Spectrochimica Acta Part A 54 (1998) 983 988

Letter
Binding of disodium cromoglycate to human serum albumin
Eduardo Ochoa de Aspuru, Ana M.L. Zaton *
Dept. of Biochemistry and Molecular Biology, Faculty of Pharmacy, Uni6ersity 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 antiallergic drugs with a cromoglycate-like mecha-

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
nism of action and most of them, like sodium
nedocromil, are topical agents, not effective by
intravenous administration [510] 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 [1114]. After inhalation, some 810%
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 mg 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. Zaton / Spectrochimica Acta Part A 54 (1998) 983988
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 TrisHCl 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 25C. 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 2.510 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 200450 nm we
calculated the increase in absorbance at the maxi-
mum absorption wavelength (DA lmax) in all
cases. We obtained the concentrations of drug
bound (Cb) according to the following equation:
Cb = DA lmax/(obof) 1 (1)
where ob and of are the extinction coefficients for
drug bound and free, respectively, lmax 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
Fig. 1. Difference absorption spectra generated on the binding
of coumarin to HSA ( ) and DSCG (); [coumarin]= 2
10 4 M and [HSA] =5 10 5 M. Difference absorption
spectrum of coumarin in 20% ethanol (- - - ); [coumarin] = 5
10 4 M.
Table 1
Drug binding to human serum albumin (HSA) when HSA concentration was 5105 M

[Drug] (104 M) [Drug]/[HSA] Coumarin Dicoumarol Cromoglycate

DA280 [Coum]b (106 M) DA280 [Dicoum]b (106 M) DA285 [DSCG]b (106 M) DA320 [DSCG]b (106 M)

0.25 0.5 0.010 2.38 0.024 7.52 0.0096 9.98 0.0080 8.32
0.50 1 0.014 3.3 0.028 8.16 0.0080 8.32 0.0088 9.15
1.0 2 0.020 4.76 0.024 7.52 0.0088 9.15 0.0088 9.15
1.5 3 0.160 38.08 0.036 10.28 0.0080 8.32 0.0080 8.32
2.0 4 0.340 80.92 0.028 8.44 0.0080 8.32 0.0112 11.65
2.5 5 0.520 123.76 0.040 11.92 0.0096 9.98 0.0112 11.65
5.0 10 1.000 238.00 0.100 29.80

For coumarin ob and of values were 15.4103 and 11.2103 cm2 mmol1, respectively; for dicoumarol, 12.32103 and 8.96103 cm2 mmol1 and for cromoglycate,
5.44103 and 4.48103 cm2 mmol1.
E. Ochoa de Aspuru, A. M.L. Zaton / Spectrochimica Acta Part A 54 (1998) 983988
985
986 E. Ochoa de Aspuru, A. M.L. Zaton / Spectrochimica Acta Part A 54 (1998) 983988

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. Zaton / Spectrochimica Acta Part A 54 (1998) 983988
spectrophotometric measurements of cromogly-
cate, coumarin and dicoumarol dissolved in
ethanol at 1020 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 (lmax =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.
Fig. 3.
987
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. Zaton / Spectrochimica Acta Part A 54 (1998) 983988
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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