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Review Article *Corresponding author Yukiko Uekaji, CycloChem Bio Co., Ltd., 7-4-5 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047 Enhancement of Oral Japan, Tel: 81-(0)78-302-7073; Fax: 81-(0)78-302-7220; Email: Submitted: 23 March 2017 of Functional Accepted: 04 April 2017 Published: 06 April 2017 ISSN: 2379-089X Ingredients by Complexation Copyright © 2017 Uekaji et al. with Cyclodextrin OPEN ACCESS Yukiko Uekaji1*, Naoko Ikuta2, Gerald Rimbach3, Seiichi Keywords Matsugo4, and Keiji Terao1,2 • γ-Cyclodextrin • Inclusion complex 1CycloChem Bio Co., Ltd., Japan • Bioavailability 2Graduate School of Medicine, Kobe University, Japan • Solubility 3Institute of Human Nutrition and Food Science, University of Kiel, Germany • Stability 4School of Natural System, Kanazawa University, Japan • Coenzyme Q10 • R-α- Abstract Natural lipophilic bioactives that possess human health benefits, such as coenzyme Q10 (CoQ10) and R-α-lipoic acid (RALA), often have undesirable characteristics that limit their use as nutraceuticals and cosmeceuticals. These bioactives are usually unstable against oxygen, ultraviolet light, low pH and heat. Furthermore, their water- solubility is low because of their hydrophobic nature or instability and this leads to low bioavailability. Therefore, systematic studies have been performed to investigate improvements in the stability, water-solubility and bioavailability of lipophilic bioactives through complexation with cyclodextrins (CDs). The solubility of CoQ10 in water is extremely low, resulting in low bioavailability when administered orally. Bioavailability of CoQ10 was enhanced significantly by complexation with γ-CD. CoQ10 generally agglutinates, but the dissociated CoQ10 from γ-CD was captured by acid to form micelles without aggregation and therefore both solubility and bioavailability were enhanced. RALA is available as a functional food ingredient but is unstable to heat or acid. To stabilize RALA, complexation with γ-CD was investigated. RALA was unstable molecule, whereas RALA-γCD complex was stable under the acidic conditions of the stomach and was easily absorbed in the intestine. CD complexation is a promising technology as a formulation aid for oral delivery of insoluble or unstable ingredients such as CoQ10 and RALA.

ABBREVIATIONS CD: Cyclodextrin; CoQ10: Coenzyme Q10; RALA: R units and γ-CD consists of eight glucose units. α-CD is widely Acid; SALA: S used as because it is not enzymatically digested -α-Lipoic GZK2 and thus has no nutritional value. In contrast, γ-CD is degraded -α-Lipoic Acid; TCNa: Sodium Taurocholate; into monosaccharides by α-amylase and therefore functions as : Dipotassium Glycyrrhizate; Cmax: Maximum Plasma 1/2: an energy source. CDs are capable of forming complexes with Concentration; AUC: Area Under the Plasma Concentration-time max a variety of ionic and lipophilic substances by taking the entire Curve; T : Time to Reach Maximum Plasma Concentration; T molecule or part of the molecule into their cavity. The formation INTRODUCTION Half-life of such molecular complexes affects many of the physicochemical properties of the guest molecules, such as their aqueous solubility, stability or bioavailability [1-3]. Cyclodextrins (CDs) are non-reducing, chiral cyclic oligosaccharides, in which D-(+)-glucopyranose units are linked Because the inside of CDs is hydrophobic and the outer surface by α-(1,4)-glycosidic bonds to form a ring structure. Depending is hydrophilic, CDs can enclose various hydrophobic substances on the number of D-(+)-glucopyranose units, and thus also on in their cavities and form inclusion complexes. α-CD forms the size of the ring, a distinction is made between α-CD, β-CD inclusion complexes with relatively smaller-sized molecules such and γ-CD: α-CD consists of six units, β-CD consists of seven as carbon dioxide gas and short-chain fatty acids (SCFAs) such as Cite this article: Uekaji Y, Ikuta N, Rimbach G, Matsugo S, Terao K (2017) Enhancement of Oral Bioavailability of Functional Ingredients by Complexation with Cyclodextrin. J Drug Des Res 4(3): 1043. Uekaji et al. (2017) Email:

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acetate, propionate and butylate. SCFAs stimulate colonic blood flow, and fluid and electrolyte uptake [4]. β-CD forms inclusion complexes with middle-sized molecules including and flavonoids such as hesperidin, which is found abundantly in citrus fruits and acts as an antioxidant to contribute to the integrity of the blood vessels, and reduce LDL cholesterol and blood pressure [5,6]. γ-CD forms inclusion complexes with 2 larger-sized moleculesR- such as macrocyclic compounds and lipophilic vitamins including vitamins A, D3, E, and K , coenzyme Q10 (CoQ10) and α-lipoic acid (RALA), which are used as nutraceutical ingredients in supplements and health foods having Figure 1 various human health benefits. Chemical structures of CoQ10 (a), RALA (b) and SALA (c). Irrespective of whether the guest molecule is in a gaseous, The chiral center is marked with an asterisk (*). liquid, or solid state, the resultant inclusion complexes are always solid powders. Stable powders are much easier to handle than unstable volatile substances such as aromatic oils. RALA is the naturally occurring compound. Converting volatile and unstable active substances into inclusion Further, RALA and its dihydro-form, which is produced via complexes facilitates the process of adding precise amounts of metabolic reduction, are powerful antioxidants because of their the substances and storing them stably until use in medicinal scavenger properties and their synergistic interaction food and cosmetic products. with other antioxidants. Dihydro-RALA, the reduced form of CDs are generally used in nutraceuticals, pharmaceuticals RALA, reduces glutathione, which is an important antioxidant for and• cosmetics• for the following purposes: many physiological processes. As an amphiphilic molecule, RALA is soluble in both aqueous and non-aqueous media. ALA is widely Solubilizing hydrophobic bioactive compounds in water- used as a pharmaceutical or nutraceutical ingredient. •• phase systems. Although it is possible to separate the ALA enantiomers (the Enhancing bioavailability for nutraceuticals used as bioactive RALA and SALA), commercially available ALA is the preventive medicines, such as vitamins, CoQ10, curcumin •• chemically synthesized racemate with the mixture containing and α-lipoic acid (ALA). the same amount of RALA and SALA. This occurs because the Stabilization of unstable bioactive compounds like pure bioactive enantiomer RALA is unstable when exposed unsaturated fatty acids and against oxidation, to low pH, light or heat [10]. RALA decomposes gradually at hydrolysis, photoreaction and thermal decomposition room temperature and easily polymerizes at temperatures •• during storage. higher than its melting point, which is 46–49 °C. Low pH also causes RALA to polymerize so quickly that it decreases RALA Reduction of the bitter taste and unpleasant smell of bioavailability. Therefore, finding solutions to stabilize RALA various plants extracts containing bioactive compounds is of industrial interest and several studies aiming to stabilize such as catechin from green tea and from chili •• RALA have been carried out. Among these attempts to stabilize peppers. RALA, for example, is complex formation or encapsulation with Long lasting controlled release of medically active chitosan [11,12], which appears to be a promising approach. substances. Unfortunately, encapsulation efficiency of chitosan with RALA has proven to be limited. Recently, it was found that complex CoQ10 (Figure 1a) is a fat-soluble, vitamin-like, benzoquinone formation with CDs was more efficient to stabilize RALA and compound that functions as an antioxidant, as a membrane enhance the bioavailability. The aim of this work was review stabilizer and as a cofactor in the production of adenosine of systematic studies in the stability, water-solubility, and triphosphate (ATP) by oxidative phosphorylation. The solubility bioavailability of lipophilic bioactives as like CoQ10 and RALA of CoQ10 in water is extremely low, resulting in very poor RESULTSthrough complexation AND DISCUSSION with CDs. bioavailability when administered orally. Recently, it was found that the bioavailability of CoQ10 was enhanced by complexation Coenzyme Q10 with γ-CD, yielding the CoQ10-γCD complex, and the plasma 1/2 CoQ10 level after a single oral administration of the CoQ10-γCD complex was extended (half-life, T increased) [7]. This long- The biological absorption of CoQ10 after oral administration lasting high CoQ10 concentration in plasma can provide various of a CoQ10-γCD complex was greatly enhanced despite the health benefits. poor soluble characteristics of the complex in water. The most RALA (Figure 1b) is a cofactor for mitochondrial enzymes reasonable explanation of this phenomenon is an increase in such as pyruvate dehydrogenase and alpha-keto-glutarate the water-solubility of CoQ10 by sodium taurocholate (TCNa), which is a major component of in the small intestine. By dehydrogenase, andR thus S plays a central role in energy metabolism [8,9]. ALA has a chiral center at its C6 carbon leading adding TCNa to a water suspension of the CoQ10-γCD complex, to two enantiomers: - and -ALA (SALA, (Figure 1c)), of which the guest molecule, CoQ10 is replaced with TCNa and forms a water-soluble TCNa-γCD complex. This occurs because TCNa J Drug Des Res 4(3): 1043 (2017) 2/15 Uekaji et al. (2017) Email:

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has a higher association constant with γ-CD than that of CoQ10. Generally, CoQ10 molecules agglutinate in water to form visible particles. However, a CoQ10 molecule dissociated from the γ-CD cavity can be soluble in water by micelle formation with TCNa. Here, the dissociated CoQ10 in water is captured by a TCNa micelle and locates to the central region of the micelle and is thus solubleBioavailability [13,14]. enhancement of CoQ10 and its mechanism

The bioavailability of CoQ10 was enhanced by complexation with γ-CD showing a unique profile. The CoQ10-γCD complex shows excellent pharmacokinetic properties with a significantly Figure 2 higher area under the CoQ10 concentration curve in blood plasma from 0–48 hours (AUC) and higher maximum plasma Time course of plasma level of CoQ10 after oral max administration of CoQ10 with three different formulations to 72 concentration (C ) [7]. This complex also shows mean plasma healthy adult subjects: the CoQ10-γCD complex (solid line), CoQ10 levels even after 24 and 48 hours that are significantly higher with oil emulsifier (dashed line) and CoQ10-MCC mixture (dotted after administration of CoQ10-γCD to healthy adult volunteers line). CoQ10 intake was 30 mg per subject. when compared with the same administration of commercially available CoQ10 formulation with dietary fatty oil-based 2 emulsifiers (so-called “Water Soluble CoQ10”) and CoQ10 CoQ10 absorption into a human epidermis structure model by formulation with microcrystalline cellulose (MCC) (Figure 2). formulation of a CoQ10-γCD complex with GZK was observed 1/2 Moreover, administration of CoQ10-γCD also gives much longer when compared with other cosmetic formulations using liposome T values when compared with the other two administered Studyor fatty oil-based on water-solubility emulsifiers. of CoQ10 captured in γ-CD formulations. This study was performed using 72 healthy TCNa micelles after complete degradation of γ-CD by human subjects and statistically calculated. Why was a significant α-amylase enhancement of the bioavailability of CoQ10 observed despite the poor aqueous solubility of the CoQ10-γCD complex? The mechanism, as mentioned in the last section, is due to the The water solubility and bioavailability of CoQ10 is enhanced significant increase in the aqueous solubility of CoQ10 with the significantly by combining the CoQ10-γCD complex with TCNa. aid of TCNa [13,14], as described in more detail below. The hypothetical mechanism is described above and presented According to the study, by adding TCNa to a water suspension in Figure (4). If the hypothesis is correct γ-CD would not affect of the CoQ10-γCD complex, a water-soluble TCNa-γCD complex the water-solubility of CoQ10 in the system. Therefore, to was formed by substitution of the guest molecule from CoQ10 confirm this hypothesis, the water-solubility change in CoQ10 to TCNa, which has a higher association constant with γ-CD was evaluated after confirming a water-solubility increase in CoQ10 by adding TCNa to the CoQ10-γCD complex followed by than CoQ10. The association–1 constants–1 of γ-CD with TCNa and CoQ10 are 4800 M and 2200 M , respectively [15]. The CoQ10 the complete degradation of γ-CD by α-amylase [14]. molecule dissociates from the γ-CD complex and this molecule To a buffer solution (pH 6.0), α-CD, γ-CD and pancreatic is captured by a TCNa micelle to form a “nanometer molecular α-amylase were added. The solution was kept for 1 h at 37°C. captured micelle”. In aqueous solution, the hydrophobic CoQ10 More than 80% of the non-complexed γ-CD was degraded by molecule generally agglutinates to form visible particles. pancreatic α-amylase, whereas α-CD (control) was not degraded. The aqueous solubility of CoQ10 through the combination On the other hand, γ-CD encapsulating CoQ10 was not degraded of the CoQ10-γCD complex with TCNa was ~100 times higher after 24 h incubation in a solution containing the CoQ10-γCD than that of a commercially available oil emulsifier formulation, complex and pancreatic α-amylase. Presumably the pancreatic “Water Soluble CoQ10”, whose particle size was controlled to be α-amylase could not approach the γ-CD encapsulating CoQ10 around 100–200 nm in diameter [13,14]. The formation of the to perform the enzymatic reaction because γ-CD did not exist “nanometer molecular captured micelle” was found to facilitate in the liquid phase but in the solid phase owing to the insoluble a significantly higher AUC via effective absorption into intestinal characteristics of the CoQ10-γCD complex in water. epithelial cells. The same bioavailability-enhancing effect of However, by adding TCNa to the solution, γ-CD encapsulating complexing γ-CD with various hydrophobic nutraceuticals with CoQ10 was completely degraded under the same conditions after human health benefits, such as curcumin and tocotrienols, was 24 h. Then, pancreatic α-amylase can readily hydrolyze γ-CD also achieved. because of the high water-solubility of the complex. Since γ-CD We applied this technique which combine CoQ10-γCD is converted into linear dextrins by this ring cleavage reaction, 2 complex with TCNa in nutraceuticals to cosmeceuticals. Instead formation of a complex between CoQ10 and γ-CD can no longer of TCNa, dipotassium glycyrrhizate (GZK ) was used, because it occur. Therefore, CoQ10 that has dissociated from the CoQ10- had also high associate constant with γ-CD, and similar chemical γCD complex with the aid of TCNa could be sustainably absorbed structure with TCNa (Figure 3) [16,17]. Here, a much higher in the small intestine because of the very slow degradation J Drug Des Res 4(3): 1043 (2017) 3/15 Uekaji et al. (2017) Email:

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Figure 3

Structure of TCNa and GZK2-γCD complexes.

Figure 4

CD complex formation and molecular micelle formation.

Effect of adding GZK2 to the CoQ10-γCD complex:

1/2 reaction of γ-CD with pancreatic α-amylase while releasing CoQ10 at a slow rate. This most likely explains why the T of The structure of TCNa consists of a hydrophilic region and CoQ10 plasma concentration after oral administration of the a hydrophobic region. The hydrophobic region is entrapped StudiesCoQ10-γCD oncomplex the was solubility-enhancing prolonged. effects of by an excellent fit in the γ-CD cavity. The hydrophilic region is hydrophobic bioactives by adding TCNa or GZK2 to the GZKplaced outside of the cavity and is accordingly thought to be the γ-CD complex 2 reason for the high water-solubility of the TCNa-γCD complex. Effect of adding TCNa to the CoQ10-γCD complex: CoQ10 is also known to form a water-soluble complex with γ-CD owing to its similar structure to TCNa, as described in Figure2 (3). Therefore, to examine this water-solubility feature of GZK , is poorly soluble in water because of its lipophilic side chain GZK2 water-solubility changes in CoQ10 following the addition of composed of 10 mono-unsaturated trans-isoprenoid units. Even to the CoQ10-γCD2 complex was compared with a mixture after sonication, the water-solubility of CoQ10 is only 0.3μg/mL. of pure CoQ10 and GZK . The 2water-solubility of CoQ10 was not Complex formation with γ-CD only enhances the water-solubility 2 of CoQ10 by modest amount, with the CoQ10 concentration in enhanced by addition of GZK to the suspension of pure CoQ10 in water. However, addition of GZK to the CoQ10-γCD complex the complex reaching 2.9 μg/mL. In contrast, the addition of 2 TCNa to the CoQ10-γCD complex enhanced the solubility of suspension increased the water-solubility of CoQ10, and this CoQ10 significantly. The addition of TCNa to a cloudy water increase correlated with increasing amounts of added GZK . 2 suspension of CoQ10-γCD complex led to the solution appearing Significantly high CoQ10 water-solubility (more than 2000 μg/ as transparent clear solution. The CoQ10 concentration of the mL) was achieved with a higher GZK /CoQ10 molecular ratio than GZK2 2 solution obtained from the formulation of CoQ10-γCD complex 10 (Figure 6) [14]. The results suggest that only one molecule of with TCNa was surprisingly high at 1147.5 μg/mL, which is more is required for the2 formation of the GZK -γCD complex, but than 100 times higher than that of the “Water Soluble CoQ10” ~10GZK molecules of GZK are essential for solubilizing one molecule formulation using the amino acid cationic surfactant, CAE (11.4 of dissociated2 CoQ10 by the formation of a molecular micelle of μg/mL), as shown in Figure (5) [13,14]. that entraps CoQ10. J Drug Des Res 4(3): 1043 (2017) 4/15 Uekaji et al. (2017) Email:

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2 formed followed by CoQ10 molecular captured micelle formation with GZK . These results suggest that the CoQ10-γCD complex is the most effective natural CD complex for performing solubility and bioavailability enhancement of CoQ10 and other lipophilic bioactives in the small intestine because of the existence of bile 2 acid, which n has a high association constant with γ-CD as well as GZK . In fact, Takahashi et al. investigated CoQ10 absorption Figure 5 in humans ( = 5) by oral administration of its β-CD and γ-CD Water solubility changes by various CoQ10 formulations. complexes containing 0.3 g of CoQ10 compared with uncomplexed CoQ10. This study showed that the highest bioavailability of CoQ10 was achieved when administered as a γ-CD complex [19].

Figure 6 2

Water solubility enhancement of CoQ10 by adding GZK to a suspension of the CoQ10-γCD complex in water.

Effect of adding GZK2 and 1-adamantane carboxylic acid to three CoQ10-natural CD complexes:

The water-solubility 2 changesGZK of CoQ10 in complex with three CDs following addition of GZK2 are showing Figure (7A). An increasing molecular ratio of to CoQ10 gave a substantially higher CoQ10 concentration2 for the CoQ10-γCD complex (CoQ10γ) mixed with GZK , whereas2 no significant increase was observed when adding GZK to the CoQ10-αCD (CoQ10α) or the CoQ10-βCD complexes2 (CoQ10β). This excellent solubility-enhancing effect of GZK2 is probably Figure 7 2 2 2 because of the high association constant of GZK toward γ-CD to Water solubility changes of CoQ10 by combination of the form the GZK -γCD complex by substituting the guest molecule three CoQ10-natural CD complexes with GZK (A) and with GZK and from CoQ10 followed by the2 formation of a CoQ10 molecular2 AdCA (B). captured micelle with GZK . The association constants of GZK with α-CD and β-CD2 are too weak to substitute CoQ10 for the formation of GZK -CD complexes. 1-Adamantane carboxylic acid (AdCA) is known to have an extremely high association constant2 with β-CD as well as a high association constant between GZK and γ-CD [18]. Therefore, we GZKevaluated the effect of AdCA on the water-solubility changes of CoQ102 by the combination of three natural CD complexes with [13,14]. As shown in Figure (7B), a significant increase in the CoQ10 solubility was2 observed by the addition of AdCA to GZKthe CoQ10β with GZK , with almost the same CoQ10 solubility Figure 8 increase2 observed when the γ-CD complex is combined with . We believe that this2 observation supports our mechanism, CoQ10 uptake monitored using the human epidermis structure model. in which, initially a GZK -γCD complex or AdCA-βCD complex is J Drug Des Res 4(3): 1043 (2017) 5/15 Uekaji et al. (2017) Email:

Central Bringing Excellence in Open Access In vitro study on the epidermis absorption-enhancing effect of CoQ10 by adding GZK2 to its γ-CD complex

A CoQ10 absorption study using an in vitro digestion Caco-2 cell model was reported by Bhagavan et al [20]. In their study, various commercially available CoQ10 formulation products were subjected to simulated digestion to mimic their passage through the gastrointestinal tract in order to generate micelles containing CoQ10 and bile acids such as taurocholate. The micelles prepared from the CoQ10 formulation products were added to monolayers of Caco-2 cells to determine the amounts of CoQ10 uptake. Their data demonstrated a significant enhancement of uptake of CoQ10 from the CoQ10-γCD complex when compared with that of pure CoQ10 powder and other formulations. Here, similar results were obtained using an in vitro human epidermis structure model. Importantly, high CoQ10 uptake by human epidermis 2 cells (keratinocytes) was observed when a solution prepared from CoQ10-γCD complex with GZK was applied. In comparison, other standard cosmetic formulations using liposome and fatty oil-based emulsifiers showed negligible uptake (Figure 8). The processes of aging and photo-aging are associated with an increase in cellular oxidation. This is partly due to a decline in Figure 9 the levels of the endogenous cellular antioxidant CoQ10. Hoppe SEM images of RALA+CD physical mixtures and RALA-CD et al., demonstrated that topical application of CoQ10 is effective complexes. (a) α-CD, physical mixture, (b) β-CD, physical mixture, (c) against UVA-mediated oxidative stress inhuman keratinocytes γ-CD, physical mixture, (d) RALA-αCD, (e) RALA-βCD,and (f) RALA- [21]. Furthermore, the topical application of CoQ10 was able to γCD. suppress significantly the expression of collagenase in human complexes dermal fibroblasts and was effective in reducing wrinkle depth. However, in cosmetic formulations, a higher concentration of CoQ10 than 0.3 wt% was required to prevent many of the RALA-CD complexes were prepared as described previously detrimental effects of photo-aging; despite the upper limit of [25]. Pure RALA was dissolved in water and mixed with a CoQ10 concentration in cosmetics being set to 0.03 wt% in Japan. corresponding molar amount of α-CD, β-CD and γ-CD to yield Accordingly, this finding may contribute to the development of a 1:1 ratio. The solution was mixed well and the pH adjusted. effectiveR-α-lipoic photo-aging acid care products containing CoQ10. The freshly prepared suspension was frozen overnight and lyophilized the next day. For the physical mixtures, RALA and the corresponding molar amounts of the different CDs were mixed to We have used α-CD, β-CD or γ-CD to stabilize RALA by complex yield a 1:1 ratio. formation. There are reports on how the physicochemical properties or characteristics of the lipophilic guest molecules We analyzed the prepared complexes using SEM with 300, such as the CoQ10, curcumin, astaxanthin and docosahexaenoic 500, 1000 and 5000 times magnification. SEM experiments acid change when using γ-CD as a host molecule [22,23]. There showed that the shape and aspect of the complex particles is a study on the physicochemical properties of racemic ALA-CD differed considerably and depended on the CD used for complex complexes that shows that complex formation with CD improves formation. Figure (9) shows SEM images with the highest ALA solubility and stability towards heat exposure [24]. However, magnification of RALA+CD physical mixtures and RALA-CD in other stabilization technique for ALA such as complex complexes. RALA+CD physical mixtures contain particles that formation with chitosan [21] the racemic ALA was analyzed, appear cracked and wrinkled, and shapes that are uneven and while in our studies we focus on the bioactive enantiomer RALA. particle sizes that vary considerably in the physical mixtures with the maximum RALA+CD physical mixture particle size exceeding In our studies, we prepared RALA-CD complexes to 50μm. There was no considerable change observed between the stabilize RALA and have examined their formation, shape and physical mixtures with RALA in SEM analysis. In contrast, the stability under heated conditions using high-pressure liquid particle size distribution of RALA-αCD, RALA-βCD and RALA- chromatography (HPLC) and scanning electron microscopy γCD complexes appeared to be more homogenous than in the (SEM). Furthermore, focusing on the use of the RALA-CD RALA+CD physical mixtures (Figure 10). Particles of RALA-CD complexes for oral consumption, we mimicked the stomach complexes form larger aggregates that appear to stack. environment and evaluated the stability of the complexes under acidic conditions. Furthermore, the bioavailability of RALA-CD Additionally, the RALA-βCD and RALA-γCD complexes complexesPhysicochemical was evaluated properties in animals and ofhealthy the human RALA-CD subjects. are shaped like prisms with parallel sides and in the case of the rod-shaped RALA-γCD particles, there are tetragonal and orthorhombic particles observed in the SEM images. While the J Drug Des Res 4(3): 1043 (2017) 6/15 Uekaji et al. (2017) Email:

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the stomach. In order to imitate the acidic environment of the stomach, we exposed free RALA and the RALA-CD complexes to pH1.2 and incubated the samples at 37 °C for 1 h. While free RALA was very unstable (43% of the incubated RALA remained), complex formation with any of the tested CDs improved the residual RALA significantly. However, RALA-αCD and RALA-γCD showed the highest stabilization of the incorporated RALA, with stability values of almost 100%. From our physicochemical data including stability, we conclude that γ-CD is the best suited CD for RALA complex formation (Table 1). The RALA-γCD complex was the most stable towards heat, humidity and low pH, as determined by quantifying the residual RALA by HPLC. The morphology of the RALA-CD complexes changes upon complex formation and differs depending on the CD used for complex formation. The RALA-γCD complex formed the most distinctive type of particle, namely rod- shaped particles. in vivo Further studies including experiments are required to explore the bioavailability and biological activity of the RALA-CD complexes in order to evaluate their potential use as nutraceuticals, pharmaceuticals and cosmeceuticals. We are currently studying the absorption mechanism of orally administrated RALA-CD in rats. McCormick14 and co-workers investigated the metabolism of dl-[1, 6- C] lipoic acid in rats and found that most of the racemate is metabolized via β-oxidation of the valeric acid side chain [26]. Having found a way to feed enantiopure RALA, it would also be interesting to investigate whether this or its administration as a complexAbsorption affects after its metabolism. single oral administration of RALA- CD complexes in animals

Cremer et al. calculated that the 50% lethal dose and the no-observed-adverse-effect level for racemic ALA in rats to be more than 2000 mg/kg and 61.9 mg/kg/day, respectively, based on acute and subchronic toxicity studies [27]. Therefore, racemic ALA has been administered at doses between 600 and Figure 10 1800 mg/day in many clinical trials [28-31]. On the other hand, Gal reported that SALA was more toxic than RALA in thiamine Particle size distributions of the RALA-αCD, -βCD and -γCD deficient rats [32]. Thus, RALA would be preferred to racemic complexes (COM), their physical mixtures (PM) and CDs. (a) α-CD, PM ALA as a nutritional supplement owing to safety concerns. In and RALA-αCD, (b) β-CD, PM and RALA-βCD, and (c) γ-CD, PM and RALA-γCD. this study, we stabilized RALA through complex formation with α-, β- and γ-CD, and the bioavailability of the prepared RALA-CD complexes were evaluated in rats. The plasma concentrations of RALA-βCD and RALA-γCD complex particles have rather smooth RALA were measured after a single oral administration of RALA or surfaces, the particles that constitute the RALA-αCD complex RALA-CDs (20 mg R-LA/kg, 2 mL/kg, (Figure 11)) to rats, and the are rougher and the shapes formed by these particles seldom Table 1: R show square and parallel surfaces. These results show that the Summarized stability test results of the analyzed -α-lipoic Thermal stability Acidic stability morphological characteristics of the RALA-CD complexes are acid/cyclodextrin(Sample) complexes. different from RALA+CD physical mixtures. (70 °C, 48h) (pH1.2, 37 °C, 1h) In the thermal stability test, RALA and RALA-CD complexes were exposed to 100% relative humidity for 30 min, 1 h, 2h, 5h, RALA-αCD 98 ± 1.25%* ~100%** 24 h or 48 h at 70°C, which is above the melting point of RALA. RALA-βCD 69 ± 1.83%* 95 ± 0.14%* After incubation, the remaining RALA was measured by HPLC. RALA-γCD ~100%** ~100%** Apart from studying the stability of the RALA-CD complexes * mean ± S.D.; ** Almost complete recovery. RALA-αCD: R-α-lipoic towards heat exposure, we also analyzed their stability under acid/α-cyclodextrin inclusion complex; RALA-βCD: R-α-lipoic acid/ acidic conditions. Similar to heat and humidity, low pH also β-cyclodextrin inclusion complex; RALA-γCD: R-α-lipoic acid/γ- cyclodextrin inclusion complex induces RALA polymerization, leading to poor absorption in J Drug Des Res 4(3): 1043 (2017) 7/15 Uekaji et al. (2017) Email:

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Table 2: R R R

PharmacokineticFormulation parameters of -α-lipoic acidRALA after oral administrationRALA-αCD of -α-lipoic acid or -α-lipoicRALA-βCD acid/cyclodextrin complexesRALA-γCD to rats.

Route 20po 20po 20po 20po C or C Dose (mg/kg)0 max (µg/mL) 1.7 ± 0.9 1.4 ± 0.6 1.6 ± 1.9 3.4 ± 2.5 max 0-tT (min) 11.8 ± 14.1 10.7 ± 10.7 33.3 ± 44.0 9.0 ± 10.7 * * * n R R AUC (μg·min/mL) 56 ± 35 56 ± 12 50 ± 19 121 ± 24 R Pharmacokinetic parameters are shown as mean ± S.D. ( = 6). RALA: -α-lipoic acid; RALA-αCD: -α-lipoic acid/α-cyclodextrin inclusion complex; 0 0-t max RALA-βCD: -α-lipoic acid/β-cyclodextrin inclusionP complex; RALA-γCD: γR-α-lipoic acid/γ-cyclodextrin inclusion complex; C : maximum plasma concentration; C : initial concentration; Tmax: time of maximum drug concentration; AUC : area under the plasma concentration versus time curve (from initial to last points); po: per os; and *: < 0.05 compared with RALA- CD. Statistical analysis was performed using analysis of variance followed by Tukey’s multiple comparison tests.

Figure 11

Plasma concentration-time profiles of RALA after oral administration of RALA (A), RALA-αCD complex (B), RALA-βCD complex (C) and RALA-γCD complex (D) and after intravenous administration of RALA sodium salt (RALA-Na) (E) to rats. Data are shown as mean ± S.D. (n = 6). pharmacokinetic parameters were calculated (Table 2). Although with respect to oral absorption. Then, the detailed mechanisms there were no significant differences in the Cmax and time to reach by which complex formation with γ-CD could enhance absorption 0-t maximum plasma concentration (Tmax) among the groups after of RALA were evaluated. oral administration, the AUC of RALA after administration of p To examine the absorption from the stomach, non-complexed the RALA-γCD complex sample was higher than that of the other RALA or the RALA-γCD complex was administered to rats complexes ( <0.05, (Table 2)). after pylorus ligation; plasma concentrations in excess of the We reported that RALA-γCD was suitable for pharmaceutical endogenous level of RALA were detected only 2 min after dosing formulation when considering pharmaceutical processing with either formulation [37,38]. This result indicates that RALA is [25]. On the other hand, there have been many reports that rapidly absorbed from the stomach regardless of the formulation CD complexes can achieve good oral bioavailability of poorly in rats. Furthermore, the concentration profiles of the two absorbable drugs [33]. Specifically, several studies have revealed formulations were almost equal in extent and velocity. A similar that γ-CD complexes enhanced the bioavailability of drugs such as observation was reported in an in situ absorption study by Peter digoxin in dogs, diazepam in rabbits, and artemisinin and CoQ10 and Borbe [39]. Moreover, the gastric pH of humans in the fasted in humans [7,34-36], but there have been no studies on RALA. state is ~1.2, whereas that of rats is ~4.0 [40,41]. Therefore, In this study, comparing the AUC 0-120min of RALA after oral RALA in the rat stomach was sufficiently stable, so that there was administration of α-, β- and γ-CD complexes to rats, it was found no difference in the absorption from the two formulations. When that RALA-γCD showed the highest plasma exposure among the administered to humans, RALA-γCD might be more stable in the groups (Figure 11), (Table 2). This result demonstrated that RALA stomach than RALA and may achieve higher exposure. by γ-CD inclusion was the preferable complex to the other CDs On the other hand, the absorption of RALA was greater J Drug Des Res 4(3): 1043 (2017) 8/15 Uekaji et al. (2017) Email:

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p after intraduodenal administration of RALA-γCD than after Plasmaenhancement of RALA absorption. of RALA-CD in healthy administration of non-inclusion RALA ( <0.05). Furthermore, human subjects results of X-ray imaging using a contrast medium (data not shown) suggested that both formulations reach the small intestine within a few minutes after oral administration. These It is well documented that 600 mg of racemic ALA has been results showed that the difference in absorption between RALA- dosed in pharmacokinetic or pharmacology studies [46-48], γCD and non-complexed RALA occurred mainly in the small and in many clinical trials, racemic ALA has been administered intestine, even at <5 min after oral administration (data not 0-t at doses between 600 and 1800 mg/day to diabetic patients Cshown). As anticipated, a large portion of the administered dose [28-31]. Breithaupt-Grögler et al. monitored the plasma ALA was retained in the stomach at 30 min, leading to AUC and concentration 10 min after dosing and demonstrated that the max values after oral administration that were lower than those area under the AUC showed dose linearity after single doses of values after intraduodenal administration. These results indicate 50 to 600 mg ALA in the fasted volunteers. They also showed that some factors in the small intestine enhance RALA absorption that there were no drug-induced effects in healthy male subjects by γ-CD complex formation. [47]. Gleiter et al., showed that food intake influenced the In 2012, Uekaji et al., reported that CoQ10, as a guest molecule bioavailability of ALA and the mean plasma ALA concentration of in γ-CD complexes, was liberated by bile acids to form micelles the fed healthy volunteers who received a single dose of 600 mg with bile acids and represents a mechanism for enhancing racemic ALA was lower than that of the fasted healthy volunteers absorption of CoQ10 [13]. If the same process also occurs in the [46]. Gleiteret et al., and Breithaupt-Grögleret et al., also reported rat intestine in the case of RALA, liberation of RALA molecules in 1996 and 1999, respectively, that the bioavailability of the could increase and be absorbed effectively. However, the AUC RALA enantiomer was higher than that of SALA when a racemic of RALA after intraduodenal administration of RALA-γCD was mixture of ALA (RALA:SALA=50:50) was administrated [46,47]. no different between the bile duct ligation (BDL) group and the Our recent animal study showed similar enantioselective respective sham operation group, indicating that bile acid did not pharmacokinetics of ALA [49]. Carlson et al., (2007) conducted associate with RALA after liberation from γ-CD. a clinical study to determine the pharmacokinetics of RALA in healthy human subjects administered with 600 mg RALA as its The complex form with a guest molecule and its liberation sodium salt [50]. This was the first reported study to demonstrate from CD was reversible and in equilibrium [42]. Therefore, the pharmacokinetics of enantiopure RALA. Since then, several γ-CD, which releases the guest molecule, would be present at pharmacokinetic clinical studies of RALA have been conducted, defined proportions in the small intestine. If γ-CD in the small primarily in Germany and the United States, but there has been intestine were digested to maltose or glucose by pancreatic no scientific research conducted in Japanese subjects. In this amylase, liberation of RALA molecules from γ-CD would likely study, we were aiming to evaluate the bioavailability of RALA-CD be promoted and the plasma RALA concentration could increase. in healthy human subjects and analyze the pharmacokinetics of Therefore, we elucidated whether α-amylase activity had an 0-t the plasma RALA levels after a single oral administration of 600 effect on the liberation of RALA from γ-CD and on its absorption. mg RALA or 6 g RALA-CD (equivalent amount of 600 mg of RALA) However, the AUC of RALA after intraduodenal administration as described in our report [57]. of RALA-γCD was no different between groups treated with and without acarbose, an α-amylase inhibitor (data not shown). This The mean plasma RALA concentration versus time profiles result indicated that α-amylase activity was also not associated after oral administration of RALA or RALA-γCD is shown in Figure with the absorption of RALA after its liberation from γ-CD. (12). The pharmacokinetic (PK) parameters are listed in Tables (3,4). For all subjects, the plasma concentrations of RALA after a The pharmacokinetic parameters after intraduodenal single oral dose of RALA-γCD were significantly higher than those administration of RALA-γCD and the water-soluble RALA- sodium salt were almost comparable, i.e., RALA-γCD dissolved immediately in small intestinal fluid and RALA might be liberated continuously from the complex. From the study by Trentin on the stability constant of the lipoate anion with CDs [43], γ-CD had a lower stability constant than α- or β-CD. This lower stability is because γ-CD has the biggest cavity among the CDs. According to these results we consider the dissolution process to be a key factor in the mechanism for enhancement of RALA-γCD, and not the liberation of RALA from γ-CD. Furthermore, we also considered whether CD increases the paracellular permeability of intestinal membranes by opening the tight junctions. Several reports have revealed that the Figure 12 permeability of nasal or cutaneous membranes to drugs was enhanced by β-CD [44,45]. Those studies, however, required pre- Plasma concentrations of RALA following a single oral dose treatment for a few hours to open the tight junctions. However, of 600 mg RALA or 6 g RALA-γCD in healthy volunteers. Values are the RALA was rapidly absorbed after administration and thereby mean ± S.D. from six subjects. Note: 6 g RALA-γCD is the equivalent this mechanism was thought to be not associated with the amount of 600 mg RALA. J Drug Des Res 4(3): 1043 (2017) 9/15 Uekaji et al. (2017) Email:

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Table 3: R R

Pharmacokinetic parameters of individual subjects administrated with a single oral dose of 600 mg -α-lipoic acid or 6 g -α-lipoic acid/γ- RALA RALA-γCD cyclodextrin. AUC AUC Subject C (µg/mL) 0–180min C (µg/mL) 0-180min max (µg min/mL) max (µg min/mL) 1 · · 2 1.02 43.6 2.62 186.7 1.03 46.7 4.45 214.7 3 1.83 74.9 5.08 216.7 4 1.02 53.8 4.93 197.3 5 3.62 158.8 4.23 189.4 R R 6 1.55 90.5 3.30 170.5 RALA-γCD: -α-lipoic acid/γ-cyclodextrin; RALA: -α-lipoic acid; Cmax: maximum plasma concentration; and AUC: area under the plasma concentration-time curve. Table 4: R R

Pharmacokinetic parameters for subjects orally administered RALAwith 600 mg -α-lipoic acid or 6g -α-lipoic acid/γ-cyclodextrin.RALA-γCD

Cmax (µg/mL) AUC (µg min/mL) 0–180min 1.68 ± 1.01 4.10 ± 0.96 ** T (min) max · 78.0 ± 43.5 195.9 ± 17.7 ** T (min) 1/2 20.8 ± 10.7 17.5 ± 6.1 p R R 38.9 ± 12.2 23.3 ± 10.3 1/2 Values are the mean ± S.D. from six subjects, ** <0.01; RALA-γCD: -α-lipoic acid/γ-cyclodextrin; RALA: -α-lipoic acid; Cmax: maximum plasma concentration; AUC: area under the plasma concentration-time curve; Tmax: time to the maximum plasma concentration; and T : half-life.

of RALA at 5, 15, 30, 45 and 60 min after oral administration. αCD complex and CoQ10-βCD complex were® prepared by a At every measured point, the mean plasma concentrations of conventional spray-dry® method. RALA was purchased® from RALA after an oral dose of RALA-γCD were higher than those Toyo Hakko Co., Ltd (Obu, Japan). CAVAMAX W6 FOOD (α- of RALA. The mean Cmax values were 1.68 ± 1.01 and 4.10 ± 0.96 CD), CAVAMAX W7 FOOD (β-CD) and CAVAMAX W8 FOOD 2 µg/mL (mean ± S.D.) for RALA and RALA-γCD administration, (γ-CD) were purchased from Wacker Chemie AG (München, respectively. The Cmax value of RALA-γCD was significantly higher Germany). GZK was donated by Tokiwa Co., Ltd. than that of RALA. The mean area under the plasma concentration- (Chiba, Japan). TCNa was purchased from Wako Pure Chemical time curve from time zero to the last sampling time (AUC0–180min) Industries, Ltd., (Osaka, Japan). AdCA was purchased from Tokyo values were 78.0 ± 43.5 and 195.9 ± 17.7 µg·min/mL (mean ± Chemical Industry Co., Ltd., (Tokyo, Japan). The human three- S.D.) for RALA and RALA-γCD administration, respectively. The dimensional cultured epidermal model “LabCyte EPI-MODEL” 1/2 mean AUC0-180min of RALA-γCD was 2.5 times higher than that of was purchased from Japan Tissue Engineering Co., Ltd. (Aichi, RALA and was also statistically significant. The Tmax and the T Japan). This is multi-layered epidermal model cultured by using were not different between RALA and RALA-γCD. At 180 min, the normal human skin cells. It consisted of multiple and viable cell mean plasma RALA concentrations had returned to nearly base layers and contained basal layer, stratum spinosum epidermidis, line levels for both RALA and RALA-γCD. granularBioavailability layer and stratum enhancement corneum. of CoQ10 and its mechanism RALA-CD was stable under acidic conditions found in the stomach and could be easily absorbed in the intestine. The mean AUC0–180min of RALA in the subjects orally administered 6 g of RALA- CD (equivalent amount of 600 mg RALA) was 2.5 times higher This open-label, single-dose crossover study was conducted than that of the subjects administered 600 mg of RALA. Thus, our as previously described [7]. Three different formulations, results suggest that complexation of RALA with γ-CD significantly CoQ10-γCD complex, CoQ10 with fatty oil based emulsifier enhanced its bioavailability in healthy human volunteers, making so called “Water Soluble CoQ10” and CoQ10-MCC mixture it a promising technology for delivering functional but unstable were comparative. The concentrations of plasma CoQ10 were ingredients like RALA. Additionally, there were no drug-induced calculated by changing all of the contents of CoQ10 into reduced side effects observed. These results indicate that 6 g of RALA-CD form (ubiquinol), which was determined using an HPLC with an isMATERIALS suitable for nutraceutical AND METHODS purposes. electrochemicalStudy on water-solubility detector and online of reduction CoQ10 system captured [7]. in TCNa micelles after complete degradation of γ-CD by α-amylase CoQ10 was obtained from Mitsubishi Gas Chemical Company, Inc. (Tokyo, Japan). CoQ10-γCD complex ®containing 20 % (wt/ wt) CoQ10 was supplied from Wacker Chemie AG (München, 500 mg of CoQ10-γCD complex and 90 mg of α-CD were Germany) as product named CAVAMAX W8 CoQ10. CoQ10- added to 15 mL of MES buffer with 165 mg of TCNa in vials. α-CD J Drug Des Res 4(3): 1043 (2017) 10/15 Uekaji et al. (2017) Email:

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2 is known as a non-degradable dextrin by α-amylase and is hence were preincubated for 18 hours at 37°C in a 5% CO environment. used as a standard substance to evaluate the degradation of γ-CD. Then the tissues were placed into a well plate with 1 mL assay After shaking at room temperature for a 1 hour, the resultant medium dispensed. After each 0.2 mL of test materials had been 2 suspension was filtrated through a 0.2 μm filter. 12 mL of the added to the tissues, they were incubated for more than 6 hours filtrated solution was pre-incubated at 37°C for a few minutes. at 37°C in a 5% CO environment. The tissues were rinsed five Then 200 μL of pancreatic α-amylase reagent was diluted five times with 1 mL of 0.1 M phosphate-buffered saline (PBS). The times beforehand with MES buffer, and was added to the solution content of absorbed CoQ10 in the tissues was extracted with 5 at 37°C in a water bath. At 0 and 60 minutes after the addition of mL of chloroform : methanol (1 : 1) by shaking for 30 minutes. diluted pancreatic α-amylase, each 0.5 mL of the suspension was Then the extracted solvent was evaporated using a centrifugal taken into 1 mL of dimethylformamide in micro tubes. They were concentrator. After drying, to the evaporated residue was added heated at 90°C for 5 minutes, and filtrated through a 0.2 μm filter. 0.7 mL ethanol including 0.1 mL of iron chloride in ethanol A Shimadzu HPLC system (LC-2010C, Shimadzu Corporation, solution (1 mg/mL) as an oxidizing reagent. This was filtered Kyoto, Japan) was used for the content measurement of α-CD and through a 0.2 μm PTFE filter, and its CoQ10 content in the tissue γ-CD. An X-bridge HPLC column (amide: 4.6 mm i.d. × 150 mm) culture was analyzed with a Shimadzu LCMS system (LCMS- was used. Column temperature was set at 30°C. The mobile phase 2020, Shimadzu Corporation, Kyoto, Japan). A Phenomenex of acetonitrile : distilled water = 65 : 35 was used at a flow rate of HPLC column (Luna 5μ C18(2) 100Å: 4.6 mm i.d. × 150 mm) was 0.9Effect mL/min. of adding CDs were TCNa detected to the using CoQ10-γCD a RI detector complex [14]. used. Column temperature was set at 35°C. The mobile phase was used with a mixture of acetonitrile and isopropanol (8 : 7) containing 0.5% formic acid and 0.1% trisodium citrate aqueous CoQ10 samples were prepared as previously described solution (1 mg/mL), with a flow rate of 0.2 mL/min. The mass [13,14]. A Shimadzu HPLC system (LC-2010C, Shimadzu spectrometer fitted with electro-spray ionization (ESI) source Corporation, Kyoto, Japan) was used for the measurement of was used for analysis. It was operated in the positive ion mode CoQ10 concentration in the aqueous solution. A Phenomenex with the following parameters: probe voltage +4.50 kV (+ESI), HPLC column (Luna 5μ C18(2) 100A: 4.6 mm i.d. × 150 mm) nebulizer gas flow 1.5 L/min, drying gas flow 15.0 L/min, block was used. Column temperature was set at 35°C. A mixture of heaterMeasurement 200°C, DL oftemperature RALA content 250°C. by HPLC methanol, ethanol, and distilled water (60: 40: 1) was used as the mobile phase with a flow rate of 0.8 mL/min. CoQ10 was detected usingEffect an of UV adding detector GZK at 2752 to nm.the CoQ10-γCD complex RALA contents in the RALA-CD complexes were analyzed by / HPLC using a chiral column (CHIRALPAK AD-RH Daicel; 4.6 mm 3 4 GZK2 CoQ10-γCD complex (60 mg, containing 12 mg of CoQ10) and I.D. x 150 mm) with a mobile phase consisting of 5 mM H PO (molar ratios against CoQ10 are 0, 0.5, 1, 2.5, 5, 10 and 20) acetonitrile (70:30, v/v) at a flow rate of 0.6 mL/min and at a 2 was added to the vials, followed by the addition of Milli-Q water temperature of 25°C. Lipoic acid was detected at 215 nm based (3 mL). Pure CoQ10 (12 mg) and GZK (molar ratios against on current literature [25] and the injection volume was 10 μl. Racemic DL-alpha lipoic acid (Fluka, Sigma-Aldrich, MO, USA) CoQ10 are 0, 0.5, 1, 2, 4 and 8) was added to the vials, followed by 2 the addition of Milli-Q water (3 mL). These resultant suspensions was used as a standard; the stock solution was prepared at 0.5 4 were sonicated for 30 minutes, and filtered through a 0.2 μm mg/mL in 25 mM KH PO buffer (pH3.5) / acetonitrile (50:50, PTFE filter to obtain a transparent solution containing CoQ10. v/v)Measurement and filtered (ADVANTECof plasma RALA DISMIC-25, content 0.2 μm). by LC-MS/MS The concentrations of CoQ10 for all samples were measured by HPLC. The same Shimadzu HPLC system (LC-2010C, Shimadzu Corporation, Kyoto, Japan) was used as in the above experiment Plasma concentrations of RALA were determined using an API Effect[14]. of adding GZK2 and AdCA to three CoQ10-natural 3200™ (AB SCIEX, Framingham, MA, USA) liquid chromatography CD complexes coupled with tandem mass spectrometry (LC-MS/MS) system interfaced with a Shimadzu Prominence HPLC system (Shimadzu, Kyoto,Morphological Japan) as previously characterization described [37].via scanning electron 2 The CoQ10-natural CD complex (10 mg) was weighed in a microscopy (SEM) analysis vial. GZK (molar ratios against CoQ10 are 0, 3, 5, 7, 10, 20, 30, and 40) and AdCA (0 or 10 mg) were added to the vials, followed by the addition of Milli-Q water (5 mL). The resultant suspensions For scanning electron microscopy analysis, the RALA-CD were sonicated for 30 minutes, and filtered through a 0.2 μm complexes were sprinkled onto conductive glue on a palladium PTFE filter to obtain a transparent solution containing CoQ10. SEM stub and sputter coated with gold for 3 min. Then, the RALA- The concentrations of CoQ10 for all samples were measured by CD complexes were measured at 15 kV with the SEM S-4500 HPLC. The same Shimadzu HPLC system (LC-2010C, Shimadzu (HITACHI, Tokyo, Japan), for morphology analysis [25]. Three Corporation, Kyoto, Japan) was used as in the above experiment different fields within each sample were randomly chosen and In[13,14]. vitro study on the epidermis absorption-enhancing 4 images of each field were taken at the magnifications 300, 500, effect of CoQ10 by adding GZK2 to its γ-CD complex 1000Particle and 5000size distributiongiving a total number of RALA-CD of 12 images complexes per sample.

CoQ10 samples containing 1000 μg/mL of CoQ10 were The particle size analysis of RALA-CD complexes using prepared as previously described [13,14]. The tissue cultures SEM data was conducted by software Scandium (OLYMPUS, J Drug Des Res 4(3): 1043 (2017) 11/15 Uekaji et al. (2017) Email:

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Figure 13

Molecular micelle formation as an innovative nanotechnology for solubility, bioavailability and epidermis permeability enhancements.

Figure 14

Three types of bioavailability enhancement: absorption (solid line) and sustention (dotted line) in blood.

Tokyo, Japan). The unit length was set on the SEM picture and by micelle formation or encapsulation using liposome or dietary calibration was done. Particles were automatically detected emulsifiers such as fatty acid base surfactants. The bioavailability using the contrast by the software. Then the detected particles of lipophilic bioactives, e.g.CoQ10, are enhanced by improving were painted and the area of the painted particles was calculated their dissolution rates using such nano-encapsulation byCONCLUSIONS the software. technologies [51]. On the other hand, this new nanotechnology (“nanometer 2 molecular captured micelle formation” by the combination of Natural lipophilic bioactives possessing human health benefits, TCNa (bile acid) or GZK with γ-CD complexes of bioactives) such as CoQ10 and RALA, often have unfavorable characteristics is a completely different approach from the preparation of 2 with respect to their use as nutraceuticals and cosmeceuticals. nanoparticles starting from a single molecular capsule, a They are usually unstable against oxygen, ultraviolet light, low nanometer host-guest complex. The addition of TCNa or GZK pH and heat. Their low water-solubility or instability leads to to the water suspensions of lipophilic bioactives does not help low bioavailability to the human body. Therefore, studies have solubilize them because of their sizable and visual particle investigated improvements in the stability, water-solubility and formation characteristics in water. However, bioactives can be bioavailability of lipophilic bioactives through complexation complexed with γ-CD for molecular separation, which leads to with CDs. As a breakthrough in a series of such studies, a new the formation of nanometer molecular captured micelles (Figure nanotechnology for nanomedicinal food and personal care 13). As a result, the bioavailability-enhancing effect of CoQ10 via applications, “nanometer molecular captured micelle formation”, GZK2 intake of a CoQ10-γCD complex was much higher. has been developed using the combination of TCNa (bile acid) or with a γ-CD complex for CoQ10 delivery. This innovative molecular captured micelle formation nanotechnology, introduced here, which is useful for the Previously known micro- or nano-encapsulation technologies enhancement of bioavailability and epidermis permeability, in the food and cosmetic fields are generally based on approaches is applicable not only for CoQ10 but also for other lipophilic that discover how to minimize particle size physically, followed J Drug Des Res 4(3): 1043 (2017) 12/15 Uekaji et al. (2017) Email:

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Table 5: PK change Physicochemical change via CD CompoundComparison name of the plasmaType concentrationCD and PK response after oral administrationReference of nutraceuticals.Species (fold) complexation C

max: 3.4 * AUC: 18.4 * Coenzyme Q10 max1/2 [7] human TC : no change T : prolonged Type 3 γ Solubility max: increased AUC: increased max1/2 [22] dog C T : – T : – max: 96.7 ** AUC: 39.1 ** Curcumin Type 3 γ 1/2max [52] human Solubility T C: shortened T C: prolonged # 1h: 2.3 ## 3hC [55] rat : 2.1 ## Solubility max: 1.4 mice γ-tocotrienol Type 3 γ Stability AUC: 1.4 max1/2 [56] (in the digestive tract) T C: shortened T : prolonged max: 2.4 AUC: 2.5 R max1/2 [57] human T C: no change T : no change Stability -α-lipoic acid Type 1 γ max: 2.0 (heat, low pH) AUC: 2.2 max1/2 [37] rat T C: no change T : no change max: 1.4 AUC: 1.6 Astaxanthin Type 3 γ 1/2 max [58] rat Solubility T : – T : prolonged 1/2 Abbreviations: Cmax, maximum plasma concentration; AUC, area under the plasma concentration-time curve; Tmax, time to the maximum plasma concentration; T , half-life. * data was calculated with baseline-adjusted PK. ** data was calculated for total curcuminoids. # suggested from plasma concentration time curves in reference paper. ## calculated using plasma concentrations measured at 1 h and 3 h after single oral administration. bioactives such as curcumin and tocotrienols. The complexation effects of natural α-CD, β-CD and γ-CD on water-solubility and bioavailability were evaluated for particular The absorption of RALA appears to differ to that of CoQ10 hydrophobic nutraceuticals such as CoQ10 [7,22], curcumin absorption. The plasma RALA profile after a single administration [52-54], tocotrienol [55,56], RALA [37,57] and astaxanthin of RALA-CD in healthy human subjects changed from that [58] (Table 5). Formation of complexes for these hydrophobic observed after a single administration of non-complexed RALA, nutraceuticals with natural CDs did not generally enhance which is different to the change observed with CoQ10. RALA in aqueous solubility. However, the bioavailability of most of the the absence of a host carrier is unstable, whereas RALA-CD is examined nutraceuticals is significantly enhanced when γ-CD stable under the acidic conditions found in the stomach and was •• 0–180min was used. easily absorbed in the intestine. The mean AUC of RALA in the subjects orally administered 6 g of RALA-CD (equivalent There are three types of approaches for bioavailability C 1/2 amount of 600 mg RALA) was 2.5 times higher than that of the enhancement of bioactives, which focus on achieving the 1/2 subjects administered 600 mg of RALA. On the other hand, we max of the bioactives and T of the Tmax of bioactives in observed no changes in Tmax and T in response to a single oral blood plasma (Figure 14). The first (Type 1) is the Cmax dose of RALA or RALA-CD. Our results suggest that complexation enhancer. RALA-CD falls into this category. A commercially of RALA with γ-CD significantly enhances RALA bioavailability available “Water Soluble CoQ10” formulation using a 1/2 in healthy human volunteers, making it a promising technology dietary fatty oil-based emulsifier is a typical example for max for delivering functional but unstable ingredients like RALA. •• enhancing C , but usually does not prolong T [59]. 1/2 Additionally, there were no drug-induced side effects observed. The second approach (Type 2) functions to prolong T . These results indicate that 6 g of RALA-CD is suitable for 1/2 For example, glucosidated bioactives such as ascorbic nutraceutical purposes. acid 2-glucoside give T prolongation of the plasma J Drug Des Res 4(3): 1043 (2017) 13/15 Uekaji et al. (2017) Email:

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ascorbic acid concentration because of the slow cleavage H, Shahidi F, editors. Bio-Nanotechnology: A revolution in food, š Š of the glucose bond by glycosidase in the intestine [60]. biomedical and health Sciences. U.K.: Wiley & Sons. 2013; 179-211. However, owing to the slow release of the bioactive into 15. Fir MM, Milivojević L. Pro ek M. midovnik A. Property studies of the bloodstream, Cmax becomes lower and no increase in coenzyme Q10–cyclodextrins complexes. Acta Chimica Slovenica. Á úñ •• the AUC is observed. 2009; 56: 885-891. 16. Cabrer PR, lvarez-Parrilla E, Al-Soufi W, Meijide F, N ez ER, Tato JV. In comparison with the above two approaches, the 1/2 Complexation of bile salts by natural cyclodextrins. Supramol Chem. inclusion complex formation of hydrophobic substances 2003; 15: 33-43. with γ-CD functions to both enhance Cmax and prolong T and therefore gives the highest AUC (Type 3). The CoQ10- 17. Koide M, Ishii R, Ukawa J, Tagaki W, Tamagaki S. 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Cite this article Uekaji Y, Ikuta N, Rimbach G, Matsugo S, Terao K (2017) Enhancement of Oral Bioavailability of Functional Ingredients by Complexation with Cyclodextrin. J Drug Des Res 4(3): 1043.

J Drug Des Res 4(3): 1043 (2017) 15/15