WO2019171107A1

WO2019171107A1 - Curcumin nanomicelles for oral administration

Info

Publication number: WO2019171107A1
Application number: PCT/IB2018/051370
Authority: WO
Inventors: Mahmoud Reza Jaafari
Original assignee: Exir Nano Sina Company
Priority date: 2018-03-04
Filing date: 2018-03-04
Publication date: 2019-09-12

Abstract

Disclosed herein is a method for producing curcuminoid nanomicelles with high stability and significantly higher oral absorption. The method may include forming a curcuminoid solution by mixing curcuminoid powder with a surfactant, forming a first mixture by mixing vitamin E and natural oils with the curcuminoid solution, forming an ascorbic acid solution by dissolving ascorbic acid in water, forming a second mixture by mixing the ascorbic acid solution with the first mixture, and homogenizing the second mixture.

Description

CURCUMIN NANOMICELLES FOR ORAL ADMINISTRATION

TECHNICAL FIELD

[0001] The present disclosure relates to nanomicellar formulations containing curcuminoids and particularly to nanomicelles containing curcumin and a method for producing the same.

BACKGROUND ART

[0002] Curcuminoids are dietary polyphenols extracted from the dried rhizomes of Curcuma longa L. (turmeric). Curcuminoids include curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC). Curcuminoids are among the most widely studied natural products owing to their promising biological and pharmacological activities. Notably, curcumin is considered as a generally recognized as safe (GRAS) compound by the United States Food and Drug Administration (FDA).

[0003] Modulatory effects of curcuminoids are known on numerous biomolecules that may serve as key elements in the pathways regulating inflammation, oxidative stress, immune responses and cellular proliferation and homeostasis. The antioxidant, anti-inflammatory, anti tumor, analgesic, anti-arthritic, immunoregulatory, and lipid-modifying activities of curcuminoids have been confirmed by in vivo and clinical studies. However, biological activity of curcuminoids in in vivo studies and clinical settings are hampered by the low oral bioavailability of the curcuminoids. The low systemic bioavailability following oral ingestion of curcuminoids is due to the low aqueous solubility, instability at physiological pH, as well as rapid metabolism and clearance. Therefore, there is a need in the art to improve the pharmacokinetic characteristics of curcuminoids to enhance their oral bioavailability and consequently pharmacological effects. SUMMARY OF THE DISCLOSURE

[0004] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings of exemplary embodiments.

[0005] According to one general aspect, the present disclosure is directed to a method for producing curcumin nanomicelles. The method may include forming a curcuminoid solution by mixing curcuminoid powder with a surfactant, forming a first mixture by mixing vitamin E and natural oils with the curcuminoid solution, forming an ascorbic acid solution by dissolving ascorbic acid in water, forming a second mixture by mixing the ascorbic acid solution with the first mixture, and homogenizing the second mixture.

[0006] According to one or more exemplary embodiments, forming a curcuminoid solution by mixing curcuminoid powder with a surfactant may include mixing curcuminoid powder with a polysorbate surfactant. According to an exemplary embodiment, the polysorbate surfactant may be polysorbate 80, polysorbate 20, polysorbate 40, or mixtures thereof.

[0007] According to one or more exemplary embodiments, forming a curcuminoid solution may include heating a polysorbate surfactant to a temperature between 50°C and 60°C; mixing curcuminoid powder with the heated polysorbate surfactant at a stirrer speed between 40 and 60 rpm for 15 to 30 minutes, heating the mixture of curcuminoid powder and the polysorbate surfactant to a temperature between 80°C and 90°C, and stirring the mixture of curcuminoid powder and the polysorbate surfactant at a stirrer speed between 75 and 100 rpm for 30 to 60 minutes. According to an exemplary embodiment, the curcuminoid powder may include curcumin, dimethyl curcumin (DMC), and bisdemethoxy curcumin (BDMC). [0008] According to one or more exemplary embodiments, forming the first mixture may include mixing vitamin E and the natural oil with the curcuminoid solution at a temperature between 50°C and 60°C with a stirrer speed of 40 to 60 rpm for 10 to 20 minutes. According to an exemplary embodiment, the natural oil may be selected from the group consisting of soya bean oil, sesame oil, olive oil, almond oil, and combinations thereof.

[0009] According to one or more exemplary embodiments, forming an ascorbic acid solution includes dissolving ascorbic acid in water at a temperature between 50°C and 60°C with a stirrer speed of 40 to 60 rpm for 15 to 30 minutes.

[0010] According to one or more exemplary embodiments, forming a second mixture may include mixing the ascorbic acid solution with the first mixture in a mixer with a stirrer speed of 40 to 60 rpm, at a temperature between 50°C and 60°C, for a period of 10 to 20 minutes, heating the obtained mixture of the ascorbic acid solution and the first mixture to a temperature between 80°C and 90°C, and stirring the heated mixture at a stirrer speed between 75 and 100 rpm for 30 to 60 minutes.

[0011] According to one or more exemplary embodiments, homogenizing the second mixture may include homogenizing the second mixture in a rotor stator homogenizer at 5000-7000 rpm at 80°C to 90°C for 15-30 minutes to obtain nanomicelles with an average size of 10 nm.

[0012] According to another general aspect, the present disclosure is directed to a nanomicelle formulation that may include a curcuminoid with a concentration between 1 and 10% by weight of the total nanomicelle formulation; a polysorbate surfactant; vitamin E with a concentration between 0.1 and 2% by weight of the total nanomicelle formulation; ascorbic acid with a concentration between 0.1 and 3% by weight of the total nanomicelle formulation; and a natural oil with a concentration between 2 and 10% by weight of the total nanomicelle formulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawing figures depict one or more implementations in accord with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements.

[0014] FIG. 1 illustrates a method for producing curcumin nanomicelles, consistent with one or more exemplary embodiments of the present disclosure;

[0015] FIG. 2 illustrates a transmission electron microscope (TEM) image of a prepared nanomicelle formulation containing curcuminoids, consistent with an exemplary embodiment of the present disclosure;

[0016] FIG. 3 is a curcumin release percentage versus time plot for nanomicellar curcuminoids, free curcuminoids, and two commercial formulations CP1 and CP2, according to an exemplary embodiment of the present disclosure;

[0017] FIG. 4A is a residual curcumin content of the prepared nanomicelle formulation during a long-term stability test at 30 °C ± 2 °C, with a relative humidity of 65 % ± 5%, consistent with an exemplary implementation of the present disclosure;

[0018] FIG. 4B is a residual curcumin content of the prepared nanomicelle formulation during an accelerated stability test at 40 °C ± 2 °C, with a relative humidity of 75% ± 5%, consistent with an exemplary implementation of the present disclosure;

[0019] FIG. 5A illustrates the release profile of curcuminoids nanomicelles in a simulated gastric fluid, consistent with an exemplary embodiment of the present disclosure;

[0020] FIG. 5B illustrates the release profile of curcuminoids nanomicelles in water, consistent with an exemplary embodiment of the present disclosure; and

[0021] FIG. 5C illustrates the release profile of curcuminoids nanomicelles in a simulated intestinal fluid, consistent with an exemplary embodiment of the present disclosure. [0022] FIG. 6A illustrates the concentration-time profile of curcumin after oral gavage of curcuminoids-loaded nanomicelles, CP1 and CP2, as well as free curcuminoids at an equal dose of 35 mg/mouse, according to an exemplary embodiment of the present disclosure.

[0023] FIG. 6B illustrates the concentration-time profile of DMC after oral gavage of curcuminoids-loaded nanomicelles, CP1 and CP2, as well as free curcuminoids at an equal dose of 35 mg/mouse, according to an exemplary embodiment of the present disclosure.

[0024] FIG. 6C illustrates the concentration-time profile of BDMC after oral gavage of curcuminoids-loaded nanomicelles, CP1 and CP2, as well as free curcuminoids at an equal dose of 35 mg/mouse, according to an exemplary embodiment of the present disclosure.

DESCRIPTION OF EMBODIMENTS

[0025] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well-known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0026] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0027] The disclosed formulations and methods are directed to improving the solubility and stability of curcuminoids by encapsulating the curcuminoids in nanomicellar structures along with vitamins and natural oils. Encapsulation of curcuminoids in nanomicellar structures improve their water-solubility and the presence of vitamins and natural oils helps improve the stability of the final nanomicellar formulation.

[0028] FIG. 1 illustrates a method 100 for producing curcuminoid nanomicelle formulation, consistent with one or more exemplary embodiments of the present disclosure. Method 100 may include step 101 of forming a curcuminoid solution by mixing curcuminoid powder with a surfactant; a step 102 of forming a first mixture by mixing vitamin E and natural oils with the curcuminoid solution; a step 103 of forming an ascorbic acid solution by dissolving ascorbic acid in water; a step 104 of forming a second mixture by mixing the ascorbic acid solution with the first mixture; and a step 105 of homogenizing the second mixture.

[0029] Referring to FIG. 1, according to one or more exemplary embodiments, step 101 of forming a curcuminoid solution by mixing curcuminoid powder with a surfactant may include mixing curcuminoid powder with a polysorbate surfactant in a stirred vessel. In an exemplary embodiment, the polysorbate surfactant may be heated up to a temperature between 50°C and 60°C. Curcumin powder may be mixed with the heated polysorbate surfactant in the stirred vessel with a stirrer speed between 40 and 60 rpm. Mixing the curcumin powder and the polysorbate surfactant with a stirrer speed of 40 to 60 rpm may be carried out for 15 to 30 minutes depending on the volume of production. After that, the mixture of curcumin and polysorbate surfactant may be further heated up to a temperature between 80°C and 90°C. The mixture may then be stirred at a rate between 75 to 100 rpm for 30 to 60 more minutes, depending on the volume of production, to obtain the curcuminoid solution. Then curcuminoid solution may be cooled down to a temperature between 50°C and 60°C.

[0030] According to an exemplary embodiment, step 101 of forming a curcuminoid solution by mixing curcuminoid powder with a surfactant may include mixing curcuminoid powder with a polysorbate surfactant selected from the group consisting of polysorbate 80, polysorbate 20, polysorbate 40, and mixtures thereof. According to an exemplary embodiment, the curcuminoid powder may contain curcumin, dimethyl curcumin (DMC), and bisdemethoxycurcumin (BDMC). The curcuminoid powder may have a purity of at least 95%.

[0031] Referring to FIG. 1, according to one or more exemplary embodiments, step 102 of forming a first mixture by mixing vitamin E and natural oils with the curcuminoid solution may include mixing vitamin E and a natural oil with the curcuminoid solution at a temperature between 50°C and 60°C with a stirrer speed of 40 to 60 rpm for about 10 to 20 minutes to completely dissolve the vitamin E and the natural oil into the curcuminoid solution. According to an exemplary embodiment, the natural oil may be soya bean oil, sesame oil, olive oil, almond oil, etc.

[0032] The presence of vitamin and natural oils is necessary for improving the stability of the final nanomicellar formulation containing curcuminoids. The presence of Vitamin E as a lipid soluble antioxidant, which resides in the hydrophobic core of nanomicelles, prevents oxidation of curcuminoids in the hydrophobic core of nanomicelles and also prevents oxidation of polysorbate surfactant and natural oil and improves stability of nanomicellar formulation. On the other hand, natural oils in the formulation improve the stability of nanomicelles against acidic conditions of gastric fluid. The presence of water soluble antioxidant ascorbic acid in the outside of nanomicelles neutralizes any oxidants presents in the outside of nanomicelles which may help the stability of formulation in general. Ascorbic acid also provides an acidic pH of around 3.5 that helps in the stability of curcuminoids which are more stable in the acidic pH.

[0033] Referring to FIG. 1, according to one or more exemplary embodiments, step 103 of forming an ascorbic acid solution by dissolving ascorbic acid in water may include heating up the water to a temperature between 50°C and 60°C and slowly adding ascorbic acid to the heated water in a mixer with a stirrer speed of 40 to 60 rpm. In an exemplary embodiment, for a complete dissolution of ascorbic acid in water, mixing may be carried out for 15 to 30 minutes.

[0034] Referring to FIG. 1, according to one or more exemplary embodiments, step 104 of forming a second mixture by mixing the ascorbic acid solution with the first mixture may include mixing the ascorbic acid solution with the first mixture in a mixer with a stirrer speed of 40 to 60 rpm, at a temperature between 50°C and 60°C, for a period of for example 10 to 20 minutes. After that, the obtained mixture may be further heated up to a temperature between 80°C and 90°C and the mixing process may continue at this temperature range for 20 to 30 more minutes.

[0035] Referring to FIG. 1, according to one or more exemplary embodiments, step 105 of homogenizing the second mixture may include homogenizing the second mixture in a rotor stator homogenizer at 5000-7000 rpm at 80°C to 90°C for 15-30 minutes to obtain nanomicelles with an average size of 10 nm. Then the homogenized second mixture may further be cooled down to room temperature while being stirred with a rate of 10 to 20 rpm.

[0036] According to one or more exemplary embodiments, the curcuminoid nanomicelle formulation that may be produced as described in detail in connection with the method 100 of FIG. 1 may include a curcuminoid, a polysorbate surfactant, ascorbic acid, vitamin E, a natural oil, and balancing amounts of distilled water. According to an exemplary embodiment, the curcuminoid nanomicelle formulation may include a curcuminoid with a concentration of about 1 to 10 wt. % based on the total weight of the curcuminoid nanomicelle formulation. According to an exemplary embodiment, concentration of the polysorbate surfactant may be between 15 and 85 wt. % based on the total weight of the curcuminoid nanomicelle formulation. The concentration of the polysorbate surfactant may be determined based on the concentration of the curcuminoids in the final curcuminoid nanomicelle formulation. For example, for producing a curcuminoid nanomicelle formulation containing a curcuminoid with a concentration of about 10 wt. %, about 80 to 85 wt. % of a polysorbate surfactant may be present in the formulation; while, for producing a curcuminoid nanomicelle formulation containing a curcuminoid with a concentration of about 1 wt. %, about 15 to 20 wt. % of a polysorbate surfactant may be present in the formulation.

[0037] According to one or more exemplary embodiment, Table 1 reports the constituents of the curcuminoid nanomicelle formulation that may be produced as described in detail in connection with the method 100 of FIG. 1 and their respective concentration ranges. The concentrations are based on the total weight of the curcuminoid nanomicelle formulation.

[0038] Table 1

[0039] Example 1: Particle size and morphology analysis [0040] For example, nanomicelle containing 7% curcuminoids was prepared as follows. First polysorbate 80 (82%) was transferred to a suitable container and warmed up to 55 °C. Then the curcuminoid powder was added to the surfactant slowly with low rate mixing (20 RPM). Then mixing at higher rate (80 RPM) was continued at higher temperature (80 °C) to completely dissolve the curcuminoid powder in polysorbate. Then the temperature of solution was reduced to 60 °C; and vitamin E (1%) and soya bean oil (5%) were added and mixed at 60 RPM for 20 minutes to completely dissolve these two ingredients in curcuminoids solution (first mixture). In a separate suitable container the water warmed up to 55 °C. Ascorbic acid (2%) was slowly added to water (3%) at 55 °C and the solution was mixed at 60 RPM to dissolve the ascorbic acid. Then add ascorbic acid solution was added to the first mixture and mixed at 60 RPM in 55 °C for 10 minutes. Then the temperature was increased to 80 °C and the mixing was continued for 20 minutes (second mixture). Finally, the second mixture was homogenized using a Rotor Stator homogenizer at 5,000 RPM in 80 °C for 15 minutes (Ultra- Turrax IKA T10 homogenizer, IKA Werke GmbH & Co. KG, Staufen, Germany).

[0041] The measurement of the particles size of nanomicelles was performed using dynamic light scattering (Zeta sizer, Malvern, UK). The light source was a diode pumped solid-state laser with a wavelength of 633 nm and a scattering angle of 90°. Size measurements were carried out in triplicate and the average of recordings was reported. The size of particles was also measured during long-term stability study and accelerated condition for 6 months. Samples were taken at 0, 3, 6, 9, 12, 15, 18, 21 and 24 months for long-term [25 °C ± 2 °C, 60% relative humidity ± 5%] and at 0, 1, 2, 4 and 6 months for accelerated condition [40 °C ± 2 °C, 75% relative humidity ± 5%] studies.

[0042] Table 2 reports the size of nanomicelles during the long-term stability study for 24 months and accelerated condition for 6 months. Referring to Table 2, the mean diameter of nanomicelles was around 10 nm, according to dynamic light scattering. The size of nanomicelles remained constant during long-term stability study for 24 months and 6 months accelerated condition.

[0043] FIG. 2 illustrates a transmission electron microscope (TEM) image of a prepared nanomicelle formulation containing curcumin, consistent with an exemplary embodiment of the present disclosure. Referring to FIG. 2, the spherical shape of nanomicelles is observed as was expected.

[0044] Table 2:

Long-term Stability Test Accelerated Stability Test

Time (month) z-average (nm) Time (month) z-average (nm)

0 9.9±0.l 0 9.l±0.l

3 9.2±0.l 1 8.8±0.l

6 9.6±0.l 2 9.7±0.l

9 9.7±0.l 4 l0.8±0.l

12 9.2±0.l 6 9.0±0.l

15 9.4±0.2

18 9.2±0.l

21 9.4±0.2

24 l0.2±0.l

[0045] Example 2: Drug loading and encapsulation efficiency

[0046] The amount of the curcuminoid that is encapsulated in the nanomicelles was determined by ultrafiltration method on a centrifugal filter unit with a molecular weight cut-off of 12 kDa (Merck Millipore; Massachusetts, USA). Briefly, nanomicelles were diluted with dextrose 5% in a 1 :9 ratios, added to a centrifugal filter unit and then centrifuged at 4000 g for 30 min. After centrifugation, the amount of free curcuminoids in the flow-through was assayed using HPLC. Unfiltered nanomicelles were heated, dissolved in DMSO and sonicated to extract the drug. The resulting solution was then diluted with methanol and drug concentration was measured using HPLC. Encapsulation efficiency was calculated as follows:

[0047] EE% =

Amount ofcurcuminoids in unfiltered nanomicelles- amount of curcuminoid in filtarated

X 100 Amount ofcurcuminoids in unfiltered nanomicelles

[0048] The amount of free curcuminoids in the flow-through, which was assayed using HPLC, was zero. Therefore, the encapsulation efficiency of curcuminoids in nanomicelles was calculated as 100%.

[0049] Example 3: In vitro dissolution study

The dissolution properties of nanomicellar curcuminoids, free curcuminoids, and two commercial formulations, namely CP1 and CP2 were investigated according to USP35 using apparatus 2 of the USP (paddle apparatus, PTWS3F, PharmaTest, Hainburg, Germany). For comparison, two commercial products of curcuminoids, one from Europe (CP1) and the other one from North America (CP2) were used. These commercial products are frequently used as a curcuminoids supplement in Europe and North America. Dissolution of curcuminoids were determined in 900 mL water containing 1% sodium lauryl sulfate (SLS) at 37.0 ± 0.5 ^°C and the release was investigated at 100 rpm. Samples (5 mL) were obtained at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min time points for analysis, and medium solution was immediately replaced after each sampling. Samples were passed through a 0.22 pm filter (Sartorius AG, Goettingen, Germany), diluted with mobile phase, and subjected to HPLC (Shimadzu, Japan). Dissolution study was carried out according to USP35

[0050] FIG. 3 is a curcumin release percentage versus time plot for nanomicellar curcuminoids, free curcuminoids, and two commercial formulations CP1 and CP2, according to an exemplary embodiment of the present disclosure. Referring to FIG. 3, 100% of the encapsulated curcuminoids in nanomicelles were dissolved in water containing 1% SLS after 20 min while free curcuminoids were sparingly soluble (approximately 7% release after 60 min). Only 22 and 25% of CP1 and CP2 were dissolved after 60 minutes, respectively.

[0051] Example 4: Stability Test

[0052] Stability of nanomicelles was assessed according to the ICH (The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) guidelines (2003) code Q1A (R2) (stability testing of new drug substances and products). Nanomicelles were stored in impenetrable tube containers and preserved from light. Samples were taken at 0, 3, 6, 12, 15, 18, 21 and 24 months for long-term [30 °C ± 2 °C, 65 % relative humidity ± 5%] and at 0, 1, 2, 4 and 6 months for accelerated condition [40 °C ± 2 °C, 75% relative humidity ± 5%] studies. Twenty pL of nanomicelles (diluted with the HPLC mobile phase to a final concentration of 30 to 50 pg/mL) was then injected to HPLC column in triplicate, to determine the content of curcuminoids in each sample.

[0053] FIG. 4A is a residual curcumin content of the prepared nanomicelle formulation during a long-term stability test, consistent with an exemplary implementation of the present disclosure. FIG. 4B is a residual curcumin content of the prepared nanomicelle formulation during an accelerated stability test, consistent with an exemplary implementation of the present disclosure. Referring to FIGS. 4A and 4B, no change was observed in content of curcuminoids in the prepared nanomicelle formulations in either long-term or accelerated stability studies.

[0054] Example 5: Release studies in simulated gastric fluid and simulated intestinal fluid

[0055] The release profile of curcuminoids nanomicelles was investigated in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) in order to determine the stability of nanomicelles in SGF and SIF. To prepare the SGF, hydrochloric acid solution (0.2 N, 39 ml) was added to sodium chloride solution (0.2 N, 250 ml), 600 ml deionized water (DW) was added and the pH was adjusted at 2.2, then the final volume was adjusted to 1000 ml with DW. SIF was prepared by dissolving KH2P04 (6.8 g) in 250 ml of DW. This solution was mixed with NaOH solution (0.2 N, 77 ml), 600 ml deionized water (DW) was added and the pH was adjusted at 6.8, then the final volume was adjusted to 1000 ml with DW.

[0056] Nanomicelles were diluted in the SGF and SIF in a ratio of 1 : 10 and incubated at 37±l.0°C, followed by sampling at 0, 1, 2, 4, 6, 12, 24, 48 and 72 h time points. To separate the released curcuminoids, the samples filtered through 0.22 m microbial filter to separate the precipitated curcuminoids. Since the solubility of curcuminoids in water is too low, they precipitate right after the disruption and release from nanomicelles. Then the determination of curcuminoids in purified nanomicelles was accomplished by HPLC. For HPLC determinations, 20 pL of nanomicelles (diluted with the HPLC mobile phase to a final concentration of 30 to

50 pg/mL) was injected to HPLC column in triplicate.

[0057] FIG. 5A illustrates the release profile of curcuminoids nanomicelles in a simulated gastric fluid, consistent with an exemplary embodiment of the present disclosure. FIG. 5B illustrates the release profile of curcuminoids nanomicelles in water, consistent with an exemplary embodiment of the present disclosure. FIG. 5C illustrates the release profile of curcuminoids nanomicelles in a simulated intestinal fluid, consistent with an exemplary embodiment of the present disclosure.

[0058] Referring to FIGS. 5A-5C, the level of curcumin, DMC and BDMC did not change significantly in the first 4 h (-100%) in SGF and SIF, reached to 98% of original concentration at time 0. However, there was a reduction in curcumin, DMC and BDMC concentrations following 72 h incubation in SGF, which reached to 82, 81 and 84%, respectively. The stability of nanomicelles was higher in SIF as compared to SGF. In SIF, the concentrations of curcumin, DMC and BDMC decreased from 98% at 6 h to 89, 87 and 87%, after 72 h, respectively.

[0059] Example 6: Pharmacokinetic study [0060] Female BALB/c mice (weight: 20±2 g) were obtained from the Pasteur Institute of Tehran, Iran. All animal studies were done in compliance with the Institutional Ethical Committee and Research Advisory Committee of Mashhad University of Medical Sciences (dated May 2, 2012; proposal code 910042). Animals were acclimatized to the laboratory conditions (temperature of 25 ± 2 °C and natural light/dark cycles) for at least 24 h before oral administration. Mice were starved for 12 h prior to the experiment. Mice were randomly assigned to either of the following groups: the nanomicellar curcuminoids, commercial product 1 (CP1), commercial product 2 (CP2) or free curcuminoids groups (n=3 in each group). Each mouse received a single dose (35 mg) of curcuminoids via oral gavage. Blood samples (0.5 mL) were taken at 30 min, 1, 2, 4 and 6 h after dosing via heart puncture and were transferred into heparinized tubes. Plasma was separated by centrifugations at 10000 g for 10 min and stored at -20 °C prior to analysis. For analysis, plasma samples were first deproteinated using methanol precipitation method. The clear methanolic layer was separated by centrifugation at 12000 g for 10 min and methanol was then evaporated under a gentle stream of nitrogen. The residue was dissolved in the mobile phase and 20 pL aliquots were injected to the HPLC system. Concentration of each curcuminoids versus time and the pharmacokinetic parameters analysis were performed with PKsolver (An add-in program for pharmacokinetic data).

[0061] FIG. 6A illustrates the concentration-time profile of curcumin after oral gavage of curcuminoids-loaded nanomicelles, CP1 and CP2, as well as free curcuminoids at an equal dose of 35 mg/mouse, according to an exemplary embodiment of the present disclosure. FIG. 6B illustrates the concentration -time profile of DMC after oral gavage of curcuminoids-loaded nanomicelles, CP1 and CP2, as well as free curcuminoids at an equal dose of 35 mg/mouse, according to an exemplary embodiment of the present disclosure. FIG. 6C illustrates the concentration-time profile of BDMC after oral gavage of curcuminoids-loaded nanomicelles, CP1 and CP2, as well as free curcuminoids at an equal dose of 35 mg/mouse, according to an exemplary embodiment of the present disclosure. Referring to FIGS. 6A-6C, there is a clear decrease in the level of curcuminoids over time in both nanomicelles and CP1 and CP2. Nevertheless, the rate of reduction in curcuminoids level in nanomicelles-treated mice is much lower which shows apparent difference between these formulations which are in clinical use. [0062] Table 3 reports pharmacokinetic parameters of curcumin, DMC and BDMC following oral administration of nanomicelles, commercial products 1 (CP1) and 2 (CP2) and also their free form in mice (n = 3).

[0063] Table 3

Curcumin

Nanomicelle Cpl Cp2 Free

Pharmacokinetic

Unit Value Value Value Value

Parameter

Lambda z 1/h 048 0.53 0.95 1.45 tl/2 h 1.44 1.30 0.73 0.48

Tmax h 0.50 0.50 1.00 0.50

Cmax rnnol/L 2540.62 346.08 119.87 59.07

Tlag h 0.00 0.00 0.00 0.00

Clast obs/Cmax 0.09 0.12 0.07 0.00

AUC 0-t rnnol/L *h 6573.12 351.36 308.23 118.97 AUC 0-inf_obs rnnol/L *h 7040.42 427.26 316.91 118.98 AUC 0-t/0-

0.93 0.82 0.97 1.00 inf obs

AUMC 0-inf_obs 17199.37 888.35 517.12 195.67 MRT 0-inf_obs 2.44 2.08 1.63 1.64

Vz/F_obs 0.01 0.15 0.12 0.20 Cl/F obs

0 00 _ 0 08 0.11 0.29

_ PMC _

Nanomicelle CPI CP2 Free

Pharmacokinetic

Unit Value Value Value Value

Parameter

Lambda z ITh 0.46 0.82 1.17 Missing tl/2 h 1.52 0.84 0.59 Missing

Tmax h 0.50 0.50 0.50 1.00

Cmax rnnol/L 773.63 392.91 144.23 1.53

Tlag h 0.00 0.00 0.00 0.00

Clast obs/Cmax 0.07 0.04 0.02 0.17

AUC 0-t rnnol/L *h 1759.01 286.41 213.78 1.84

AUC 0-inf_obs rnnol/L *h 1885.38 306.04 215.63 Missing AUC 0-t/0-

0.93 0.94 0.99 Missing inf obs AUMC 0-inf_obs 4443.98 349.97 281.19 Missing MRT 0-inf_obs 2.36 1.14 1.30 Missing Vz/F_obs 0.04 0.14 0.14 Missing Cl/F obs

0 02 0.11 0.16 Missing

_ BDMC

Nanomicelle Cpl Cp2 Free

Pharmacokinetic

Unit Value Value Value Value

Parameter

Lambda z i/h 0.37 2.98 1.05 Missing tl/2 h 1.86 0.23 0.66 Missing

Tmax h 0.50 0.50 0.50 1.00

Cmax rnnol/L 137.48 134.88 115.06 0.16

Tlag h 0.00 0.00 0.00 0.00

Clast obs/Cmax 0.12 0.01 0.19 1.00

AUC 0-t rnnol/L *h 331.25 93.79 105.49 0.07 AUC 0-inf_obs rnnol/L *h 374.87 94.32 126.80 Missing AUC 0-t/0-

0.88 0.99 0.83 Missing inf obs

[0064] Referring to Table 3, pharmacokinetic parameters such as maximum plasma concentration (Cmax) and time to reach the maximum concentration (Tmax) were significantly improved in the nanomicellar versus free curcuminoids. Following oral administration of nanomicelles, the curcumin Cmax of approximately 2540.62 nmol/L was reached after 30 min, while the values were 346.08 and 59.07 for CP1 and free curcumin, respectively. Free curcumin was rapidly metabolized, results in a short t ½ of 0.48, as compared to nanomicelle (t ½: 1.44 h). Tmax for all three curcuminoids components, i.e. curcumin, DMC and BDMC achieved at 30 min following administration of nanomicelles. Nanomicelle formulation significantly increased the AUC 0-t and AUC 0-inf of curcumin as compared to CP1, CP 2 free curcumin. The AUC 0-inf of curcumin for nanomicelles was 16.5, 22.3 and 59.2 times more than CP1, CP2 and free curcuminoids. The bioavailability of other ingredients including DMC and BDMC was also superior following administration of nanomicelles as compared to their free form or CPs. [0065] While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0066] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0067] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0068] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0069] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms“comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by“a” or“an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0070] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0071] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

WHAT IS CLAIMED IS:

1. A method for producing curcumin nanomicelles, the method comprising:

forming a curcuminoid solution by mixing curcuminoid powder with a surfactant; forming a first mixture by mixing vitamin E and natural oils with the curcuminoid solution; forming an ascorbic acid solution by dissolving ascorbic acid in water; forming a second mixture by mixing the ascorbic acid solution with the first mixture; and homogenizing the second mixture.

2. The method according to claim 1, wherein forming a curcuminoid solution by mixing curcuminoid powder with a surfactant includes mixing curcuminoid powder with a polysorbate surfactant.

3. The method according to claim 2, wherein the polysorbate surfactant is selected from the group consisting of polysorbate 80, polysorbate 20, polysorbate 40, and mixtures thereof.

4. The method according to claim 1, wherein forming a curcuminoid solution comprises: heating a polysorbate surfactant to a temperature between 50°C and 60°C; mixing curcuminoid powder with the heated polysorbate surfactant at a stirrer speed between 40 and 60 rpm for 15 to 30 minutes; heating the mixture of curcuminoid powder and the polysorbate surfactant to a temperature between 80°C and 90°C; ai stirring the mixture of curcuminoid powder and the polysorbate surfactant at a stirrer speed between 75 and 100 rpm for 30 to 60 minutes.

5. The method according to claim 1 , wherein the curcuminoid powder includes, curcumin, dimethyl curcumin (DMC), and bisdem ethoxy curcumin (BDMC).

6. The method according to claim 1, wherein forming the first mixture comprises mixing vitamin E and the natural oil with the curcuminoid solution at a temperature between 50°C and 60°C with a stirrer speed of 40 to 60 rpm for 10 to 20 minutes.

7. The method according to claim 6, wherein the natural oil is selected from the group consisting of soya bean oil, sesame oil, olive oil, almond oil, and combinations thereof.

8. The method according to claim 1, wherein forming an ascorbic acid solution includes dissolving ascorbic acid in water at a temperature between 50°C and 60°C with a stirrer speed of 40 to 60 rpm for 15 to 30 minutes;

9. The method according to claim 1, wherein forming a second mixture comprises:

mixing the ascorbic acid solution with the first mixture in a mixer with a stirrer speed of 40 to 60 rpm, at a temperature between 50°C and 60°C, for a period of 10 to 20 minutes;

heating the obtained mixture of the ascorbic acid solution and the first mixture to a temperature between 80°C and 90°C; and

stirring the heated mixture (second mixture) at a stirrer speed between 75 and 100 rpm for 30 to 60 minutes.

10. The method according to claim 1, wherein homogenizing the second mixture includes homogenizing the second mixture in a rotor stator homogenizer at 5000-7000 rpm at 80°C to 90°C for 15-30 minutes to obtain nanomicelles with an average size of 10 nm.

11. A nanomicelle formulation, comprising:

a curcuminoid with a concentration between 1 and 10% by weight of the total nanomicelle formulation;

a polysorbate surfactant;

vitamin E with a concentration between 0.1 and 2% by weight of the total nanomicelle formulation;

ascorbic acid with a concentration between 0.1 and 3% by weight of the total nanomicelle formulation; and

a natural oil with a concentration between 2 and 10% by weight of the total nanomicelle formulation.