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J. Med. Chem. 2007, 50, 2896-2902
Relaxation Dynamics of Piroxicam Structures within Human Serum Albumin Protein Maged El-Kemary,† Michał Gil, and Abderrazzak Douhal* Departamento de Quı´mica Fı´sica, ICAM, and Seccio´ n de Quı´micas, Facultad de Ciencias del Medio Ambiente, UniVersidad de Castilla-La Mancha, AVda. Carlos III, S.N., 45071, Toledo, Spain ReceiVed December 11, 2006
We report on steady-state and ps-time-resolved emission studies of piroxicam (1) drug within human serum albumin (HSA) protein in cyclodextrin and in neat solvents. The steady-state results indicate that 1 binds to HSA protein and that two binding sites are involved. The fluorescence decays corresponding to site I in subdomain IIA and to site II in subdomain IIIA have time constants of ∼60 ps and ∼360 ps, respectively. The results suggest that the anion forms bind to site I, whereas the zwitterionic ones bind to site II. The energy-transfer process from excited tryptophan to 1 can occur with moderate efficiency (50%). The rotational time of 1 encapsulated by HSA indicates diffusion within the protein. These findings can be used for a better understanding of piroxicam and HSA interactions. 1. Introduction Studying structural dynamics of drug-protein complexes is crucial for understanding the biological effects and functions of drugs in the body.1 The nature of the interaction forces involved in drug-protein complexes plays a significant role in drug delivery and action.2-6 Elucidating the nature of the interactions and the time scale involved provide insights into the mechanism of molecular recognition and the role of binding in protein dynamics and function.7-10 Recently, femtosecond studies of dynamical solvation and local rigidity in a series of reversible conformations of human serum albumin (HSAa) have been reported at different pH values.11 The results revealed that the changes in the solvation dynamics with pH are correlated with the conformational transitions and are related to their structure integrity.11 Piroxicam (1, Figure 1) is a nonsteroidal anti-inflammatory drug (NSAID). Depending on the pH of the medium, it can adopt different structures.12 At neutral pH, under biologically relevant conditions, the anionic structure predominates (1d, Figure 1).13 At the ground state it may exist in various conformations because of its ability to form inter- and intramolecular H-bonds. Upon electronic excitation of the closed enol structure (1) in nonpolar solvents, an intramolecular protontransfer reaction takes place, transforming it to the keto-type form (1b, Figure 1).14 The keto form has a ps-lifetime in the first electronically excited state (S1) and a short-lived triplet state (7.5 ns).15 In protic solvents, a second species called an open conformer (1c, Figure 1) is formed.16 Furthermore, piroxicam caged within cyclodextrins (CD) and micelles was studied.14,17,18 In CD, depending on the concentration of the guest relative to that of the host and on pH of the medium, it forms 1:1, 1:2, and 2:2 guest/host stoichiometry complexes.14,17,19 At pH ) 4, 1 in CD exists as the closed conformer 1b.14 In reverse micelles containing water, the anionic species predominates in addition to the presence of open and closed conformers.18 * To whom correspondence should be addressed. Tel.: +34-925-265717. Fax: +34-925-268840. E-mail:
[email protected]. † Present address: Department of Chemistry, Faculty of Science, Kafr ElSheikh University, 33516 Kafr ElSheikh, Egypt. a Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; HSA, human serum albumin; CD, cyclodextrin; DM-β-CD, dimethyl-β-cyclodextrin; Trp214, tryptophan 214; EG, ethylene glycol; THF, tetrahydrofuran; CHX, cyclohexane; IRF, instrumental response function.
HSA protein is the most abundant one in blood plasma and it transports about 80% of a wide variety of fatty acids, metal ions, steroid hormones, vitamins, and drugs.1-3 It also regulates the osmotic blood pressure.1-3 The large plasticity of HSA is essential for the albumin molecule to accommodate a variety of ligands and to perform the transport function in the circulatory system.11 When transporting a drug, the nature of the interactions involved in the stability of the drug-protein plays a significant role in drug delivery.1-5 From the structural point of view, HSA is a single polypeptide chain consisting of 585 amino acids. Crystallographic studies indicate that at pH ≈ 7, it adopts a heart-shaped three-dimensional structure with three homologous domains I-III (Figure 1). Each domain contains two subdomains A and B.6,20 The ligand binds to HSA in regions located in hydrophobic cavities in subdomains IIA (binding site I) and IIIA (binding site II). Binding to site I is dominated by the strong hydrophobic interactions with most neutral, bulky, heterocyclic compounds, while binding to site II mainly involves ion (dipole)-dipole, van der Waals, and/or H-bonding interactions in the polar cationic group of HSA.6,21 Therefore, a detailed characterization of its effect on the structure and stability of a caged drug is essential for understanding the relevant keys for its physiological functions, delivery, and efficiency. The interaction between piroxicam and HSA has been previously studied. It is spontaneous, exothermic, and involves H-bonds and hydrophobic forces.22 It has been shown that 1 preferentially binds to site I (like warfarin) and has lesser affinity to site II (diazepam locus) of HSA.23 The binding constants for each site were determined using equilibrium dialysis and are K1 ) 2.3 × 105 M-1 and K2 ) 3.3 × 104 M-1, respectively.22 Although from the previous studies22-24 it was not clear which structures of 1 binds to HSA sites and no photophysical study of the complexes has been yet published. Here, we report on the excited-state dynamics of 1 in solution, in CD, and in HSA. The results provide information on the structural/conformational changes of 1 within HSA. We found that within the protein the anionic structure 1d of piroxicam binds to site I and that possibly the zwitterionic form (1e, Figure 1) binds to site II of HSA. The anisotropy measurements show that the drug diffuses within the protein. Furthermore, upon excitation of HSA, energy transfer may occur from Trp214 of the protein to the caged 1. Some neutral open structure 1c binds to the surface of the protein and probably via H-bonding.
10.1021/jm061421f CCC: $37.00 © 2007 American Chemical Society Published on Web 05/17/2007
Relaxation Dynamics of Piroxicam Structures
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2897
Figure 1. Crystalline structure of HSA (from ref 6) and possible structures of piroxicam (1): enol (1a), keto (1b), open (1c), anionic (1d), and zwitterionic (1e) forms.
Figure 2. UV-visible absorption (left) and emission (right) spectra of 1 (2 × 10-5 M) in water buffer solution (pH 7.1) in the absence (- - -) and presence (s) of HSA (20 µM) and upon excitation at 355 nm.
2. Experimental Section Piroxicam (1,2-benzothiazine-3-carboxamide-4-hydroxy-2-methyl-N-(2-pyridyl)-1,1-dioxide; 98%), HSA protein (99%, SigmaAldrich), and dimethyl-β-CD (DM-β-CD, 99%, Acros Organics) were used as received. All the solvents were spectroscopic grade (Sigma-Aldrich). We used a buffer of 50 mM sodium phosphate in Milli-Q water giving a pH ∼7.1. Steady-state UV-visible absorption and emission spectra were recorded on Varian (Cary E1) and Perkin-Elmer (LS 50B) spectrophotometers, respectively. Emission decays were measured by a time-correlated single photon counting system described before.25 The sample was excited by a 40 ps pulsed (20 MHz) laser centered at 371 nm, and the emission signal was collected at six wavelengths at magic angle. The instrumental response function (IRF) of the apparatus was typically 65 ps. Multiexponential functions convoluted with the IRF signal were fitted to the emission decays using the Fluofit package (PicoQuant). The quality of the fits was characterized in terms of residual distribution and reduced χ2 value. All measurements were done at HSA concentration of 20 µM and at 293 ( 1 K.
3. Results and Discussion 3.1. Steady-State Observation. Figure 2 shows the UVvisible absorption and emission spectra of 1 in neutral water and in the presence of 20 µM of HSA. Upon addition of HSA, the absorption maximum at ∼355 nm slightly shifts to 358 nm
Figure 3. (A) Fluorescence spectra of HSA (20 µM) and piroxicam solutions in water at pH ) 7.1 and at different drug concentrations (excitation at 280 nm). The emission intensity (I) of the spectra was corrected from reabsorption and inner filter effects, Icorr ) Iobs × antilog [(A(λexc) + (A(λem))/2], where A is the optical density at the excitation or emission wavelength. (B) Fluorescence excitation spectra of HSA (20 µM) in water (- - -) observed at 360 nm, piroxicam (20 µM) in water (- ‚ -) observed at 480 nm and piroxicam in the presence of HSA (solid line) observed at 480 nm.
without change in the intensity, whereas the emission intensity is enhanced significantly with a shift from ∼457 to 485 nm. These spectral changes indicate that 1 binds to HSA protein. Figure 3A shows the change in the fluorescence spectra of the protein in the presence of different concentrations of 1 upon excitation at 280 nm. In neutral buffer solution, we observed a strong fluorescence band having a peak at 337 nm due to
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Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
El-Kemary et al. Table 1. Values of the Emission Lifetimes (τi) and Normalized Pre-Exponential Factors (ai) from the Multiexponential Fit to the Fluorescence Decays of 1 in Different Media Observed at 460 nma medium neutral buffer alkaline bufferb CHX THF EGc EGd HSA DM-β-CD
Figure 4. Normalized absorption spectra of 1 in EG at different concentrations: (A) 6 × 10-4 M (green); (B) 6 × 10-5 M (blue); (C) 1.5 × 10-6 M (red); (D) 6 × 10-4 M in alkaline EG (black).
tryptophan (Trp214) emission.26 Addition of 1 causes a concentration-dependent quenching of Trp214 emission with a concomitant increase in the emission at ∼490 nm, which is due to the bound 1. The emission spectrum of HSA has the maximum at 337 nm and shifts to ∼308 nm when increasing piroxicam concentration. This might be due (i) to a change in the local environment of Trp214 in HAS or (ii) that the remaining emission comes from the tyrosines of HSA. Another possible source of the blue shift that we cannot exclude is the increase of absorption of 1 when increasing its concentration. We believe that the decrease in Trp214 emission and the simultaneous increase in the piroxicam one suggest an energytransfer process between HSA and the caged drug. The inset of Figure 3A shows an iso-emissive point at ∼445 nm. Figure 3B shows that upon encapsulation of 1, the S0 f S1 transition of piroxicam shifts to longer wavelengths by 26 nm (1920 cm-1). At pH 7.1 (and 9-12) the absorption at 355 nm is due to the anion of 1 (phenolate-type structure, 1d). Figure 3B shows the excitation spectra of free and HSAcaged 1 recorded at 480 nm. For the piroxicam spectrum, it is clear that the ratio between the intensity of the bands at ∼360 and ∼280 nm decreases (from 1.6 to 1) in presence of HSA. The change at 280 nm is due to Trp214 absorption when gating the emission of caged 1. Therefore, an energy transfer process can occur upon excitation of Trp214 to produce an excited caged 1 emitting at 480 nm. To understand the extent of the H-bonding interactions of 1, with its restricting-motion environment, we have studied piroxicam in ethylene glycol (EG). The UV-absorption spectra at three different concentrations of 1 are shown in Figure 4. At a concentration of ∼10-5 M or larger, the absorption maximum of the first electronic transition shifts to shorter wavelengths and it stays at 330 nm when [piroxicam] g 5 × 10-5 M. For lower concentrations (e6 × 10-6 M), the maximum appears at 361 nm. The presence of dimers/oligomers is unlikely in this range of concentrations and can be safely excluded. Another possible source of such a large blue shift is the ground-state hydrogen-bonding interaction of 1 with EG and formation of a conjugate anion of 1, favored at low concentrations of the drug (Ostwald law for weak acid equilibrium). To support this explanation, we recorded the absorption spectrum of 1 in neutral and alkaline EG solutions (made by adding one KOH pellet to pure EG). After adding a small drop of alkaline EG to 6 × 10-6 M solution of 1, we observed a spectral change similar to the effect of dilution (spectrum D in Figure 4). To further support this explanation, we added a small amount of water to a concentrated solution of 1 in EG and we observed no change
λA/nm λF/nm τ1/ps
a1% 99.8
355
455
