FIELD OF THE INVENTION
[001] The present disclosure relates to micellar compositions with high bioavailability. In particular, it relates to a micellar composition comprising an active ingredient encapsulated in poloxamer 407, and at least one solubilizer. 5
BACKGROUND OF THE INVENTION
[002] Flavonoids are a group of secondary metabolites which are polyphenols. They exhibit a wide range of biochemical properties including antioxidant activity, inhibition of tyrosine kinases, cAMP phosphodiesterases, and induction of phase II
10 metabolizing enzyme in in-vitro conditions (David et al., 2015, Int. J. Chem. Sci.: 13(1), 459-500). Xanthohumol (2′, 4′, 4-trihydroxy-6′-methoxy-3′-prenylchalcone, XN), is prenylflavonoid, present in female inflorescence of hops (Humulus lupus L.) plant which long been used in medical and brewing industry. Xanthohumol holds a broad range of health benefits such as antioxidant, anti-inflammatory, antimicrobial,
15 immune modulatory activity etc. (Legette et al. 2014, Mol Nutr Food Res. 58:248-255).
[003] US 2008/0207928 A1 describes the use of xanthohumol for hair and scalp care. But still the usage of xanthohumol is very less due to its poor solubility, instability, and bioavailability (Legette et al. 2014, Mol Nutr Food Res. 58:248-255).
20 Furthermore, the oral bioavailability of xanthohumol is less than 1% (Avula et al., J Chromatogr Sci. 2004; 42:378-382).
[004] US9006293 B2 demonstrates that xanthohumol shows increased level of water solubility and stability with cyclodextrin complex as compared to xanthohumol alone.
25 [005] Thus, enhancing the bioavailability of xanthohumol has been and remains a challenge and there is a need to devise better formulations to remedy this problem to better utilize the properties of this flavonoid.
2

SUMMARY OF THE INVENTION
[006] In an aspect of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles
5 comprising: (i) poloxamer 407; and (ii) at least one solubilizer.
[007] In an aspect of the present disclosure, there is provided a method of preparing micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, said method comprising: (i)
10 contacting said at least one active ingredient, poloxamer 407 and at least one
solubilizer, in the presence of at least one organic solvent to obtain a mixture; (ii) homogenising said mixture to obtain a clear and transparent solution; (iii) removing said at least one organic solvent from said solution at a temperature in the range of 30-50 °C to obtain film of micelles or clusters thereof encapsulating said active
15 ingredient; and (iv) contacting said film with water to obtain a solution of micelles or
clusters thereof.
[008] In an aspect of the present disclosure, there is provided a method of increasing xanthohumol bioavailability in cells, said method comprising: (a) obtaining a composition comprising micelles or micellar clusters thereof comprising: (a) at least
20 one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (b) contacting said composition with cells, wherein said composition increases xanthohumol bioavailability in cells. [009] These and other features, aspects, and advantages of the present subject matter
25 will be better understood with reference to the following description and appended
claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of
3

the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
5 [010] The following drawings form part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may be
better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[011] Figure 1 depicts that stability of xanthohumol micelles.
10 [012] Figure 2 depicts the cellular uptake of xanthohumol (XN)-loaded micelles in
HaCaT cells, in accordance with an embodiment of the present disclosure.
[013] Figure 3 depicts the quantification of fluorescence intensity obtained through
confocal laser scanning microscopy for various preparations and free xanthohumol in
accordance with an embodiment of the present disclosure.
15 [014] Figure 4 depicts the effect of free xanthohumol and xanthohumol loaded
micelles on inhibition of nitric oxide (NO) production in HaCaT cells, in accordance
with an embodiment of the present disclosure.
[015] Figure 5 depicts the effect of the free xanthohumol and xanthohumol loaded
micelles on inhibition of pro-inflammatory cytokine production. Figure 5A depicts
20 the effect on the percentage inhibition of interleukin 6 (IL-6), and Figure 5B depicts
the effect on the percentage inhibition of interleukin 8 (IL-8), in accordance with an
embodiment of the present disclosure.
[016] Figure 6 depicts inhibitory effect of free xanthohumol and xanthohumol
loaded micelles on UVB-induced phosphorylation of p65 (Ser536) in HaCaT cells, in
25 accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
4

[017] Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
understood that the present disclosure includes all such variations and modifications.
The disclosure also includes all such steps, features, compositions, and compounds
5 referred to or indicated in this specification, individually or collectively, and any and
all combinations of any or more of such steps or features.
Definitions
[018] For convenience, before further description of the present disclosure, certain
terms employed in the specification, and examples are delineated here. These
10 definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the meanings
recognized and known to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth below.
[019] The articles “a”, “an” and “the” are used to refer to one or to more than one
15 (i.e., to at least one) of the grammatical object of the article.
[020] The terms “comprise” and “comprising” are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed
as “consists of only”.
[021] Throughout this specification, unless the context requires otherwise the word
20 “comprise”, and variations such as “comprises” and “comprising”, will be understood
to imply the inclusion of a stated element or step or group of element or steps but not
the exclusion of any other element or step or group of element or steps.
[022] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
25 [023] Carriers are substances that serve as mechanisms to improve the delivery and
the effectiveness of drugs.
[024] A diluent (also referred to as filler, dilutant, or thinner) is a diluting agent.
5

[025] An excipient is an inactive substance that serve as the vehicle or medium for a drug or other active substance. Excipients include colouring agents, humectants, preservatives, emollients, and combinations thereof.
[026] Unless defined otherwise, all technical and scientific terms used herein have 5 the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
10 [027] Xanthohumol is a prenylated flavonoid obtained from the female inflorescence of hops. It is reputed to possess many therapeutic properties including anti-carcinogenic, and anti-inflammatory effects among others. However, the bioavailability of this compound is very poor and one of the possible reasons is its ability bind to cystolic proteins. A possible solution to increase the bioavailability of
15 this compound is using micellar compositions to enhance the solubility of this compound. Although many micellar compositions are available in the market, most of them are synthetic in nature, and are prepared from non-biodegradable polymers which could potentially accumulate in various body tissues and have toxic effects. Hence, what is required is a biodegradable polymer with enhanced longevity, stability
20 and bioavailability. Poloxamer 407 is a biopolymer approved by United Nations Food and Drug Authority (USFDA), and is one of the most attractive options due to its biocompatibility, biodegradability, and low toxicity. It is a block copolymer of ethylene oxide (EO) and propylene oxide (PO) which are arranged in a basic EOx-POy-EOx structure. Likewise, D-α-Tocopheryl polyethylene glycol 1000 succinate
25 (TPGS), a derivative of natural vitamin E (α-tocopherol) and polyethylene glycol 1000, has been widely applied in the food and drug industry as solubilizer, absorption enhancer, and a vehicle for lipid-based drug delivery formulations. It has also been found that TPGS enhances the solubility, and increases the oral bioavailability of
6

anticancer drugs. The present disclosure attempts to enhance the systemic
bioavailability of xanthohumol using a polymeric micelle formulation at specific
weight/ weight ratios, which increases the cellular uptake and bioavailability of the
active when compared with the active alone. Further, it does so using biodegradable
5 and biocompatible compounds.
[028] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
10 [029] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer. [030] In an embodiment of the present disclosure, there is provided a composition
15 comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one active ingredient is xanthohumol. [031] In an embodiment of the present disclosure, there is provided a composition
20 comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol
25 octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters,
polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol, polyethoxylated tallow amine,
7

cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof.
[032] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
5 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters,
10 polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers
of polyethylene glycol and polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof, wherein said at least one active ingredient is xanthohumol.
15 [033] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate.
20 [034] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate, and said at
25 least one active ingredient is xanthohumol.
[035] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
8

comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6,
at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8.
[036] In an embodiment of the present disclosure, there is provided a composition
5 comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8,
10 and at least one active ingredient is xanthohumol.
[037] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
15 one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6,
at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8, and said at least one solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers,
20 glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl
esters, block copolymers of polyethylene glycol and polypropylene glycol,
polyethoxylated tallow amine, cocamide MEA, cocamide DEA,
dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof.
25 [038] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
9

one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6,
at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8,
said at least one solubilizer is selected from the group consisting of polyethylene
glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers,
5 polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers,
glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl
esters, block copolymers of polyethylene glycol and polypropylene glycol,
polyethoxylated tallow amine, cocamide MEA, cocamide DEA,
dodecyldimetpylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations
10 thereof, and said active ingredient is xanthohumol.
[039] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
15 one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6,
at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8, and said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate. [040] In an embodiment of the present disclosure, there is provided a composition
20 comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8,
25 said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate,
and said active ingredient is xanthohumol.
[041] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active
10

ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 –
1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range
5 of 1:0.1 – 1:0.5.
[042] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
10 one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 –
1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, and at least one active ingredient is xanthohumol.
[043] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active
15 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, and said at least one solubilizer is selected from the group consisting
20 of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl
ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl
ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan
alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol,
polyethoxylated tallow amine, cocamide MEA, cocamide DEA,
25 dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations
thereof.
[044] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active
11

ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 –
1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range
5 of 1:0.1 – 1:0.5, and said at least one solubilizer is D-α-tocopheryl polyethylene
glycol 1000 succinate.
[045] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
10 comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, said at least one solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl
15 ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl
ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan
alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol,
polyethoxylated tallow amine, cocamide MEA, cocamide DEA,
dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations
20 thereof, and said active ingredient is xanthohumol.
[046] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
25 one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 –
1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate, and said active ingredient is xanthohumol.
12

[047] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
5 one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, and said at least
one solubilizer to said at least one active ingredient w/w ratio is 1:0.4.
[048] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
10 comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, and at least one active ingredient is xanthohumol. [049] In an embodiment of the present disclosure, there is provided a composition
15 comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient is 1:0.4, and said at least one solubilizer is
20 selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene
glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
25 DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and
combinations thereof.
[050] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active
13

ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least
one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one
solubilizer to at least one active ingredient is 1:0.4, said at least one solubilizer is
5 selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene
glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
10 DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and
combinations thereof, and said active ingredient is xanthohumol.
[051] In an embodiment of the present disclosure, there is provided a composition comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one
15 solubilizer, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said
micelle is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, and said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate. [052] In an embodiment of the present disclosure, there is provided a composition
20 comprising: (a) at least one active ingredient encapsulated in the interior of the
micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000
25 succinate, and said active ingredient is xanthohumol.
[053] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
14

comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-80%, and w/w
percentage of the at least one solubilizer in said composition is in the range of 12-
5 40%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is
in the range of 1:0.1 – 1:0.8, and at least one active ingredient is xanthohumol.
[054] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
10 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-80%, w/w
percentage of the at least one solubilizer in said composition is in the range of 12-
15 40%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is
in the range of 1:0.1 – 1:0.8, said at least one solubilizer is selected from the group
consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers,
glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol
20 alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl
esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and
polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and
combinations thereof, and said active ingredient is xanthohumol.
25 [055] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
15

percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-80%, w/w
percentage of the at least one solubilizer in said composition is in the range of 12-
40%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
5 is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is
in the range of 1:0.1 – 1:0.8, said at least one solubilizer is D-α-tocopheryl
polyethylene glycol 1000 succinate, and said active ingredient is xanthohumol.
[056] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
10 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-80%, w/w
percentage of the at least one solubilizer in said composition is in the range of 12-
15 40%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient
w/w ratio is in the range of 1:0.1 – 1:0.5, and at least one active ingredient is
xanthohumol.
[057] In an embodiment of the present disclosure, there is provided a composition
20 comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-80%, w/w
25 percentage of the at least one solubilizer in said composition is in the range of 12-
40%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, said at least one solubilizer is selected from
16

the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl
ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene
glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan
alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and
5 polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof, and said active ingredient is xanthohumol.
[058] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
10 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-80%, w/w
percentage of the at least one solubilizer in said composition is in the range of 12-
15 40%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient
w/w ratio is in the range of 1:0.1 – 1:0.5, said at least one solubilizer is D-α-
tocopheryl polyethylene glycol 1000 succinate, and said active ingredient is
xanthohumol.
20 [059] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
25 percentage of poloxamer 407 in said composition is in the range of 55-80%, w/w
percentage of the at least one solubilizer in said composition is in the range of 12-
40%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
17

is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, and at least one active ingredient is xanthohumol.
[060] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
5 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-80%, w/w
percentage of the at least one solubilizer in said composition is in the range of 12-
10 40%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is 1:2.9, at least one solubilizer to at least one active ingredient is 1:0.4, said at least
one solubilizer is selected from the group consisting of polyethylene glycol alkyl
ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol
octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters,
15 polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers
of polyethylene glycol and polypropylene glycol, polyethoxylated tallow amine,
cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, D-α-tocopheryl
polyethylene glycol, and combinations thereof, and said active ingredient is
xanthohumol.
20 [061] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) at least one active ingredient encapsulated in the interior of the
micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one
solubilizer, wherein w/w percentage of the active ingredient in said composition is in
the range of 5-10%, w/w percentage of poloxamer 407 in said composition is in the
25 range of 55-80%, w/w percentage of the at least one solubilizer in said composition is
in the range of 12-40%, wherein said at least one solubilizer to poloxamer 407 w/w
ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient
18

w/w ratio is 1:0.4, said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate, and said active ingredient is xanthohumol.
[062] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
5 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-70%, w/w
percentage of the at least one solubilizer in said composition is in the range of 17-
10 35%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is
in the range of 1:0.1 – 1:0.8, and at least one active ingredient is xanthohumol.
[063] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
15 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-70%, w/w
percentage of the at least one solubilizer in said composition is in the range of 17-
20 35%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is
in the range of 1:0.1 – 1:0.8, said at least one solubilizer is selected from the group
consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers,
glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol
25 alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl
esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and
polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
19

DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof, and said active ingredient is xanthohumol.
[064] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
5 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-70%, w/w
percentage of the at least one solubilizer in said composition is in the range of 17-
10 35%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is
in the range of 1:0.1 – 1:0.8, said at least one solubilizer is D-α-tocopheryl
polyethylene glycol 1000 succinate, and said active ingredient is xanthohumol.
[065] In an embodiment of the present disclosure, there is provided a composition
15 comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-70%, w/w
20 percentage of the at least one solubilizer in said composition is in the range of 17-
35%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient
w/w ratio is in the range of 1:0.1 – 1:0.5, and at least one active ingredient is
xanthohumol.
25 [066] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
20

percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-70%, w/w
percentage of the at least one solubilizer in said composition is in the range of 17-
35%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
5 is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient
w/w ratio is in the range of 1:0.1 – 1:0.5, said at least one solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan
10 alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and
polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof, and said active ingredient is xanthohumol. [067] In an embodiment of the present disclosure, there is provided a composition
15 comprising micelles or micellar clusters thereof comprising: (a) at least one active
ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w percentage of the active ingredient in said composition is in the range of 5-10%, w/w percentage of poloxamer 407 in said composition is in the range of 55-70%, w/w
20 percentage of the at least one solubilizer in said composition is in the range of 17-
35%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate, and said active ingredient is
25 xanthohumol.
[068] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
21

comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-70%, w/w
percentage of the at least one solubilizer in said composition is in the range of 17-
5 35%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, and
at least one active ingredient is xanthohumol.
[069] In an embodiment of the present disclosure, there is provided a composition
comprising micelles or micellar clusters thereof comprising: (a) at least one active
10 ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is in the range of 5-10%, w/w
percentage of poloxamer 407 in said composition is in the range of 55-70%, w/w
percentage of the at least one solubilizer in said composition is in the range of 17-
15 35%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle
is 1:2.9, at least one solubilizer to at least one active ingredient is 1:0.4, said at least
one solubilizer is selected from the group consisting of polyethylene glycol alkyl
ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol
octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters,
20 polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers
of polyethylene glycol and polypropylene glycol, polyethoxylated tallow amine,
cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, D-α-tocopheryl
polyethylene glycol, and combinations thereof, and said active ingredient is
xanthohumol.
25 [070] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) at least one active ingredient encapsulated in the interior of the
micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one
solubilizer, wherein w/w percentage of the active ingredient in said composition is in
22

the range of 5-10%, w/w percentage of poloxamer 407 in said composition is in the
range of 55-70%, w/w percentage of the at least one solubilizer in said composition is
in the range of 17-35%, wherein said at least one solubilizer to poloxamer 407 w/w
ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient
5 w/w ratio is 1:0.4, said at least one solubilizer is D-α-tocopheryl polyethylene glycol
1000 succinate, and said active ingredient is xanthohumol.
[071] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
10 comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is of 9%, w/w percentage of poloxamer 407 in said composition is 68%, w/w percentage of the at least one solubilizer in said composition is 23%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least
15 one active ingredient w/w ratio is 1:0.4, and at least one active ingredient is
xanthohumol.
[072] In an embodiment of the present disclosure, there is provided a composition comprising micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
20 comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
percentage of the active ingredient in said composition is 9%, w/w percentage of poloxamer 407 in said composition is 68%, w/w percentage of the at least one solubilizer in said composition is 23%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least
25 one active ingredient is 1:0.4, said at least one solubilizer is selected from the group
consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl
23

esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and
polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and
combinations thereof, and said active ingredient is xanthohumol.
5 [073] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w percentage of the active ingredient in said composition is 9%, w/w percentage of poloxamer 407 in said composition is 68%, w/w percentage
10 of the at least one solubilizer in said composition is 23%, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate, and said active ingredient is xanthohumol.
15 [074] The disclosure also relates to a method of preparing micelles or micellar
clusters thereof comprising: (a) contacting said at least one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to obtain a clear and transparent solution; (c) removing said at least one organic solvent from the said
20 solution at a temperature in the range of 30-50 °C to obtain film of micelles or
clusters thereof encapsulating said active ingredient; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof, wherein said composition comprises micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
25 comprising: (i) poloxamer 407; and (ii) at least one solubilizer.
[075] In an embodiment of the present disclosure, there is provided a method of preparing micelles or micellar clusters thereof comprising: (a) contacting said at least one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of
24

at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to
obtain a clear and transparent solution; (c) removing said at least one organic solvent
from the said solution at a temperature in the range of 30-50 °C to obtain film of
micelles or clusters thereof encapsulating said active ingredient; and (d) contacting
5 said film with water to obtain a solution of micelles or clusters thereof, wherein said
composition comprises micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, and at least one active ingredient is xanthohumol.
10 [076] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or micellar clusters thereof comprising: (a) contacting said at least one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to obtain a clear and transparent solution; (c) removing said at least one organic solvent
15 from the said solution at a temperature in the range of 30-50 °C to obtain film of
micelles or clusters thereof encapsulating said active ingredient; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof, wherein said composition comprises micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
20 comprising: (i) poloxamer 407; and (ii) at least one solubilizer, and said at least one
solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers
25 of polyethylene glycol and polypropylene glycol, polyethoxylated tallow amine,
cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof.
25

[077] In an embodiment of the present disclosure, there is provided a method of
preparing micelles or micellar clusters thereof comprising: (a) contacting said at least
one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of
at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to
5 obtain a clear and transparent solution; (c) removing said at least one organic solvent
from the said solution at a temperature in the range of 30-50 °C to obtain film of micelles or clusters thereof encapsulating said active ingredient; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof, wherein said composition comprises micelles or micellar clusters thereof comprising: (a) at least
10 one active ingredient encapsulated in the interior of the micelles; and (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer, and said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate.
[078] In an embodiment of the present disclosure, there is provided a method of preparing micelles or micellar clusters thereof comprising: (a) contacting said at least
15 one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of
at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to obtain a clear and transparent solution; (c) removing said at least one organic solvent from the said solution at a temperature in the range of 30-50 °C to obtain film of micelles or clusters thereof encapsulating said active ingredient; and (d) contacting
20 said film with water to obtain a solution of micelles or clusters thereof, wherein said
at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 - 1:6, and said at least one solubilizer to said at least one active ingredient w/w ratio is in the range of 1:0.1- 1:0.8. [079] In an embodiment of the present disclosure, there is provided a method of
25 preparing micelles or micellar clusters thereof comprising: (a) contacting said at least
one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to obtain a clear and transparent solution; (c) removing said at least one organic solvent
26

from the said solution at a temperature in the range of 30-50 °C to obtain film of
micelles or clusters thereof encapsulating said active ingredient; and (d) contacting
said film with water to obtain a solution of micelles or clusters thereof, wherein said
at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of
5 1:1.5- 1:4, and said at least one solubilizer to said at least one active ingredient w/w
ratio is in the range of 1:0.1- 1:0.5.
[080] In an embodiment of the present disclosure, there is provided a method of preparing micelles or micellar clusters thereof comprising: (a) contacting said at least one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of
10 at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to
obtain a clear and transparent solution; (c) removing said at least one organic solvent from the said solution at a temperature in the range of 30-50 °C to obtain film of micelles or clusters thereof encapsulating said active ingredient; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof, wherein said
15 at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9 and said at
least one solubilizer to said at least one active ingredient w/w ratio is 1:0.4. [081] In an embodiment of the present disclosure, there is provided a method of preparing micelles or micellar clusters thereof comprising: (a) contacting said at least one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of
20 at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to
obtain a clear and transparent solution; (c) removing said at least one organic solvent from the said solution at a temperature in the range of 30-50 °C to obtain film of micelles or clusters thereof encapsulating said active ingredient; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof, wherein said
25 composition comprises micelles or micellar clusters thereof comprising: (a) at least
one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein said one active agent is xanthohumol, said at least one solubilizer is D-α-tocopheryl
27

polyethylene glycol 1000 succinate, at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, and said at least one solubilizer to said at least one active ingredient w/w ratio is 1:0.4.
[082] In an embodiment of the present disclosure, there is provided a method of
5 preparing micelles or micellar clusters thereof comprising: (a) contacting said at least
one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to obtain a clear and transparent solution; (c) removing said at least one organic solvent from the said solution at a temperature in the range of 30-50 °C to obtain film of
10 micelles or clusters thereof encapsulating said active ingredient; and (d) contacting
said film with water to obtain a solution of micelles or clusters thereof, wherein said composition comprises micelles or micellar clusters thereof comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; and (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer, wherein w/w
15 percentage of the active ingredient in said composition is 9%, w/w percentage of
poloxamer 407 in said composition is 68%, w/w percentage of the at least one solubilizer in said composition is 23%, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, said at least one solubilizer is D-α-tocopheryl
20 polyethylene glycol 1000 succinate, and said active ingredient is xanthohumol.In an
embodiment of the present disclosure, there is provided a method of preparing micelles or micellar clusters thereof comprising: (a) contacting said at least one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to obtain a
25 clear and transparent solution; (c) removing said at least one organic solvent from the
said solution at a temperature in the range of 30-50 °C to obtain film of micelles or clusters thereof encapsulating said active ingredient; and (d) contacting said film with water to obtain a solution of micelles or clusters thereof, wherein said at least one
28

organic solvent is selected from the group consisting of ethanol, chloroform, methanol, tertiary butanol, trimethylamine, diethyl ether, dichloromethane, trichloromethane, cyclohexane, acetonitrile and combinations thereof.
[083] In an embodiment of the present disclosure, there is provided a method of
5 preparing micelles or micellar clusters thereof comprising: (a) contacting said at least
one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to obtain a clear and transparent solution; (c) removing said at least one organic solvent from the said solution at a temperature in the range of 30-50 °C to obtain film of
10 micelles or clusters thereof encapsulating said active ingredient; and (d) contacting
said film with water to obtain a solution of micelles or clusters thereof, wherein said at least one organic solvent is acetonitrile.
[084] In an embodiment of the present disclosure, there is provided a method of preparing micelles or micellar clusters thereof comprising: (a) contacting said at least
15 one active ingredient, poloxamer 407, and at least one solubilizer, in the presence of
at least one organic solvent to obtain a mixture; (b) homogenising the said mixture to obtain a clear and transparent solution; (c) removing said at least one organic solvent from the said solution at a temperature in the range of 30-50 °C to obtain film of micelles or clusters thereof encapsulating said active ingredient; and (d) contacting
20 said film with water to obtain a solution of micelles or clusters thereof, wherein
removal of said solvent is carried out at a temperature of 40 °C.
[085] In an embodiment of the present disclosure, there is provided a method of increasing xanthohumol bioavailability in cells, said method comprising: (a) obtaining a composition comprising micelles or micellar clusters thereof comprising:
25 (i) at least one active ingredient encapsulated in the interior of the micelles; and (ii)
the micelles comprising: (ii-a) poloxamer 407; and (ii-b). at least one solubilizer; and (b) contacting said composition with cells, wherein said composition increases xanthohumol bioavailability in cells.
29

[086] In an embodiment of the present disclosure, there is provided a method of
increasing xanthohumol bioavailability in cells, said method comprising: (a)
obtaining a composition comprising micelles or micellar clusters thereof comprising:
(i) at least one active ingredient encapsulated in the interior of the micelles; and (ii)
5 the micelles comprising: (ii-a) Poloxamer 407; and (ii-b) at least one solubilizer; and
(b) contacting said composition with cells, wherein said composition increases xanthohumol bioavailability in cells, wherein said at least one active ingredient is xanthohumol. [087] In an embodiment of the present disclosure, there is provided a method of
10 increasing xanthohumol bioavailability in cells, said method comprising: (a)
obtaining a composition comprising micelles or micellar clusters thereof comprising: (i) at least one active ingredient encapsulated in the interior of the micelles; and (ii) the micelles comprising: (ii- a) poloxamer 407; and (ii-b). at least one solubilizer; and (b) contacting said composition with cells, wherein said composition increases
15 xanthohumol bioavailability in cells, wherein said at least one solubilizer is selected
from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol
20 and polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof.
[088] In an embodiment of the present disclosure, there is provided a method of increasing xanthohumol bioavailability in cells, said method comprising: (a)
25 obtaining a composition comprising micelles or micellar clusters thereof comprising:
(i) at least one active ingredient encapsulated in the interior of the micelles; and (ii) the micelles comprising: (ii- a) poloxamer 407; and (ii-b). at least one solubilizer; and (b) contacting said composition with cells, wherein said composition increases
30

xanthohumol bioavailability in cells, wherein said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate.
[089] In an embodiment of the present disclosure, there is provided a method of
increasing xanthohumol bioavailability in cells, said method comprising: (a)
5 obtaining a composition comprising micelles or micellar clusters thereof comprising:
(i) at least one active ingredient encapsulated in the interior of the micelles; and (ii) the micelles comprising: (ii-a). poloxamer 407; and (ii-b) at least one solubilizer; and (b) contacting said composition with cells, wherein said composition increases xanthohumol bioavailability in cells, wherein said at least one solubilizer to
10 poloxamer 407 w/w ratio in said micelle is in the range of 1:1 - 1:6, and said at least
one solubilizer to said at least one active ingredient w/w ratio is in the range of 1:0.1-1:0.8.
[090] In an embodiment of the present disclosure, there is provided a method of increasing xanthohumol bioavailability in cells, said method comprising: (a)
15 obtaining a composition comprising micelles or micellar clusters thereof comprising:
(i) at least one active ingredient encapsulated in the interior of the micelles; and (ii) the micelles comprising: (ii- a) poloxamer 407; and (ii-b). at least one solubilizer; and (b) contacting said composition with cells, wherein said composition increases xanthohumol bioavailability in cells, wherein said at least one solubilizer to
20 poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 - 1:4, and said at least
one solubilizer to said at least one active ingredient w/w ratio is in the range of 1:0.1-1:0.5.
[091] In an embodiment of the present disclosure, there is provided a method of increasing xanthohumol bioavailability in cells, said method comprising: (a)
25 obtaining a composition comprising micelles or micellar clusters thereof comprising:
(i) at least one active ingredient encapsulated in the interior of the micelles; and (ii) the micelles comprising: (ii-a) poloxamer 407; and (ii-b) at least one solubilizer; and (b) contacting said composition with cells, wherein said composition increases
31

xanthohumol bioavailability in cells, wherein at least one solubilizer to poloxamer 407poloxamer 407 w/w ratio in said micelle is 1:2.9 and said at least one solubilizer to said at least one active ingredient w/w ratio is 1:0.4.
[092] In an embodiment of the present disclosure, there is provided a composition
5 micelles or micellar clusters thereof for use in formulations, comprising: (a) at least
one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6, at
10 least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8.
[093] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
15 additive, at least one diluent, and at least one excipient, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8, and at least one active ingredient is xanthohumol. [094] In an embodiment of the present disclosure, there is provided a composition
20 micelles or micellar clusters thereof for use in formulations, comprising: (a) at least
one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6, at
25 least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8,
and said at least one solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers,
32

glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl
esters, block copolymers of polyethylene glycol and polypropylene glycol,
polyethoxylated tallow amine, cocamide MEA, cocamide DEA,
dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations
5 thereof.
[095] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
10 additive, at least one diluent, and at least one excipient, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8, said at least one solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers,
15 polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers,
glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl
esters, block copolymers of polyethylene glycol and polypropylene glycol,
polyethoxylated tallow amine, cocamide MEA, cocamide DEA,
dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations
20 thereof, and said active ingredient is xanthohumol.
[096] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
25 additive, at least one diluent, and at least one excipient, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8,
33

and said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate.
[097] In an embodiment of the present disclosure, there is provided a composition
micelles or micellar clusters thereof for use in formulations, comprising: (a) at least
5 one active ingredient encapsulated in the interior of the micelles; (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1 – 1:6, at least one solubilizer to at least one active ingredient is in the range of 1:0.1 – 1:0.8,
10 said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate,
and said active ingredient is xanthohumol.
[098] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles
15 comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5.
20 [099] In an embodiment of the present disclosure, there is provided a composition
micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one additive, at least one diluent, and at least one excipient, wherein said at least one
25 solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at
least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, and at least one active ingredient is xanthohumol.
34

[100] In an embodiment of the present disclosure, there is provided a composition
micelles or micellar clusters thereof for use in formulations, comprising: (a) at least
one active ingredient encapsulated in the interior of the micelles; (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
5 additive, at least one diluent, and at least one excipient, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, and said at least one solubilizer is selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl
10 ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl
ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan
alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol,
polyethoxylated tallow amine, cocamide MEA, cocamide DEA,
dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations
15 thereof.
[101] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
20 additive, at least one diluent, and at least one excipient, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, and said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate.
25 [102] In an embodiment of the present disclosure, there is provided a composition
micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
35

additive, at least one diluent, and at least one excipient, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at
least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1
– 1:0.5, said at least one solubilizer is selected from the group consisting of
5 polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl
ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl
ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan
alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol,
polyethoxylated tallow amine, cocamide MEA, cocamide DEA,
10 dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations
thereof, and said active ingredient is xanthohumol.
[103] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles
15 comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5 – 1:4, at least one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1 – 1:0.5, said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000
20 succinate, and said active ingredient is xanthohumol.
[104] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
25 additive, at least one diluent, and at least one excipient, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, and said at least one solubilizer to said at least one active ingredient w/w ratio is 1:0.4.
36

[105] In an embodiment of the present disclosure, there is provided a composition
micelles or micellar clusters thereof for use in formulations, comprising: (a) at least
one active ingredient encapsulated in the interior of the micelles; (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
5 additive, at least one diluent, and at least one excipient, wherein said at least one
solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, and at least one active ingredient is xanthohumol. [106] In an embodiment of the present disclosure, there is provided a composition
10 micelles or micellar clusters thereof for use in formulations, comprising: (a) at least
one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer
15 to at least one active ingredient is 1:0.4, and said at least one solubilizer is selected
from the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol
20 and polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof.
[107] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least
25 one active ingredient encapsulated in the interior of the micelles; (b) the micelles
comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer
37

to at least one active ingredient is 1:0.4, said at least one solubilizer is selected from
the group consisting of polyethylene glycol alkyl ethers, polypropylene glycol alkyl
ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene
glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan
5 alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and
polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and combinations thereof, and said active ingredient is xanthohumol. [108] In an embodiment of the present disclosure, there is provided a composition
10 micelles or micellar clusters thereof for use in formulations, comprising: (a) at least
one active ingredient encapsulated in the interior of the micelles; (b) the micelles comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer
15 to at least one active ingredient w/w ratio is 1:0.4, and said at least one solubilizer is
D-α-tocopheryl polyethylene glycol 1000 succinate.
[109] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles
20 comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
additive, at least one diluent, and at least one excipient, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate, and said active ingredient is
25 xanthohumol.
[110] In an embodiment of the present disclosure, there is provided a composition micelles or micellar clusters thereof for use in formulations, comprising: (a) at least one active ingredient encapsulated in the interior of the micelles; (b) the micelles
38

comprising: (i) poloxamer 407; and (ii) at least one solubilizer; and (c) at least one
additive, at least one diluent, and at least one excipient, wherein w/w percentage of
the active ingredient in said composition is 9%, w/w percentage of poloxamer 407 in
said composition is 68%, w/w percentage of the at least one solubilizer in said
5 composition is 23%, wherein said at least one solubilizer to poloxamer 407 w/w ratio
in said micelle is 1:2.9, at least one solubilizer to at least one active ingredient w/w ratio is 1:0.4, said at least one solubilizer is D-α-tocopheryl polyethylene glycol 1000 succinate, and said active ingredient is xanthohumol. [111] Although the subject matter has been described with reference to specific
10 embodiments, this description is not meant to be construed in a limiting sense.
Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the
15 present subject matter as defined.
EXAMPLES
[112] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined
20 otherwise, all technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which this disclosure
belongs. Although methods and materials similar or equivalent to those described
herein can be used in the practice of the disclosed methods and compositions, the
exemplary methods, devices and materials are described herein. It is to be
25 understood that this disclosure is not limited to particular methods, and experimental
conditions described, as such methods and conditions may vary.
39

Example 1
Materials and methods
[113] Xanthohumol was procured as hop (Humulus lupulus) from Sigma having the
purity of more than 96%, L-α-lysophosphatidylcholine from Sigma, USA, sodium
5 deoxycholate, polyvinylpyrrolidone K 30 and D-α-Tocopherol polyethylene glycol
1000 succinate (TPGS) were procured from Sigma, USA, poloxamer 407 was procured from BASF India Limited, ethanol and acetonitrile (HPLC grade) were procured from Fisher Scientific, India.
[114] The HaCaT cell lines, which are an immortalized human keratinocyte cells
10 were obtained from NCCS, Pune, India and cultured in complete Dulbecco's
Modified Eagle's medium (DMEM) (Sigma Aldrich) with 10% fetal bovine serum (FBS, Gibco) and 1% of penicillin/streptomycin (Invitrogen, USA) All cells were incubated at 37°C with a humidified 5% CO2 atmosphere.
Example 2
15 Preparation of micelles
[115] Method 1: Thin-Film Dispersion: The xanthohumol (XN) loaded micelle were prepared using thin-film dispersion method. For this, 0.5 mg of XN, 40 mg of L-α-lysophosphatidylcholine, 30 mg of sodium deoxycholate and 30 mg of polyvinylpyrrolidone K 30 were dissolved in 2 ml of ethanol with ultrasonic
20 treatment (sonication with one cycle of five seconds at 25% power for 5 cycles) to
form a clear and transparent solution. The organic solvent was then evaporated by incubating at 50 ºC to obtain a thin film loaded with XN. Subsequently, the obtained micelles mixture was dissolved using double distilled water and lyophilized. [116] Result: XN loaded micelles were successfully prepared using thin-film
25 dispersion method. The ethanol was evaporated by incubating the mixture at 50ºC.
Thus obtained thin film was dissolved using double distilled water while vortexing. Then the formed XN loaded micelles were lyophilized using sublimation method and stored for further use.
40

[117] Method 2: Thin Film Hydration: The process of making the micelles
composition is a step wise reaction which is described as under. For instance, 0.5 mg
of XN was dissolved in 3.5 ml of acetonitrile which contains different ratios of TPGS
and poloxamer 407 under mild stirring. The ratios of TPGS: poloxamer 407 (w/w)
5 was in the range of Prep 1– 1: 5 (0.86 mg :4.3 mg), Prep 2 - 1 :2.9 9 (1.3mg :3.8mg),
and Prep 3 - 1: 1.9 (1.7mg : 3.25mg). The mixture was then ultra-sonicated with one cycle of five seconds at 25% power for 5 cycles. Then the solvent was evaporated using a rotary evaporator maintained at 40°C. Subsequently, the solid XN/copolymer matrix was dissolved in Milli Q water, filtered and lyophilized.
10 [118] Result: XN loaded mixed polymeric micelles were successfully prepared by
thin film hydration method. The prepared solid XN/copolymer was dissolved in Milli Q water to form core-shell structured XN-loaded micelles. Then the excess of unincorporated XN molecules were removed by filtration using 0.22μm syringe filter. Thus obtained XN polymeric micelles solution was lyophilized using sublimation
15 method and stored for further use.
Example 3
Study of the stability of the micelles
[119] In order to analyze the stability of the micelles prepared using different
techniques, appropriate amount of lyophilized XN loaded micelles were dissolved in
20 Milli Q water. Then the aqueous solution of XN loaded micelles were stored at room
temperature and monitored periodically (once a week; for 2 months) for any changes in appearance. When the hydrophobic drugs are completely solubilized in the core of the micelle, then a clear solution will be observed. Therefore, any change in the appearance of the solution indicates instability.
25 [120] Figure 1 depicts the stability of the micelles prepared using different
techniques. XN indicates the stability of xanthohumol as such without use of any micelles, though it is stable for longer duration however its bioavailability is very poor.. The middle panel of Figure 1 indicates the micellar compositions prepared
41

using thin film dispersion method. The right most panel shows the stability of mi cellar compositions prepared using thin film hydration method. All the three ratios of micelles prepared using this method were stable for prolonged period of time as compared to the control. Thus, it can be inferred that the micelles prepared using thin 5 film dispersion method were stable for longer duration of time and thus can be used in different formulations.
Example 4
Determination of Drug Loading Entrapment Efficiency
[121] Drug loading % (DL) and encapsulation efficiency % (EE) of the XN micelles 10 were estimated using high performance liquid chromatography (HPLC; Agilent 1200
with PDA Detector). For this, an appropriate amount of lyophilized XN micelles was
dissolved in a required volume of methanol. Then 20μL of the mixture was injected
automatically in the injection port and analyzed using the mobile phase consisting of
1% acetic acid/ methanol. The flow rate was set at 1 mL/min with Inertsil ODS C-18, 15 250*4.6mm, 5µm column. The detection wave-length for XN was set at 370 nm, and
the elution time for XN was 21mins under chromatographic conditions described
above.
[122] Drug loading (DL %) and encapsulation efficiency (EE %) of the XN micelles
were calculated using the equation (a) and (b) below:

[123] Results: Drug loading (DL %) and encapsulation efficiency (EE %) of the
XN-loaded polymeric micelles are summarized in Table 1. As evident from Table 1,
when the method of preparation was compared, thin film hydration method was
giving much better results. While calculating drug loading capacity of the micelles
25 formed, it was found that there was about 7 to 11 fold increase in the drug loading
42

capacity of the micelles when prepared using thin film hydration method. It increased
from 0.78 % to 8.93 % when prep 2 with ratio of 1:2.9 of solubilizer to poloxamer
407 was used, which had active to poloxamer ratio of 1:0.5. There is also significant
increase in the encapsulation efficiency when the two methods of preparation of
5 micellar composition were compared. As we can see from Table 1 encapsulation
efficiency increased from 52 % when micelle was prepared using thin film dispersion
method to 96 % when the best ratio of thin film hydration was used. Based on these
results, it can be inferred that micelles prepared using thin film hydration method
with at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9 and
10 said at least one solubilizer to said at least one active ingredient w/w ratio is 1:0.4 has
the best drug loading and encapsulation efficiency.
43

Table 1: Determination of Drug Loading Entrapment Efficiency of different micellar compositions

Preparation Method Drug Loading (DL %) Encapsulation efficiency (EE %)
Thin Film Dispersion
Prep 1 0.78 52
Thin Film Hydration
Prep 1 5.84 64
Prep 2 8.93 96
Prep 3 6.64 74
Example 5
5 Cellular Uptake of XN Micelles
[124] Cellular uptake of XN micelles by HaCaT cells was quantitatively measured using confocal laser scanning microscopy (CLSM). Briefly, HaCaT cells were plated in 6 well plate (5 × 105 cells per well) containing complete DMEM medium and incubated overnight. Then the cells were washed and treated with an equivalent dose
10 (10μg/ml) of free XN and aqueous XN loaded micelles prepared by thin film
dispersion and thin film hydration method. After 18 h, the cells were washed thrice with 1X PBS (phosphate buffer saline) for 5 mins and imaged immediately using Zeiss LSM 710 confocal microscope with 488 nm laser and 40X objective. The cellular fluorescence intensity was also quantitative measured using Zeiss 2010
15 software.
[125] Results: The cellular uptake of free XN and XN micelles prepared by dispersion or hydration methods was evaluated using CLSM. In Figure 2 Panel A shows the CLSM images of HaCaT cells treated with free XN, Panel B shows the cells exposed to XN micelles prepared by dispersion method and Panel C, D, E shows
20 the cells exposed to XN micelles prepared by hydration method. After 18 h, very
less/moderate level of fluorescence intensity (Green colour) was observed in HaCaT
44

cells that are exposed with free XN (Panel A) or XN micelles (dispersion method; Panel B). On the other hand, the HaCaT cells treated with XN polymeric micelles (hydration method; Panel C, D, E) showed increased level of fluorescence intensity. The increase in the level of fluorescence inside the cytoplasm confirms that micelles 5 prepared by hydration method facilitate the diffusion of XN across the cellular membrane and increases the cellular bioavailability.
[126] Furthermore, the mean fluorescence intensity of HaCaT cells treated with free XN and XN micelles was measured and illustrated in Figure 3. The increased level of mean fluorescence intensity was observed in cells treated with XN micelles prepared
10 by hydration method (prep1-15.04 %, prep 2-99.92 % and prep 3 - 89.56%) when compared to free XN or XN micelles (20%) prepared by thin-film dispersion method. [127] In order address the poor aqueous solubility and bioavailability of XN here, a micellar composition has been developed comprising of poloxamer 407, and D-α-Tocopheryl polyethylene glycol 1000 succinate polymeric micelles that are capable
15 of solubilizing a broad range of poorly water-soluble drugs like xanthohumol and enhance their systemic bioavailability.
Example 6
Nitric Oxide Production
[128] The poor bioavailability xanthohumol has rendered it difficult to conduct
20 detailed analyses of the effects of xanthohumol. As micellar compositions of xanthohumol were formulated, the effects of this active on proinflammatory responses was analysed. For analysing nitric oxide production, HaCaT cells were seeded at a density of 5 × 105 cells per well in DMEM medium and incubated at 37 °C with a humidified 5% CO2 atmosphere. After 18 h, to induce inflammation, the
25 cells were exposed to160µW of UVB for 60 seconds. Immediately the cells were washed and treated with equal concentrations (10μg/ml) of aqueous free XN or XN loaded micelles prepared by thin film dispersion and thin film hydration. The cells were then incubated in serum-free DMEM medium for 24 h.
45

[129] The concentration of Nitric Oxide (NO) in culture supernatants was
determined as nitrite using Griess reagent. Briefly, 100μL of cell culture medium
with an equal volume of Griess reagent in a 96-well plate was incubated at room
temperature for 10 min. Then the absorbance was measured at 540 nm in a microplate
5 reader (Thermo Fisher, Germany). The amount of nitrite in the media was calculated
from sodium nitrite (NaNO2) standard curve.
[130] Results: To evaluate the effects of XN micelles on nitric oxide (NO) production, UVB stimulated HaCaT cells were treated with free XN and XN loaded polymeric micelles and incubated at 37°C for 24 h. The NO production was
10 significantly suppressed in HaCaT cells treated with XN micelles prepared by thin-
film hydration method prep 1 (28.9%), prep 2 (48%) and prep 3 (32.5%) (Figure 4). The inhibition of NO production was much higher in cells treated with prep 2 XN micelles (48%) when compared with the cells treated with free XN (26.1%) or XN micelles (27%) prepared by thin-film dispersion method. This indicates that XN
15 micelles prepared by thin film hydration using prep 2 as composition of the micelle,
has a significant enhancement in the bioavailability of xanthohumol.
Example 7
Effect of micellar composition on IL-6 and IL-8 cytokine production
[131] HaCaT cells were seeded at a density of 5 × 105 cells per well in DMEM
20 medium and incubated at 37°C. After 18 h, the IL-6 and IL-8 secretion was
stimulated by treating the cells with 160µW of UVB for 60 seconds. Immediately the cells were washed and treated with an equivalent dose (10μg/ml) of aqueous free XN, or XN loaded micelles (prepared by thin film dispersion method and thin film hydration method) and incubated in serum-free DMEM medium for 24 h. The culture
25 supernatant was collected and the levels of secreted cytokines was determined using
an ELISA kit (Krishgen BioSystems, India).
[132] Results: The IL-8 and IL-6 cytokine production in HaCaT cells was significantly reduced in cells treated with XN micelles prepared by thin-film
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hydration method prep 1 (IL-6: 21%; IL-8: 64.3%), prep 2 (IL-6: 37.9%; IL-8: 74.3%), and prep 3 (IL-6: 29.4%; IL-8: 68.8%) (Figure 5A and B). The increased level of IL-6 and IL-8 inhibition was observed in cells treated with XN micelles prepared by thin-film hydration method prep 2 (IL-6: 37.9%, IL-8: 74.3%) when 5 compared with cells treated with free XN (IL-6:26.2 %; IL-8:51%) or XN micelles prepared through thin-film dispersion method (IL-6:27.3%; IL-8:59.4%). In this case also, higher bioavailability of xanthohumol was observed when a micellar composition using thin film hydration was observed.
Example 8
10 NFκB p65 (RelA) ser536 phosphorylation assay
[133] HaCaT cells (5 × 105 cells per well) were seeded in 12-well plates until 80% confluent. After 18 h, NFκB p65 ser 536 phosphorylation was stimulated by treating the cells with 160µW of UVB for 60 seconds. Immediately after, the cells were washed and treated with an equivalent dose (10μg/ml) of aqueous free XN, or XN
15 loaded micelles prepared by thin fim dispersion method or thin film hydration method, and incubated in serum-free DMEM medium for 24 h. [134] Detection of NFκB p65 ser536 phosphorylation was performed using sandwich ELISA (Cell Signalling Inc). Briefly, the cells were washed using ice cold 0.1M phosphate buffer saline and lysed using 0.5 ml ice cold 1X cell lysis buffer
20 (20mM Tris-HCl (pH 7.5); 150mM NaCl; 1mM Na2EDTA; 1mM EGTA; 1% triton; 2.5mM sodium pyrophosphate; 1mM beta-glycerophosphate; 1mM Na3VO4; 1µg/mL leupeptin; 1mM PMSF (phenylmethylsulfonyl fluoride) (immediately before use) and incubated on ice for five minutes. The cell lysate was collected and lysed using probe sonication (sonication with one cycle of five seconds at 25% power). Then the cell
25 lysate was centrifuged at 14,000 RPM for 10 minutes at 4ºC. Adequate amount of lysate was transferred to antigen coated 96 well plates and incubated at 37ºC overnight. After washing the wells with 1X wash buffer, 100μL of NF-κB p65 detection antibody was added and incubated at 37 ºC. After 1 h, the wells were
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washed using 1X wash buffer and 100 µL of horseradish peroxidase (HRP) -linked secondary antibody was added and incubated at 37ºC. After 30 min, again the wells were washed using 1X wash buffer and 100 µL of 3, 3′, 5, 5′-tetramethylbenzidine (TMB) buffer was added. The stop solution was added to each well after 30 mins and 5 the absorbance was recorded at 450 nm.
[135] Results: Stimulation of HaCaT cells with UVB (160µW, 1 min) rapidly caused NF-κB p65 phosphorylation, which was significantly reduced when cells were treated with XN micelles prepared by thin-film hydration method prep 1 (22.9%) prep 2 (44.4%) and prep 3 (32.5%) (Figure 6). Increased level of inhibition was
10 achieved in cells treated with XN micelles prepared by thin-film hydration method prep 2 (44.4%) when compared with cells treated with free XN (24 %) or XN micelles (30.5%) prepared by thin-film dispersion method.
[136] Although many studies have reported the protective effects of xanthohumol, the present investigation studied the effect of XN-loaded polymeric micelle and free
15 XN on the inflammatory factors such as nitric oxide production, proinflammatory cytokines such as IL 6 and IL 8 and NF-κB using UVB stimulated human keratinocyte cell line, HaCaT.
[137] Overall, the present disclosure provides a micellar composition comprising xanthohumol as an active ingredient and poloxamer 407, and D-α-tocopheryl
20 polyethylene glycol 1000 succinate as the biodegradable micellar composition. Bioavailability and solubility of xanthohumol in said micellar composition was confirmed with the help of different cellular assays, which itself was an unexpected finding. Thus, a combination of xanthohumol in this micellar composition, shows synergism in the bioavailability of xanthohumol as confirmed with the different assay
25 performed. Further, identification of this specific composition that can aid in different diseases cannot be arrived without undue experimentation, and is thus not obvious to a person skilled in the art.
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I/We claim:
1. A composition comprising micelles or micellar clusters thereof comprising:
(a) at least one active ingredient encapsulated in the interior of the micelles; and
(b) the micelles comprising:
5 i. poloxamer 407; and
ii. at least one solubilizer.
2. The composition as claimed in claim 1, wherein said at least one active ingredient
is xanthohumol.
3. The composition as claimed in claim 1, wherein said at least one solubilizer is
10 selected from the group consisting of polyethylene glycol alkyl ethers, polypropylene
glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octyl phenyl ethers,
polyethylene glycol alkyl phenyl ethers, glycerol alkyl esters, polyoxyethylene glycol
sorbitan alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol
and polypropylene glycol, polyethoxylated tallow amine, cocamide MEA, cocamide
15 DEA, dodecyldimethylamine oxide, D-α-tocopheryl polyethylene glycol, and
combinations thereof.
4. The composition as claimed in claim 1, wherein said at least one solubilizer is D-α-
tocopheryl polyethylene glycol 1000 succinate.
5. The composition as claimed in claim 1, wherein said at least one solubilizer to
20 poloxamer 407 w/w ratio in said micelle is in the range of 1:1 - 1:6, and said at least
one solubilizer to at least one active ingredient w/w ratio is in the range of 1:0.1-1:0.8.
6. The composition as claimed in claim 1, wherein said at least one solubilizer to
poloxamer 407 w/w ratio in said micelle is in the range of 1:1.5- 1:4, and said at least
25 one solubilizer to said at least one active ingredient w/w ratio is in the range of 1:0.1-
1:0.5.
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7. The composition as claimed in claim 1, wherein said at least one solubilizer to poloxamer 407 w/w ratio in said micelle is 1:2.9 and said at least one solubilizer to said at least one active ingredient w/w ratio is 1:0.4.
8. A method of preparing micelles or micellar clusters thereof as claimed in any of the
5 claims 1-7, said method comprising:
(a) contacting said at least one active ingredient, poloxamer 407, and said at least one solubilizer in the presence of at least one organic solvent to obtain a mixture;
(b) homogenising said mixture to obtain a clear and transparent solution;
(c) removing said at least one organic solvent from said solution at a temperature in
10 the range of 30-50°C to obtain film of micelles or clusters thereof encapsulating
said active ingredient; and
(d) contacting said film with water to obtain a solution of micelles or clusters
thereof.
9. The method as claimed in claim 8, wherein said at least one organic solvent is
15 selected from the group consisting of ethanol, chloroform, methanol, tertiary butanol,
trimethylamine, diethyl ether, dichloromethane, trichloromethane, cyclohexane, acetonitrile, and combinations thereof.
10. The method as claimed in claim 8, wherein said at least one organic solvent is
acetonitrile.
20 11. The method as claimed in claim 8, wherein removal of said at least one organic
solvent in step (c) is carried out at a temperature of 40 °C.
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12. A method of increasing xanthohumol bioavailability in cells, said method comprising:
(a) obtaining a composition as claimed in any of the claims 1-7; and
(b) contacting said composition with cells,
5 wherein said composition increases xanthohumol bioavailability in cells.