BSA (blue) in aerated PBS at λexc = 308 nm (all concentrations were 2.5 × 10−5 M). (A) Transient absorption spectra obtained 0.04 μs after the laser pulse and (B) decays monitored at the maximum of the absorption bands (460 or 560 nm).

transient absorption spectra of FA in PBS solution and in the presence of HSA and BSA obtained 0.04 μs after the laser pulse. As it has been previously observed, the triplet absorption of FA in PBS consisted of a broad band in the 350−700 nm region.11 By contrast, in the presence of protein an absorption spectrum with two maxima at ca. 350 and 560 nm was detected (see also Figure S4), which is similar to that of the benzophenone ketyl radical.31 Besides, a residual absorption was observed in the 400−500 nm and 600−700 nm ranges, which can be assigned to the triplet state of the noncomplexed FA. This indicates a moderate degree of binding of the drug to SAs at the employed concentrations. Interestingly, the decay kinetics at the absorption maximum (Figure 2B) followed a monoexponential law for FA in PBS, assigned to the triplet excited state free in solution (τ = 0.88 ± 0.02 μs). By contrast, two lifetime components were necessary to obtain a good fitting for the FA/SA mixtures; their values and the weight of the corresponding pre-exponential factors were τ1 = 0.14 ± 0.01 μs (76%) and τ2 = 0.91 ± 0.23 μs (24%) for HSA, and τ1 = 0.11 ± 0.04 μs (65%) and τ2 = 0.82 ± 0.08 μs (35%) for BSA. The shorter lifetime components were assigned to the benzophenone ketyl radical formed within the protein cavities, while the longer ones were in the order of the triplet excited state lifetime of the noncomplexed FA. The relative weights of the two lifetimes in the case of HSA support a higher affinity of FA to this protein. This was confirmed by fluorescence measurements; thus, changes in the protein emission by addition of increasing amounts of FA revealed a moderate quenching that was more remarkable for HSA than for BSA (see Figure S5). The main FA photoproducts are also able to interact with biomolecules producing photosensitized damage.14,15 In fact, photoproducts 1 and 3 absorb in the UVB−UVA range (see

Figure 1. (A) Photodegradation of FA (10−4 M) in PBS (red) and in the presence of equimolar amounts of HSA (black) and BSA (blue). (B) Formation of photoproduct 1 (solid circles) and 2 (open circles) in the presence of HSA (black) and BSA (blue).

Photoproduct yields were strongly dependent on the investigated protein. Photoproducts 1 and 2 were obtained in higher yields, while 3 was always a minor photoproduct (see Figure S3). Interestingly, at short irradiation times 1 and 2 were detected in higher amounts in the presence of HSA. Besides, at long irradiation times the concentrations of 1 and 2 started to decrease due to photobinding to the protein, a process that was faster in the presence of BSA than in HSA (see Figure 1B). In order to check the feasibility of covalent photobinding of FA to the proteins, steady-state photolysis of intermolecular FA/Tyr and FA/Trp mixures was performed in deaerated PBS at λmax = 300 nm through Pyrex. Subsequent analysis by UPLCMS/MS evidenced the formation of photoproducts arising from coupling between the drug and the amino acids. Thus, in the case of FA/Tyr, the photoadduct of direct coupling was detected through the MH+ and MH+-H2O ions (MH+ exact mass value of 500.1495 and molecular formula of C26H27ClNO7; MH+-H2O of 482.1368 and molecular formula of C26H25ClNO6). Likewise, for FA/Trp, the photoproduct of direct coupling was identified through the MH+-H2O ion (exact mass value for MH+-H2O of 505.3322 and molecular formula of C28H26ClN2O5). 43


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transient at ca. 400 nm was attributed to the Tyr radical.39 These signals were clearly observed after subtraction of the FATyr spectrum from that of FA-Trp; thus, in the difference spectrum a positive band due to the Trp radical and a negative band corresponding to the Tyr radical were distinguishable (see Figure S10). It is worth noting the short lifetime of the biradical in both dyads (Figure 3B); a higher decay rate constant value was obtained for FA-Tyr (3.3 × 107 s−1) compared to that for FA-Trp (1.4 × 107 s−1). The structures of the biradicals are shown in Figure 3C and D. In the FA/protein systems, this process would give rise to caged radical pairs, where C−C coupling would afford fuchsone-like covalent photoadducts similar to those previously described for tiaprofenic acid.40

Figure S6) and are more hydrophobic than FA. This could result in stronger interaction with biomolecules, leading to enhanced photosensitizing properties. As a matter of fact, the photoproducts are sparingly soluble in aqueous buffer, while they are efficiently solubilized in the presence of SAs (see Figure S7). Since photoproduct 1 was formed in higher yields in all investigated media, we studied its photophysical properties in the presence of HSA and BSA. Fluorescence quenching studies revealed that the binding of 1 to SAs was higher than that of FA (see Figure S5), with higher affinity to HSA than to BSA. The obtained LFP spectra were compatible with the formation of the benzophenone ketyl radical (see Figure S8). Experiments of LFP were also performed with photoproducts 1, 2, and 3 in organic MeCN solutions (see Figure S9); as they maintain the benzophenone chromophore, the typical triplet−triplet absorption band with maxima at ca. 340 and 530 nm was detected. The longest triplet lifetime was observed for photoproduct 2. In order to gain deeper insight into the photosensitizing properties of protein-bound FA, two model dyads containing FA covalently linked to Tyr or Trp through an amide bond were synthesized (Chart 1). In this context, it has been demonstrated in previous reports that dyads composed of a drug covalently linked to an amino acid may provide key information on the processes that occur in the binding sites between the two chromophores (such as energy transfer, electron transfer, exciplex formation, etc.).32−36 Because of the poor solubility of the FA-amino acid linked systems in aqueous media, LFP measurements were performed at λexc = 355 nm in acetonitrile under N2. Figure 3A shows the

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CONCLUSIONS The photoreactivity of FA in the presence of HSA and BSA was lower than that in the bulk solution; hence, photoproduct formation occurs to a lesser extent within the protein cavities. Once formed, the photoproducts migrate from the solution to the intraprotein microenvironment, due to their markedly higher hydrophobicity. Covalent photobinding to the protein is revealed through the decrease in photoproduct yields at long irradiation times, a process that is faster for BSA than for HSA. Accordingly, intermolecular FA/Trp, FA/Tyr, and FA/HSA irradiations give rise to the formation of photoproducts arising from coupling between the drug and the biomolecule; this interaction has implications in the photoallergy elicited by FA. Moderate dark, noncovalent binding of FA to SAs is evidenced by fluorescence and LFP experiments, which demonstrates a higher affinity to HSA. This is in agreement with the faster photodegradation of FA in the presence of BSA compared to that of HSA. Mechanistic studies using model dyads suggest that the primary photochemical process underlying photoallergy is formal hydrogen atom transfer from an amino acid residue present in the binding site to the excited benzophenone chromophore. This results in the formation of tightly bound radical pairs that undergo C−C coupling to give covalent photoadducts.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00357. Spectroscopic measurements, 1H- and 13C NMR spectra of 4, FA-Tyr, and FA-Trp (PDF)

Figure 3. (A) Laser flash photolysis spectra of FA (red), FA-Tyr (violet), and FA-Trp (green) recorded 0.03 μs after the laser pulse, and (B) LFP decays for FA at 550 nm (red) and FA-Tyr (violet) and FA-Trp (green) at 570 nm, in MeCN/N2 (λexc = 355 nm). Structures of biradicals resulting from the intramolecular photoreaction in (C) FA-Tyr and (D) FA-Trp.

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AUTHOR INFORMATION

Corresponding Authors

*(M.C.J.) E-mail: [email protected]. *(M.A.M.) E-mail: [email protected].

LFP absorption spectra of FA, FA-Trp, and FA-Tyr obtained 0.03 μs after the laser pulse. As anticipated, FA displayed the typical triplet−triplet absorption band with maxima at 340 and 550 nm, whereas the spectra of the dyads showed a quite different picture. In both cases, bands peaking at ca. 340 and 570 nm (5:1 relative intensity) corresponding to the benzophenone ketyl radical31 were observed. However, in the case of FA-Trp a significant absorption at ∼500 nm was noticed and assigned to the Trp radical,37,38 whereas for FA-Tyr a

Funding

Financial support from the Spanish Government (CTQ201347872-C2-1-P, JCI-2011-09926, and Miguel Servet CP11/ 00154), EU (PCIG12GA-2012-334257) and Generalitat Valenciana (PROMETEOII/2013/005) is gratefully acknowledged. Notes

The authors declare no competing financial interest. 44


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ACKNOWLEDGMENTS The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia, which belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001, of the PE I+D+i 2013-2016, funded by ISCIII and FEDERPT13/ 0001.

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ABBREVIATIONS

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REFERENCES

BSA, bovine serum albumin; BtOH, 1-hydroxybenzotriazole; DTT, dithiothreitol; EDC, 1-(3-(dimethylamino)propyl)-Nethylcarbodiimide hydrochloride; FA, fenofibric acid; FB, fenofibrate; HSA, human serum albumin; IAM, iodoacetamide; LFP, laser flash photolysis; PBS, phosphate buffer solution; SAs, serum albumins; TFA, trifluoroacetic acid; Trp, tryptophan; Tyr, tyrosine

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Mechanistic Studies on the Photoallergy Mediated by Fenofibric Acid

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