Enhancement of intestinal absorption of coenzyme Q10 using emulsions containing oleyl polyethylene acetic acids

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Enhancement of intestinal absorption of coenzyme Q10 using emulsions containing oleyl polyethylene acetic acids Yuki Sato , Sayaka Yokoyama , Yoshiaki Yamaki , Yuta Nishimura , Mami Miyashita , Shingo Maruyama , Yoh Takekuma , Mitsuru Sugawara PII: DOI: Reference:

S0928-0987(19)30417-8 https://doi.org/10.1016/j.ejps.2019.105144 PHASCI 105144

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European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

9 August 2019 8 October 2019 10 November 2019

Please cite this article as: Yuki Sato , Sayaka Yokoyama , Yoshiaki Yamaki , Yuta Nishimura , Mami Miyashita , Shingo Maruyama , Yoh Takekuma , Mitsuru Sugawara , Enhancement of intestinal absorption of coenzyme Q10 using emulsions containing oleyl polyethylene acetic acids, European Journal of Pharmaceutical Sciences (2019), doi: https://doi.org/10.1016/j.ejps.2019.105144

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European Journal of Pharmaceutical Sciences Research articles Enhancement of intestinal absorption of coenzyme Q10 using emulsions containing oleyl polyethylene acetic acids

Yuki Sato a, 1 †, Sayaka Yokoyama a, 1, Yoshiaki Yamaki a, 1, Yuta Nishimura a, Mami Miyashita a

, Shingo Maruyama b, Yoh Takekuma a, Mitsuru Sugawara a, *

a

Faculty of Pharmaceutical Sciences, Hokkaido University

Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo 060-0812, Japan b

MORESCO Corp.

5-5-3, Minatojimaminamimachi, Chuo-ku, Kobe 650-0047, Japan

1

†

*

Equally contributed to this work Present address: Kyoto University Hospital

To whom correspondence should be addressed

Mitsuru Sugawara, Ph. D., Laboratory of Pharmacokinetics, Faculty of Pharmaceutical Sciences, Hokkaido University

1

Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo 060-0812, Japan Tel/Fax: +81-11-706-3923/ +81-11-706-4984 E-mail: [email protected]

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Abstract Emulsions have often been prepared to improve absorption of lipophilic compounds that have poor solubility. Coenzyme Q10 (CoQ10) is a lipophilic compound that has been used as an anti-aging supplement. We focused on oleyl polyethyleneoxy acetic acid, an oxa acid derivative, to prepare emulsions of CoQ10 with the expectation of application to oral pharmaceutics. Oxa acids were purified and classified into four groups based on the average length of the ethylene oxide chain. The emulsion that were prepared using the four oxa acid groups were administered to rats and the plasma concentration profiles of CoQ10 were analyzed. The absorption of CoQ10 was improved in all emulsion groups compared with that in the powder group. The emulsion using oxa acid (n=9.0) greatly increased the plasma concentration of CoQ10. Absorption was also improved by using emulsions containing larger percentage of oxa acids (6%, 15% and 23%) to compared with the same oxa acid (n=9.0). The effects of oxa acids on cell viability were almost the same as those of conventional surfactants such as polyoxyethylene (20) sorbitan monooleate (Tween 80). The results showed that oxa acids are useful to prepare emulsions for oral administration and that the absorption of CoQ10 using oxa acids is significantly improved by using our formulations. Keywords: emulsion; oxa acids; absorption; coenzyme Q10; intestine.

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1. Introduction

Coenzyme Q (CoQ) has a benzoquinone ring linked to a polyisoprenyl chain of 9 or 10 units in mammalian species. CoQ10, also known as ubiquinone, is a ubiquitous component that is essential for various activities related to energy metabolism. CoQ10 also functions in its reduced form as an antioxidant that prevents lipid peroxidation of biological membranes and serum low-density lipoprotein (LDL) (Stocker et al., 1991; Nohl et al., 1998). The use of CoQ10 as a therapeutic cardiovascular agent was based on its fundamental roles in mitochondrial function and cellular bioenergetics (Overvad et al., 1999; Fotino et al., 2013). Many studies have also indicated a beneficial antioxidant effect of CoQ10 as supplementation, and CoQ10 has received much attention due to its antioxidant activities (Lee et al., 2012; Östman et al., 2012; Sohet and Delzenne, 2012). In addition to these researches, benefits of intake of CoQ10 in some systematic reviews have recently been recognized (Bakhshayeshkaram et al., 2018; Mehrabani et al., 2019). On the other hand, Saboori et al. reported that the CoQ10 supplementation showed no beneficial effects (Saboori et al., 2019). Attention must be paid when we read some articles on clinical trials because of the difference of each population of these studies. CoQ10 is present in some foods such as sardines, mackerels, green leafy vegetables such as spinach, soybeans, peanuts and beef liver. We usually intake 3-5 mg of CoQ10 per a

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day from these foods (Weber et al., 1997). However, absorption of CoQ10 from the gastrointestinal tract is poor despite its high degree of permeability. Song et al. reported that coenzyme Q10 is classified into BCS (biopharmaceutics classification system) 2, a category of low solubility-high permeability drugs (Song et al., 2016; Amidon et al., 1995; Yu et al., 2002), or BCS 4, a category of low solubility–low permeability drugs (Ochiai et al., 2007). We previously reported that the bioavailability of CoQ10 was very low, less than 10% in rats (Ochiai et al., 2007). We also reported that its low absorption was improved by using an emulsion formulation containing the surfactant Tween 20 or Tween 80 (Sato et al., 2013) and that the low absorption of other lipophilic compounds such as lutein was improved by using emulsion formulations (Sato et al., 2011). We have studied the components of emulsion formulations and how the low absorption is improved. We focused on new materials, oleyl polyethyleneoxy acetic acid, called oxa acids. Oxa acids are generic terms that include lipophilic [(CH2CH2O)n] and hydrophilic [COOH] structures. They are uniquely structured high-tech compounds with several advantages that make them effective in various applications in pharmaceuticals, nanotechnology, cosmetics and chemistry (Fedeli et al., 2015, Mejia-Ariza and Huskens, 2016). However, the application of oxa acids to pharmaceutics of emulsion formulations as a surfactant has not been studied. There are various surfactants to prepare emulsions. We hypothesized that it is easier to develop a strategy to improve the absorption by emulsification when structures of surfactants are standardized.

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In this study, we investigated emulsification using oleyl polyethyleneoxy acetic acid as an oxa acid and assessed its properties compared with the properties of other surfactants. This is the first showing that oxa acids are useful for preparation of emulsions to improve the absorption of lipophilic compounds such as CoQ10.

2. Materials and Methods 2.1. Chemicals and reagents

CoQ10 powder (MW 863.34, PubChem CID: 5281915) and other reagents were purchased from Wako Pure Chemical Corp. (Osaka, Japan). Oleyl polyethyleneoxy (n=1.9/4.9/9.0/10.5) acetic acids were obtained from MORESCO (Kobe, Japan). The average molecular weights of the oleyl polyethyleneoxy (n=1.9/4.9/9.0/10.5) acetic acids were 441, 573, 752 and 819, respectively (Fig. 1). All of the reagents except for oleyl polyethyleneoxy acetic acids were of the highest grade available and used without further purification.

2.2. Animals

Male Wistar rats, aged 5 or 6 weeks (160-180 g in weight), were obtained from Jla (Tokyo, Japan). All of the rats were housed in plastic cages. The housing conditions were the

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same as those described previously (Sato et al., 2018). The experimental protocols were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the “Guide for the Care and Use of Laboratory Animals” (Approval number 18-0044).

2.3. Cell culture

Caco-2 cells were obtained from RIKEN (Ibaraki, Japan) The medium used for growth of Caco-2 cells was Dulbecco‟s modified Eagle‟s medium (Sigma, St. Louis, MO, U.S.A) with 10% fetal bovine serum (Corning, NY, U.S.A), 1% non-essential amino acids (Gibco-Invitrogen, Grand Island, NY, USA), 4 mM glutamine (Invitrogen, Grand Island, NY, USA) and 100 IU/mL penicillin-100 μg/mL streptomycin (Sigma). Caco-2 cells were maintained as described previously (Takekawa et al., 2016).

2.4. Preparation of suspension and emulsion formulations

METOLOSE® (Shin-Etsu Chemical Co. Ltd., Tokyo, Japan) was used to prepare a suspension of CoQ10 as described previously (Sato et al., 2013). For emulsions, we prepared emulsions formulations with four different compositions. For the first emulsions, six milligrams of CoQ10 was dissolved in 120 mg (130 μL) of

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soybean oil at 37ºC. Thirty-six milligrams of each oleyl polyethyleneoxy (n=1.9/4.9/9.0) acetic acid and 444 μL of distilled water were added to the mixture and the mixture was stirred at 70-80ºC until a uniform consistency was obtained. For cooling, the mixture was transferred to another vial container and stirred with cooling until limpidity was obtained. The final compositions of soybean oil and each oleyl polyethyleneoxy acetic acid containing 2.5% of CoQ10 in the first emulsion were 20% and 6.0%, respectively. This emulsion was defined as „emulsion 6% oxa acids (n=1.9/4.9/9.0)‟. For the second emulsion, sixty milligrams of CoQ10 was dissolved in 650 mg (652 μL) of soybean oil at 37ºC. Nine-hundred milligrams of oleyl polyethyleneoxy (n=9.0) acetic acid and 4.5 mL of distilled water were added to the mixture and the mixture was stirred at 70-80ºC until a uniform consistency was obtained. For cooling, the mixture was transferred to another vial container and stirred with cooling until limpidity was obtained. The final compositions of soybean oil and each oleyl polyethyleneoxy acetic acid containing 2.5% of CoQ10 in the second emulsion were 10% and 15%, respectively. This emulsion was defined as „emulsion 15% oxa acids (n=9.0)‟. For third and fourth emulsions, one-hundred fifty milligrams of CoQ10 was dissolved in 150 or 300 mg (163 or 326 μL) of soybean oil at 37ºC. Then 1.38 grams of oleyl polyethyleneoxy (n=9.0/10.5) acetic acid and 4.32 or 4.17 mL of distilled water were added to the mixture and the mixture was stirred at 70-80ºC until a uniform consistency was obtained. For cooling, the mixture was transferred to another vial container and stirred with cooling until limpidity was

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obtained. The final compositions of soybean oil and each oleyl polyethyleneoxy acetic acid containing 2.5% of CoQ10 in the third and fourth emulsions were 2.5% or 5.0% and 23%, respectively. These emulsions were defined as „emulsion 2.5/5% oil and 23% of oxa acids (n=9.0/10.5)‟.

2.5. Assay for prediction of biorelevant dissolution in the intestine

The method for the assay was determined according to a previous study with some modifications [Shono et al., 2009]. FaSSGF (fasted state simulated gastric fluid) and FaSSIF (fasted state simulated intestinal fluid) were used for measurement of particle sizes in the body. The emulsions prepared without CoQ10 were diluted 50 times water (control) or FaSSGF or FaSSIF and were incubated at 37ºC for 3 h with continuous stirring.

2.6. Measurement of particle sizes in emulsions

For confirmation of the properties of our emulsions prepared by the methods described in 2.4. and tested by the methods described in 2.5., the average particle sizes (z-average diameters) were measured by using a quasi-elastic light scattering method (Zeta Nano ZS; Malvern Instruments, Herrenberg, Germany) described previously (Takekawa et al.,

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2016).

2.7. Comparison of the effects of oxa acids on cell viability with other surfactants in an in vitro system

Caco-2 cells, human carcinoma cells, were used as a digestive model. The cells were seeded at a density of 1.0-2.0 × 105 cells/mL on a 96-well plastic plate (Corning) until reaching confluency as described previously [Takekawa et al., 2016]. After removal of the growth medium, 0.2 mL of each emulsion diluted with distilled water (control) or with FaSSGF or FaSSIF was added and incubated for 1 or 3 h at 37ºC. Each cell monolayer was mixed gently during the incubation. After incubation, seventy microliters of the culture fluid was obtained and the average particle sizes were measured as described in 2.6..

2.8. Oral administration, collection of plasma samples and analytical procedures

The rats were fasted for 14-16 h before the experiments. CoQ10 was orally administered in powder (in 0.5% methylcellulose) or as emulsions. The dose of CoQ10 was 25 mg/kg body weight (1 mL/kg body weight) in all groups. Blood samples were collected and plasma samples were obtained as described

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previously (Sato et al., 2011). All samples were kept at -20ºC until the assay. The concentration of CoQ10 was determined using an HPLC system equipped with an LC-20AD pump and an SPD-10AV UV-VIS detector (SHIMADZU, Kyoto, Japan). The conditions for extraction of CoQ10 and the HPLC conditions were the same as those described previously (Sato et al., 2013).

2.9. Data analysis and statistical analysis

To analyze the pharmacokinetics of CoQ10, area under the curve (AUC), maximum concentration (Cmax) and time at maximum concentration (Tmax) were calculated. The value of ABS0-48 [AUC0-48h-C0×48h], an index of absorption, was calculated deducting an endogenetic concentration of CoQ10. Student‟s t-test was used to determine the significance of differences between two group means. Statistical significance among means of more than two groups was determined by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test. Data are expressed as means with standard deviation (S.D.). Statistical significance was defined as P<0.05.

3. Results

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3.1. Properties of emulsions containing oxa acids

In the first part of this study, we prepared emulsions using oxa acids. In emulsion 6% oxa acids (n=1.9/4.9/9.0), the three emulsions had a white turbid appearance. The average particle sizes of emulsion 6% oxa acids (n=1.9/4.9/9.0) were 114.4 ± 9.2, 280.1 ± 19.3 and 208.2 ± 21.6 nm, respectively. For comparison of the properties of oxa acids (n=1.9/4.9/9.0), we made a ternary phase diagram comprising water/oil/oxa-acid (n=9.0) (Fig. 2). For examples, semi-transparent emulsions could be prepared when the percentage of oxa acid (n=9.0) was less than 10% even without oil (closed triangles) (Fig. 2B). On the other hand, gel or highly viscous gel were produced when the percentage of oxa acid (n=9.0) was more than 15% (inverted open triangles). We then focused on the three different appearances and average particle sizes of uniform emulsions. The appearances of emulsions 6%, 15% and 23% oxa acids (n=9.0) were white turbid, semi-transparent and transparent, respectively (Fig. 2B and supplemental figure 1). On the other hand, the average particle sizes of emulsions 6%, 15% and 23% oxa acids (n=9.0) were 208.2 ± 21.6, 52.5 ± 14.2 and 129.7 ± 1.7 nm, respectively.

3.2. Plasma concentration profile of CoQ10 formulation after oral administration

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Next, the plasma concentrations of CoQ10 were investigated after oral administration of each prepared formulation. As expected, the absorption of CoQ10 in the powder (control) group was poor (Fig. 3A). There was a slight increase in the concentration of CoQ10 at 24 h after administration in the powder group. We then investigated the concentrations of CoQ10 up to 48 h after oral administration in all groups. The plasma concentrations of CoQ10 in the emulsion 6% oxa acids (n=1.9/4.9/9.0) groups were slightly higher than the concentrations in the control group (Figs. 3B, C and D). The Cmax values in these emulsions 6% oxa acids (n=1.9/4.9/9.0) groups were 136.2 ± 98.0, 125.2 ± 41.2 and 177.5 ± 94.2 ng/mL, respectively (Table 1). In these three groups, the concentration of CoQ10 was highest in the emulsion 6% oxa acid (n=9.0) group. We next prepared another emulsion containing 15% of oxa acid (n=9.0) to obtain a higher concentration of CoQ10 after oral administration. The average particle size of the emulsion 15% oxa acid (n=9.0) was 52.5 ± 14.2 nm. On the other hand, the average particle sizes of emulsions 6% oxa acids (n=1.9/4.9/9.0) were 114.4 ± 9.2, 280.1 ± 19.3 and 208.2 ± 21.6 nm, respectively. It was shown that the plasma concentration of CoQ10 in the emulsion 15% oxa acid (n=9.0) was higher than that in the emulsion 6% oxa acid (n=9.0) (Fig. 3E). We also investigated the plasma concentration of CoQ10 after oral administration of an emulsion formulation using 23% oxa acid. There was a higher plasma concentration profile than those for 6% and 15% oxa acids (n=9.0). The largest value of ABS, 18,442 ± 3247.5, was also obtained in the emulsion using 23% oxa acid (n=9.0) (Fig. 3F).

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3.3. Particle size of each emulsion in the simulated digestive tract

The results showed that the use of these compounds for preparation of emulsions was effective for improving intestinal absorption of CoQ10, and the largest value of ABS was obtained with the emulsion using 23% oxa acid (n=9.0). We next focused on the particle sizes of emulsions in the simulated digestive tract. After incubation with distilled water or with FaSSGF or FaSSIF, the average particle size of each emulsion was determined. The results are shown in Table 2.

3.4. Effects of longer chain size of oxa acids on the absorption and particle size of a CoQ10 emulsion formulation We next investigated the effects of a longer chain of oxa acid on the absorption and particle size of a CoQ10 emulsion formulation since we developed another oxa acid (n=10.5) with longer ethylene oxide chains than those of oxa acid (n=9.0). We prepared two kinds of emulsion using oxa acid (n=10.5). One is the same composition as that of the emulsion using oxa acid (n=9.0) shown in Fig. 3F: 23% oxa acid (n=10.5), 2.5% oil and 72% distilled water (and 2.5% CoQ10) (emulsion 2.5% oil and 23% oxa acid (n=10.5)). The other composition is 23% oxa acid (n=10.5), 5.0% oil and 69.5% distilled water (and 2.5% CoQ10) (emulsion

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5.0% oil and 23% oxa acid (n=10.5)). Almost the same plasma concentration of both emulsions using 23% oxa acid (n=10.5) was obtained as the concentration of CoQ10 in emulsion 23% oxa acid (n=9.0) (Fig. 4). Table 1 shows the pharmacokinetic parameters of the emulsion containing 2.5% oil and 23% oxa acid (n=10.5) for comparison with those of the emulsion containing 2.5% oil and 23% oxa acid (n=9.0). The values of Cmax and Tmax were almost the same in the emulsions 23% oxa acids (n=9.0 and 10.5). On the other hand, the value of ABS was largest for the emulsion 2.5% oil and 23% oxa acid (n=9.0). The particle sizes of these emulsions in the simulated digestive tract were also investigated. The average particle sizes of administered emulsions using 23% oxa acids and of emulsions incubated in water or in FaSSGF or FaSSIF were measured (Table 2). As shown in Table 2, the average particle sizes of emulsions after incubation with FaSSGF and FaSSIF did not change except for the emulsion 5.0% oil and 23% oxa acid (n=10.5).

3.5. Comparison of the effects of oxa acids with other surfactants We also investigated the effects of oxa acids on cell viability to compare the properties with that of other surfactants. After incubation with dilution solutions (control or FaSSGF or FaSSIF), the viability of Caco-2 cells was almost the same as that with conventional surfactants such as Tween 80 (supplemental figure 2).

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4. Discussion

We focused on oleyl polyethyleneoxy acetic acid, an oxa acid derivative, to prepare emulsions of CoQ10 with the expectation of application to oral pharmaceutics. This is the first report that absorption of CoQ10, a typical example of poorly water-soluble drug, was improved by emulsion using oxa acids. Generally, poorly water-soluble components with poor membrane permeability classified into BCS 4 show very low bioavailability [Yu et al., 2002]. Some pharmaceutical devices are often needed to improve the absorption of not only components classified into BCS 4 but also components classified into BCS 2. We prepared some O/W emulsions using oleyl polyethyleneoxy acetic acid as an oxa acid and assessed these properties. We also compared some properties of other surfactants in order to improve the absorption of CoQ10 as a compound belonging to BCS 4 or BCS 2. When administered orally, the absorption of W/O emulsions were better than O/W emulsions (Hu et al., 2014). The oil phase of O/W emulsions would act as a carrier of the lipophilic active compound in this study, CoQ10, and a dispersion phase in the continuous phase. Fig. 2 shows a ternary phase diagram of oxa acid (n=9.0), water and soybean oil in our emulsions. These emulsions showed mainly transparent, semi-transparent and white turbid appearance. Gel or highly viscous gel were produced when the percentage of oxa acid (n=9.0) was more than 15% in case of emulsions without oil and CoQ10. Emulsions without oil could

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be prepared when the percentage of oxa acid (n=9.0) was less than 10%. On the other hand, uniform emulsions would not be obtained when the composition was without oil (0%) and 23% of oxa acid and 2.5% of CoQ10. Even when the emulsion was obtained in the composition temporarily, CoQ10 precipitated soon (data not shown). So we consider that CoQ10 in emulsion is mainly dissolved in the oil and the percentage of oil and surfactant is important for the preparation of uniform and stable emulsions. The average particle sizes of our emulsions were about 100-250 nm. There was no significant relationship between particle sizes of emulsions and ethylene oxide chain lengths of oxa acids though we prepared various emulsions. On the other hand, emulsions using oxa acids could be prepared for oral administration in the same way of using other conventional surfactants. We then administered these uniform emulsions to rats for the absorption study. It was shown that the absorption of CoQ10 was significantly improved by the emulsion formulation using oxa acids compared with the suspension up to 48 h (Fig. 3). It was also confirmed that little absorption of CoQ10 was observed in suspension (powder) group. The maximum concentration (Cmax) was significantly increased in each emulsion group compared with powder group. On the other hand, there were no significant differences of the values of each Tmax in emulsion groups except for the powder group (Table 1). Comparing the effects pf oxa acids with different ethylene oxide chain lengths (n=1.9, 4.9, 9.0), it was shown that the value of AUC was larger when oxa acid with a longer ethylene oxide chain was used

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to prepare the emulsion despite the fact that there was no significant alteration of other parameters, Cmax and Tmax (Figs. 3B-D and Table 1). Absorption was also improved by using emulsions containing larger percentages of oxa acids (6%, 15% and 23%) compared with the oxa acids (n=9.0) (Figs. 3D-F and Table 1). The largest value of AUC was obtained when the emulsion using 23% oxa acid (n=9.0) was used (Table 1). This value was larger than that of other research groups (Onoue, et al., 2012; Qin, et al., 2017). In addition, emulsions using oxa acid improved the absorption of CoQ10 larger than our conventional emulsion using Tween 20 or Tween 80 (Sato et al., 2013). We also investigated the relationship between length of the ethylene oxide chain in oxa acid and absorption of CoQ10. The absorption of CoQ10 was improved by using an emulsion with oxa acid (n=10.5) (Fig. 4). However, the emulsion using oxa acid with a longer chain did not show the larger absorption (Figs. 3F, 4 and Table 1). In this study, we considered that oxa acid (n=9.0) would be the best appropriate emulsifier for preparation of emulsion. Emulsions used in experiments for which results are shown in Figs. 3F and Fig. 4 were all transparent. As in a ternary phase diagram, it was suggested that a transparent emulsion, i.e., an emulsion with an average particle size <100 nm, improves the absorption of CoQ10. Considering the better absorption of CoQ10 emulsion using oxa acid with ternary phase diagram, a better absorption would be obtained when transparent emulsions, 2.5-4% of oil and more 20% of oxa acid (n=9.0), were used. We also consider that smaller particles even in an average particle size <100 nm contribute to be easier to permeate

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the membrane and to improve the absorption. We then considered the adverse effect of oxa acids. Tween 80 and Tween 20 are well-known surfactants, emulsifiers in polysorbates (Yu and Huang, 2012; Yeom et al., 2017). Cremophor EL is also used for emulsification. Each value of LD50 (lethal dose 50%) of Tween 80 and Tween 20 was 38,000 and 39,000 mg/kg (rats, single oral administration) (World Health Organization, 1974). The value of LD50 of Cremophor EL would be 2,613 mg/kg (mice, single oral administration) (Park et al., 2009). The value of LD50 of oleyl polyethylene acetic acids are estimated to >2,000 mg/kg (rats, single oral administration) by European Chemicals Agency (2006). Our maximum dose of oxa acids was about 230 mg/kg in this study. There were no dead rats during the experiment due to a potential acute toxicity of oxa acid. We confirmed the effects of oxa acids on cell viability. After incubation with dilution solutions (control or FaSSGF or FaSSIF), the viability of Caco-2 cells was almost the same as that with Tween 80 or Tween 20 (supplemental figure 2) at the concentration of 1% of oxa acid (n=9.0). At least, the cell viability after incubation with oxa acid (n=9.0) was better than that with oxa acid (n=10.5). These results suggested that oxa acid (n=9.0) is available for ingredient of oral formulation similar as Tween 80 though more detail studies about the safety are needed. In the present study, a clear relationship between particle size of the emulsion and the value of AUC was not found. An emulsion with smaller size in the preparation would not

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always improve the absorption in spite of the tendency. Oxa acids, in particular oxa acid (n=9.0), are useful for the preparation of emulsions to improve intestinal absorption. Further investigations to clarify these relationships are in progress.

Acknowledgments

This work was supported in part by Grants-in-Aid from Regional R&D Proposal-Based Program from Northern Advancement Center for Science & Technology of Hokkaido and Grants-in-Aid for Scientific Research (C) (Grant number 16K00842) from the Japan Society for the Promotion of Science (JSPS).

Conflict of interest

The authors report no conflicts of interest in this work.

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Figure legends Fig. 1. Chemical structures of oleyl polyethyleneoxy (n=1.9/4.9/9.0/10.5) acetic acid.

Fig. 2. Ternary phase diagram of oxa acid (n=9.0), a surfactant, an oil, water. Emulsions were prepared with oxa acid (9.0), soybean oil and water. Over all view (A) and enlarged view were shown. Closed circle and triangle show transparent and semi-transparent emulsion, respectively. Open lozenge, square and inverted-triangle show white turbid emulsion, phase separation and highly viscous liquid or gel, respectively.

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Fig. 3. Plasma concentration profile of CoQ10 after oral administration of CoQ10 suspension (A), emulsions 6% oxa acids (n=1.9/4.9/9.0) (B-D), emulsion 15% oxa acids (n=9.0) (E) and emulsion 23% of oxa acids (n=9.0) (F) Each point represents the mean with S.D. of 5-6 measurements. All rats were fasted for 14-16 h before the experiments. Powder in 0.5% methylcellulose as a suspension (A: white square) or some emulsions (B-F) of CoQ10 (25 mg/kg weight) was administered and blood samples were obtained after administration up to 48 h. Figs. 3B-D (B: white, C: light gray and D: dark gray rhombus) show plasma concentration profiles of administration of emulsions 6% oxa acids (n=1.9/4.9/9.0), respectively. Fig. 3E (dark gray triangles) and 3F (dark gray circles) show plasma concentration profiles of administration of emulsion 15% oxa acid (n=9.0) and emulsion 23% oxa acid (n=9.0) and 2.5% oil, respectively.

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Fig. 4. Difference of plasma concentration profiles of CoQ10 after oral administration of emulsion containing oxa acids (n=10.5) in oil percentage of emulsion. Each point represents the mean with S.D. of 5-6 measurements. All rats were fasted for 14-16 h before the experiments. Emulsions of CoQ10 (25 mg/kg weight) containing 23% of oxa acid (n=10.5) and 2.5% (A: black circles) or 5% (B: black dot circles) soybean oil were administered and blood samples were obtained after administration up to 48 h.

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Graphical abstract

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Table 1 Pharmacokinetic parameters of CoQ10 after oral administration of each formulation Cmax (μg/mL)

Powder (suspension)

Emulsion 6% oxa acid (n=1.9) Emulsion 6% oxa acid (n=4.9) Emulsion 6% oxa acid (n=9.0) Emulsion 15% oxa acid (n=9.0) Emulsion 2.5% oil and 23% oxa acid (n=9.0) Emulsion 2.5% oil and 23% oxa acid (n=10.5) Emulsion 5.0% oil and 23% oxa acid (n=10.5)

Tmax (h)

ABS0-48 h (μg×h/mL)

31.02 ± 12.9

24 ± 0

632.2 ±301.0

136.2 ± 98.0

4.6 ± 1.1

4,897 ±1,594

125.2 ± 41.2

3.3 ± 1.8

4,308 ± 1,927

177.5 ± 94.2

3.2 ± 1.5

6,465 ± 1,107

318.7 ± 125

3.5 ± 1.8

7,914 ± 1,345

818.1 ± 253

6.8 ± 2.2

18,442 ± 3,247.5

923.8 ± 340

4.6 ± 2.4

12,140 ± 3,248

519.8 ± 208

5.4 ± 3.7

9,273 ± 3,037

Each parameter represents the mean ± S.D. of 5-6 measurements. The value of ABS was calculated by the trapezoidal method from the data Figs. 3 and 4.

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Table 2 Average particle sizes (nm) of each formulation of CoQ10 after incubation with FaSSGF and FaSSIF

Emulsion 2.5% oil and 23% oxa acid (n=9.0) Emulsion 2.5% oil and 23% oxa acid (n=10.5) Emulsion 5.0% oil and 23% oxa acid (n=10.5)

3 h after incubation

3 h after incubation

3 h after incubation

with water (control)

with FaSSGF

with FaSSIF

4.9 ± 0.8

37.0 ± 6.9

102.7 ± 13.8

7.2 ± 0.8

15.4 ± 1.2

75.0 ± 3.2

9.9 ± 1.2

18.3 ± 0.2

38.6 ± 1.7

Each parameter represents the mean ± S.D. of 3-4 measurements. Each emulsion was diluted 50 times water (control) or FaSSGF or FaSSIF and were incubated at 37ºC for 3 h with continuous stirring. The average particle sizes (z-average diameters) were measured by using a quasi-elastic light scattering method.

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