Cyclodextrins and ternary complexes : technology to improve solubility of poorly soluble drugs

Cyclodextrins (CDs) are cyclic oligosaccharides composed of D-glucopyranoside units linked by glycosidic bonds. Their main property is the ability to modify the physicochemical and biological characteristics of low-soluble drugs through the formation of drug:CD inclusion complexes. Inclusion complexation requires that host molecules fit completely or partially within the CD cavity. This adjustment is directly related to the physicochemical properties of the guest and host molecules, easy accommodation of guest molecules within the CD cavity, stoichiometry, therapeutic dose, and toxicity. However, dosage forms may achieve a high volume, depending on the amount of CD required. Thus, it is necessary to increase solubilization efficiency in order to use smaller amounts of CD. This can be achieved by adding small amounts of water-soluble polymers to the system. This review addresses aspects related to drug complexation with CDs using water-soluble polymers to optimize the amount of CD used in the formulation in order to increase drug solubility and reduce dosage form volume.


INTRODUCTION
Among several factors, solubility in water is of paramount importance in the development of a sufficiently safe and effective dosage formulation, because preparation, absorption and even the biological activity of a drug are all dependent on its solubility.However, the amount of lipophilic molecules used in treatment is relatively high and tends to increase, considering that many different drugs have low solubility (Lipinski, 2000;Grant, Zhang, 2011).Thus, use of cyclodextrins (CDs) is one of several technologies available to improve the solubility of poorly water-soluble drugs.The most remarkable property of CDs is their ability to modify the physicochemical characteristics of molecules that are accommodated within their internal cavity to form the so-called inclusion complexes (Loftsson, Brewster, 1997;Tsai et al., 2010).
Typical characteristics of formulations containing inclusion complexes include a faster dissolution rate and shorter drug release time, as well as more efficient absorption.This translates into greater oral bioavailability of the drugs involved and an increase in biological activity, which may result in a reduction in drug dosage (Valle, 2004;Garnero et al., 2010).
However, the use of CDs is limited in some cases, because guest molecules need to fit completely or partially within the CD cavity.This adjustment is directly related to the physicochemical properties of the guest and host molecules, easy accommodation of guest molecules within the CD cavity, stoichiometry, therapeutic dose, and CD toxicity (Loftsson, Brewster, 1997).
An increase in formulation volume represents a critical stage in the applicability of CD inclusion complexes.We can consider that 1 g of a solid complex corresponds to 100-250 mg of a drug (when the molecular weights of the drug and the CD are 200-400 g/mol and 1200-1500 g/mol, respectively).Therefore, the use of CDs in oral solid dosage forms is limited to drug doses less than 200 mg that have good complexation properties (Loftsson, Brewster, 1996).
A strategy often used to improve complexation between drugs and CDs is the addition of small amounts of water-soluble polymers to the system, which causes an increase in solubilization efficiency, while requiring smaller amounts of CD (Loftsson, Fridriksdóttir, 1998;Mura et al., 2001).These results can be attributed to the synergistic effect of polymer and CD solubilization on the formation of drug:CD:water-soluble polymer ternary complexes (Carrier et al., 2007).
Water-soluble polymers are able to interact with drugs, CD molecules, and even with drug:CD complexes (Loftsson et al., 1996).The mechanism involved in increasing CD complexation efficiency in the presence of water-soluble polymers is not yet fully understood; however, it is believed that water-soluble polymers can reduce CD mobility and increase the complex solubility (Veiga et al., 2006).The addition of water-soluble polymers has been shown to increase drug bioavailability and cause an up to 80% reduction in the amount of CD required (Loftsson, Fridriksdóttir, 1998;Mura et al., 2001).
The purpose of this study is to outline the relevance of using CDs to improve the solubility of poorly watersoluble drugs, with special emphasis on their structural characteristics, physicochemical properties, productive processes, toxicity, derivatives, and use in the pharmaceutical industry.Despite the recognized benefits of ternary (drug:CD:water-soluble polymer) complexes, there have been no reviews on the subject in the scientific literature.This review addresses aspects related to drug complexation with CDs using water-soluble polymers to increase drug solubility and reduce dosage form volume.
CDs with less than 6 units of glucose do not exist for stoichiometric reasons and those with more than 8 units offer low yields and weak complexing properties, thus making them unsuitable for the pharmaceutical industry (Loftsson, Brewster, 1997;Jug et al., 2011).
According to Szejtli (2004), the history of CDs can be divided into three distinct periods (Figure 2 groups can be found at the broadest end, bonded to the C2 and C3 atoms of the glucose units, while the primary hydroxyl groups are located at the narrower opposite end, bonded to the C6 atoms of the glucose units (Bekers et al., 1991;Loftsson et al., 2004;Veiga et al., 2006).
The molecular arrangement of CDs is a result of the free rotation of primary hydroxyl groups, which reduces the diameter of the cavity at its narrowest end, i.e., the end with the smallest molecular diameter.CH groups bonded to the H1, H2 and H4 hydrogen atoms can be found on the outside of the molecule, while the hydroxyls find their way outside the truncated cone, thus becoming the external layer of hydrophilic CDs (Brewster, Loftsson, 2007).
In the internal layer, CH groups are bonded to the H3 and H5 hydrogen atoms by glycosidic oxygen bridges.Intramolecular hydrogen bonds between the C2-OH groups of a glucose unit and the C3-OH groups of an adjacent glucose unit stabilize the CD structure, making it rigid (Loftsson, Brewster, 1997;Brewster, Loftsson, 2007).

Properties
The most important property of CDs is their ability to modify the physicochemical and biological characteristics of drugs.Their cavity can establish interactions through intermolecular forces with molecules, ions or radicals, acting as a host substance.The resulting molecular complex is called an inclusion compound or a supramolecular compound (Loftsson, Brewster, 1997;Li et al., 2010).
Table I details several characteristics of natural CDs.Of all natural CDs, β-CD has the lowest solubility, due to the high number of intramolecular hydrogen bonds among secondary hydroxyl groups within the molecule.These interactions make the structure rigid and prevent hydration by water molecules (Szejtli, 1994;Loftsson et al., 2005b).(treating the starch with Bacillus macerans), the final product was a mixture of a-CD (60%), b-CD (20%) and g-CD (20%), as well as small amounts of CDs with more than 8 units of glucose.However, purity was a major hurdle, becoming a critical issue that had to be overcome before the use of CDs could be made possible (Loftsson, Duchêne, 2007).
An alternative to address the issue of impurity was the use of biotechnological processes, which, along with other innovations, led to an increased purity of the resulting CD, thus making their use as pharmaceutical excipients feasible (Loftsson et al., 2005b).

Cyclodextrin structure
Due to the lack of free rotation about the glycosidic bonds and chain conformation of glucose units, CDs display a torus-like or hollow truncated cone shape.In this peculiar structure (Figure 3), the secondary hydroxyl In α-CD, only 4 of 6 possible hydrogen bonds can be established, because one of the glucose units is in a distorted position.The γ-CD is a noncoplanar, more flexible structure, thus being the most soluble of the three CDs (Loftsson et al., 2005b).
CDs are stable in alkaline medium, hydrolyze in strongly acidic medium and are resistant to enzymatic degradation by β-amylase, although CDs, particularly γ-CD, are susceptible to attack by α-amylase.CDs can form stable hydrates and their stability is identical to that of starch; thus, they can be stored for years without suffering any degradation (Szejtli, 1994).

Toxicity
The safety profile of natural CDs and their derivatives has been widely studied, and they have generally proven to be atoxic, because they only manage to cross biological membranes with some degree of difficulty.Thus, oral administration of CDs should not be regarded as a problem (Valle, 2004).

CYCLODEXTRINS AND THEIR DERIVATIVES
It is possible to introduce chemical modifications into the primary and secondary hydroxyl groups of natural CDs through the bonds of several functional groups, thus  : Szejtli, 1994;Veiga et al., 2006;Brewster, Loftsson, 2007;Wintgens and Amiel, 2010   improving solubility, toxicity and increasing the inclusion capacity of original CDs and their derivatives (Uekama, Irie, 2004).CD derivatives can be obtained by substitution with methyl, ethyl, carboxymethyl, hydroxyethyl, hydroxypropyl, sulfobutyl, or saccharide groups or even by polymerization of CDs.Many derivatives of natural CDs have been synthesized and characterized, but only a few are being used in studies involving new pharmaceutical excipients, including derivatives with methyl, hydroxypropyl and sulfobutyl ether substitutes (Mosher, Thompson, 2002;Uekama, Irie, 2004;Veiga et al., 2006).

HYDROPHILIC DERIVATIVES Methylated derivatives
These derivatives can be obtained by selective methylation of all secondary hydroxyl groups in C2 and all primary hydroxyls in C6, methylation of all hydroxyl groups, including those in C3, or even randomly, in the C2, C3 or C6 positions (Imai et al., 1984;Veiga et al., 2006).
The methylated derivatives show alterations in their physical and chemical properties, as well as structural alterations when compared to natural CDs (Table III).Solubility in water and organic solvents is significantly greater; however, water solubility decreases as the temperature increases (a reaction similar to that of nonionic surfactants).These derivatives exhibit reasonable stability in alkaline medium and are hydrolyzed by strong acids, giving rise to linear oligosaccharides (Uekama, Irie, 2004;Veiga et al., 2006).
Dimethyl-β-CD is the least vulnerable to acid hydrolysis.At the opposite extreme, trimethyl-γ-CD is the most susceptible, due to severe distortion in the configuration of the CD ring (Uekama, Irie, 2004).
Obtaining hydroxyalkyl derivatives is a non-selective process that occurs by the condensation of hydroxyalkylating agents (hydroxypropyl and hydroxyethyl) in alkaline medium.The product of the condensation reaction is invariably a mixture of the respective derivatives, with various degrees of substitution.These mixtures not only prevent recrystallization, but also result in the conversion of the drug from a crystalline state into an amorphous state (Uekama, Otagari, 1998;Uekama et al., 2006).
The degree of substitution (S) expresses the number of hydroxyl groups replaced in a unit of glucose, which may range from 1 to 3, and the average degree of substitution (DS) expresses the average number of hydroxyls replaced per unit of glucose, which is between 0 and 3.The average molar substitution (MS) expresses the number of hydroxypropyl groups per unit of glucose (Veiga et al., 2006).
Hydroxyalkyl derivatives have high water solubility and low hygroscopicity compared to the original CD; thus, in the presence of high humidity (> 90%), they dissolve in water adsorption.They have a surface tension identical to that of natural CDs, but this characteristic is altered in derivatives with high degrees of substitution (Uekama, Otagari, 1998;Uekama et al., 2006).

Ramified derivatives
This class of CDs is obtained by chemical or enzymatic synthesis, where the substitution of primary or secondary hydroxyl groups for mono-or disaccharides through α-(1.6)bonds results in the formation of ramified CDs with high water solubility and chemical purity (Veiga et al., 2006).
Although ramified CDs have physical and chemical properties similar to those of natural CDs, such as surface tension and complexation capacity (Table IV), their solubility in water, as well as in aqueous solutions of ethanol, methanol, acetone, formaldehyde and ethylene glycol, is superior (Duchêne Wouessidjewe, 1990b).

HYDROPHOBIC DERIVATIVES
CDs and their derivatives are mainly used in the pharmaceutical industry to improve the solubility and dissolution speed of poorly soluble drugs by means of inclusion complexation.However, some CD derivatives act in an opposite manner, with the main function of controlling the dissolution speed of water-soluble drugs.These derivatives are represented by ethylated and acylated CDs (Uekama et al., 2006).

Ethylated derivatives
The aqueous solubility of CDs is reduced when their hydroxyl groups are replaced with alkyl groups larger than methyl, through an ether or ester bond.Solubility decreases proportionally to the rate of substitution, which increases in less polar solvents, thus presenting fewer hygroscopic characteristics and lower surface tension (Uekama et al., 2006;Mosher, Thompson, 2002).

Acylated derivatives
These are obtained by substitution of all β-CD hydroxyl groups for different alkyl chains, resulting in reduced aqueous solubility, melting point and rate of alkaline hydrolysis as the respective alkyl chain increases.
In concentrated solutions of β-CD derivatives in organic solvents (ethanol, acetone or chloroform), the viscosity increases due to a gelation process occurring after the solvent has evaporated (Mosher, Thompson, 2002).
Differently from the ethylated β-CD derivatives, acylates are easily eliminated from the organism after alkaline hydrolysis, yielding the original CD (β-CD).This is an important factor in the event of enteral administration, because this CD is not toxic when administered by this route (Mosher, Thompson, 2002).

Ionizable derivatives
The substitution of CD hydroxyl groups for ionizable groups imparts hydrophilic characteristics to the new structure, as well as pH-dependent complexation capacity.In other words, solubility is low in acidic medium, becoming greater in neutral or alkaline media.This pH-dependent characteristic is a result of the ionization of the carboxylic groups that show a pKa value around 3.5 (Ma et al., 2000).Among all ionizable CDs, one in particular stands out: SBE-β-CD.This is a polyanionic CD formed when the 2, 3 and 6 hydroxyl groups of β-CD glucose units are substituted for sulfobutyl ether groups, which are totally ionized over a broad pH range.They provide a negatively  charged polar head, attached to a hydrophobic tail, which is connected to the internal cavity (Stella et al., 2002).SBE-β-CD has a peculiar structure, where substitute groups that exercise mutual electrostatic repulsion are in a favorable position for entry into the CD cavity.As a result, there is an increase in its hydrophobic proper-ties and complexation capacity, which is the reason for its wide-ranging pharmaceutical application.Another relevant attribute is the fact that the charge of the CD molecule is located at a site as far as possible from the hydrophobic cavity, thus intensifying its solubilizing capacity (Zia et al., 2001).

FORMATION OF INCLUSION COMPLEXES
The truncated cone structure of CDs, which are open at both ends, enables the inclusion of a wide variety of organic molecules (apolar drugs) in their central cavities.Host-guest complexes, or drug-CD complexes also known as inclusion complexes or compounds, result from the association between host molecules (CDs) and encapsulated molecules (drugs) (Szejtli, 1998;Tsai et al., 2010).
The formation of a complex (Figure 4) in an aqueous solution takes place when water molecules are removed from the apolar cavity of CDs (which are in an energetically unfavorable environment due to the nature of the polar-polar interaction) and substituted for a guest molecule or lipophilic group with polarity, size and shape compatible with that of the CD structure (Szejtli, 1998;Rafati et al., 2009).
This process is energetically favorable and contributes to an increase in complex stability, because it causes changes in enthalpy and a reduction in the total energy of the system (Saenger, 1980;Veiga et al., 2006).
Furthermore, other forces are involved in the formation and stabilization of inclusion complexes, such as van der Waals interactions (dipole-dipole interaction and London dispersion forces), 3-center, 2-electron bonds (between guest molecule and CD hydroxyl groups), hydrophobic interactions, release of deformation energy from the macromolecular ring of CDs, and steric effects (Saenger, 1980;Bekers et al., 1991;Szejtli, 1998;Bibby et al., 2000;Flasinski et al., 2010).
The complexes formed are usually more water soluble than the active ingredients they contain as well as more stable in solution form.They also dissociate easily in order to release the drug molecule (Lofttson et al., 2005 a, b;Wintgens, Amiel, 2010).
Obtaining complexes with CDs may occur in the liquid, semi-solid or solid phases.In the liquid phase, the following methods have been suggested: coprecipitation, coevaporation, neutralization, freeze-drying, and drying by pulverization.In the solid phase, the most common methods are grinding or supercritical fluid technology, while malaxation is employed in the semi-solid phase (Valle, 2004).
Drug-CD complexes have an extremely rapid and dynamic formation and dissociation kinetics in solution form, continually forming and dissociating by covalent bonds.Complex dissociation is expressed quantitatively by the dissociation constant (Kc), where [drug-CD], [drug] and [CD] are the concentrations of the complexed drug, the free drug and the free CD, respectively.This dissociation constant ranges from 0 to 10 5 , where 0 indicates that the drug is incapable of forming a complex with CD and 10 5 indicates the upper limit of drug-CD complexes (Tompson, 1997;Stella, Rajewski, 1997;Veiga et al., 2006;Loftsson et al., 2007;Rafati et al., 2009). (1) The dissociation kinetics will be inversely proportional to the strength of the bond between the CD and the drug, i.e., the slower the dissociation kinetics, the stronger the drug-CD bond (Kc).Even in this situation, the dissociation velocity of the complexes is considered to be practically instantaneous (Loftsson et al., 2007).
Molecular modeling studies have gained strong emphasis in the investigation of complexation with CDs.This allows the construction of three-dimensional models of drug-CD complexes, visualization of structural integrity, and intra-and intermolecular interactions (Seridi, Boufelfel, 2011;Leila et al., 2011;Eid et al., 2011;Ge et al., 2011;Mishur et al., 2011).

CYCLODEXTRINS IN THE PHARMACEUTICAL INDUSTRY
CDs and their derivatives are present in several areas, most notably in the pharmaceutical industry, where they are extensively used because of their complexing

FORMATION OF TERNARY COMPLEXES
When a water-soluble polymer, a CD and a drug are mixed together in a solution to obtain the so-called ternary complexes, it is possible to increase drug solubilization, when compared to the polymer and CD separately, which is a result of the synergistic effect between these components (Loftsson et al., 1994).An example is the synergistic effect resulting from the addition of hydroxypropyl methylcellulose (HPMC) to the complex formed by SBE-β-CD and carbamazepine, with a consequent increase in drug solubility in the resulting ternary complex (Smith et al., 2005).
Formulations containing drug:CD complexes with the addition of a water-soluble polymer have proven to be capable of increasing the bioavailability of formulations while reducing the amount of CD by up to 80% (Loftsson, Fridriksdóttir, 1998;Mura et al., 2001).In the presence of water, the polymer aids in the wettability of particles, resulting in accelerated dissolution and increased amount of drug delivered in vitro (Lahiani-skiba et al., 2006).
The interaction of water-soluble polymers with drug molecules may occur by means of ion-ion, ion-dipole and dipole-dipole electrostatic bonds, van der Waals force, or 3-center, 2-electron bonds (Ribeiro et al., 2003).Similarly, the interaction between polymers and CDs and drug:CD complexes begins to occur on the external surface of the CD molecule.CDs, polymers and drug:CD complexes form aggregates capable of solubilizing drugs and other hydrophobic molecules (Loftsson et al., 2007), as shown in Figure 5.
Several types of interactions between polymers and drugs may be established as a result of the structural difference and polarity of CD molecules, which may give rise to various complexation efficiencies.Povidone (PVP) and HPMC polymers were evaluated in the complexation of vinpocetine with β-CD and SBE-β-CD.The best complexation efficiency results were obtained for PVP with β-CD and for HPMC with SBE-β-CD (Ribeiro et al., 2003).
The resulting chemical structure of the drug is still unknown, as is the nature of the interaction between CDs and the water-soluble polymer, but it is recognized that, in aqueous solutions, polymers stabilize micelles and other types of aggregates, reduce CD mobility and increase the solubility of complexes by changing the hydration properties of CD molecules (Loftsson et al., 2005b).
This process can be accelerated by heating the ternary system.Thus, it is possible to activate the bonds between system components during the preparation of complexes by heating them in an autoclave (120 to 140 ºC) for 20 to 40 minutes, in an ultrasound bath (over 30 ºC) for 1 hour, or even with microwaves at 40 ºC for 5 minutes (Loftsson et al., 2005b).
Thermodynamic parameters (entropy and enthalpy) prove that different forces and/or mechanisms are at play in the formation of the complex, depending on the presence or absence of a polymer.Addition of polymers changes the entropy (ΔSº) of the system, which becomes more negative, indicating the formation of a more organized structure with greater enthalpy (Loftsson et al., 1994).
Studies have proven that HPMC and PVP increase the complexation of hydrocortisone, dexamethasone and naproxen with β-CD (Ammar et al., 2006).Valero and colleagues (2003) observed that, at low PVP concentrations, the complexation process occurs entropically, and in larger proportions, it occurs enthalpically.

COMPLEXATION EFFICIENCY AND THE STA-BILITY CONSTANT
The stability constant (K C ), calculated from the phase solubility diagram (drug concentration x CD concentration), can be considered an apparent stability constant for several complexes, describing the combined effect of various structures on the solubility of a drug.Accordingly, a definition for complexation efficiency (CE) as a more precise method for evaluating the solubilizing effect of CDs has been proposed (Loftsson et al., 2007).
The stability constant of a complex is determined from the slope of the phase diagram and the intrinsic solubility of a drug (S 0 ) (Equation 1).Theoretically, the intersection (S int ) of the phase solubility diagram should be identical to S 0 .However, drugs with an aqueous solubility of less than 0.1 mM show an intersection in the phase solubility diagram that is generally much greater than S 0 ,  thus resulting in imprecise K C values.Therefore, CE is calculated from the slope of phase solubility diagrams and is independent of S 0 and S int , in accordance with Equation 2 (Loftsson et al., 2007).In Equation 3, K 1:1 represents the stability constant 1:1 ratio between drug and CD, and D represents drug concentration. (2) (3) Some researchers consider that complexes with K C values ranging between 200 and 5000 M -1 are applicable to dosage formulations, while K C values between 7 and 100 M -1 were deemed sufficient by others, because they were able to improve the physical and chemical properties of drugs compared to non-complexed forms (Veiga et al., 2006).
K C values are widely used to determine the stoichiometry of complexes, as well as to compare the affinity of drugs for CDs, thus determining whether the addition of water-soluble polymers to the system actually results in greater interaction between the components (Loftsson et al., 2007).

POLYMERS USED TO OBTAIN TERNARY COMPLEXES
Obtaining complexes with CDs, drugs and watersoluble polymers has gained greater acceptance due to the relatively low cost of polymers (Lahiani-Skiba et al., 2006).The most important requirements in choosing polymers to form inclusion complexes with drugs and CDs are water solubility and absence of biological activity.The most commonly used polymers for this purpose may be classified as natural, semi-synthetic and synthetic (Veiga et al., 2006), as detailed in Table VII.
There is no pre-established range of ideal polymer concentration for obtaining ternary complexes.However, it is known that, at high concentrations, the viscosity of the medium increases, thus impairing complexation.The amount of polymer must be such that the solubilizing effect is maximized, but not sufficient to cause a significant increase in viscosity (Ribeiro et al., 2003).
In studies by Loftsson and colleagues (1994) with hydrocortisone, 17β-estradiol and triamcinolone in an HPβ-CD 10% (p/v) aqueous solution, the ideal concentration of polymers ranged from 0.05 to 0.25% (p/v) and greater concentrations led to a reduction in drug solubility.Table VIII details the complexation solubility and efficiency of some drugs in their free form and in ternary complexes.

PHARMACEUTICAL APPLICATIONS OF TER-NARY INCLUSION COMPLEXES
Most drugs with low aqueous solubility have organic solvents, emulsifiers and extreme pH conditions in their formulations, which can cause irritation and other adverse reactions (Del Valle, 2004).The drug:CD:polymer complexes can be administered in any dosage form for the treatment of a variety of ailments, depending on the biological activity of the complexed drug.Research on ternary complexes has gained prominence in recent decades, and it is therefore possible to find a considerable number of studies in which drug:CD:water-soluble polymers obtained for several drugs are described (Table IX).

CONCLUSION
Improving the solubility of poorly soluble drugs is one of the main applications of CDs and their derivatives, which have the ability to encapsulate organic molecules in their cavities, thus forming inclusion complexes, which    in turn modify the physicochemical characteristics of such drugs.The drug:CD:water-soluble polymer complex represents an attractive alternative, especially in cases where a high amount of CD is required for complexation, which significantly increases the volume of dosage forms.Thus, it is possible to obtain solid-form medications with an optimized dissolution profile, which may result in improved bioavailability.

FIGURE 2 -
FIGURE 2 -Timeline of relevant events in the history of cyclodextrins (CDs).

FIGURE 3
FIGURE 3 -β-cyclodextrin (β-CD) structure, with representations of its size and position of hydroxyl groups.
Table II lists the solubility of natural CDs in water and other organic solvents.

TABLE I -
Properties of natural cyclodextrins (CDs) (Adapted from

TABLE III -
Physicochemical properties of cyclodextrins (CDs) and their methylated derivatives.Source: Duchêne and Wouessidjewe,

TABLE V -
Details of some characteristics of cyclodextrin (CD) derivatives

TABLE VII -
Polymers most commonly used to obtain ternary complexes

TABLE IX -
Ternary complexes between drugs, cyclodextrins (CDs) and water-soluble polymers, as described in the scientific literature

TABLE IX -
Ternary complexes between drugs, cyclodextrins (CDs) and water-soluble polymers, as described in the scientific literature (cont.)