Anal. Chem. 1994,66, 1198-1203
Electrochemical Study of Amiodarone Charge-Transfer Complexes Rowena V. Ball,t George M. Eckert,* Felix Gutmann,t and Danny K. Y. Wong’gt School of Chemistry, Macquarie University, Sydney, New South Wales 2 109, Australia, and Department of Physiology and Pharmacology, University of New South Wales, P.O. Box 1, Kensington, New South Wales 2033, Australia
The electron-donatingpropertiesof the drug amiodarone(also known as cordarone), have been studied by conductimetry. Amiodarone was found to form a charge-transfer complex in vitro with the electron acceptor iodine, which is involved in human thyroid metabolism. Amiodarone was also found to interact with some other biological molecules with the capacity to behave as electron acceptors, such as dopamine hydrochloride, (-)-epinephrine, serotonin hydrochloride, coenzyme Qw and @-nicotinamideadenine dinucleotide. An electronic absorption band in the visible region of the spectrum due to chargetransfer complex formation between iodine and amiodarone was also observed, supporting the conductimetric results. The primary event in the action of many drugs is often a reversible association between a drug molecule and a receptor to form a complex. In such reactions, some drugs can behave as electron donors (or acceptors in somecases), forming chargetransfer complexes by electron transfer from an occupied orbital to an empty orbital of an acceptor molecule.1 The formation of a charge-transfer complex is often characterized by an intense, broad, electronic absorption band either in the visible or in the UV r e g i ~ n . ~However, ,~ charge-transfer reactions sometimes demonstrate no color change, no obvious charge-transfer band in the absorption spectrum, no electron spin resonance signal, and no change in dipole moment. By contrast, if these interactions are carried out in vitro as electrode reactions within an “active space” or within an electrical double layer, significant changes occur which may be detected electrochemically, e.g., by conductimetry (vide infra). Thus, electrochemistry provides a supplementary technique for charge-transfer systems not discernible spectroscopically. Conductimetric titrations have often been employed to study interactions of charge-transfer complexes. 1+8 The theoretical background on conductimetrictitrations of charge~~
~~
~~~~~~
+ Maquarie University.
*
University of New South Wales. (1) Eckert, G. M.; Gutmann, F.; Keyzcr, H. In Electrophurmucology; Eckert, G. M., Gutmann, F., Keyzer, H., Eds.; CRC Press: Boca Raton, FL, 1990; pp 1-23. (2) Mulliken. R. S. J. Phys. Chem. 1952, 56, 801-822. (3) Murrcll, J. N. Q. Reo. 1961, 15 (Z), 191-206. (4) Gutmann, F.; Smith, L. C.; Sliflrin, M. A. In The Phenothiazines and Srrucrurully Reluted Drugs; Forrest, I. S., Carr, J., Usdin, E., Eds.; Raven Press: New York, 1974; pp 15-31. ( 5 ) Gutmann, F.; Keyzer, H. Electrochim. Acto 1966, I ! , 555-568. (6) Gutmann, F.; Keyzer, H. Electrochim. Acta 1966, 11, 1163-1169. (7) Eckert, G. M.; Gutmann, F. J. Elecfrounul.Chem. Interfuciul Electrochem. 1975, 62, 267-272. (8) Eckert, G. M.;Gutmann, F.; Kabos, M. J. Eiol. Phys. 1982, 10, 51-63.
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transfer complexesin solution has been reviewed by Gutmann.9 Briefly, a complex (DA) formed from the association of a donor (D) and an acceptor (A) may, in the presence of a solvent of high permittivity, dissociate into ions giving rise to appreciable ionic conductivity:
D+A=DA
DA+D++AThe formation of charge-transfer complexes can thus be followed by measuring changes in the conductivity of a solution of, say, the donor in an inert solvent of sufficiently high permittivity, consequent upon addition of a solution of the acceptor in the same solvent, or vice versa. This amounts to a conductimetric titration in which the donor may be titrated with the acceptor. The titration can then be repeated by titrating the acceptor solution with the donor solution. In the absence of interaction, theconductivity of the titrant is linearly related to the mole fraction of donor. However, in the case where charge-transfer complexation occurs, followed by its dissociation into ions, as represented by the scheme above, the theory predicts a fourth-power relation between the conductivity and the mole fraction of donor in the solution. In a plot of the conductivity of titrant against the mole fraction of donor, the value of a conductivity peak above a baseline connecting the conductivities of pure donor and acceptor solutions is a measure of the excess conductivity caused by the formation and subsequent dissociationof the complex. The stoichiometry of the complex may then be deduced from the mole fraction at the conductivity peak and the known concentrations of the stock solutions. Conversely, if association of ionic species occurs, the interaction is accompanied by a decrease in the concentration of free charge carriers, giving rise to a conductance decrease. Extensive conductimetric and spectroscopic studies on charge-transfer interactions of drugs, especiallythose involving chlorpromazine, have been performed by Gutmann and coworkers.l@ Some of these results are of direct clinical significance such as the interaction of chlorpromazine with heparin (which exerts an anticoagulant action in blood plasma) indicated by a conductivity minimum at a 1:l stoichiometry.* Conductivity titration has also been carried out to investigate the charge-transfer interactions between chlorpromazine and dopamine, serotonin, and acetylcholine, re~pectively.~ As well (9) Gutmann, F. J. Sei. I d . Res. 1967, 26 (1). 19-28.
0003-2700/94/0366-1198$04.50/0
0 1994 Amerlcan Chemlcal Society
Flguro 1. Structure of amiodarone.
as providing supporting evidence for charge-transfer complexation,the unexpectedlylarge capacitance increasesin these donor-acceptor systems have been attributed to a strong adsorption phenomenon on the electrode surface. This has been used to explain certain drug effects invivoa4For example, in the treatment of schizophrenia, it has been hypothesized that endogenously formed dopamine complexes with chlorpromazine adsorbed on synaptic membranes, which act as an electrode surface, hence inhibiting access by dopamine to the nerve endings. More recently, certain undesirable side effects of some drugs, such as melanosislo and xanthomata,’ have been suggested to occur via interactions involving exciplexes (Le., light-activated charge-transfer complexes). In our laboratory, we are interested in the pharmacochemical study of the cardiac antiarrhythmic agent, amiodarone, also known as cordarone. The structure of amiodarone is depicted in Figure 1. Administration of amiodarone to patients is restricted to specialist physicians, owing to its high toxicity and the frequent occurrence of side effects such as neurological effects, impaired vision, and hyper- and hypothyroidism.12 Although amiodarone has been in use since the early 1960s, fundamental physical quantities such as its pK,, solubility, molar absorption coefficient in the UV region, and partition coefficients were only reported in 1984.13 The exact electrophysiologicalmechanism of action of amiodarone on cardiac tissues is unknown and is the subject of active research, as is the distribution of the drug through organs and tissues. In this work, conductimetric and spectroscopic methods have been employed to examine the interaction of amiodarone as a possible electron donor with several small molecules of biological importance including iodine, coenzyme Qo, &nicotinamide adenine dinucleotide (@-NAD), (-)epinephrine, serotonin hydrochloride, and dopamine hydrochloride. Iodine is well-known for its electron-accepting properties, which may be deduced from molecular orbital ~onsiderations.1~It has been used in the past as a model acceptor to investigate the electron-donating properties of organic molecules.’ In human biology, iodine is required for the biosynthesis of the thyroid hormones triiodothyronine and thyroxine, which regulate metabolic rate. A number of other drugs which have antithyroid action, such as disulfram, tetramethylthiourea, and 2-mercapto- 1-methyl- 1-methylimidazole, have been shown to form charge-transfer complexes with iodine.” Coenzyme Q, synonymous with ubiquinone, and the dinucleotide @NAD are universal biological electron (10) Forrest, I. S.;Gutmann, F.; Keyzer, H. Reo. Agressol. 1966, 7, 147-153. (11) Eckert, G. M.; Gutmann, F.; Keyzer, H. Xenobiotica 1989, 19, 561-579. (12) Mason, J. W. N.Engl. J . Med. 1987,.316,455-466. (13) Bonati, A.; Gaspari, F.; Daranno, V.; Benfenati, E.; Neyroz, W.; Galletti, F.; Tognoni, G. J . Pharm. Sci. 1984, 73, 829-830. (14) Dewar, M. J. S.;Lepley, A. R. J. Am. Chem. Soc. 1961,83,4560-4563. (15) Raby, C.; Lagorce, J.; Jambut-Absil, A.; Buxeraud, J.; Catanzano, G. Endocrinology 1990,126, 1683-1691.
carriers, which accept reducing equivalents in vivo from a variety of reduced substrates, such as carbohydrates and fatty acids, which are ultimately transported to molecular oxygen. Thus it is possible that coenzyme Q and @-NADmay behave as electron acceptors toward electron-donating drug molecules. The unsubstituted form of coenzyme Q, coenzyme Qo, was used in this work for its greater solubility in aqueous medium and to localize the electron-accepting properties to the quinone ring. Among the other small molecules listed above, serotonin and the catecholamines (-)-epinephrine and dopamine are neurotransmitters, which are known to behave as electron acceptors toward a strong donor.4 Amiodarone, which is known to have adverse neurological effects such as Parkinsonism,12 may reasonably be suspected of altering, perhaps indirectly, the actions of these neurotransmitters. A longterm goal of this work is to construct a simple model system for elucidation of the biological reactions of amiodarone. Results from these studies will provide the fundamental chemical and pharmacological information required for possible structural modification of the drug to enhance the primary antiarrhythmic action and suppress undesirable side effects.
EXPERIMENTAL SECTION Chemicals and Reagents. Amiodarone hydrochloride (2butyl- 3- [ 3,5-diiodo-4- (8-diet hylaminoet hoxy) benzoyl J benzofuran) was manufactured by Sanofi Winthrop of Manchester, United Kingdom, and was donated by Reckitt and Coleman, New South Wales, Australia. Iodine was obtained from Unilab and was resublimed before use. Dopamine hydrochloride, (-)-epinephrine, coenzyme Qo(2,3-dimethoxy5-methyl- 1,Cbenzoquinone), and 8-nicotinamide adenine dinucleotide were purchased from Sigma Chemicals. Serotonin hydrochloride was obtained from Aldrich Chemical Co. Acetonitrile and chloroform were analytical grade reagents. Deionized water from a Milli-Q water system was used. Equipment. Conductance and capacitance measurements were made using a Wayne-Kerr B224 conductivity bridge operating at 1592 Hz and accurate to 0.1 p S and 1 pF over the range of these experiments. An enclosed, opaque Teflon electrochemical cell, fitted with a pair of bright gold electrodes and thermostated at 37.0 “C, was used for the conductivity titrations. The electrodes were cleaned after every second or third titration using an electrochemical procedure.16 The cell constant was measured to be 29.67 f 0.05 m-l at 37.0 “C using thevalues for the conductivity of 0.7463 g k g l potassium chloride solutions given by Shedlovsky.” Electronic absorption spectra were recorded using a Varian DMS 90 UV-visible spectrophotometer. The temperature of the cell holders was controlled to a tolerance of f l OC. All glassware and the Teflon cell used in the experiments were dried in a stream of dry nitrogen before use. Experimental Procedure. A solution of the donor, amiodarone, was titrated into a known volume (usually 15 mL) of (16) Gileadi, E.; Kirowa-Eisner; Penciner, J. Interfacial Electrochemistry; An Experimental Approach: Addison-Wesley: Reading, PA, 1975; p 31 1. (17) Shedlovsky, T. Physical Methods of Chemistry, Part ZA. In Techniques of Chemistry; Weissberger, A., Rossiter, B. W., Eds.; Wiley-Interscience: New York, 1971; p 178. (18) Gutmann, F. In Modern Biwlectrochemistry;Gutmann, F., Keyzer, H., Eds.; Plenum Pres: New York and London, 1985.
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an acceptor in the cell. Conductance and capacitance were plotted against mole fraction of donor, XD, in the cell as the readings were taken. The titration was usually stopped after theaddition of 30-40 mL of donor solution. A reverse titration was then carried out in the same manner, with the acceptor being added to the donor in the cell. A complete range of conductance and capacitance values is thus obtained for mole fractions of donor ranging from 0 to 1. The mole fraction quantity used, XD, is nominal only, representing the mole fraction of donor in the absence of complex formation and dissociation. The cell conductivity values (K) were obtained by multiplying each conductance reading by the measured cell constant, 29.67 m-l. Relative permittivity values in this system are simply the measured capacitance values divided by the capacitance of the clean empty cell.
RESULTS AND DISCUSSION Conductivity Titration. The interactions between amiodarone and several biologically important molecules were initially investigated by conductimetry. Figure 2 shows the titration curves of cell conductivity and relative permittivity plotted against XD for a series of amiodaroneacceptor complexes. In each figure, the lower curves represent cell conductivity, scaled on the left-hand axis; the upper curves represent relative permittivity, scaled on the right-hand axis. Open symbols are for the forward titration; closed symbols are for the reverse titration. Figure 2a shows the titration curve for the amiodarone-iodine system in acetonitrile. This result indicates that amiodarone is a likely charge-transfer complexing reagent, as the curve has the classical form predicted by the theory developed by Gutmann et aL5s6 The stoichiometry of the complex may be deduced by considering the value of the horizontal coordinate at the conductivity maximum.6 Hence, in Figure 2a, the conductivity maximum occurs at a 1:1 mole ratio and this is taken as the stoichiometry of the complex. However, in order to work with electrolytes more akin to biological systems, all other titrations have been performed in aqueous solutions. Although the conductivity peak is somewhat diffuse in the amiodaroneiodine-water system (Figure 2b), the stoichiometry can still be deduced to be 1:l since the reverse titration curve displays a peak at XD 0.5. The results are summarized in Table 1. For ease of comparison among donor-acceptor-solvent systems, a normalized quantity termed an excess molar conductivity, A,, is defined such that
-
maximum conductivity above the baseline Ax = initial donor concentration at stoichiometric ratio Also, the maximum change in relative permittivity is defined as Aer =
maximum capacitance above the baseline capacitance of the clean empty cell
where the baseline in this case refers to a baseline connecting the capacitances of the pure donor and the pure acceptor solutions. For clarity, this baseline is not drawn in the capacitance plots. From Table 1 we can see, for example, that the excess molar conductivity of the amiodarone-iodine 1200
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system in acetonitrile is similar to that of the forward titration of this system in water, while the relativeconductivity increase in the amiodarone-dopamine hydrochloridewater system is comparatively small. One general feature of the titration curves (e.g., Figure 2b,c) is the vertical mismatch, within experimental error, of the forward and reverse branches. Consider the conductivity titration curve for amiodarone-iodinecomplex in water (Figure 2b). In the reverse titration, where amiodarone is initially in excess, complexation occurs with a maximum at a 1:l mole ratio. In the forward titration, we seethat complexationbegins, but at a donor mole fraction of -0.12 the slope of the curve decreases abruptly, after which the conductivity approaches the baseline almost linearly. Moreover, the relative permittivity followsthe conductivity. The prominent noncoincidence of forward and reverse titration curves has previously been attributed to preferential adsorption of one component of the charge-transfer complexonto the electrodes.I6 This may then lead toa decreaseincharge carrier mobility in the bulk solution, as the complex and the products of complex dissociationdesorb more slowly from the electrodes. Further investigations into the adsorption phenomenon of amiodarone on electrode surfaces are being carried out in our laboratory. In addition to the above explanation, we also propose that the observed conductivity may be due to two opposing effects which influence the charge concentration and mobility. It is expected that dissociation of the complex will increase the number of charge carriers, but subsequent aggregation of charged species into molecular ion clusters will promote a decrease in charge mobility, leading to a decrease in conductivity. When iodine is in excess, there appears to be a critical concentration of amidarone above which the formation of micellar structures is favored. The critical micellar concentration of amiodarone in this system was determined to be -6.0 X l e 5M. When amiodarone is initially in excess, competition for iodine seems to prevent aggregation. These two effects are also seen in the conductivity curve for amiodarone with NAD as acceptor (Figure 2d), in which an abrupt decrease in the slope of the curve at an acceptor mole fraction of -0.06 (note that this corresponds to XD = 0.94 in the figure) signals the onset of aggregation. Here it is the concentration of the acceptor, rather than that of the donor, which appears to control the switch in equilibria from simple ions to ion clusters. It is also observed that the conductivity dips below the baseline where (-)-epinephrine and coenzyme Qo are used as acceptor, as shown in Figure 2e,f. A curve which shows a conductivity minimum and a net decrease in solution conductivity over the entire donor mole fraction range may indicate that the number and/or mobility of charges already present is reduced through an association process. Although the conductivity decrease alone does not necessarily show that charge transfer is involved in this association, color changes indicate that charge-transfer complexation may have occurred in these systems. In the experiment, a colorless solution of (-)-epinephrine is observed to turn into a deep rose color when a colorlessamiodarone solution is added. Epinephrine is known to be easily oxidized to the rose-colored ortho quinone (19) Wilson, C. 0.; Gisvold, 0.; Docrgc, R. F . Textbook of Orgunic Medicinal und Phormaceuticul Chemistry, 7th ed.; Lippincott: Philadelphia, 1977.
l2r
3800
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900
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-210
-I
I 0.0
I
0.2
I
I
0.4
0.6
is
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I
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0.8
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Flgure 2. Plots of data from conductlvlty titrations carried out at 37 'C. (a) Acetonttrile and (b-g) water were used as solvent. The stock sdutlon concentratlons were each 1.0 mM In (a) and 0.50 mM In (b-g).
adrenochrome and oxidation is thought to have a role in epinephrine deactivation in vivo.I9 In this study, the species which interacts with amiodarone is therefore possibly adrenochrome, which like other quinones, such as the coenzyme Qo used here, would have the capacity to accept electrons into an electron-deficient ?r-system. Another measure of electrodeeffects is found in the excess relative permittivity values, A+ (Table 1). The measured
capacitance between the electrodes usually has a more erratic relationship to the mole fraction of donor than the conductivity, although it follows, more or less roughly, the conductivity curves. Similar large increases in relative permittivity (up to 3 orders of magnitude; see Table 1) observed for some of the donor-acceptor pairs have also been reported in other donoracceptor systems? This observation has previously been proposed by Gutmann's to be due to the formation or Analytical Chemlstw, Vol. 88, No. 7, April 1, 1994
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Table 1. Summary of Results from the ConductlVny Mratlonr In Flguro 2.
donor amiodarone HC1
acceptor iodine
amiodarone HCl amiodarone HCl amiodarone HC1 amiodarone HCl amiodarone HCl amiodarone HCl a
complexstoichiometry donor:acceptor 1:1
solvent acetonitrile
iodine
1:l
water
dopamine HCl serotonin HCl (-)-epinephrine coenzymeQo
1:l (nominal) 1:l (nominal) 1:l 1:2 1:l (nominal)
water water water water water
NAD
AI (mS m2mol-')
AG
11.04 (forward) 31.93 (reverse) 2.02 4.51 -11.28 -11.41 3.32
278 (forward) 490 (reverse) 261 (forward) 1890 (reverse) -0 172 (reverse) -654 408 229
1450
350
12.58
The maximum error in the AI values is 0.05 mS m2 mol-'.
0.8
0.6 0
8 8 5
0
0.4 I
0.2
350 1650
wavelength / nm
I550
wavelength i nm
Figve 3. Electronicabsorption spectra of amkdarone-iodine solutlons In CHCI:, at 30 O C , showing the growth of the charge-transfer band at 422 nm. The iodine concentratlon is kept constant at 1.O mM: (a) pure iodine; (b) 4.0, (c) 8.0, (d) 8.0, (e) 10.0, and (f) 12.0 mM amiodarone.
Figure 4. Temperature dependence of the absorption Intensity of the charge-transfer band at 422 nm: amiodarone, 10.0 mM; iodlne, 1.0 mM; (a) 38, (b) 30, and (c) 20 O C .
concentration of the complex within the electric double layer or on the electrode surface. A quantitative study on this phenomenon by electrochemical impedance spectroscopy is currently being conducted in our laboratory. Absorption Spectra. In the present study, visible absorption spectroscopy has also been employedto ascertain the formation of charge-transfer complexes. The crucial feature of the charge-transfer band observed in such experiments is that its intensity decreases with increasing temperature, while the intensity of absorptions due to the components increases with temperature. The effect on the electronic absorption spectrum of iodine in the region between 350 and 650 nm consequent upon the addition of solutionsof amiodarone was studied. No significant absorption intensity was observed in this region for pure amiodarone. Successively more concentrated amiodarone solutionsinduce an apparent blue-shift of the 5 10-nm transition of iodine. A new absorption band also appears in the visible region, at higher energy than pure iodine absorption in the visible region, but at lower energy than pure amiodarone absorption in the ultraviolet as shown in Figure 3. In Figure
4, it is demonstrated that the intensity of this new band decreases with temperature while that of the iodine transition increases. We may therefore tentatively assign the new band as absorption due to a charge-transfer transition. The maximum absorption of this band occurs at a wavelength of 422 nm. In contrast to the conductivity titrations, which are carried out in a moderately polar solvent to induce dissociation of the complex into ions (acetonitrile was the solvent of choice in this work), it is essential for observation of a charge-transfer band that complex dissociation into ions be minimized, and the equilibrium shifted toward complexation. Although nonpolar solventssuch as carbon tetrachloride and cyclohexane are ideal for this purpose, amiodarone is extremely insoluble in these and many other solvents. Experimentally, it was found that chloroform best fulfilled the requirements of reagent solubility and complex stability. Proposed Significance of Iodine. The evidence presented in this work that amiodarone complexes with iodine raises the possibility that the complexing behavior of the drug may be involved in its effect on thyroid metabolism. The suggestion that there may be a correlation between the antithyroid activity and the iodine-complexing ability of some drugs has been
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made previously.*S No mechanism has been proposed or tested, but it could be postulated, for example, that complexation of iodine by amiodaronemay reduce the availability of molecular iodine. Alternatively, the amount of oxidized iodine available to participate in biosynthesis could be restricted, through the reactions amiodarone.1, + amiodarone.1'
I-
+ I-
+ I, + 1,-
This binding of oxidized iodine could lead to hypothyroidism. Conversely, the reversibility of the amidoarone-iodine equilibrium provides a possible clue as to how the drug may induce hyperthyroidism. When tissue levels of amiodarone are high (the loading dose is 2-6 g per day1*),relatively large amounts of iodine could be stored as the amiodarone-12 complex. Release of the iodine under appropriate conditions may then force overproduction of the thyroid hormones triiodothyronine and thyroxine, resulting in hyperthyroidism.
CONCLUSIONS We have established that amiodarone is a complexing reagent which can form charge-transfer complexes in vitro with a variety of biologically important small molecules. Complexation with iodine, an electron acceptor, indicates that
amiodarone may be characterized as an electron donor. This does not rule out the possibility that amiodarone may also behave as an electron acceptor under some circumstances. The donor-acceptor interactions and the stoichiometryof the complexes were studied by conductivity titration. This technique has also provided preliminary results, through the experimentally repeatable decrease in conductivity at the critical micellar concentration described above, that chargetransfer interactions may be able to induce formation of molecular clusters of amiodarone and electron acceptors in aqueous solution. Supporting results for transfer of charge have been obtained by spectroscopic observation of the electronic transition corresponding to electron transfer from donor to acceptor. This work demonstrates that conductimetric titrations, carried out using a model system free of complicatingfactors, may provide valuable qualitative information on the mode of action of a drug, which would not be extracted from clinical or pharmacological studies. In future work, refinements of these techniques will be applied to obtain quantitative information on the strength and stability of amiodaroneacceptor complexes, and comparisons with other drugacceptor-solvent systems will be made. Received for review September 22, 1993. Accepted January 5, 1994.@ Abstract published in Aduance ACS Abstracts, February IS, 1994.
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