Effect of Cyclodextrin Charge on Complexation of Neutral and Charged Substrates: Comparison of (SBE)7M-β-CD to HP-β-CD
V. Zia, 1,3 R. A. Rajewski,2 and V. J. Stella1,2,4
Received January 16, 2001; accepted February 7, 2001
Scale for pharmaceutical use. These consist of the three parent cyclodextrins (α-, β-, and γ-CDs) and three classes of modified cyclodextrins, namely, methylated, hydroxypropylated and sulfobutylated cyclodextrins (1). Naturally produced cy- clodextrins, excluding γ-CDs, along with methylated cyclo- dextrins have no significant use in parenteral dosage forms due to toxicity issues (2). However, HP-β-CD and (SBE)7M- β-CD have recently gained considerable attention in the class of modified β-CDs because of their improved safety profiles (1,2).HP-β-CD and (SBE)7M-β-CD are highly soluble com- pounds capable of forming reversible inclusion complexes with a variety of molecules to form soluble complexes unionized states, Table I describes those conditions that differed from the earlier study.
Purpose. To understand the role of charge in substrate/cyclodextrin complexation by comparing the binding of neutral and charged sub- strates to a neutral cyclodextrin, such as hydroxypropyl β-CD (HP- β-CD) with 3.5 degrees of substitution, and an anionically charged cyclodextrin, such as sulfobutyl ether β-CD ((SBE)7M-β-CD) with 6.8 degrees of substitution.
Method. HP-β-CD and (SBE)7M-β-CD were evaluated in their ability to form inclusion complexes with neutral compounds, as well as to cationic and anionic substrates in their charged and uncharged forms. The complexation constants (Kc) were determined via a UV spectro- photometric technique, by monitoring the change in substrate absor- bance upon incremental addition of a concentrated cyclodextrin so- lution. The role of electrostatic interaction was probed by observing Kc as a function of solution ionic strength.
Results. Neutral molecules displayed a stronger interaction with (SBE)7M-β-CD compared to HP-β-CD. In those cases where the guest possessed a charge (positive or negative), HP-β-CD/substrate complexes exhibited a decrease in complexation strength (2 to 31 times lower) compared to the neutral forms of the same substrate. The same was true (but to a larger extent, 41 times lower) for nega- tively charged molecules binding to (SBE)7M-β-CD due to charge- charge repulsion. However, positively charged molecules interacting with the negatively charged (SBE)7M-β-CD displayed a similar bind- ing capability as their neutral counterpart, due to charge-charge at- traction. Further evaluation through manipulation of solution ionic strength revealed strong electrostatic interactions between substrate and cyclodextrin charges. In addition, the studies suggested that on average two sulfonates out of seven may be involved in forming ionic attraction or repulsion effects with the positive charges on prazosin and papaverine, or negative charges of ionized naproxen and warfarin.
Conclusions. Presence of charge on the cyclodextrin structure pro- vides an additional site of interaction compared to neutral cyclodex- trins, which may be modified using solution ionic strength.
KEY WORDS: cyclodextrins; ionic strength; coulombic interactions; sulfobutyl cyclodextrins; hydroypropyl cyclodextrin.
INTRODUCTION
Although numerous natural and modified cyclodextrins have been described in the literature, there are only six types of cyclodextrins to date that are produced on an industrial β-CD is derived by substitution of either primary or second- ary hydrogen of the hydroxyl group of β-CD with a 2-hy- droxypropyl moiety (3). As shown in Figure 1, there are 21 possible sites where hydroxypropyl groups can attach to the cyclodextrin torus. In the case of the specific HP-β-CD used in this study, an average of approximately 3.5 hydroxypropyl groups are attached to the torus. The (SBE)7M-β-CD is de- rivatized in a similar manner, with the exception that the substituents are sulfobutyl groups (4). (SBE)7M-β-CD has an average of 6.8 substituents per cyclodextrin, and therefore has about seven negative charges associated with it, which are counterbalanced with sodium ions (Fig. 1).
Because of the different substituents placed on the cy- clodextrins, HP-β-CD and (SBE)7M-β-CD have properties that differ from one another. The charged sulfonate groups of sulfobutyl ether moieties provide a charged head group in addition a hydrophobic tail group that are attached to the cyclodextrin cavity. Usually, placing a charged group on or around the cyclodextrin cavity reduces its complexation abil- ity (4–7). Neutral guests interacting with charged cyclodex- trins generally show lower stability constants relative to simi- lar neutral cyclodextrins. This decrease in apparent binding has been associated with changes in the hydrophobicity of the cavity interior and/or changes in geometry of inclusion com- plexation (5,7). Unlike most other charged cyclodextrins, (SBE)7M-β-CD often shows superior binding to most neutral substrates, due to the significant separation of the charged sulfonate moiety from the cyclodextrin torus and the interac- tion of some substrates with portions of the butyl moiety (8,9). In some cases, where oppositely charged molecules and charged cyclodextrins interacted, strong complexations have been observed (7–12). In such cases, the improvement in com- plexation has been associated with additional interaction sites provided by the cyclodextrin charge. The mode of interaction is presumed to be hydrophobic interaction with the cavity interior along with additional charge-charge interaction be- tween the charged guest and host.
In the present work, the interaction of neutral (HP-β-CD) and charged ((SBE)7M-β-CD) cyclodextrins with various neutral, anionic, and cationic molecules were studied. The two cyclodextrins resemble each other in providing not only a hydrophobic cavity, but also the potential for additional sur- faces of interaction. However, (SBE)7M-β-CD may provide supplementary binding sites for molecules capable of forming ionic interactions with the charged sulfonate moieties. Thus, the role of charge interactions between the guest and the host, was the primary focus of this study.
Ionic Strength Effects
To better determine the effects of charge interactions, solutions of varying ionic strength (I = 0.01, 0.1, 0.2, 0.3 M) were prepared at the appropriate pH values to form the neutral or charged form of the molecules under investigation. Solution ionic strength was determined by the buffer and the NaCl salt added, while assuming substrate and cyclodextrin contribution to be minimal and negligible since substrate con- centrations were generally below 10−5 M and cyclodextrin concentration ranges were 10−5–10−3 M. The complexation constants of molecules to cyclodextrins were determined at a fixed pH value with varying ionic strength.
RESULT AND DISCUSSION
Fig. 1. Structural representation of HP-β-CD and (SBE)7M-β-CD.
EXPERIMENTAL
Materials
All substrates and their sources were identical to those described and defined previously (9). HP-β-CD (Lot 92-3, MW 1338; TDS 3.5; water content = 7.4%) was obtained form Cerestar (Hammond, Indiana), previously known as American-Maize Products. The preparation and characteriza- tion of (SBE)7M-β-CD (MW 2207.6; TDS 6.8; water content = 9.8%) and its derivatives have also been described previ- ously (9). All cyclodextrins were corrected for water content prior to use. Water was deionized and charcoal filtered prior to glass distillation, using a Corning Mega-Pure™ System MP-1 (Corning, New York).
Instrumentation
Karl Fischer water analyses of cyclodextrins were per- formed on a Brinkmann 652 KF-Coulometer. UV analysis.
Complexation of Neutral Molecules to (SBE)7M-β-CD and HP-β-CD
The complexation constants of ten unionized molecules with HP-β-CD and (SBE)7M-β-CD were determined and the results are reported in Table II along with the Kc ratio of (SBE)7M-β-CD over HP-β-CD. All complexes exhibited lin- ear x-reciprocal plots, suggesting a 1:1 substrate/cyclodextrin complexation (9). The negatively charged (SBE)7M-β-CD uniformly showed larger complexation capability over HP-β- CD, but the extent of increase in complexation was not the same for all molecules. Molecules such as hydrocortisone, prednisolone, and methylprednisolone showed only a slight increase in complexation (1.2–1.4 times), while molecules such as testosterone, benzylguanine, and prazosin showed more significant improvements of 3.2–9.1 times in their bind- ing constants.
The stronger complexation ability of (SBE)7M-β-CD over HP-β-CD may be attributed to several reasons. The charged sulfonate moieties of (SBE)7M-β-CD are not ex- pected to fold back into the cyclodextrin cavity, eliminating any intra- or inter-molecular complexation similar to those associated with the HP-β-CD (1,14). In addition, the charged sulfonate groups of each cyclodextrin are likely to repel one another by extending out and away form each other, provid- ing a hydrophobic region near the cavity composed of only the alkyl ether portions of the sulfobutyl groups. Therefore,a Experiments were performed at 25°C and the appropriate pH value for the dominant presence of neutral species. The derived Kc values are the average of two experiments. b Ratio of (SBE)7M-β-CD over HP-β-CD. complexation of neutral molecules to (SBE)7M-β-CD may occur not only via the cyclodextrin cavity, but also the alkyl chains near the cavity (9). In addition to these differences, specific interactions between the various substrates and the two cyclodextrins may be important. Perhaps hydrogen bond- ing interactions with strategically placed hydrogen bond do- nor or acceptor groups on the substrates or the cyclodextrin could account for some of the differences observed.
Method
The cyclodextrin complexation constants were deter- mined by monitoring the change in substrate UV absorbance upon the incremental addition of a concentrated cyclodextrin solution (13). Complexation constants were determined via reciprocal plots. Specifics of the actual procedure were de- scribed in an earlier paper (9). The drug concentration and the pH values studied were also mentioned previously. Be- cause some of the agents were studied in both ionized and Table II. Comparison of HP-β-CD to (SBE)7M-β-CD in Forming Inclusion Complexes with Neutral Forms of Various Substrates anitude for both cyclodextrins. Naproxen was the only mol- ecule that showed a difference in complexation ability to the two cyclodextrins with increasing ionic strength. A significant increase in Kc value of naproxen/(SBE)7M-β-CD complex was observed with increasing ionic strength. However, naproxen/HP-β-CD complex did not show an effect in com- plexation strength upon increasing ionic strength.
To a certain extent, the increased binding of some mol- ecules upon increased ionic strength can be explained by salt- ing out effects. According to the hydrophobic bond concept, the aqueous solubility of hydrophobic molecules may be de- creased with an increase in salt concentration (15,16). The increase in complexation constants (Kc) of some molecules with HP-β-CD and (SBE)7M-β-CD may be attributed to the same effect, where the interaction of the molecule with the hydrophobic cyclodextrin cavity is increased with addition of electrolytes. However, this phenomenon does not seem to affect all complexes or affect them to the same extent, possi- bly due to various substrate/CD complexation positions, strengths, geometry, and specific interactions.
Ionic Strength Effects on Complexation of Neutral Molecules
The effect of solution ionic strength on the complexation constants of various neutral molecules to HP-β-CD and (SBE)7M-β-CD is presented in Table III. The effect of ionic strength on the complexation ability of each cyclodextrin was varied. Hydrocortisone and prazosin displayed no significant change in complexation to either cyclodextrin upon increas- ing ionic strength. However, complexation of both cyclodex- trins to warfarin increased as the ionic strength was increased. This increased complexation of warfarin was of similar magtion of the linear fits to the intercept gives the apparent Kc at zero ionic strength, where the effect of charge is greatest. As shown in Table V, the Kc of anionic warfarin and naproxen are 14 and 20 times stronger with HP-β-CD than (SBE)7M- β-CD, re-emphasizing the effect of charge-charge repulsion between a negatively charged substrate and (SBE)7M-β-CD. The complexation of positively charged prazosin (Table V) at zero ionic strength is about 50 time greater with (SBE)7M-β- CD than HP-β-CD, suggesting strong opposite charge attrac- tion forces between the host (SBE)7M-β-CD and the cationic guest molecule.
Table IV shows the Kc values for various anionic and cationic molecules in their charged and uncharged states with HP-β-CD and (SBE)7M-β-CD, with the Kc ratio of neutral over charged substrates in the last column. The complexation of all molecules with HP-β-CD was decreased upon substrate ionization (positive or negative), but the extent of the de- crease was not uniform for each complex. As shown in the last column of Table IV, for HP-β-CD complexes, warfarin dis- played the smallest change, while prazosin showed the largest change. For (SBE)7M-β-CD complexes, change was depen- dent on the charge of the guest molecule. Positively charged molecules maintained their complexation strength compared to their neutral forms. However, negatively charged mol- ecules showed decreased complexation. This reduction in complexation strength for anionic molecules to (SBE)7M-β- CD was larger than those seen with HP-β-CD.
It has been shown that the complexation strength of mol- ecules to cyclodextrins decreases with an increase in the hy- drophilicity of the substrate (17–19). This is attributed to the much simplified rule of “like-dissolves-like.” With an increase in the hydrophilicity of the substrate, such as the addition of a charged or hydrophilic moiety, an improved interaction with the polar solvent occurs, lowering the cyclodextrin com- plexation strength. This has been further established by changing the polarity of the solvent (20–22). The interaction of a molecule in an aqueous solution of cyclodextrin may be viewed as a ternary system, with the interior cavity of cyclo- dextrin being the “non-polar solvent”, and water being the polar solvent. Thus, molecules that cannot interact effectively with water tend to interact well with the apolar solvent (cy- clodextrin cavity) and vice versa (23,24), assuming the geom- etry is favorable. This effect is observed with the decrease in Kc of molecules complexing to the HP-β-CD upon ionization. However, the larger decreases in complexation of the nega- tively charged molecules binding to (SBE)7M-β-CD com- pared to the same complexes of HP-β-CD suggests the exis- tence of an additional ionic repulsion force between the charged sulfonates and the negative guest molecules. Con- versely, in cases where the guest molecules possess a positive charge, electrostatic attraction may occur between the posi- tive charge of the guest and the negative sulfonates of the (SBE)7M-β-CD. The presence of this charge attraction seems to counter balance the adverse effect of the increased substrate hydrophilicity discussed earlier, thus maintaining their binding potential (see Table IV).
Effect of Ionic Strength on Complexation of Charged Molecules
To further investigate the effect of charge interaction, Kc values of charged substrates were determined in solutions of varying ionic strength. The effect of increasing ionic strength on complexation of anionically charged molecules such as naproxen to HP-β-CD and (SBE)7M-β-CD is shown in Figure 2a. Negatively charged molecules display a stronger compl- exation to HP-β-CD than (SBE)7M-β-CD. Both modified cy- clodextrins showed improved binding ability with an increase in the ionic strength of the solution, however, this increase was more pronounced with (SBE)7M-β-CD than HP-β-CD. The increased Kc values upon increasing ionic strength sug- gest reduction in charge-charge repulsion due to charge shielding at higher solution ionic strengths. The effect of ionic strength on cationic molecules such as prazosin interacting with HP-β-CD and (SBE)7M-β-CD is shown in Figure 2b. In contrast to the effects seen with negatively charged molecules, the binding of cationic molecules to (SBE)7M-β-CD decreases with an increase in the solution ionic strength, and ap- proaches the Kc value seen for HP-β-CD. In such cases, the charge shielding is also evident as a reduction in Kc value of positively charged molecules complexing to negatively charged cyclodextrin at higher solution ionic strength. Unfor- tunately, the Kc of HP-β-CD/papaverine was too small to be determined accurately by our method. An increase in the solution ionic strength generally weakens ion-ion and ion- dipole interactions (25). The fact that the complexation of charged molecules to negatively charged (SBE)7M-β-CD are highly dependent on the solution electrolyte concentration verifies the significance of charge interactions in stabilizing or destabilizing a complex.
Fig. 2. A representative example of the effect of ionic strength on the complexation of (a) negatively charged substrate (naproxen in this case) and (b) positively charged substrate (prazosin in this case) to HP-β-CD (●) and (SBE)7M-β-CD (O), at 25°C and using NaCl to control ionic strength. The results are the average of two experiments with the error bars representing the upper and lower limits (in some cases the error bars are small enough to be within the symbols).
Figure 3 shows the plots of log-Kc versus the square root of ionic strength (I1/2) for anionic and cationic molecules binding to HP-β-CD and (SBE)7M-β-CD. Such plots may al- low one to determine Kc values independent of salt effect by extrapolation to zero ionic strength (16,26). Figures 3a and 3b represent the complexation of all neutral and charged mol- ecules with HP-β-CD and (SBE)7M-β-CD, respectively. Neu- tral molecules binding to either cyclodextrin in Figure 3a Based on the Debye-Hu¨ ckel description of charge inter- actions, the changing Kc as a function of ionic strength in an aqueous solution may be derived from the equilibrium reac- tion in scheme 1 (27–30). The intrinsic Kc (Kc0) is the product of the concentration (c) and activity coefficient (γ) dependent equilibriums (Eqs. 1 and 2) (13), which may be written in logarithmic terms (Eq. 3).
In the case of prazosin and papaverine the slopes are −1.6 and −2.7, respectively (see Table V), suggesting an average of 1.8 and 2.3 units of negatively charged sulfonates (roughly 2 units) interact with the positively charged substrates studied. Therefore, approximately an average of two sulfonate moi- eties of (SBE)7M-β-CD interact with the positive charge of the guest molecule in forming the complex. Similar interac- tion of 2.0 and 2.5, although of charge-charge repulsion type is seen with warfarin and naproxen, respectively. Interest- ingly, the similarity of warfarin (2.0) and prazosin (1.8) in electrostatic attraction and repulsion may be related to their structure, having their charge buried within their molecular structure. In the case of papaverine and naproxen, where their charge is more removed from the hydrophobic part of the molecule, there seems to be a stronger electrostatic inter- action of 2.3 and 2.5 units. Further studies are necessary in determining the underlying principle for the above observation.
CONCLUSION
(SBE)7M-β-CD displayed a larger binding capability compared to HP-β-CD in forming inclusion complexes with neutral substrates. Charged molecules (cationic or anionic) binding to neutral cyclodextrin, HP-β-CD, displayed a de- crease (2 to 31 times) in complexation, compared to their neutral counterparts. The presence of a negative charge on the substrate reduced its complexation to (SBE)7M-β-CD by about 40 times, which was attributed to ionic repulsion ef- fects. Positively charged substrates complexing to (SBE)7M- β-CD did not display much change in binding strength when compared to their neutral counterparts, due to opposite charge interactions of the substrate and sulfonate moieties of (SBE)7M-β-CD. Due to the presence of charge-charge inter- action, it was shown that the complexation strengths of mol- ecules may be manipulated through solution electrolyte con- centrations. Further analysis suggested that the ionic interac- tion of the charged substrates studied occurred through interactions with up to two sulfonate groups of the (SBE)7M- β-CD.
ACKNOWLEDGMENTS
The authors would like to thank the Schering-Plough foundation and Kansas Technology Enterprise Corporation for financial funding. The authors would also like to thank Donna Kowalski for performing the statistical analysis and interpretation of the data.
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