Simple circular dichroism method for selection of the optimal cyclodextrin for drug complexation
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Cyclodextrins are very important excipients in the pharmaceutical industry. Given the multitude of native and semisynthetic cyclodextrin derivatives, there is a need for a rapid and reliable method for the selection of the optimal cyclodextrins for further pharmaceutical testing. During our research, circular dichroism (CD) spectroscopy has been successfully used to describe the qualitative and quantitative complexation of model compounds with different cyclodextrins. For the appearance of a circular dichroism signal, either a chiral or a chirally perturbed chromophore is required. Achiral or racemic compounds do not have corresponding circular dichroism spectra and neither do chiral cyclodextrins due to the absence of a chromophore group. During complexation of a chromophoric guest molecule, its absorption transition becomes chirally perturbed in the proximity of a cyclodextrin molecule and an induced circular dichroism (ICD) signal appears. This phenomenon gives an inherent selectivity to the method. The sign and intensity of the induced circular dichroism signal in case of different cyclodextrins provides information about the approximate structure of the complex as well as their stability relative to each other. In this study, we report a straightforward induced circular dichroism -based approach for the rapid preselection of the optimal cyclodextrin. The distinctive features of the method were demonstrated using five azole-type antifungal drug molecules (fluconazole, miconazole, clotrimazole, bifonazole and tioconazole) along with native α-, β-, and γ-cyclodextrins, as well as dimethyl-, trimethyl-, carboxymethyl-, hydroxypropyl- and sulfobuthylether-β-cyclodextrins. In addition, with the aid of this method, 27 stability constants were determined, amongst which 16 have been unavailable in the literature previously.
KeywordsCyclodextrin Host–guest complexation Circular dichroism spectroscopy Complex stability constant
Electron spin resonance
High performace liquid chromatography
Induced circular dichroism
Nuclear magnetic resonance
Nuclear overhauser effect spectroscopy
Phase solubility studies
Polar surface area
Rotating frame nuclear overhauser effect spectroscopy
Thin layer chromatography
Cyclodextrins are being used in numerous areas of the industry for a wide range of applications [1, 2, 3, 4]. Their main property, the ability to form inclusion complexes can aid the dissolution of poorly soluble drugs, improve the retention of volatile molecules, and enhance chemical stability. However, despite having numerous advantages, cyclodextrins can cause some unexpected issues e.g.: in case of racemic drugs, cyclodextrins used in the pharmaceutical industry have different influence on the dissolution of enantiomers from the formulation [5, 6].
The selection of the most suitable cyclodextrin for a certain application can be made by using numerous analytical methods such as spectroscopic methods in solutions (UV spectrophotometry, NMR, fluorescence, ESR), separation techniques (CE, HPLC, TLC), and electroanalytical methods [7, 8]. In case of solid phase samples, IR, Raman, X-ray, and thermal methods are the most commonly used, however, these approaches are not capable of determining the liquid phase (equilibrium) stability constant.
UV spectroscopy is a relatively straightforward method having low material needs, but it provides very little information about the structure of the complex. In this method, the change in the transitions of the chromophore group signals the formation of a complex . As a result of the formation of an inclusion complex and the chromophore getting into a more apolar environment, the UV absorbance spectrum might become more structured than in an aqueous solution . This interaction might result in either a hyper- or hypochromic and hypso- or bathocromic shift of the absorption spectrum. The two disadvantages of the method are that (a) other UV-active compounds may interfere with the spectrum, (b) the complexation does not necessarily result a characteristic change in the spectrum.
NMR spectroscopy has several advantages. Presence of a chromophore in the guest molecule is not required and, unlike in CD and UV spectroscopy, it provides information about the structure of the complex and it allows for quantification from the changes in the chemical shifts . This method’s drawback is its low sensitivity and the occasional need for deuterated solvents. If the guest molecule has low solubility, long acquisition times may be needed for achieving adequate signal–noise ratios in the case of 2D NOESY or ROESY techniques, which are necessary in structure analysis. The use of other solvents might be required to ensure the proper concentration of the compound. However, this additional solvent might affect the stability and the structure of the complex. In case of native cyclodextrins—due to their symmetry—the interpretation of the spectra can be simple, but in the case of modified cyclodextrins, the presence of structural isomers (different substitution degree) complicates the structural analysis.
The biggest advantage of fluorescence spectroscopy is the quantum yield effect, making it more sensitive and usable in a wider concentration range, compared to other techniques. It has to be noted that the signal intensity, despite being in correlation with the concentration, is not linear . When using modified cyclodextrins containing fluorophore groups, the fluorescence spectrum changes due to interactions with the guest molecule. Commonly, the fluorophore is displaced from the cavity by the guest molecule in a competitive process. Based on this principle, cyclodextrins can be applied as molecular sensors .
Capillary electrophoresis requires small quantities of material, but either the guest molecule or the cyclodextrin must have a charged functional group. Neutral molecules and cyclodextrins cannot be tested directly. If the cyclodextrin is dissolved in the mobile phase, it might function as a chiral selector in case of enantiomer pairs .
HPLC methods are not restricted to charged molecules and apply cyclodextrins two ways: first, the cyclodextrin is covalently bonded to the solid phase ; second, the cyclodextrin is dissolved in the mobile phase .
When applying covalently bonded cyclodextrins, the separation can be performed using normal (NP) or reverse phase (RP) mode . Testing different cyclodextrins require purchasing a column of each type. This is costly and time-consuming, especially if optimisation of the chromatographic parameters also requires addressing mobile phase composition and temperature parameters.
When cyclodextrins are dissolved in the mobile phase, a simple RP column (RP2–RP18) may be used to identify the optimal cyclodextrin by varying the cyclodextrin concentration and analysing the reduction in the retention time of the guest molecule. With this method, an apparent stability constant can be determined as well . Problems posed by this technique are high costs, because for method optimisation large quantities of cyclodextrins might be required. In addition, optimization can be a lengthy procedure. Similarly, to capillary electrophoresis, attaining enantiomer selectivity can be set as an aim [19, 20].
Alongside the methods mentioned above, circular dichroism can come to mind as a good alternative. Its sensitivity and material needs are on par with UV spectroscopy but it provides more detailed structural information. Upon analysis of CD spectra, the formation of inclusion complexes can be confirmed and the structure of the complex can be assessed using theoretical calculations. Another benefit of this method is that, like UV spectroscopy, it can rapidly provide results, resulting in shorter experiment times. Furthermore, CD spectroscopy is less expensive than NMR or separation techniques.
The limiting factor of the method is the necessity of a chromophore on the guest molecule, which needs to be able to interact with the cyclodextrin to produce an induced CD signal (ICD) . The appearance of this ICD signal in the region of the absorption bands is a definite proof of the complex formation. The sign and intensity of the ICD band, supplemented with theoretical calculations give opportunities for further estimations regarding the complex structure [22, 23, 24]. ICD spectra can be used to distinguish between cyclodextrins having different degrees of substitution . In case of achiral or racemic substances when only the complex is CD active, it is possible to estimate the equilibrium (stability) constant, because the intensity of the ICD signal will be proportional to the complex concentration. Similar selectivity can only be achieved using NMR .
For the calculations, the following need to be taken into account:
With the condition [G]T ≥ [HG] ≥ 0, the quadratic equation gives only the solution of Eq. (5) and the other roots does not need to be taken into consideration.
Using these calculations, the stability constants of drug–cyclodextrin interactions can easily be compared and the most suitable cyclodextrin may be selected for the purpose in question. As a drawback, it needs to be mentioned that the acquired ICD signal has low intensity sometimes, resulting in disadvantageous signal–noise ratios in the case of low-stability complexes (logK < 2). Light scattering can be another occurring problem. It can be detected from the UV spectra measured along the CD, outside of the absorption region, the increase in the baseline signals the presence of solid particles. While these can be removed from the solutions by filtration, accurate calculations cannot be made from the ellipticity data acquired. In racemic substances, the stability of the two diastereomer complexes are different, so the calculated data is only average or apparent constant.
To demonstrate the method’s capability five antifungal azole molecules were tested with different cyclodextrins.
Materials and methods
The following materials are products of Cyclolab Ltd. (Budapest, Hungary): α-cyclodextrin (ACyD); β-cyclodextrin (BCyD); γ-cyclodextrin (GCyD), heptakis(2,6-di-O-methyl)-β-cyclodextrin (DMBCyD); (2-hydroxypropyl)-β-cyclodextrin (HPBCyD) (D.S~5,5); heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TMBCyD); β-cyclodextrin sulfobutyl ether sodium salt (SBBCyD) (D.S~6,3); carboxymethyl-β-cyclodextrin sodium salt (D.S~3,5) (CMBCyD); fluconazole [2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol], miconazole [(RS)-1-(2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl)-1H-imidazole] and bifonazole [(RS)-1-[Phenyl(4-phenylphenyl) methyl]-1H-imidazole] were pharmacopoeial grade (Ph. Eur. 7.8) products of Sandoz (Budapest, Hungary). Clotrimazole [1-[(2-chlorophenyl)- (diphenyl)methyl]-1H-imidazole] and tioconazole [(RS)-1-[2-[(2-Chloro-3-thienyl)methoxy]-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole] as well as the dimethyl sulfoxide (DMSO) used to dissolve the drug were purchased from Sigma-Aldrich (Budapest, Hungary). For the structures of model compounds see Supplement Fig. S1. CD and UV experiments were performed on a Jasco J-720 spectropolarimeter (Jasco Ltd., Tokyo, Japan) in Jasco and Hellma cylindrical cuvettes with pathlengths ranging from 1 to 20 mm. The nonlinear parameter fitting was performed with Origin 8.0.
Drugs were dissolved in DMSO to form 0.1 M stock solutions, which were further diluted with distilled water to 0.0016 M for the experiments. Cyclodextrin stock solutions were 0.016 M in water. Experimental cyclodextrin (CyD)-drug solutions were mixed with the following concentration ratios: 0:1, 0.5:1, 1:1, 2:1, 5:1 and 10:1. In certain occasions, 20:1 and 50:1 ratio were also prepared. To promote the solubility of the drugs, 5 µl 2 M HCl was used (pH ~ 2).
First, solutions containing only the drugs were measured. For each experiment, the cuvette was chosen so the absorption at the absorption peak would not exceed 2. All the spectra were registered with the following parameters: scan speed: 50 nm/min; slit: 2 nm; response (D.I.T.): 2 s; accumulation 3–5 depending on the signal–noise ratio.
Results and discussion
Based on literature data, the logK stability value of the complex changes depending on the protonation of the molecule. In case of β-cyclodextrin complexes, the logK of the positively charged form is 1.03, while for the unionised form it is 1.97  or 1.83 . At pH ~ 2, the positively charged form is the dominant. Our previous, unpublished data showed 1.13 (positively charged) and a 1.64 (neutral) logK for HPBCyD, 1.9 (charged) and 1.72 (neutral) for SBBCyD. During experiments ICD spectra could not be registered; see spectra in Supplement Fig. S2. Low logK values as well as low logP and high polar surface area (PSA) of FLZ is a possible explanation for this, based on previous literature  these two physical parameters, as well as the geometry of the molecule are the main factors affecting cyclodextrin stability constants.
BIZ–CyD complex stability values and regression coefficients
LogK1:1 (M−1) and (R2) (all data)
LogK1:1 (M−1) and (R2) (without the last data point)
PSA value of CLZ is similar to BIZ, however the logP is higher as a result of the chlorophenyl group. Based on these parameters, K values are expected to be similar but the geometry of the molecule is different. CLZ is more compact, resulting in lower conformational freedom, also due to its molecular shape it is not expected to sink into the cavity like the biphenyl or phenyl group of BIZ.
In the case of CLZ, structural information cannot be gained from the UV spectra, neither the structural, neither intensity changes appear after CyD complexation; see Supplement Fig. S3. If observing simply the UV absorbance as a function of cyclodextrin concentration changes, the conclusion might be false, because there are no significant changes.
CLZ–CyD complex-stability values and regression coefficients
LogK1:1 (M−1) and R2
Molecular parameters and logarithm of stability constant (M−1) of azole–βCyD complexes
MIZ–CyD complex-stability values and regression coefficients
LogK1:1 (M−1) and R2
TIZ–CyD complex-stability values and regression coefficients
LogK1:1 (M−1) and R2
Circular dichroism is a suitable method for investigating the interaction of guest molecules with cyclodextrins. The appearance of the ICD signal is a matchless and strong proof of a non-covalent host–guest type inclusion complex formation. Such unambiguous, straightaway and fast evidence cannot be achieved by any other method. The intensity, sign, structure and shifts of spectral bands along with physico-chemical properties and literature data allow for estimation of the complex structure. The CD spectroscopy may be an alternative to phase solubility studies also, because the UV and CD signals are measured in parallel, so the information about the increased solubility and the complexed guest concentration can be measured on two channels. A further possibility is the use of a third channel, if the CD spectropolarimeter is equipped with a fluorescent detector.
Taking into consideration that both the imidazole and triazole chromophores absorption maxima appear under 240 nm, the signals must arise from the phenyl groups. The LB band of a phenyl group have maximum about 250 nm, which is highly affected by several factors e.g. solvents, substituents, and the homo- and hetero-molecular interactions. The fine structure of this transition is affected by the polarity of its surroundings and by conformational freedom. The complexation ensures a more apolar media in the cavity of the cyclodextrin for the lipophilic part(s) of the molecule; therefore the ΔG (Gibbs free energy) and ΔS (entropy) changes are advantageous in regard to complex formation. These facts allow to assume that the apolar phenyl or substituted phenyl chromophores take part dominantly in the complexation. In the case of MIZ and TIZ the higher absorption wavelengths are associated with the dichlorophenyl moieties.
The method is also useful for calculating stability constants based on simple equations. The simplicity of the calculation follows from the selectivity of the method, because only the complex gives signal, while other methods give the average signal of the different coexisting species (free and complexed particles). A very stable complex may be unfavourable from the point of view of drug release. Comparing the obtained values with each other, it may provide information on which cyclodextrins are suitable for complexation, however the value of the stability constant should not be the only basis for the decision.
Summary and comparison of logK values are calculated and collected from the literature
1.03  (NMR)
3.34–3.52  (CE)
2.66  (PS)
2.91  (PS)
3.12  (CE)
3.84  (CE)
3.13–3.26  (CE)
1.13  (NMR)
3.66  (NMR)
2.65  (PS)
2.56  (PS)
2.41  (PS)
2.86  (CE)
1.90  (NMR)
3.94  (NMR)
6.45 (1:2)  (NMR)
3.04  (PS)
Open access funding provided by Semmelweis University (SE). The authors thank András Váradi, Ph.D. (Columbia University) for language editing and proofreading.
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