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Spectrochimica Acta Part A 54 (1998) 983–988 Letter Binding of disodium cromoglycate to human serum

Spectrochimica Acta Part A 54 (1998) 983–988

Spectrochimica Acta Part A 54 (1998) 983–988 Letter Binding of disodium cromoglycate to human serum albumin

Letter

Binding of disodium cromoglycate to human serum albumin

Eduardo Ochoa de Aspuru, Ana M.L. Zato´n *

Dept. of Biochemistry and Molecular Biology, Faculty of Pharmacy, Uni ersity of The Basque Country, P.O. Box 450-01080, Vitoria-Gasteiz, Spain

Received 18 December 1997; accepted 6 March 1998

Abstract

The binding of several benzopiranone derivatives to human serum albumin was determined. The antiallergic drug disodium cromoglycate binds weakly to serum albumin. However, its precursors, chromones of smaller size, were able to bind in a hydrophobic pocket in the protein, and are carried by serum albumin in blood. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Disodium cromoglycate; Chromone binding; Human serum albumin; Difference spectroscopy

1. Introduction

Disodium cromoglycate (DSCG) was first in- troduced into clinical practice for the prophylaxis of asthma more than 25 years ago, and proved to be one of the most effective drugs for the treat- ment of this disease [1,2]. The drug neither in- hibits IgE binding to most cells nor the interaction between bound IgE and a specific antigen, but suppresses the liberation of histamine due to this interaction, raising the intracellular concentration of cyclic nucleotides [3,4] and blocking the transient rise in the intracellular cal- cium concentration [3]. Nowadays, there are few

* Corresponding author. Tel.: +34 945 183100; fax: +34 945 130756; e-mail: GBPZALOA@VF.EHU.ES

antiallergic drugs with a cromoglycate-like mecha- nism of action and most of them, like sodium nedocromil, are topical agents, not effective by intravenous administration [5–10] due to their low solubility. DSCG is an amphipathic, lipid-insoluble com- pound. Consequently, following intravenous ad- ministration, the drug is cleared rapidly from the plasma and tissues, being accumulated only in the liver and kidneys and then excreted, about half in the urine and half in the bile [11]. On the other hand, the drug is very poorly absorbed from the gastrointestinal tract after oral supply (1% of the dose), and only being effective by topical adminis- tration [11–14]. After inhalation, some 8–10% penetrates deep into the lungs and is absorbed into the blood, where its half-life is about 80 min, reaching a peak level of 9 g ml 1 [12,13]. Its routes of administration are intravenous, subcuta-

1386-1425/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S1386-1425(98)00056-0

984 E. Ochoa de Aspuru, A. M.L. Zato´n / Spectrochimica Acta Part A 54 (1998) 983–988

neous or via inhalation. Aqueous solutions are available for nasal and ophthalmic uses [13,14]. Most drugs administered nowadays are dis- tributed through the circulatory system by means of their binding to some plasma protein. HSA is the serum protein that shows the highest non-spe- cific binding capacity, and is the principal carrier of drugs and endogen substances in blood. The anticoagulant drug dicoumarol, with a similar size and structure to that of cromoglycate, is carried by HSA [15,16]. The aim of the work reported here was to characterize the interaction between DSCG and HSA, in order to elucidate if DSCG or its precur- sors are carried by the protein.

2. Materials and methods

Human serum albumin (essentially fatty acid- free) and chromone-2-carboxylic acid were ob- tained from Sigma Chemical (St. Louis, MO). Disodium cromoglycate (the disodium salt of 1,3-

bis-(2-carboxychromone-5-yloxy)-2-hydroxy

propane, DSCG) was purchased from Fisons (Loughorough, UK), 2H-1-benzopyran-2-one (coumarin) and 3,3-methylene-bis-(4-hydroxy- coumarin) (dicoumarol) from Janssen Chimica (Beerse, Belgium). Ethanol and other chemicals were obtained from Merck (Darmstadt, Ger- many). Solutions were prepared in phosphate (1/ 15 M) or Tris–HCl buffer, both at pH 7.4, and in saline solution (NaCl 0.9%). Absortion spectra and absorbance difference measurements were made with a double-beam UV/VIS spectrophotometer model 3600 from Beckman Instruments, connected with a thermo- static bath from Selecta (Spain) to maintain the temperature constant at 25°C. Double-compart- ment quartz cuvettes from Hellma (Germany) permitted us to perform the difference spectra directly. The light path through each cuvette was 2 × 0.4375 cm. The data analysis was performed with the Enzfitter program from Biosoft (UK) in a Tandon computer. The optimum concentrations of HSA to determine difference spectra were 2.5 and 5.0 × 10 5 M whereas cromoglycate, cou- marin and dicoumarol concentrations varied from 2.5×10 5 to 2.5× 10 4 M.

The binding measurements were made by a previously described difference absorbance tech- nique [17]. In one of the compartments of the reference cuvette HAS was placed and in the other compartment the drug. The sample cuvette was filled with the HSA-drug mixture in one compartment and buffer solution in the other. Both cuvettes were filled and placed in the spec- trophotometer in the same way for all the tests to minimize any differences between them. The spec- trum was scanned at the most appropriate sensi- tivity value of 0.2. From the difference spectra for 200–450 nm we calculated the increase in absorbance at the maxi- mum absorption wavelength ( A max ) in all cases. We obtained the concentrations of drug bound (C b ) according to the following equation:

C b = A max /( b f ) 1

(1)

where b and f are the extinction coefficients for drug bound and free, respectively, max was 280 nm for coumarin and dicoumarol and 285 and 320 nm for disodium cromoglycate, and l the light-path. Each data point was the mean of three experiments. In addition, in order to characterize the nature of the difference spectra obtained, we carried out

nature of the difference spectra obtained, we carried out Fig. 1. Difference absorption spectra generated on

Fig. 1. Difference absorption spectra generated on the binding of coumarin to HSA (—) and DSCG (···); [coumarin]=2 × 104 M and [HSA] =5 × 105 M. Difference absorption spectrum of coumarin in 20% ethanol (- - - ); [coumarin] = 5 × 10 4 M.

For coumarin b and f values were 15.4×10 3 and 11.2×10 3 cm 2 mmol 1 , respectively; for dicoumarol, 12.32×10 3 and 8.96×10 3 cm 2 mmol 1 and for cromoglycate,

[DSCG] b (10 6 M)

8.3210.2838.08

7.522.380.0100.5 8.320.0240.25

9.150.014

4.762 9.150.0201.0

11.650.520

11.650.340

0.0112

0.0112

0.0080

0.0080

0.0088

0.0088

A 320

[DSCG] b (10 6 M)

Cromoglycate[Drug]

8.32

8.32

8.32

9.15

123.76 9.985

9.98

0.0080

0.0080

0.0080

0.0096

0.00962.5

0.0088

A 285

[Dicoum] b (10 6 M)

Table 1 Drug binding to human serum albumin (HSA) when HSA concentration was 5×10 5 M

80.92 8.444

7.52

11.92

29.800.100238.005.0

8.16

Dicoumarol

A 280

0.024

0.040

3 0.0361.5

0.028

0.0282.0

[Coum] b (10 6 M)

1 3.30.50

Coumarin[Drug]/[HSA]

A 280

1.000

0.160

5.44×10 3 and 4.48×10 3 cm 2 mmol 1 .

10

(10 4 M)

E. Ochoa de Aspuru, A. M.L. Zato´n / Spectrochimica Acta Part A 54 (1998) 983–988

985

986 E. Ochoa de Aspuru, A. M.L. Zato´n / Spectrochimica Acta Part A 54 (1998) 983–988

Zato´n / Spectrochimica Acta Part A 54 (1998) 983–988 Fig. 2. Plots of [drug] b o

Fig. 2. Plots of [drug] bound /[HSA] molar rates vs [drug] free : coumarin ( ), dicoumarol ( ), cromoglycate ( ). In all cases, [drug] =2.5 ×10 5 –5×10 5 M.

E. Ochoa de Aspuru, A. M.L. Zato´n / Spectrochimica Acta Part A 54 (1998) 983–988

987

spectrophotometric measurements of cromogly- cate, coumarin and dicoumarol dissolved in ethanol at 10–20 or 50%.

3. Results and discussion

Interaction of cromoglycate with HSA leads to the formation of complexes that exhibit an ab- sorption band with a maximum at 320 nm. This band is shifted about 5 nm in comparison to the spectrum of free DSCG ( max =325 nm). As a result of this, the difference absorption spectrum obtained is characterized by two positive peaks, at 285 and 320 nm, respectively, and an isosbestic point at 300 nm (Fig. 1). Comparison with cou- marin in ethanol, shaved that the difference spec- tra were similar to those generated with HSA.

spec- tra were similar to those generated with HSA. F i g . 3 . Therefore,

Fig. 3.

Therefore, since difference spectroscopy detects displacement of the chromophores of both drugs towards a more apolar site, DSCG and coumarin appear to interact with a site of low poPo3larity on the protein. We calculated the concentrations of drug bound to this apolar pocket of HSA, as described in Section 2. As seen in Table 1 and Fig. 2, only coumarin and chromone-2-carboxylic acid show significant binding, whereas neither cromoglycate nor dicoumarol exhibit significant binding. In order to characterize better these effects, the binding parameters were obtained from plots of [drug] bound /[HSA] versus free concentration (Fig. 2). The ratio [coumarin] bound /[HSA] increased rapidly, in a cooperative manner, up to values near five, showing a dissociation constant of 0.12 mM. There results suggest that HSA may bind 5 moles of coumarin per protein mol. With chromone-2-carboxylic acid, the drug bound in- creases according to the ligand concentration, up to 4 moles of chromone bound per HSA mol. The dissociation constant achieved was 85 mM. In contrast, the variation of [drug] bound / [HSA] was minimal for both dicoumarol and cromoglycate. Though cromoglycate showed no significant bind- ing up to a drug concentration of about 2.5 × 10 4 M, dicoumarol began to show evidence at some binding at this upper limit (Fig. 2). The greater binding capacities of HSA for cou- marin and chromone-2-carboxylic acid may be explained by their smaller size and, therefore, greater accesibility to the apolar binding site of the protein. Dicoumarol is a bis-hydroxycoumarin and DSCG is a bis-chromone (Fig. 3). Their greater molecular size may prevent access of the compound to the apolar binding site of the protein and thus its transport in the blood by HSA. Smaller drugs that can bind to HSA should be carried by the bloodstream in an efficient way. In conclusion, HSA can transport only negligi- ble quantities of cromoglycate in the blood stream at concentrations which are clinically feasible. The design of antiallergic drugs with an appropriately smaller molecular size, corresponding to that of chromone-2-carboxylic acid, would seem to be a desirable experimental approach to improve asthma therapy.

988 E. Ochoa de Aspuru, A. M.L. Zato´n / Spectrochimica Acta Part A 54 (1998) 983–988

Acknowledgements

Eduardo Ochoa de Aspuru thanks the Basque Government for economic and technical support through its programme for training of researchers. The authors also wish to thank the University of the Basque Country for the funding of this inves- tigation (UPV 042.123-0150/89).

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