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In situ Transient Absorption Spectroscopy to Assess Competition between Serum Albumin and Alpha-1-Acid Glycoprotein for Drug Transport l P Rau erez-Ruiz, Carlos J. Bueno, M. Consuelo Jim enez,* and Miguel A. Miranda* Departamento de Química, Instituto de Tecnología Química UPV-CSIC, Universidad Polit ecnica de Valencia, Camino de Vera s/n, Valencia, Spain
ABSTRACT A new methodology based on transient absorption spectroscopy has been developed to assess the distribution of a drug between two transport proteins that are present in blood. The results show that serum albumins are the major carriers for (S)- and (R)-flurbiprofen (FBP); conversely, for the corresponding FBP methyl esters (FBPMe), the process is species-dependent, with bovine alpha-1-acid glycoprotein competing favorably with bovine serum albumin when both proteins are present in the same medium. Thus, transient absorption spectroscopy constitutes a new methodology for rapid and reliable assessment of drug distribution between serum albumins and alpha-1-acid glycoproteins that can be applied in situ and does not require any previous workup or separation of the analytes. The concept can, in principle, be extended to the investigation of drug distribution between several compartments available in biological systems. SECTION Biophysical Chemistry
erum albumin (SA) and alpha-1-acid glycoprotein (AAG) are transport proteins; they carry endogenous and exogenous agents in the bloodstream, to achieve a selective delivery to specific targets. Hence, their interaction with drugs plays a crucial role in pharmacokinetics.1 Human serum albumin (HSA) is synthesized and secreted by the liver. Its primary structure consists of a single chain of 585 amino acid residues, with 17 disulfide bridges, 1 tryptophan, and 1 free cystein and has a molecular weight of 67 kDa; 67% of the secondary structure is formed by a R-helix of six turns. The three-dimensional structure of HSA can be described in terms of three domains, each of them composed of two subdomains. According to the pioneering work of Sudlow and co-workers based on the displacement of fluorescence probes, drugs bind primarily to two main high-affinity sites, called site I (warfarin site) and site II (indole-benzodiazepine site), with association constants in the range of 104-106 M-1. In addition, lower-affinity sites can also be populated, although to a lower extent.2 Bovine serum albumin (BSA) is also among the most studied proteins in biochemical research; BSA and HSA have a 76% sequence identity, but BSA contains two Trp residues instead of one. Thus, binding of small organic molecules (i.e., drugs or fatty acids) to HSA and BSA has been studied for years through different well-established techniques in order to gain deeper insight into the role of transport proteins and also in relation with the structural basis for designing new therapeutic agents.3-13 Likewise, AAGs are synthesized in the liver, although they can also be expressed in extra hepatic tissues. Being one of the major types of acute phase proteins, their serum concentration
S
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increases in response to a systemic tissue injury, inflammation, or infection. Human AAG (HAAG) consists of 1 polypeptide chain with 183 amino acids, and has a molecular weight of 44 KDa. This protein exhibits a high degree of glycosylation, with carbohydrates accounting for ∼45% of its total mass. Up to seven binding sites have been described for HAAG; however, most drugs and small molecules bind almost exclusively to one of them. It is large and flexible, so that the high-affinity binding areas for basic, acid, and neutral drugs overlap. The other binding sites have a much lower importance, and therefore, their role in the transport of substrates is negligible.14-22 As regards bovine AAG (BAAG), binding of some ligands has been studied by fluorescence techniques. The results reveal a basic drug binding site and a steroid hormone binding site, which significantly overlap and affect each other, but do not contain an acidic ligand binding region. The hydrophobic nature of the binding pockets on the two AAGs is similar, but their microviscosities are markedly different.23 While SAs are the most abundant proteins in plasma, their concentration decreases in many diseases, such as inflammatory processes. The reverse is true for AAG values under similar conditions. Thus, a noninvasive, real time investigation of the competing interactions between anti-inflammatory drugs and these two types of transport proteins simultaneously
Received Date: January 8, 2010 Accepted Date: February 2, 2010 Published on Web Date: February 10, 2010
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the prodrug FBPMe (Chart 1); in this case, a significant stereodifferentiation in the triplet lifetimes in the presence of HSA is observed (τIS =31.5 μs, τIIS =4.1 μs, τIR =157.6 μs, and τIIR= 16.6 μs).25,26 With this background, we decided to make use of the discrimination between the triplet lifetimes of FBPand FBPMe in protein binding to develop a new methodology aimed at a rapid and reliable assessment of distribution between two proteins present simultaneously in a biological medium. The strategy is based on determination of the percentage of drug or prodrug from the relative contributions of the triplet lifetimes obtained from the decay curves in the presence of SA/AAG binary systems (Scheme 1). Parallel studies were conducted with the (S)- and (R)-isomers in order to detect a possible stereodifferentiation in the process. Since the behavior of FBP and FBPMe in HSA and BSA has been previously determined,24-26 binding of the two probes to AAG was studied with a similar methodology. Thus, a series of solutions containing (S)-FBP, 2.5 10-5 M, in the presence of HAAG or BAAG at different molar ratios (from 1.0:0.5 to 1.0:2.0) were prepared and submitted to LFP. Similar experiments were performed on (S)-FBPMe. In all cases, the typical triplet-triplet absorption spectra were obtained as transient bands in the region of 300-450 nm, with maxima at λ = 360 nm (data not shown). The decay kinetics were homogeneous throughout the entire spectra. Some representative decays (at λ = 360 nm and for a drug/protein molar ratio of 1:0.5) are shown in Figure 1. It can be observed that for both FBP and FBPMe, the decay traces lengthen only slightly in the presence of the human protein, while the change is much more dramatic in the presence of BAAG. Only one τT value different from 1.5 μs (due to free FBP or FBPMe) was found in the presence of the AAGs, indicating only one type of binding site. The decay traces at 360 nm were satisfactorily fitted with eq 1
available in the same medium becomes an important issue, which is difficult to address by the existing techniques. Determination of drug binding to a single protein is usually performed by different methods, such as ultrafiltration, chromatography, circular dichroism, capillary electrophoresis, NMR spectroscopy, fluorescence spectroscopy, biochemical assays, and so forth. However, none of these analytical tools is suitable for a rapid and reliable quantification of drug distribution between two different proteins naturally occurring in the same biological medium. In this context, the development of new methodologies capable of addressing this issue would be useful to deal with a more general type of problems, where low-molecular-weight ligands interact with microheterogeneous biological systems composed of different compartments.24-28 We have recently reported the use of triplet excited states as reporters for the binding of drugs to transport proteins, which is based on the fact that the properties of these transient species are highly sensitive to the microenvironment. Thus, laser flash photolysis (LFP) measurements have been performed on (S)- and (R)-flurbiprofen (FBP, Chart 1) in the presence of HSA.24 At 266 nm (2.5 10-5 M, PBS, air), (S)-FBP exhibits a characteristic transient triplet-triplet absorption spectrum centered at 360 nm, with a lifetime (τT) of 1.5 μs. In the presence of HSA (1:1 molar ratio), two τT values are observed (11.2 and 35.9 μs) and ascribed to (S)-FBP inside of the two main HSA binding sites. At different (S)-FBP/HSA ratios, drug distribution among the bulk solution and the protein binding sites has been established from the relative contributions of the components with different τT values. The same trend, but with different triplet lifetimes (10.2 and 39 μs), has been observed for (R)-FBP/HSA systems. For experimental convenience, the above experiments have been done under ambient oxygen pressure; bubbling with nitrogen or oxygen results in formation of foams, making it difficult to obtain accurate values for the triplet lifetimes. Nevertheless, rough estimations have been done on the effect of oxygen in these systems, where triplet quenching occurs much slower than in solution.24 Similar studies have been performed with
ΔðODÞ ¼ ΔðODÞ0 þ AFree e -t=τF þ ABound e -t=τB
ð1Þ
From the preexponential factors, the percentages of free and protein-bound ligand were obtained. Such values are shown in Table 1, together with the triplet lifetimes of the bound species. The results indicate that FBP binds to a low extent to both AAGs, while inclusion of FBPMe in both proteins is more efficient. Remarkably, in the case of BAAG, the percentage of bound FBPMe was close to 50%. Once the binding degree of FBP and FBPMe in both proteins separately was determined, the situation in binary
Chart 1. Chemical Structures of the Ligands Used in This Study
Scheme 1
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Figure 1. Decays (λ = 360 nm) for FBP(Me) and FBP(Me)/AAG mixtures at a 1:0.5 molar ratio after LFP at 266 nm. (A) (S)-enantiomers. (B) (R)-enantiomers.
In summary, transient absorption spectroscopy constitutes a new methodology for rapid and reliable assessment of drug distribution between serum albumins and alpha-1-acid glycoproteins. It can be applied in situ and does not require any previous workup or separation of the analytes. The basic procedure relies on determination of the percentages of ligand in the different microenvironments from the relative contributions of the triplet lifetimes obtained by laser flash photolysis upon fitting of the decay curves in the presence of SA/AAG binary mixtures. The concept can in principle be extended to the investigation of drug distribution between several compartments available in biological systems.
Table 1. Triplet Lifetimes and Percentages of FBP and FBPMe Bound to HAAG and BAAG Determined upon LFP Experiments AAG
τT (μs)
% FBP(Me) bounda
(S)-FBP (R)-FBP
human human
25.5 22.2
5 6
(S)-FBPMe
human
23.0
14
(R)-FBPMe
human
22.0
13
(S)-FBP
bovine
30.3
5
(R)-FBP
bovine
23.5
6
(S)-FBPMe
bovine
27.8
52
(R)-FBPMe
bovine
29.3
51
METHODS
a
Percentage of FBP or FBPMe bound to human or bovine alpha1-acid glycoprotein.
Experimental details for a typical determination of binding degree for a FPB(Me)/AAG 1:0.5 M mixture is as follows. To 2963 μL of a 2.5 10-5 M solution of (S)-FBP(Me) in PBS, 37 μL of AAG 1 10-3 M in PBS was added. Then, the resulting solution (3 mL) was placed in a quartz cuvette and submitted to laser flash photolysis (10 shots for monitoring at 360 nm). This light dose did not result in any detectable decomposition of the sample, as revealed by UV-vis absorption measurements prior to and after photolysis. For the binary systems containing FBP(Me)/SA/AAG, solutions were prepared as follows. To 2926 μL of a 2.5 10-5 M solution of (S)-FBP(Me) in PBS, 37 μL of SA 1 10-3 M in PBS and 37 μL of AAG 1 10-3 M in PBS were added. The LFP experiment was performed by using a Q-switched Nd:YAG laser (Quantel Brilliant, 266 nm, 10 mJ per pulse, 5 ns fwhm) coupled to a mLFP-111 Luzchem miniaturized equipment. The absorbance of (S)-FBP(Me) was found to be ∼0.2 at the laser wavelength. The experiments were carried out in PBS (pH = 7.4, 0.01 M) at room temperature (22 °C) and under air atmosphere. The decay traces of FBP(Me)/AAG mixtures were fitted by eq 1, containing two monoexponential terms attributed to free and AAG-bound FBP(Me). For the more complex systems containing SA and AAG, eq 2 was employed to fit the decay traces. This experiment was repeated at least three times with fresh sample; triplet lifetimes and fittings of the decay traces were coincident within the experimental error margins. Under the employed experimental conditions, upon 266 nm excitation, FBP or FBPMe absorb a minor part of the incident light; however, this is enough to produce a good-quality signal in LFP. Fortunately, control experiments showed that direct absorption by the protein does not lead to any interfering
systems containing serum albumin/glycoprotein was investigated. Thus, FBP(Me)/SA/AAG mixtures (molar ratio of 1:0.5:0.5) were submitted to LFP at λexc = 266 nm. As an example, the decays monitored at λ = 360 nm for the (S)enantiomers of FBPMe are shown in Figure 2. To obtain accurate fittings, it was assumed that, for a given FBP(Me)/SA/AAG mixture, four different kinds of triplets are present; they are ascribed to FBP(Me) free, bound to AAG, and within site I or site II of SA. Then, the law for fitting the decay trace would be given by eq 2 ΔOD ¼ ΔðODÞ0 þ AFree e -t=τF þ ABound e -t=τB þ AI e -t=τI þ AII e -t=τII
ð2Þ
in which τF corresponds to the species in the bulk solution and τB to the ligand bound to AAG, and τI and τII are the triplet lifetimes in site I and site II of SA, respectively. Assuming that the AI/AII ratios found in the FBP(Me)/SA 1:0.5 mixtures remain constant and fixing the triplet lifetimes to the values determined in separate FBP(Me)/AAG and FBP(Me)/SA experiments, the fitting of each decay trace allowed us to obtain AFree, ABound, AI, and AII and hence the distribution of FBP or FBPMe within each protein and the bulk solution. The results are displayed in Figure 3. They show that SAs are the major transport proteins for the free acids, while in the case of the methyl esters, the process is species-dependent, with BAAG competing favorably with BSA when both proteins are present in the same medium.
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Figure 2. Decays (λ = 360 nm) for FBPMe/SA/AAG mixtures at a 1:0.5:0.5 molar ratio after LFP at 266 nm. (A) (S)-FBPMe. (B) (R)-FBPMe. Generalitat Valenciana (Prometeo Program) is gratefully acknowledged.
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Figure 3. Percentage of FBP(Me) free (violet), site I-bound (blue), site II-bound (green), and AAG-bound (orange) in SA/AAG binary systems at a FBP(Me)/SA/AAG 1:0.5:0.5 molar ratio, as determined by LFP. (A) (S)-enantiomers. (B) (R)-enantiomers. In general, errors have been lower than 5% of the represented values.
transient absorption. Fluorescence emission of the FBP(Me)/ AAG and FBP(Me)/SA/AAG complex perfectly matched with the calculation for the independent emission of the components, taking into account the relative absorbance. This allowed ruling out of singlet-singlet energy transfer between FBP(Me) and the proteins.
AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. E-mail: mcjimene@ qim.upv.es (M.C.J.);
[email protected] (M.A.M.).
ACKNOWLEDGMENT Financial support from the MEC (Grant CTQ2007-67010 and predoctoral fellowship to C.J.B.), the Fundaci on Caja Murcia (postdoctoral fellowship to R.P.-R.), the Carlos III Institute of Health (Grant RIRAAF, RETICS program), and the
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