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PHAREP 64 1–5 Pharmacological Reports xxx (2014) xxx–xxx
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Short communication
Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study Q1 Małgorzata
Macia˛z˙ek-Jurczyk
Medical University of Silesia, Department of Physical Pharmacy, Sosnowiec, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 February 2014 Received in revised form 18 March 2014 Accepted 18 March 2014 Available online xxx
Background: A combination of phenylbutazone (PBZ) and ketoprofen (KP) is popular in therapy of rheumatoid arthritis (RA) but could be unsafe due to the uncontrolled growth of toxicity. Methods: Quenching fluorescence of serum albumin in the presence of the both drugs has been characterized by dynamic KQ [M 1], static V [M 1] quenching constants and also association constants Ka [M 1]. Results: The quenching of tryptophanyl residues fluorescence by the KP and PBZ indicates the capability of these drugs to accept the energy from Trp-214 and Trp-135. Strong displacement of KP and PBZ bound to albumin cause by the binding of the second drug to SA close to Trp-214 (subdomain IIA) has been obtained. The displacement was also confirmed on the basis of quenching and association constants. Conclusions: The conclusion, that both PBZ and KP form a binding site in the same subdomains (IIA or/ and IB), points to the necessity of using a monitoring therapy owning to the possible increase of the uncontrolled toxic effects. ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Keywords: Competition Spectrofluorescence Serum albumin
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Introduction
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Rheumatoid arthritis (RA) results from inflammation of the joints that have been attacked because of a faulty immune system. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to reduce pain as well as inflammation in the joints. When two drugs are administered together the interaction and competition between them for the binding site on albumin may lead to the increase of unbound drug fraction in blood and evoke adverse/toxic effects. Phenylbutazone (PBZ) and ketoprofen (KP) are commonly used together in combination therapy of RA. Phenylbutazone and ketoprofen bind to plasma proteins, primarily to albumin, by 98–99%. Serum albumin (SA) is the most abundant plasma protein. The binding of drugs to plasma protein and thus formation of drug plasma protein complex is an important factor affecting their distribution and rate of metabolism. The binding mainly takes place in subdomains IIA and IIIA, known as Sudlow’s sites I and II. The interaction of drug with serum albumin is important component in understanding the mechanism of action, especially, drug distribution and interaction with SA. Binding of one drug
E-mail address: [email protected].
molecule to SA often influences simultaneous binding of another drug [1]. It can lead to the displacement of one drug by another in the binding site [2]. NSAIDs have high protein binding that may represent displacement of bound drugs especially those bound to albumin. Many studies have confirmed a competition between drugs in binding to the primary binding site on albumin [1–3]. Because both PBZ and KP show a high affinity for the serum albumin binding site, the possibility of displacement from secondary binding site to another site seems to be important to study and should be taken into account.
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Materials and methods
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Reagents
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Bovine serum albumin (BSA) and ketoprofen (KP) were purchased from MP Biomedicals (USA), human serum albumin (HSA) from ICN Biomedicals (USA). Phenylbutazone (PBZ) was provided by Sigma–Aldrich Chemical Co. (Germany). Sodium dihydrogen phosphate dehydrate (Na2HPO42H2O), dipotassium phosphate trihydrate (K2HPO43H2O) and methanol were purchased from POCH (Poland).
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http://dx.doi.org/10.1016/j.pharep.2014.03.005 1734-1140/ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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Fluorescence quenching measurements
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Emission fluorescence spectra were recorded on a Kontron Instrument AG spectrofluorimeter. To excite the SA fluorophores 295 nm wavelength was used. All solutions were prepared using 0.05 M sodium phosphate buffer (pH 7.4 0.1) and incubated overnight. Ketoprofen and phenylbutazone solutions were initially dissolved in methanol (not exceeding 1% v/v in the final concentration). To obtain the binary complexes (KP-SA, PBZ-SA) the solution of SA was titrated by ketoprofen and phenylbutazone solution. To obtain the ternary complexes (KP-[PBZ]-SA, PBZ-[KP]-SA) the solution of SA and appropriate volumes of first solution (to get molar ratio drug-albumin 1:1) was titrated by a second ligand. The intensity of fluorescence was corrected for the inner filter effect [4]. The UV–VIS absorption does not exceed 0.3 near excitation and emission wavelength. Therefore the correction for fluorescence intensity by the above equation was credible [5]. Dynamic KQ [M 1] and static V [M 1] quenching constants were calculated according to the modified Stern–Volmer nonlinear regression equation [6]. The association constants Ka [M 1] were estimated using the Scatchard method [7]. To calculate the percentage of displacement of KP (PBZ) from its binding site in albumin by PBZ (KP) the relationship was used: F0 F 100% F0
(1)
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where F0 and F are the fluorescence intensities of KP (PBZ) – SA in the absence and presence of ligands.
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Results and discussion
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Effect of phenylbutazone and ketoprofen on serum albumin fluorescence. Comparative study
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Ketoprofen (KP), an arylpropionic acid that contains a benzophenone moiety, is used as a photolabel of the binding region of Sudlow’s site II of serum albumin (SA) in subdomain IIIA [8]. For ketoprofen binding to SA the most important are the guanidine moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411, which is, protrude toward the center of site II [9]. The guanidine moiety probably interacts electrostatically with the carboxyl group of ketoprofen, the phenolic oxygen could make a hydrogen bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction (p–p) with one or both of the aromatic rings of ketoprofen [9]. A wavelength of 295 nm used in the study excites only tryptophanyl residues, i.e. Trp 135, 214 located in subdomains IB and IIA of BSA and Trp 214 in subdomain IIA of HSA. Fluorescence quenching of SA can be used to obtain many ligand-albumin binding information. When the fluorescence emission spectra of the donor and the absorption spectra of the acceptor have enough overlap and the distance between them should not exceed 10 nm, the energy transfer can occur [10]. Then, the albumin fluorescence could be quenched. The decrease of SA fluorescence intensity was observed at emission maxima for KP-SA (Fig. 1) and PBZ-SA (data not shown) systems. Addition of increasing amounts of ketoprofen and phenylbutazone causes the fluorescence of SA quenched. At KP:SA and PBZ-SA molar ratios 6.25:1 quenching fluorescence equals to almost 60–70% and was higher than that caused by PBZ. When absorbance at excitation and emission fluorescence was within 0.05 and 0.3 correction for the inner filter effect (IFE) was done (KP-SA system). Absorbance of PBZ at the concentration used was 0.05 and the fluorescence spectra have not been corrected. The quenching of tryptophanyl residues fluorescence by KP and
Fig. 1. Quenching curve of bovine (&) and human (&) serum albumin (8 10 6 [mol/L]) in the presence of KP (5 10 6 [mol/L]–5 10 5 [mol/L]), [KP]:[SA] 0.625:1–6.25:1; lex = 295 nm; T = 310 K. Error bars represent standard deviation.
PBZ indicates the capability of KP and PBZ to accept the energy from Trp-214, 135, i.e. two excited fluorophores, and thus location in human and bovine serum albumin subdomain IIA and IB, respectively. The aromatic residues of albumin form by specific stacking interaction with one or both of the aromatic rings of ketoprofen and phenylbutazone a sandwich-type complex. The quenching curves of HSA excited at lex = 295 nm in the presence of KP (Fig. 1) have been compared with those of BSA. The quenching curves of SA do not overlap. Fluorescence quenching of BSA by KP is more extended when compared to HSA. This effect points to alterations within Trp 214 and 135 microenvironments caused by the presence of KP means that besides of the primary binding site in subdomain IIIA the secondary KP-SA binding site in subdomain IIA or IB may exist. The p–p interaction between tryptophanyl residues (Trp-214, 135) and the aromatic ring of ketoprofen has been suggested basing on results presented in Fig. 1. With the increase of KP:SA molar ratio from 0.625:1 to 6.25:1 the decrease in both albumin fluorescence has been observed. The capability of phenylbutazone to accept the energy from the excited fluorophores of SA was observed by many scientists. Sułkowska et al. [1] observed that primary high affinity binding site and secondary binding site in serum albumin for PBZ are placed in subdomain IIA where Trp-214, 263 are located and IIIA of serum albumin. These sites are characteristic for a hydrophobic interaction. PBZ forms a ‘‘sandwich structure’’ with aromatic residues of SA and subdomain IIA is a major binding site for PBZ [11]. The quenching curves for PBZ-HSA and PBZ-BSA systems did not overlap. This indicates that Trp-135 located in the subdomain IB participates in the formation of PBZ-BSA complex. The results obtained show that tryptophanyl residues of albumin are accessible to KP and PBZ. Both, KP and PBZ form ‘‘sandwich-type’’ complex in the IIA subdomain, between their aromatic rings and the aromatic residues of tryptophan(s). The analysis of the simultaneous binding of these two drugs in Sudlow’s site I as well as possible competition between them in the binding within subdomain IIA (primary binding site for phenylbutazone and secondary binding site for ketoprofen) seemed to be necessary. Fluorescence intensity data in KP-SA (Fig. 2) and PBZ-SA (data not shown) systems were analyzed according to Stern–Volmer dependence [12]. The dependence of F0/F on concentration of KP (Fig. 2) when only tryptophanyl residues have been excited at 295 nm allows for description of both collisional (dynamic) and static quenching in subdomain IIA or IB. Linear dependence points to dynamic quenching. The Stern–Volmer plot showed a linear relationship between fluorescence intensity and quencher concentration [KP] below 2 10 5 [mol/L] in KP-SA (Fig. 2) and
Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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Fig. 2. The Stern–Volmer plots of F0/F vs.[KP] M for KP-HSA (&) complex (in the insert KP-BSA (&) complex); lex = 295 nm, T = 310 K. Error bars represent standard deviation.
Fig. 3. Isotherm plot of r vs. Lf [M] for KP-HSA (&) complex (in the insert KP-BSA (&) complex); lex = 295 nm, T = 310 K. Error bars represent standard deviation.
1 10 4 [mol/L] in PBZ-SA. In case of dynamic quenching the quencher must diffuse to fluorophore during the lifetime of the excited state. Then the fluorophore returns to its ground state without emission of a photon. At a higher concentration of KP in the KP-BSA (above 2 10 5 [mol/L]) and PBZ in PBZ-SA complexes (above 1 10 4 [mol/L]), positive deviation from the straight line can be explained by the fact that not all excited states are quenched by the collisional mechanism (dynamic quenching). It suggests a more complex quenching process, probably an additional presence of a static component in the quenching mechanism [13]. The phenomenon was explained by Eftink and Ghiron [6]. The static quenching is often observed if the fluorophore is adjacent to ketoprofen and phenylbutazone at the moment of excitation [14]. The upward curvature in the Stern–Volmer plot indicates that Trp214 and Trp-135 residues located in subdomain IIA and IB, respectively, are exposed to the quencher. In case of static quenching, a non-fluorescent complex between fluorophore and the quencher is formed. The ligand molecules are displaced after the quenching of fluorophore. The formation of this complex does not influence the diffusion in the excited state. The negative deviation from a straight line has been observed above concentration of KP 2 10 5 [mol/L] in the KP-HSA system. A downwardcurving plot is obtained when a fluorophore is distributed in two or more different environments that have different accessibility to fluorescence quenching (heterogeneously emitting system). It is difficult to analyze the downward curve (the insert in Fig. 2) in terms of the general treatment by Eftink and Ghiron, because their treatment contains too many parameters to be determined [15]. Therefore, in that case the simpler treatment proposed by Lehrer [16] should be used. A modified Stern–Volmer equation allowed to
obtained dynamic (KQ [M 1]) and static (V [M 1]) quenching 181 constants [13], which have been collected in Table 1. 182 The association constants Ka [M 1] and the mean number of Q2183 drug molecules bound to one molecule of albumin in the given 184 class of binding site (n) for the specific class of KP (Fig. 3) and PBZ 185 (Fig. not shown) binding sites in SA structure are presented in 186 Table 1. The shape of the isotherms indicates a mixed (specific and 187 nonspecific) nature of the interaction of KP (Fig. 3) and PBZ with 188 both serum albumins. In previous paper the formation of PBZ-SA 189 complex using spectrofluorescence and 1HNMR technique has 190 been studied [3]. Ka values determined for PBZ-SA complex in 191 present experiment are different than those reported in previous 192 study probably due to the use of lower PBZ-SA molar ratio 193 (presented PBZ:SA molar ratio 0.625:1–6.25:1 and previous 0.5:1– 194 0.625:1). As seen in Table 1, phenylbutazone creates a stronger 195 interaction with serum albumin than ketoprofen. It is noteworthy 196 because both PBZ and KP have a common binding site. 197 Phenylbutazone, often used as a marker for specific Sudlow’s site 198 I in albumin molecule, is located in subdomain IIA [17]. This place 199 is also a secondary binding site for KP. The possible competition 200 between KP and PBZ to the binding sites in albumin should be 201 taken into account. Strong PBZ binding to albumin may cause the 202 decreases of the concentration of an active drug (KP) in blood. 203 Effect of PBZ and KP on the KP-SA and PBZ-SA systems. Comparative study
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The quenching of both human and bovine serum albumin fluorescence excited at lex = 295 nm by KP and PBZ in the presence of constant concentration of the second ligand (PBZ – Fig. 4A and
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Table 1 Association Ka, quenching KQ and V constants and number of binding sites (n) for KP-SA, KP-[PBZ]-SA, PBZ-SA, PBZ-[KP]-SA systems; lex = 295 nm. KQ 104 [M A KP-HSA KP-[PBZ]-HSA PBZ-HSA PBZ-[KP]-HSA B KP-BSA KP-[PBZ]-BSA PBZ-BSA PBZ-[KP]-BSA a b
1
]
V 104 [M
1
]
2.40 0.08a 1.35 0.03a 3.03 0.06a 3.36 0.08a
0.34 0.06a,b 0.32 0.03a,b 0.25 0.08a 0.29 0.06a
2.14 0.07a 1.62 0.09a 3.41 0.04a 3.68 0.02a
0.24 0.02a 0.27 0.03a 0.32 0.04a 0.37 0.02a
KaI 104 [M 3.03 0.07a 1.46 0.10a 42.99 0.15a 96.65 0.22a 2.06 0.01a 1.89 0.01a 3.57 0.25a 7.15 0.09a
1
]
KaII 104 [M
1
]
nI
nII
0.29 0.07a 0.41 0.09a
0.47 0.02a 0.59 0.03a 0.45 0.12a 0.28 0.08a
1.83 0.21a 2.06 0.14a
0.06 0.00a 0.43 0.07a
0.30 0.01a 0.42 0.01a 0.95 0.05a 0.17 0.05a
2.66 0.17a 1.06 0.11a
Standard error. Negative deviation.
Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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Fig. 5. Effect of phenylbutazone and ketoprofen on fluorescence of ketoprofen (KP-[PBZ]-SA) and phenylbutazone (PBZ-[KP]-SA) bound to serum albumin. Percentage displacement of KP and PBZ in the ternary systems against drug:SA molar ratio. Error bars represent standard deviation.
Fig. 4. Comparison of quenching of HSA fluorescence by KP (&) and KP in the presence of PBZ (*) (A) and BSA fluorescence by PBZ (&) and in the presence of KP (*) (B); F/F0 is the ratio of the fluorescence of serum with and without quencher(s), &ex = 295 nm, T = 310 K. Error bars represent standard deviation.
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KP – Fig. 4B) was analyzed and compared in order to estimate the information about the effect of PBZ on KP (KP-[PBZ]-SA) and KP on PBZ (PBZ-[KP]-SA) secondary and primary binding site in SA, respectively. After addition of PBZ and KP, the quenching fluorescence of serum albumin in KP-SA complexes decreases (Fig. 4A and B). It means that PBZ influences the quenching of SA fluorescence by KP (Fig. 4A) and PBZ influences the quenching of SA fluorescence by KP (Fig. 4B). This phenomenon points to a possible effect of competitor (PBZ, KP) on KP and PBZ binding site within Trp-214 in subdomain IIA of SA, which is the primary for PBZ [1] and secondary for KP binding site in SA. This can be explained by the release of drugs from their binding sites. Second drug (PBZ or KP) reduces the affinity of first drug to subdomain IIA of SA, displaces the drug molecule and hinders the transfer energy due to the hydrophobic interactions stabilizing the complex. Therefore reciprocal interactions of both drugs (PBZ and KP) within the subdomain, which is common for this kind of binding of PBZ and KP to BSA, should not be excluded. Displacement of ketoprofen and phenylbutazone from their biding site in albumin was also studied by determining the percentage of displacement of bound KP and PBZ according to Eq. (1) (Fig. 5). A strong displacement of ketoprofen (>50%) and phenylbutazone (>30%) bound to albumin cause by the binding of the second drug to SA close to Trp-214 (subdomain IIA) was observed. The decrease of tryptophan fluorescence is the result of conformational changes of albumin molecule due to the interaction with the second drug. The dependence of F0/F on KP and PBZ concentration without and in the presence of PBZ and KP allowed for description of both
collisional (dynamic) and static quenching. The Stern–Volmer plot showed a linear relationship (dynamic quenching) between fluorescence intensity and quenchers (KP and PBZ) concentration below 2 10 5 [mol/L] and 1 10 4, respectively in both ternary complexes, similarly as it was in the binary KP-SA and PBZ-SA systems. Above these concentrations the positive deviation from the straight lines shows that static and dynamic quenching occur simultaneously. Effect of phenylbutazone and ketoprofen on their fluorescence was also confirmed on the basis of dynamic KQ [M 1] and static V [M 1] quenching constants and association constants Ka [M 1] (Table 1). In the high affinity binding sites of KP in serum albumin the presence of PBZ does not affect the static quenching constants V [M 1] for the p–p interaction between aromatic rings of KP and aromatic residues of serum albumin in subdomain IIA and IB. Similarly to the KP-[PBZ]-SA complex the presence of KP does not influence the static quenching constants V [M 1]. The dynamic quenching constants KQ [M 1] determined for binary system KP-SA are lower than that for the ternary KP-[PBZ]-SA. This phenomenon shows that in the presence of PBZ the interaction between KP and SA becomes weaker within subdomain IIA and IB, in the vicinity of Trp-214 and Trp-135. PBZ probably weakens the collision between excited state of SA and the quencher (KP). The comparison of the dynamic quenching constants KQ [M 1] of binary PBZ-SA and ternary PBZ-[KP]-SA complexes suggests that the presence of KP results in the increase of its values. This phenomenon shows as opposed to the quenching curves (Fig. 4B), that the presence of KP in the system makes formation of PBZ-SA complex easier. The interaction between PBZ and SA in the presence of KP becomes stronger within subdomain IIA and IB, in the vicinity of Trp-214, 135. KP probably makes the collision between excited state of SA and the PBZ more stable. The contrary relation between the results of the analysis of quenching curves (Fig. 4B) and dynamic quenching constants (Table 1) is probably caused by the fact that the qualitative analysis of the quenching effect allows for the assessment of the affinity of PBZ and KP to the total surface of SA macromolecule while quantitative analysis of the quenching parameters shows the changes in the specific binding sites with the static and dynamic quenching. The comparison of the association constants of binary with ternary complexes suggests that an addition of PBZ at constant concentration to KP-SA binary complex makes the values of Ka lower while addition of KP makes Ka higher in comparison with the corresponding binary complexes (Table 1). This leads to the conclusion that the presence of bound
Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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molecules of PBZ induces the weakness of affinity between PBZ and tryptophanyl residues located outside the subdomain IIA in the vicinity of Trp-214 due to the hydrophobic interaction and PBZ high affinity to SA. It is noteworthy that the presence of PBZ causes the increase of free biologically active fraction of KP. PBZ administered before KP may change affinity of the latter to the albumin. Trp-214 cannot be quenched since the KP cannot approach and accept its energy. Changes in parameters characterizing low affinity binding site are probably a result of the reorientation of drug molecules toward the mentioned fluorophore in the binding site. The published data indicate that PBZ interacts also with tyrosyl residues in subdomain IIA (Tyr-263) and subdomain IIIA (Tyr-401, 411, 452, 497), where a secondary weaker PBZ-SA binding site can be located [3]. Although presented paper does not describe the data on the participation of tyrosyl residues, it can be assumed that PBZ competes with the KP in the binding site in subdomain IIIA. Muirden et al. [18] found that an addition of phenylbutazone causes a displacement of a significant amount of salicylate bound to albumin. Curry et al. [19] confirmed the interaction of aromatic ring of drugs having an aromatic ring and carboxylic group with hydrophobic cavity of serum albumin. It was concluded that the complex is stabilized by hydrophobic interaction [19]. The presence of KP at the constant concentration dramatically affects the affinity between protein and PBZ in its secondary binding site. The increase of association constant in the ternary complex suggest that in the presence of KP the interaction between PBZ and SA becomes stronger within subdomain IIA, in the vicinity of Trp-214. KP induces distinct differences of binding parameters in the primary binding site of PBZ in subdomain IIA where Trp-214 plays a major role in the formation of the complex and can stabilize the interaction between aromatic ring of PBZ and aromatic residues of SA. The presence of KP in the system probably simplifies the formation of the PBZ-SA complex and may cause the decrease of free, biologically active fraction of ketoprofen. Moreover the rise of Ka observed for both systems can be explained by the formation of an additional interaction (e.g. electrostatic or hydrogenous) as a result of the conformational changes of the binding site. In analogy to the KP-[PBZ]-SA system, KP administered before PBZ may change affinity of the latter to the albumin. The analysis of association constants allows observing the inverse relation between Ka and n. The decrease of one parameter with the rise of the second parameter is observed. The increased number of bound molecules of PBZ or KP makes the energy transfer to the quencher difficult and this leads to the decrease of KQ and Ka values.
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Conclusions
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The competitive interaction as the mechanism leading to the decrease of KP affinity and increase of PBZ affinity toward the secondary binding site have been analyzed. The observed changes are undesirable from the pharmacological point of view. When KP and PBZ are administered at the same time, the bound drug can be displaced by another drug from its binding site, what may result in increase of the concentration of free biologically active fraction of
5
the released drug. Consequently this may lead to serious side/toxic effects. Therefore such conclusions may relieve to determine the appropriate dosage of both KP and PBZ to reach the therapeutic concentration.
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Conflict of interests
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I inform, that there is no conflict of interests. Acknowledgements
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This work was supported by grant from Medical University of Q3342 Silesia KNW-1-001/K/3/0 and KNW-2-001/N/3/K. 343 References
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Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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Short communication
Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study Q1 Małgorzata
Macia˛z˙ek-Jurczyk
Medical University of Silesia, Department of Physical Pharmacy, Sosnowiec, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 February 2014 Received in revised form 18 March 2014 Accepted 18 March 2014 Available online xxx
Background: A combination of phenylbutazone (PBZ) and ketoprofen (KP) is popular in therapy of rheumatoid arthritis (RA) but could be unsafe due to the uncontrolled growth of toxicity. Methods: Quenching fluorescence of serum albumin in the presence of the both drugs has been characterized by dynamic KQ [M 1], static V [M 1] quenching constants and also association constants Ka [M 1]. Results: The quenching of tryptophanyl residues fluorescence by the KP and PBZ indicates the capability of these drugs to accept the energy from Trp-214 and Trp-135. Strong displacement of KP and PBZ bound to albumin cause by the binding of the second drug to SA close to Trp-214 (subdomain IIA) has been obtained. The displacement was also confirmed on the basis of quenching and association constants. Conclusions: The conclusion, that both PBZ and KP form a binding site in the same subdomains (IIA or/ and IB), points to the necessity of using a monitoring therapy owning to the possible increase of the uncontrolled toxic effects. ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Keywords: Competition Spectrofluorescence Serum albumin
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Introduction
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Rheumatoid arthritis (RA) results from inflammation of the joints that have been attacked because of a faulty immune system. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to reduce pain as well as inflammation in the joints. When two drugs are administered together the interaction and competition between them for the binding site on albumin may lead to the increase of unbound drug fraction in blood and evoke adverse/toxic effects. Phenylbutazone (PBZ) and ketoprofen (KP) are commonly used together in combination therapy of RA. Phenylbutazone and ketoprofen bind to plasma proteins, primarily to albumin, by 98–99%. Serum albumin (SA) is the most abundant plasma protein. The binding of drugs to plasma protein and thus formation of drug plasma protein complex is an important factor affecting their distribution and rate of metabolism. The binding mainly takes place in subdomains IIA and IIIA, known as Sudlow’s sites I and II. The interaction of drug with serum albumin is important component in understanding the mechanism of action, especially, drug distribution and interaction with SA. Binding of one drug
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molecule to SA often influences simultaneous binding of another drug [1]. It can lead to the displacement of one drug by another in the binding site [2]. NSAIDs have high protein binding that may represent displacement of bound drugs especially those bound to albumin. Many studies have confirmed a competition between drugs in binding to the primary binding site on albumin [1–3]. Because both PBZ and KP show a high affinity for the serum albumin binding site, the possibility of displacement from secondary binding site to another site seems to be important to study and should be taken into account.
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Materials and methods
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Reagents
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Bovine serum albumin (BSA) and ketoprofen (KP) were purchased from MP Biomedicals (USA), human serum albumin (HSA) from ICN Biomedicals (USA). Phenylbutazone (PBZ) was provided by Sigma–Aldrich Chemical Co. (Germany). Sodium dihydrogen phosphate dehydrate (Na2HPO42H2O), dipotassium phosphate trihydrate (K2HPO43H2O) and methanol were purchased from POCH (Poland).
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http://dx.doi.org/10.1016/j.pharep.2014.03.005 1734-1140/ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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Fluorescence quenching measurements
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Emission fluorescence spectra were recorded on a Kontron Instrument AG spectrofluorimeter. To excite the SA fluorophores 295 nm wavelength was used. All solutions were prepared using 0.05 M sodium phosphate buffer (pH 7.4 0.1) and incubated overnight. Ketoprofen and phenylbutazone solutions were initially dissolved in methanol (not exceeding 1% v/v in the final concentration). To obtain the binary complexes (KP-SA, PBZ-SA) the solution of SA was titrated by ketoprofen and phenylbutazone solution. To obtain the ternary complexes (KP-[PBZ]-SA, PBZ-[KP]-SA) the solution of SA and appropriate volumes of first solution (to get molar ratio drug-albumin 1:1) was titrated by a second ligand. The intensity of fluorescence was corrected for the inner filter effect [4]. The UV–VIS absorption does not exceed 0.3 near excitation and emission wavelength. Therefore the correction for fluorescence intensity by the above equation was credible [5]. Dynamic KQ [M 1] and static V [M 1] quenching constants were calculated according to the modified Stern–Volmer nonlinear regression equation [6]. The association constants Ka [M 1] were estimated using the Scatchard method [7]. To calculate the percentage of displacement of KP (PBZ) from its binding site in albumin by PBZ (KP) the relationship was used: F0 F 100% F0
(1)
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where F0 and F are the fluorescence intensities of KP (PBZ) – SA in the absence and presence of ligands.
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Results and discussion
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Effect of phenylbutazone and ketoprofen on serum albumin fluorescence. Comparative study
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Ketoprofen (KP), an arylpropionic acid that contains a benzophenone moiety, is used as a photolabel of the binding region of Sudlow’s site II of serum albumin (SA) in subdomain IIIA [8]. For ketoprofen binding to SA the most important are the guanidine moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411, which is, protrude toward the center of site II [9]. The guanidine moiety probably interacts electrostatically with the carboxyl group of ketoprofen, the phenolic oxygen could make a hydrogen bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction (p–p) with one or both of the aromatic rings of ketoprofen [9]. A wavelength of 295 nm used in the study excites only tryptophanyl residues, i.e. Trp 135, 214 located in subdomains IB and IIA of BSA and Trp 214 in subdomain IIA of HSA. Fluorescence quenching of SA can be used to obtain many ligand-albumin binding information. When the fluorescence emission spectra of the donor and the absorption spectra of the acceptor have enough overlap and the distance between them should not exceed 10 nm, the energy transfer can occur [10]. Then, the albumin fluorescence could be quenched. The decrease of SA fluorescence intensity was observed at emission maxima for KP-SA (Fig. 1) and PBZ-SA (data not shown) systems. Addition of increasing amounts of ketoprofen and phenylbutazone causes the fluorescence of SA quenched. At KP:SA and PBZ-SA molar ratios 6.25:1 quenching fluorescence equals to almost 60–70% and was higher than that caused by PBZ. When absorbance at excitation and emission fluorescence was within 0.05 and 0.3 correction for the inner filter effect (IFE) was done (KP-SA system). Absorbance of PBZ at the concentration used was 0.05 and the fluorescence spectra have not been corrected. The quenching of tryptophanyl residues fluorescence by KP and
Fig. 1. Quenching curve of bovine (&) and human (&) serum albumin (8 10 6 [mol/L]) in the presence of KP (5 10 6 [mol/L]–5 10 5 [mol/L]), [KP]:[SA] 0.625:1–6.25:1; lex = 295 nm; T = 310 K. Error bars represent standard deviation.
PBZ indicates the capability of KP and PBZ to accept the energy from Trp-214, 135, i.e. two excited fluorophores, and thus location in human and bovine serum albumin subdomain IIA and IB, respectively. The aromatic residues of albumin form by specific stacking interaction with one or both of the aromatic rings of ketoprofen and phenylbutazone a sandwich-type complex. The quenching curves of HSA excited at lex = 295 nm in the presence of KP (Fig. 1) have been compared with those of BSA. The quenching curves of SA do not overlap. Fluorescence quenching of BSA by KP is more extended when compared to HSA. This effect points to alterations within Trp 214 and 135 microenvironments caused by the presence of KP means that besides of the primary binding site in subdomain IIIA the secondary KP-SA binding site in subdomain IIA or IB may exist. The p–p interaction between tryptophanyl residues (Trp-214, 135) and the aromatic ring of ketoprofen has been suggested basing on results presented in Fig. 1. With the increase of KP:SA molar ratio from 0.625:1 to 6.25:1 the decrease in both albumin fluorescence has been observed. The capability of phenylbutazone to accept the energy from the excited fluorophores of SA was observed by many scientists. Sułkowska et al. [1] observed that primary high affinity binding site and secondary binding site in serum albumin for PBZ are placed in subdomain IIA where Trp-214, 263 are located and IIIA of serum albumin. These sites are characteristic for a hydrophobic interaction. PBZ forms a ‘‘sandwich structure’’ with aromatic residues of SA and subdomain IIA is a major binding site for PBZ [11]. The quenching curves for PBZ-HSA and PBZ-BSA systems did not overlap. This indicates that Trp-135 located in the subdomain IB participates in the formation of PBZ-BSA complex. The results obtained show that tryptophanyl residues of albumin are accessible to KP and PBZ. Both, KP and PBZ form ‘‘sandwich-type’’ complex in the IIA subdomain, between their aromatic rings and the aromatic residues of tryptophan(s). The analysis of the simultaneous binding of these two drugs in Sudlow’s site I as well as possible competition between them in the binding within subdomain IIA (primary binding site for phenylbutazone and secondary binding site for ketoprofen) seemed to be necessary. Fluorescence intensity data in KP-SA (Fig. 2) and PBZ-SA (data not shown) systems were analyzed according to Stern–Volmer dependence [12]. The dependence of F0/F on concentration of KP (Fig. 2) when only tryptophanyl residues have been excited at 295 nm allows for description of both collisional (dynamic) and static quenching in subdomain IIA or IB. Linear dependence points to dynamic quenching. The Stern–Volmer plot showed a linear relationship between fluorescence intensity and quencher concentration [KP] below 2 10 5 [mol/L] in KP-SA (Fig. 2) and
Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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Fig. 2. The Stern–Volmer plots of F0/F vs.[KP] M for KP-HSA (&) complex (in the insert KP-BSA (&) complex); lex = 295 nm, T = 310 K. Error bars represent standard deviation.
Fig. 3. Isotherm plot of r vs. Lf [M] for KP-HSA (&) complex (in the insert KP-BSA (&) complex); lex = 295 nm, T = 310 K. Error bars represent standard deviation.
1 10 4 [mol/L] in PBZ-SA. In case of dynamic quenching the quencher must diffuse to fluorophore during the lifetime of the excited state. Then the fluorophore returns to its ground state without emission of a photon. At a higher concentration of KP in the KP-BSA (above 2 10 5 [mol/L]) and PBZ in PBZ-SA complexes (above 1 10 4 [mol/L]), positive deviation from the straight line can be explained by the fact that not all excited states are quenched by the collisional mechanism (dynamic quenching). It suggests a more complex quenching process, probably an additional presence of a static component in the quenching mechanism [13]. The phenomenon was explained by Eftink and Ghiron [6]. The static quenching is often observed if the fluorophore is adjacent to ketoprofen and phenylbutazone at the moment of excitation [14]. The upward curvature in the Stern–Volmer plot indicates that Trp214 and Trp-135 residues located in subdomain IIA and IB, respectively, are exposed to the quencher. In case of static quenching, a non-fluorescent complex between fluorophore and the quencher is formed. The ligand molecules are displaced after the quenching of fluorophore. The formation of this complex does not influence the diffusion in the excited state. The negative deviation from a straight line has been observed above concentration of KP 2 10 5 [mol/L] in the KP-HSA system. A downwardcurving plot is obtained when a fluorophore is distributed in two or more different environments that have different accessibility to fluorescence quenching (heterogeneously emitting system). It is difficult to analyze the downward curve (the insert in Fig. 2) in terms of the general treatment by Eftink and Ghiron, because their treatment contains too many parameters to be determined [15]. Therefore, in that case the simpler treatment proposed by Lehrer [16] should be used. A modified Stern–Volmer equation allowed to
obtained dynamic (KQ [M 1]) and static (V [M 1]) quenching 181 constants [13], which have been collected in Table 1. 182 The association constants Ka [M 1] and the mean number of Q2183 drug molecules bound to one molecule of albumin in the given 184 class of binding site (n) for the specific class of KP (Fig. 3) and PBZ 185 (Fig. not shown) binding sites in SA structure are presented in 186 Table 1. The shape of the isotherms indicates a mixed (specific and 187 nonspecific) nature of the interaction of KP (Fig. 3) and PBZ with 188 both serum albumins. In previous paper the formation of PBZ-SA 189 complex using spectrofluorescence and 1HNMR technique has 190 been studied [3]. Ka values determined for PBZ-SA complex in 191 present experiment are different than those reported in previous 192 study probably due to the use of lower PBZ-SA molar ratio 193 (presented PBZ:SA molar ratio 0.625:1–6.25:1 and previous 0.5:1– 194 0.625:1). As seen in Table 1, phenylbutazone creates a stronger 195 interaction with serum albumin than ketoprofen. It is noteworthy 196 because both PBZ and KP have a common binding site. 197 Phenylbutazone, often used as a marker for specific Sudlow’s site 198 I in albumin molecule, is located in subdomain IIA [17]. This place 199 is also a secondary binding site for KP. The possible competition 200 between KP and PBZ to the binding sites in albumin should be 201 taken into account. Strong PBZ binding to albumin may cause the 202 decreases of the concentration of an active drug (KP) in blood. 203 Effect of PBZ and KP on the KP-SA and PBZ-SA systems. Comparative study
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The quenching of both human and bovine serum albumin fluorescence excited at lex = 295 nm by KP and PBZ in the presence of constant concentration of the second ligand (PBZ – Fig. 4A and
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Table 1 Association Ka, quenching KQ and V constants and number of binding sites (n) for KP-SA, KP-[PBZ]-SA, PBZ-SA, PBZ-[KP]-SA systems; lex = 295 nm. KQ 104 [M A KP-HSA KP-[PBZ]-HSA PBZ-HSA PBZ-[KP]-HSA B KP-BSA KP-[PBZ]-BSA PBZ-BSA PBZ-[KP]-BSA a b
1
]
V 104 [M
1
]
2.40 0.08a 1.35 0.03a 3.03 0.06a 3.36 0.08a
0.34 0.06a,b 0.32 0.03a,b 0.25 0.08a 0.29 0.06a
2.14 0.07a 1.62 0.09a 3.41 0.04a 3.68 0.02a
0.24 0.02a 0.27 0.03a 0.32 0.04a 0.37 0.02a
KaI 104 [M 3.03 0.07a 1.46 0.10a 42.99 0.15a 96.65 0.22a 2.06 0.01a 1.89 0.01a 3.57 0.25a 7.15 0.09a
1
]
KaII 104 [M
1
]
nI
nII
0.29 0.07a 0.41 0.09a
0.47 0.02a 0.59 0.03a 0.45 0.12a 0.28 0.08a
1.83 0.21a 2.06 0.14a
0.06 0.00a 0.43 0.07a
0.30 0.01a 0.42 0.01a 0.95 0.05a 0.17 0.05a
2.66 0.17a 1.06 0.11a
Standard error. Negative deviation.
Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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Fig. 5. Effect of phenylbutazone and ketoprofen on fluorescence of ketoprofen (KP-[PBZ]-SA) and phenylbutazone (PBZ-[KP]-SA) bound to serum albumin. Percentage displacement of KP and PBZ in the ternary systems against drug:SA molar ratio. Error bars represent standard deviation.
Fig. 4. Comparison of quenching of HSA fluorescence by KP (&) and KP in the presence of PBZ (*) (A) and BSA fluorescence by PBZ (&) and in the presence of KP (*) (B); F/F0 is the ratio of the fluorescence of serum with and without quencher(s), &ex = 295 nm, T = 310 K. Error bars represent standard deviation.
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KP – Fig. 4B) was analyzed and compared in order to estimate the information about the effect of PBZ on KP (KP-[PBZ]-SA) and KP on PBZ (PBZ-[KP]-SA) secondary and primary binding site in SA, respectively. After addition of PBZ and KP, the quenching fluorescence of serum albumin in KP-SA complexes decreases (Fig. 4A and B). It means that PBZ influences the quenching of SA fluorescence by KP (Fig. 4A) and PBZ influences the quenching of SA fluorescence by KP (Fig. 4B). This phenomenon points to a possible effect of competitor (PBZ, KP) on KP and PBZ binding site within Trp-214 in subdomain IIA of SA, which is the primary for PBZ [1] and secondary for KP binding site in SA. This can be explained by the release of drugs from their binding sites. Second drug (PBZ or KP) reduces the affinity of first drug to subdomain IIA of SA, displaces the drug molecule and hinders the transfer energy due to the hydrophobic interactions stabilizing the complex. Therefore reciprocal interactions of both drugs (PBZ and KP) within the subdomain, which is common for this kind of binding of PBZ and KP to BSA, should not be excluded. Displacement of ketoprofen and phenylbutazone from their biding site in albumin was also studied by determining the percentage of displacement of bound KP and PBZ according to Eq. (1) (Fig. 5). A strong displacement of ketoprofen (>50%) and phenylbutazone (>30%) bound to albumin cause by the binding of the second drug to SA close to Trp-214 (subdomain IIA) was observed. The decrease of tryptophan fluorescence is the result of conformational changes of albumin molecule due to the interaction with the second drug. The dependence of F0/F on KP and PBZ concentration without and in the presence of PBZ and KP allowed for description of both
collisional (dynamic) and static quenching. The Stern–Volmer plot showed a linear relationship (dynamic quenching) between fluorescence intensity and quenchers (KP and PBZ) concentration below 2 10 5 [mol/L] and 1 10 4, respectively in both ternary complexes, similarly as it was in the binary KP-SA and PBZ-SA systems. Above these concentrations the positive deviation from the straight lines shows that static and dynamic quenching occur simultaneously. Effect of phenylbutazone and ketoprofen on their fluorescence was also confirmed on the basis of dynamic KQ [M 1] and static V [M 1] quenching constants and association constants Ka [M 1] (Table 1). In the high affinity binding sites of KP in serum albumin the presence of PBZ does not affect the static quenching constants V [M 1] for the p–p interaction between aromatic rings of KP and aromatic residues of serum albumin in subdomain IIA and IB. Similarly to the KP-[PBZ]-SA complex the presence of KP does not influence the static quenching constants V [M 1]. The dynamic quenching constants KQ [M 1] determined for binary system KP-SA are lower than that for the ternary KP-[PBZ]-SA. This phenomenon shows that in the presence of PBZ the interaction between KP and SA becomes weaker within subdomain IIA and IB, in the vicinity of Trp-214 and Trp-135. PBZ probably weakens the collision between excited state of SA and the quencher (KP). The comparison of the dynamic quenching constants KQ [M 1] of binary PBZ-SA and ternary PBZ-[KP]-SA complexes suggests that the presence of KP results in the increase of its values. This phenomenon shows as opposed to the quenching curves (Fig. 4B), that the presence of KP in the system makes formation of PBZ-SA complex easier. The interaction between PBZ and SA in the presence of KP becomes stronger within subdomain IIA and IB, in the vicinity of Trp-214, 135. KP probably makes the collision between excited state of SA and the PBZ more stable. The contrary relation between the results of the analysis of quenching curves (Fig. 4B) and dynamic quenching constants (Table 1) is probably caused by the fact that the qualitative analysis of the quenching effect allows for the assessment of the affinity of PBZ and KP to the total surface of SA macromolecule while quantitative analysis of the quenching parameters shows the changes in the specific binding sites with the static and dynamic quenching. The comparison of the association constants of binary with ternary complexes suggests that an addition of PBZ at constant concentration to KP-SA binary complex makes the values of Ka lower while addition of KP makes Ka higher in comparison with the corresponding binary complexes (Table 1). This leads to the conclusion that the presence of bound
Please cite this article in press as: Macia˛z˙ek-Jurczyk M. Phenylbutazone and ketoprofen binding to serum albumin. Fluorescence study. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.005
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molecules of PBZ induces the weakness of affinity between PBZ and tryptophanyl residues located outside the subdomain IIA in the vicinity of Trp-214 due to the hydrophobic interaction and PBZ high affinity to SA. It is noteworthy that the presence of PBZ causes the increase of free biologically active fraction of KP. PBZ administered before KP may change affinity of the latter to the albumin. Trp-214 cannot be quenched since the KP cannot approach and accept its energy. Changes in parameters characterizing low affinity binding site are probably a result of the reorientation of drug molecules toward the mentioned fluorophore in the binding site. The published data indicate that PBZ interacts also with tyrosyl residues in subdomain IIA (Tyr-263) and subdomain IIIA (Tyr-401, 411, 452, 497), where a secondary weaker PBZ-SA binding site can be located [3]. Although presented paper does not describe the data on the participation of tyrosyl residues, it can be assumed that PBZ competes with the KP in the binding site in subdomain IIIA. Muirden et al. [18] found that an addition of phenylbutazone causes a displacement of a significant amount of salicylate bound to albumin. Curry et al. [19] confirmed the interaction of aromatic ring of drugs having an aromatic ring and carboxylic group with hydrophobic cavity of serum albumin. It was concluded that the complex is stabilized by hydrophobic interaction [19]. The presence of KP at the constant concentration dramatically affects the affinity between protein and PBZ in its secondary binding site. The increase of association constant in the ternary complex suggest that in the presence of KP the interaction between PBZ and SA becomes stronger within subdomain IIA, in the vicinity of Trp-214. KP induces distinct differences of binding parameters in the primary binding site of PBZ in subdomain IIA where Trp-214 plays a major role in the formation of the complex and can stabilize the interaction between aromatic ring of PBZ and aromatic residues of SA. The presence of KP in the system probably simplifies the formation of the PBZ-SA complex and may cause the decrease of free, biologically active fraction of ketoprofen. Moreover the rise of Ka observed for both systems can be explained by the formation of an additional interaction (e.g. electrostatic or hydrogenous) as a result of the conformational changes of the binding site. In analogy to the KP-[PBZ]-SA system, KP administered before PBZ may change affinity of the latter to the albumin. The analysis of association constants allows observing the inverse relation between Ka and n. The decrease of one parameter with the rise of the second parameter is observed. The increased number of bound molecules of PBZ or KP makes the energy transfer to the quencher difficult and this leads to the decrease of KQ and Ka values.
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Conclusions
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The competitive interaction as the mechanism leading to the decrease of KP affinity and increase of PBZ affinity toward the secondary binding site have been analyzed. The observed changes are undesirable from the pharmacological point of view. When KP and PBZ are administered at the same time, the bound drug can be displaced by another drug from its binding site, what may result in increase of the concentration of free biologically active fraction of
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the released drug. Consequently this may lead to serious side/toxic effects. Therefore such conclusions may relieve to determine the appropriate dosage of both KP and PBZ to reach the therapeutic concentration.
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Conflict of interests
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I inform, that there is no conflict of interests. Acknowledgements
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This work was supported by grant from Medical University of Q3342 Silesia KNW-1-001/K/3/0 and KNW-2-001/N/3/K. 343 References
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