A Novel Amorphous Curcumin Preparation Improved Oral Absorption Efficiency in Healthy Volunteers: A Single-Dose, Double-Blind, Two-Way Crossover Study


 Background: Curcumin has diverse biological activities such as anti-cancer, antioxidant, and anti-inflammatory properties and is assumed to exhibit beneficial effects in the prevention and treatment of various diseases. Although curcumin is known to be safe in humans, its therapeutic efficacy is limited owing to its poor bioavailability. To overcome this problem, we prepared a novel curcumin preparation curcuRougeTM using the amorphous solid dispersion method. In this study, we aimed to investigate the oral absorption efficiency of curcuRougeTM and compare its efficiency with that of Theracurmin®, a highly absorptive curcumin preparation dispersed with colloidal submicron-particles exhibiting improved bioavailability, in rats and healthy volunteers. Methods: In the animal experiment, male Sprague–Dawley rats were orally administered curcuRougeTM or Theracurmin® (10 mg/kg of curcumin). The plasma curcumin levels were measured at 0.25, 0.5, 1, 2, 4, and 6 h after administration. In addition, we performed a single-dose, double-blind, two-way crossover study to compare plasma curcumin levels after the administration of curcuRougeTM or Theracurmin® in humans. Twelve healthy volunteers were administered curcuRougeTM or Theracurmin® containing 30 mg curcumin. The plasma curcumin concentrations at 0.5, 1, 2, 4, and 8 h after ingestion were determined. Results: The area under plasma concentration–time curve (AUC0-6 h) and maximum plasma concentration (Cmax) of curcuRougeTM in rats were 3.7- and 9.6-fold higher than those of Theracurmin®, respectively. Twelve healthy volunteers were orally administered 90 mg of curcuRougeTM or Theracurmin® in a randomized double-blind crossover study. In these volunteers, the AUC0-8 h and Cmax of curcuRougeTM were 3.4-fold and 5.4-fold higher than those of Theracurmin®, respectively. Conclusion: These findings indicate that curcuRougeTM shows better bioavailability than other highly absorptive curcumin preparations, such as Theracurmin®. Hence, curcuRougeTM is assumed to exhibit clinical efficacy for managing various diseases at a low dose.Trial registration: The trial was registered with the UMIN Clinical Trials Registry (January 8, 2020, UMIN000039083, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000044573).


Introduction
The morbidity and mortality of lifestyle-related diseases, including type 2 diabetes, cardiovascular disease, and cancer, has increased in developed countries. As these diseases are caused by abnormalities in multiple signaling pathways because of environmental, genetic, and aging factors, it is di cult to prevent the development of such diseases by blocking only one of these signaling pathways [1]. Recently, it has been revealed that food ingredients exhibit diverse functions that help prevent various diseases associated with biological regulatory system abnormalities. Foods with these actions are known as functional foods. Functional foods with multi-faceted actions are inexpensive and play an important role in increasing healthy life expectancy and improving quality of life [2].
Curcumin is a natural polyphenol extracted from the rhizome of Curcuma longa. It has been used as a traditional medicine to treat various diseases, in India and China. Recently, curcumin has received considerable attention owing to its multiple pharmacological activities, such as anti-in ammatory, antioxidant, anti-viral, anti-tumor, and cell protective effects [3][4][5], and it is expected to be used as a supplement to prevent lifestyle-related diseases [6]. Despite its bene cial effects and low toxicity in humans, the poor water solubility and extremely low bioavailability of curcumin limit its widespread application [7]. Lao et al. reported that the maximum plasma concentration (C max ) of curcumin was only 50.5 and 57.6 ng/mL after 10 g and 12 g curcumin intake, respectively, in humans [7]. Even if a high dose of curcumin is orally administered, only a small quantity of curcumin is detected in blood plasma.
Moreover, curcumin in plasma is rapidly metabolized and excreted in feces and urine [8].
Many studies have been conducted to overcome the limitations of existing curcumin delivery systems, such as particles, micelles, emulsions, and liposomes [9][10][11][12]. In our previous study, we prepared an effective curcumin preparation Theracurmin®, which is a submicron-particle colloidal dispersion [13]. Theracurmin® achieved a 27-fold higher area under the plasma concentration-time curve (AUC) at 0-6 h than native curcumin in humans. The C max of Theracurmin® was 10.7-and 5.6-fold higher than that of the commercial curcumin preparations BCM-95® and Meriva®, respectively [14].
In this study, we prepared a new amorphous solid dispersion of curcumin and named it curcuRouge™. curcuRouge™ showed better oral bioavailability than Theracurmin® in rats. Moreover, we determined the oral absorption e ciency of curcuRouge™ and compared it with that of Theracurmin® by performing a randomized, double-blind, two-way crossover clinical study in healthy volunteers.
Theracurmin® was purchased from a market.
Preparation of curcuRouge™ curcuRouge™ was prepared and supplied by Therabiopharma Inc. Brie y, curcumin was amorphized by heating, coarsely ground, and then mixed with a dispersant to produce a ne powder. X-ray diffraction (XRD) analysis was performed using a MiniFlex diffractometer (Rigaku Co., Japan). Microscopic images were taken using a Primovert inverted microscope (Zeiss, Deutschland).

Experimental design in rats
The animal experiment conformed to the Guide for the Care and Use of Laboratory Animals by the Institute of Laboratory Animals, University of Shizuoka (US#176279).
The experiment was performed according to a previous study (15). Brie y, 10 male Sprague-Dawley rats (8-week-old; 250-290 g) were randomly assigned to the Theracurmin® (N = 5) and curcuRouge™ (N = 5) groups. The curcumin preparations were orally administered at a dose of 10 mg/kg through direct stomach intubation using a sonde. Blood samples were collected from the tail of rats at 0, 0.25, 0.5, 1, 2, 4, and 6 h after administration; then, placed in dark-colored heparinized tubes to protect from light, centrifuged at 1,500 × g for 10 min, and stored at -20 °C until analysis.

Clinical study in humans
The clinical study was approved by the Clinical Research Ethics Committees of Shizuoka General Hospital (SGHIRB#2019071), University of Shizuoka (IRB#1-25), and Kyoto Medical Center (IRB#19-056). The study was conducted in accordance with the ethical principles based on the Helsinki Declaration, and it was registered with the UMIN Clinical Trials Registry (UMIN000039083). This singledose, double-blind, two-way crossover study was performed from 8 January 2020 to 2 February 2020 at Shizuoka General Hospital and the University of Shizuoka in Japan. All volunteers provided written informed consent prior to participation in compliance with the Declaration of Helsinki. Twelve healthy volunteers were enrolled in the study. The screening procedures included medical history examination, physical examination, hematologic pro ling, and blood chemistry analysis. The volunteers were not taking any medications or any dietary or herbal supplements. Women who were pregnant or breastfeeding were excluded from the study.
The clinical study was performed according to a previous study (16). The participants were divided into two groups. The components of Theracurmin® and curcuRouge™ are shown in Table 1. Both the preparations were labeled; however, the volunteers and researchers were blinded to the preparations. The volunteers did not consume curcumin-containing food for more than one week before this study. The day before the study, they nished dinner by 9 PM, and fasted overnight (water intake was not restricted). The next morning, the volunteers consumed either of the curcumin preparations (90 mg) with a sip of mineral water. Blood specimens were collected immediately before and at 0.5, 1, 2, 4, and 8 h after taking the preparations. After blood sampling at 4 h, the volunteers were fed a rice ball containing pickled plum for lunch. All blood specimens were collected in 5 mL blood-collecting vessels containing heparin, immediately centrifuged at 1,500 × g for 10 min, and stored at -20 °C until analysis. After a washout period of 1-2 weeks, the alternate curcumin preparation was administered using the same protocol. After the measurement of plasma curcumin concentration, these data were xed, and the labels were opened.

Power analysis
Power analysis was performed using G*Power 3.1.9.2 software [17]. A previous crossover study examined the effects of Theracurmin® in nine healthy volunteers (dropout rate: 0%) [14,16]. Based on the results of that study, a power analysis was performed with an effective dose of 0.5, a power of 0.7, and a signi cance level of 0.05. Assuming a dropout rate of 5%, the sample size for the current study was determined as 12 volunteers.
Sample preparation and measurement of plasma curcumin levels The measurement of plasma curcumin levels was performed as reported previously [13]. Brie y, each plasma sample was reacted with 1,000 U β-glucuronidase at 37 °C for 1 h to hydrolyze the curcumin conjugates. After extraction with chloroform, the dried extracts were reconstituted in 50% acetonitrile and injected into an HPLC-MS/MS system comprising the Elute UHPLC system (Bruker Japan, Japan) and micrOTOF compact (Bruker Japan, Japan) with (+) electrospray ionization.

Pharmacokinetics
The AUC was calculated using the trapezoidal method [13]. The C max and time to reach maximum concentration (T max ) were obtained directly from the measured data. The relative values among native curcumin, Theracurmin®, and curcuRouge™ were calculated using individual data, and the mean values were then represented.

Statistical analysis
Data are expressed as the mean ± standard deviation (S.D.). For evaluation of curcumin absorption, nonparametric Kruskal-Wallis test followed by the Steel-Dwass test was performed. p < 0.05 was considered statistically signi cant.

XRD analysis and microscope images of curcuRouge™
The physical formulation of curcuRouge™ was characterized using the XRD system. As can be seen in Fig. 1, the XRD pattern of native curcumin showed several intense peaks, which indicated crystal forms of curcumin. In contrast, the XRD pattern of curcuRouge™ showed a typical halo form without characteristic peaks. The photographic images of native curcumin and curcuRouge™ are shown in Fig. 2. Native curcumin is yellow in color, whereas curcuRouge™ has a light red shade ( Fig. 2A and 2B). Small particles of curcumin (< 10 µm) were observed in curcuRouge™ (Fig. 2C). These data indicated that curcuRouge™ is an amorphous formulation.

Volunteer characteristics and disposition
Twelve healthy volunteers (eight men and four women) were enrolled in this study; their mean age was 22.8 ± 2.1 years. The mean body mass index was 20.9 ± 2.0 kg/mm 2 . The hemodynamic and blood analysis parameters are presented in Table 3. No volunteer discontinued the study, and no adverse effects were observed during this study.

Discussion
In this study, we developed curcuRouge™, an amorphous curcumin preparation, to improve the oral absorption of curcumin. Oral absorption experiments in rats showed that the C max and AUC 0− 6 h of curcuRouge™ were 9.6-and 3.7-fold higher than those of Theracurmin®, respectively. The crossover study comparing the oral absorption of Theracurmin® and curcuRouge™ in healthy volunteers revealed that the C max and AUC 0− 8 h of curcuRouge™ were 5.4-and 3.4-fold higher than those of Theracurmin®, respectively. These results demonstrated that the novel curcumin preparation curcuRouge™ exhibited more than 3-fold higher oral absorption and bioavailability than Theracurmin®.
Curcumin has been expected to be applied for the prevention of various diseases [18,19]. Currently, some of the highly absorbed curcumin preparations are commercially available and being applied clinically to address the poor oral absorption of curcumin. Meriva® is a mixture of curcumin and phosphatidylcholine derived from soybean lecithin and contains 20% curcuminoids [20]. It has been shown to have 29 times higher absorption than curcumin. Longvida® is also a solid lipid curcumin particle-based formulation with soy lecithin containing puri ed phospholipids [21]. A comparative pharmacokinetic study in healthy volunteers demonstrated that the bioavailability of Longvida® is 100 times higher than that of curcuminoids (AUC 0 − t : 95.3 ng·h/mL/650 mg curcuminoids). BCM-95® is a mixture of curcumin and turmeric-derived essential oils, and its bioavailability has been shown to be 6.9 times higher than that of curcumin [22]. CurcuWin® is a water-soluble formulation comprising polyvinyl pyrrolidine as a hydrophilic carrier, cellulosic derivatives, and natural antioxidants [23]. The relative absorption of curcumin in CurcuWin® is 136 times higher than that of curcumin (AUC 0− 12 h : 307 ng·h/mL/376 mg curcuminoids). NovaSol® is a micelle formulation with Tween-80 as a nonionic surfactant [24]. Its bioavailability is 185 times higher than that of unformulated curcumin. These preparations have demonstrated superior oral absorption to native curcumin in humans; however, these studies used high doses of curcumin formulation (300-2000 mg). A high plasma level of curcumin may cause for potential adverse effects. Thus, a comparative study using a low dose should be performed to determine the effective and optimal blood levels of curcumin that do not pose any risks. Theracurmin®, which we developed previously, showed 27-fold higher oral absorption than native curcumin in humans at a dose of 30 mg curcumin equivalent, which is lower than the doses reported in other clinical studies [13]. A comparison among Theracurmin® and the existing preparations Meriva® and BCM-95® demonstrated that Theracurmin® had the highest oral absorption [14]. To develop a curcumin preparation with improved absorption e ciency at a low dose when compared with Theracurmin®, we prepared amorphous curcumin. Generally, the bioavailability and stability of poorly water-soluble drugs is improved in their amorphous forms [25]. curcuRouge™ particles were approximately < 10 µm in diameter, and XRD revealed that they are amorphous. The results of animal and human studies showed that the amorphous form of curcuRouge™ had > 3-fold higher oral absorption than Theracurmin®. The amorphous form of curcuRouge™ is considered to contribute to its improved bioavailability. Comparison of curcuRouge™ with other types of curcumin formulations was di cult because the clinical study conditions were different. However, our ndings suggest that curcuRouge™ possesses higher bioavailability than Theracurmin®.
Curcumin preparations with improved absorption characteristics can help exert the bene cial physiological effects of curcumin. To date, highly absorbed curcumin preparations have been demonstrated to have the following effects on various illnesses: improved QOL in patients with end-stage pancreatic cancer [26]; improved memory function and reduced amyloid-β/tau protein accumulation in patients with mild cognitive impairment [27]; improvement of moderate Crohn's disease [28]; alleviation of pain in patients with knee osteoarthritis [29][30][31]; and decreased levels of α1-antitrypsin-LDL, a form of oxidized LDL in blood, and possible reduction of cardiovascular risk in patients with moderate chronic obstructive pulmonary disease [32]. Many animal experiments have revealed the potential of curcumin as a therapeutic agent for hypertension and myocardial infarction-induced heart diseases [33,34], diabetes [35], obesity [36], and atherosclerosis [37]. curcuRouge™ developed in this study is considered to have a higher bioavailability than Theracurmin® and other absorption-improved preparations, such as BCM-95® and Meriva®. Our ndings suggest that curcuRouge™ as well as other highly absorbed preparations have the potential to demonstrate bene cial effects in patients with the abovementioned diseases even at low doses.
As curcuRouge™ can readily reach high blood levels that native curcumin cannot, adverse reactions that have previously remained unnoticed may emerge. To con rm the safety of curcuRouge™, we conducted a 28-day repeated dose safety study of 300 mg/kg curcumin in rats. The results indicated that repeated administration of curcuRouge™ at a high dose caused no abnormalities in general well-being and ndings from observing internal organs after autopsy (Additional le 1). No adverse reactions occurred in humans after a single administration of curcuRouge™ (90 mg curcumin) in this study. However, additional careful investigation of adverse reactions is necessary for its clinical application.

Conclusion
In this study, we demonstrated that the amorphous form of curcuRouge™ had superior oral absorption to Theracurmin® in rats and humans. Therefore, this novel curcumin preparation can effectively exert the physiological activities of curcumin at low doses. CurcuRouge™ is expected to be used as a highly functional food material for managing various diseases, including lifestyle diseases.

Funding
This work was supported by a grant from the Japan Science and Technology Agency (T. Morimoto; 17K08279, Hasegawa; 18K08121) and joint research funding from Therabiopharma Inc.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.