Compositions and methods of preparation of bioactive triterpenoid milk fat globule formulations

Abstract

The present invention relates to compositions for oral or topical administration to humans and methods for their preparation. The present invention relates to pentacyclic triterpenoid compounds, in which the triterpenoid compounds are incorporated into lipid vesicles surrounded by a triple-layer membrane, preferably milk fat globules. The triterpenoid compounds are preferably betulinic acid, betulin, lupeol, oleanolic acid, and / or ursolic acid, most preferably betulinic acid. Incorporation of the triterpenoid compounds into milk fat globules or lipid vesicles at a high payload of at least 10-50 mg / ml is preferably achieved by high-energy emulsification techniques, most preferably sonication. In a preferred embodiment, the composition is present as a liquid emulsion formulation. In another preferred embodiment, the composition is a powder, which can be prepared from a liquid embodiment of the composition by, for example, spray drying or freeze-drying techniques. The composition may be used as a pharmaceutical, dietary supplement, or functional food for the treatment or prevention of cancer, type 2 diabetes, obesity, and cardiovascular disease.

Description

[Technical Field]

[0001] The present invention relates to particular formulations of bioactive pentacyclic triterpenoids, such as betulinic acid and betulin, especially for use as medicines or functional foods, and also for the treatment or prevention of cancer. [Background technology]

[0002] 1. Background of the Invention Plant-derived molecules, either directly or as lead compounds, form the basis of important chemotherapeutic agents for cancer and other diseases [1,2] and have great potential as novel drugs [3]. Pentacyclic triterpenoids are one of the largest classes of natural plant secondary metabolites, possessing highly beneficial biological effects and pharmacological potential. These compounds possess a 30-carbon skeleton containing five ring structures: either five six-membered rings (ursane and oleanane subtypes) or four six-membered rings and one five-membered ring (lupane subtype).

[0003] The phytochemical compound betulinic acid is a member of the lupane-type pentacyclic triterpenoids and has established anticancer activity in vitro and in animal studies. Betulin, another bioactive member of this class, is a direct analog of betulinic acid, differing only in that it lacks the carboxyl group at C-28 of the lupane skeleton. Lupeol, a third member of the lupane-type pentacyclic triterpenoids, also possesses significant anticancer and anti-inflammatory biological activities [4]. Additionally, oleanolic acid and ursolic acid, isomeric analogs of two pentacyclic triterpenoids of the oleanane and ursane types, respectively, have established favorable biological activities, particularly anticancer activity [5]. The chemical structures of these bioactive pentacyclic triterpenoids are shown in Figure 1.

[0004] Betulinic acid, betulin, lupeol, oleanolic acid, and ursolic acid are all available in large quantities in many plant species. For example, the bark of the white birch tree (Betula sericea albae) contains abundant betulin, up to 30% by dry weight [6], and approximately one-tenth that amount of betulinic acid [7]. In particular, betulinic acid is known to exhibit broad-spectrum activity against all major cancer types in vitro [8] and inhibits tumor growth of major cancer types in in vivo preclinical studies [9]. Other activities of this compound have also been established, including anti-inflammatory, anti-HIV, and beneficial effects against type 2 diabetes [10-12].

[0005] Importantly, betulinic acid has been shown to be non-cytotoxic to non-cancerous healthy cells in vitro, and there is no evidence of toxicity even at concentrations (up to 500 mg / kg body weight) that effectively inhibit cancer growth in animal studies [9,13], indicating its favorable therapeutic index for clinical use.

[0006] Mechanistically, betulinic acid's anticancer potential has been pinpointed to diverse, partially interrelated effects, pathways, and molecules. The selective cytotoxicity of betulinic acid toward cancer cells can be explained by its specific mechanistic effects on metabolic pathways adapted in dividing cancer cells compared with healthy cells, rendering them vulnerable and selective targets for drug intervention.

[14] For example, betulinic acid has been shown to inhibit the activity of stearoyl-CoA desaturase-1 (SCD-1), an enzyme whose correct function in cellular lipid metabolism is essential for cancer cell division and tumor growth.

[15] Similarly, the effects of betulinic acid on glucose metabolism in cancer cells

[16] are altered compared with healthy cells (the so-called Warburg effect

[17] ), likely contributing to the compound's selectivity toward cancer cells and, more broadly, its polypharmacology.

[18]

[0007] Taken together, these bioactive pentacyclic triterpenoids, and betulinic acid in particular, are promising drug candidates for the aforementioned diseases. Due to their lack of toxicity to healthy tissues, their preclinical established synergistic and priming effects in combination with standard cancer therapeutics [19-23], and their proposed role in the prevention of cancer [24-26] and other diseases, such as hepatotoxicity

[27] , type 2 diabetes

[28] , and diabetes-related disorders [29,30], betulinic acid in particular is not only a potential cancer adjuvant therapy but also a promising functional food or nutraceutical.

[0008] However, despite their promising activity profile and lack of toxicity, preclinical development of these pentacyclic triterpenoids for clinical application has been significantly hampered by significant untoward properties.

[0009] First, the bioactive triterpenoids of interest herein are extremely hydrophobic and consequently have very low water solubility, which poses a major challenge to making these compounds fully bioavailable.

[0010] Second, the molar concentrations required for the desired potent biological effects are relatively high (e.g., compared to some established anticancer drugs), ranging from 10–20 μM in vitro in studies with cancer cell lines, highlighting the importance of developing formulations that not only allow good solubility of triterpenoids but also incorporate these compounds at high payloads

[31] .

[0011] To address the low water solubility of triterpenoids of interest, there are essentially two available strategies in the art that have been extensively explored in numerous studies: semisynthetic derivatives with higher solubility than their natural triterpenoid analogues have been explored [11,32-35], or pharmaceutical carriers and delivery systems have been developed aiming at high solubility and efficient bioavailability of the compounds.

[0012] However, despite the identification of several interesting derivatives, none of the compounds have reached clinical use as pharmaceuticals or functional foods. Therefore, considering that the water solubility of prior art triterpene derivatives has only improved slightly, if at all, coupled with the abundant occurrence of natural compounds in plant species (e.g., birch trees are rich in betulinic acid and betulin), natural pentacyclic triterpenoid compounds must still be considered as prime candidates for development for human use.

[0013] 1.1 Desirable properties of triterpenoid formulations A prerequisite for the use of natural pentacyclic triterpenoids, especially betulinic acid and betulin, is the development of drug carrier systems that allow their efficient solubilization. Importantly, formulations that address the two unfavorable properties of pentacyclic triterpenoids addressed herein—their low solubility and the required high in vivo bioavailability—by efficiently incorporating triterpenoids at high concentrations must not only have a high payload of compounds, but also enable their efficient intestinal delivery, incorporation, and absorption after oral administration [36,37].

[0014] Oral administration is the preferred route of administration for triterpenoid compounds when used as adjuvants in combination with cancer treatment [19,20], and when used as dietary supplements or functional foods for anticancer chemoprevention

[25] , as well as for the chemoprevention of other disease states and to achieve other beneficial health effects.

[0015] Therefore, a drug carrier ideally meets three requirements. First, it must efficiently solubilize triterpenoids in the carrier system with a high drug loading capacity, where drug loading capacity is defined as the mass fraction of the drug carrier to be encapsulated. Second, the carrier system must ensure efficient in vivo intestinal absorption of the encapsulated triterpenoid compounds. Third, it is desirable that the triterpenoid delivery system itself be devoid of excipients that may cause adverse toxic side effects, especially when used pharmaceuticalally in combination with standard cancer chemotherapy or as a functional food.

[0016] Taken together, developing such a delivery system is a major challenge and is an ongoing issue that must be resolved for the human application of these promising pentacyclic triterpenoids.

[0017] 1.2 Formulation of triterpenoids in the art, detailed for betulinic acid The field of drug delivery has developed rapidly over the past decade, with various organic nanoparticle-based systems being developed to solubilize drugs with poor bioavailability. Various of these nanoparticle-based drug carriers have been investigated for their ability to solubilize and transport the pentacyclic triterpenoids betulinic acid, betulin, lupeol, oleanolic acid, and ursolic acid, or their semisynthetic derivatives. These novel formulations, primarily incorporating triterpenes, have been tested in different model systems, both in vitro and in vivo [38,39]. Various delivery systems, particularly for betulinic acid, have been devised and tested, the most important of which are discussed herein and summarized in Table 1.

[0018] Nanoscale delivery systems developed for betulinic acid include, among others, carbohydrate-based polymer nanoparticles, e.g., cyclodextrin

[40] and chitosan

[41] , dendrimers

[41] , poly(lactic-co-glycolic acid) (PLGA) nanoparticles [42, 43], and betulinic acid-containing PLGA nanoparticles copolymerized with polyethylene glycol (PEG)

[44] .

[0019] Lipid-based nanoparticles are another broad class of carriers applied to the delivery of lipophilic anticancer compounds

[45] . Lipid carriers generally have fairly good loading capacities. Their large-scale production from natural sources is often possible at reasonable cost, depending on the specific lipid carrier type. Furthermore, lipid-based formulations are generally considered beneficial for the intestinal absorption of lipophilic active pharmaceutical ingredients contained in the carrier

[46] . Within this category, betulinic acid has been tested in vitro and in vivo as a carrier encapsulated in liposomes [9, 47] and in water-in-oil nanoemulsions

[48] .

[0020] Despite many efforts in the field of betulinic acid drug delivery, none of the formulations reported to date has proven to be particularly favorable. Generally, liposomes, which exist in a wide variety of forms, are the lipid (nano)particles most commonly used as drug carriers

[49] . Although liposomes have excellent potential as drug carriers, they also have drawbacks associated with them, such as their moderate loading capacity for hydrophobic compounds and the high manufacturing costs of high-performance liposome types

[50] .

[0021] For the delivery of bioactive triterpenoids in pharmaceutical and nutraceutical contexts, the carrier ideally should encapsulate a sufficiently high amount of the active compound, and the formulation should possess additional advantageous properties when administered orally. Liposomes likely do not fully meet these requirements, as studies have shown. For example, the maximum loading capacity of betulinic acid in large liposomes appears to be 5 mg of betulinic acid per ml of liposomes in studies where betulinic acid was tested in vivo [9]. This indicates that an 80 kg individual would require 12 g of betulinic acid per week, as extrapolated from the weekly oral betulinic acid dose of 150 mg per kg body weight required to achieve the antitumor effect observed in mice [9]. Therefore, a human would require a weekly intake of 2.4 liters of such a liposomal formulation (at 5 mg / ml betulinic acid). Such an intake is not feasible. Patient (or client) compliance rates would be very low, and furthermore, such extremely high daily doses of lipid liposome vehicles are not healthy.

[0022] Recently developed proliposomes, powdered carbohydrate carriers coated with phospholipids, can be reconstituted into liposomes upon addition of water

[51] , but they suffer from the same problem: excessive amounts of excipients are required to achieve sufficient bioavailability of the active triterpenoids.

[0023] The cancer preventive (i.e., chemopreventive) effect of betulinic acid against cancer may be achieved at relatively low in vivo concentrations, and extrapolation from mouse studies to humans may require a somewhat lower human dose per kg body weight due to the high metabolic rate in mice.

[52] However, the effective oral use of pentacyclic triterpenoid preparations for human health promotion will likely depend heavily on the payload of the active pharmaceutical ingredient in the preparation.

[0024] The initial focus in developing a suitable formulation for oral administration of triterpenoids is therefore their high payload, preferably at a concentration significantly exceeding the aforementioned 5 mg active ingredient / ml formulation (which is the highest liposomal payload achieved in the prior art), which is the main problem to be solved in this field.

[0025] Furthermore, any oral formulation should ensure good intestinal absorption of the triterpenoids, and the excipients of the delivery system should be devoid of any toxicity and should preferably be approved as food additives, e.g., listed in the FDA's "Generally Recognized as Safe" (GRAS) category, and preferably themselves constitute natural foods or ingredients thereof.

[0026] Liposomal formulations are generally not considered optimal drug carriers for oral administration [53,54]. Liposomal and other triterpenoid formulations developed to date all have the aforementioned weaknesses, which are described in detail in the case of betulinic acid, and the key literature is summarized in Table 1. Therefore, there is a continuing need for suitable formulations for bioactive triterpenoids that allow their oral administration in feasible dosage forms and at active compound dosages that achieve high bioavailability.

[0027] SUMMARY OF THE INVENTION It is an object of the present invention to overcome one or more of the above problems, as well as possible related problems. Summary of the Invention

[0028] 2. Summary of the invention In one aspect, the present invention provides a pharmaceutical composition of a particulate carrier system for the oral administration of high doses of extremely hydrophobic bioactive pentacyclic triterpenoids, such as betulinic acid, betulin, lupeol, oleanolic acid, and ursolic acid. The present invention also provides methods for their preparation.

[0029] The triterpenoid formulations of the present invention preferably consist of natural (food) ingredients, in particular milk fat globules, as the main excipient and can be applied as functional foods or pharmaceuticals for the improvement of general health and for the treatment, chemoprevention, or amelioration of common disease states such as cancer, cardiovascular disease, and type 2 diabetes without harmful side effects.

[0030] High-energy solubilization and emulsification methods, particularly high-shear mixing combined with sonication, can be used in the present invention to solubilize and load triterpenoids at high concentrations into milk fat globules, which are preferably the primary excipient of the particulate drug carrier system.

[0031] 2.1 Needs to be addressed in this field The bioactive pentacyclic triterpenoids of the present invention (the chemical structures of which are shown in Figure 1), particularly betulinic acid and betulin, and more particularly betulinic acid, have great potential as drugs or nutraceutical compounds for the treatment, chemoprevention, and amelioration of common and serious disease states, as fully supported by numerous preclinical in vitro and animal studies.

[55] For human use, however, important properties remain lacking in this field, which is the main reason why few clinical trials of bioactive triterpenoids have been conducted.

[0032] First, the pharmacologically active doses of these triterpenoids are usually relatively high (e.g., compared with many established anticancer drugs). As estimated from animal studies, high doses are beneficial for the production of effective triterpenoid bioactivity. [9] Such high doses can be administered because they are not toxic to healthy cells and tissues. [9] Therefore, the most important point is that for clinical use, the formulation composition and its manufacturing process must enable efficient solubilization and achieve very high incorporation concentrations of triterpenoids in drug delivery formulations.

[0033] Ideally, the triterpenoid carrier should also be efficiently taken up and absorbed in the gastrointestinal tract after oral administration, making the triterpenoid available at high in vivo doses.

[0034] Furthermore, and a third important consideration, the excipients in a drug carrier system are ideally biocompatible, preferably composed of food-grade ingredients, and necessarily free of adverse effects at high administered doses, and more desirably, the excipients themselves provide additional health benefits.

[0035] Finally, the high stability of the formulation and its cost-effective manufacture are highly desirable and beneficial properties of the preferred formulation.

[0036] The invention disclosed herein is the result of comprehensive research to discover a solubilization and carrier system that meets all the above-mentioned key points, thereby providing a solution to the need in the art.

[0037] This study examined various candidate drug delivery compositions and their possible formulation methods for the pentacyclic triterpenoids addressed herein, particularly betulinic acid. In addition to liposomes, which are currently a highly established drug carrier platform, particularly for hydrophilic compounds, but also for lipophilic compounds, various other organic systems, particularly lipid-based carrier systems, were analyzed. Furthermore, many different methods for successfully solubilizing, assembling, and incorporating triterpenoids into these candidate drug carrier systems were analyzed.

[0038] 3. Overview of the Invention 3.1 Drug Delivery Compositions of Triterpenoids Assembled with Milk Fat Globules The present invention discloses a microparticle formulation that is composed of target bioactive five-ring triterpenoids combined with milk fat globules, and is preferably assembled using high-energy emulsification methods, particularly ultrasonication.The formulation of the present invention is a highly preferred composition for solubilizing and encapsulating a high dose of these bioactive five-ring triterpenoids, and their subsequent non-toxic oral delivery results in predicted high bioavailability.The triterpenoid is preferably either betulinic acid or betulin, and in the most preferred embodiment, betulinic acid.

[0039] Compositions of lipophilic pentacyclic triterpenoids solubilized and assembled with milk fat globules appear to be preferred drug carriers and delivery systems in all of the above desirable embodiments.

[0040] In particular, the high payload of triterpenoids in this delivery system is the result of the combination of specific properties of bioactive triterpenoid compounds on the one hand and milk fat globules on the other hand. Taken together, this set of combined properties allows for efficient solubilization of high loading levels of triterpenoids in milk fat globule-based triterpenoid carriers.

[0041] 3.2 Overview of milk components and milk fat globule structure Milk is a unique, evolutionarily developed source of energy, structure-forming molecules, and protection for newborns. Animal milk, particularly that of bovine origin, and its derivative dairy products are common foods worldwide. Essentially, milk is a colloidal, multiphase, polydisperse system containing protein particles and lipid vesicular structures. The major proteins in milk are casein, organized in micellar particles [56, 57], and whey proteins

[58] . With regard to the lipid content of milk, milk can be considered a water-in-oil macroemulsion.

[0042] Milk, before any processing, contains 2%–5% lipids.

[59] These lipids are organized into milk fat globules, which have a highly structured structure that is only present in milk.

[0043] The size of milk fat globules in raw milk ranges from 0.1 to 15 μm in diameter

[60] . Milk fat globules consist of a triglyceride core enclosed in a three-layer lipid membrane that assembles during secretion of milk fat globules from the epithelial cells of the mammary gland

[61] .

[0044] More specifically, the generally accepted physiological process is that a triglyceride core is first enveloped in the cytoplasm by a monolayer of polar lipids and proteins. Then, during milk fat globule secretion, a bilayer membrane derived from the secreting epithelial cell is added, which contains glycosylated and non-glycosylated proteins, glycerophospholipids, sphingolipids, enzymes, and cholesterol. An additional interstitial layer of protein is formed between the monolayer and the bilayer lipid membrane during secretion. Together, these form a 10–50 nm thick multilayer envelope that constitutes the triply structured milk fat globule membrane (MFGM)

[60] .

[0045] 3.3 Emulsifying function of components in the milk fat globule membrane Importantly, the amphiphilic properties of polar lipids in MFGM along with proteins in the membrane are crucial for preventing aggregation of triglycerides in the core of milk fat globules, thereby ensuring the water-in-oil emulsion properties of milk

[62] . Thus, membrane components function as important emulsifiers to hold milk fat globules in the emulsion, and thus different roles exist for polar lipids and proteins in MFGM

[63] .

[0046] Homogenization is a common process performed on consumer milk industrially processed from bovine sources to increase its fat stability, significantly reduce the average size of milk fat globules, and also enhance their emulsifying properties by inducing the attachment of casein micelles to the milk fat globule membrane [64-67].

[0047] 3.4 Aspects of Triterpenoid-Milk Fat Globule Formulations The present invention discloses a solubilization and carrier system for target triterpenoids in a particulate (also known as microvesicle) composition that is fully integrated with and integrated into milk fat globules.

[0048] The triterpenoid formulations of the present invention have the properties of lipid vesicles which, in a preferred embodiment, can be contained in a high viscosity emulsion.

[0049] Furthermore, the triterpenoid-milk fat globule compositions of the present invention can be present in or processed into various adapted forms, such as, for example, an emulsion which is further diluted in a dairy-based beverage, an ointment or cream for topical administration, or in powdered form by spray drying, freeze drying or similar techniques, which powder can then be used, for example, in a dietary supplement or beverage.

[0050] Depending on the initial dairy source of the milk fat globules used for its preparation (i.e., either raw milk or one of several possible processed dairy products, such as homogenized milk), the compositions of the present invention may contain certain amounts of additional milk-derived components. In particular, casein micelles and / or whey proteins may be present in certain amounts, or these milk components may be absent or almost absent in the compositions of the present invention.

[0051] In some embodiments of the present invention, these additional milk-derived components may be intentionally added to the milk fat globules used in the preparation of the triterpenoid composition because they may impart beneficial properties. For example, casein micelles that attach to milk fat globules may further improve the emulsifying properties of the milk fat globule membrane, and similarly, whey proteins may help to further improve emulsion stability

[68] .

[0052] 3.5 Efficient drug loading in triterpenoid-milk fat globule formulations compared to nanocarrier systems The development of drug delivery systems has progressed dramatically over the past few decades, revolutionizing the field. In particular, nanosized colloidal carriers, either lipid- or polymer-based, have attracted much attention and have been developed in a variety of forms. Nanocarriers are defined as particles with a diameter of less than 100 nm, although particles up to several hundred nm (and even 500 nm) are often referred to as nanosized

[69] . Nanocarriers have several advantageous properties compared to microsized delivery systems.

[0053] The high surface area-to-volume ratio of nanocarriers promotes their solubility, bioavailability

[65] , and stability, while their small size helps prevent immune-mediated clearance by macrophages of the reticuloendothelial system.

[70] Thus, nano-sized particles exhibit long circulation times in cancer, and depending on the tumor microenvironment, a passive targeting mechanism known as the enhanced permeation and retention (EPR) effect can advantageously result in higher accumulation of carrier-loaded drugs in tumors.

[71]

[0054] Nanocarriers have been developed in a variety of forms and can be classified according to the origin of their excipients (natural or synthetic), whether they are organic or inorganic (the latter being, for example, diagnostic gold nanoparticles), the basis of their chemical matrix (lipid- or polymer-based) and their physicochemical particle composition (solid particles or core-shell vesicles with a water-soluble core) [71-73].

[0055] Generally, and as recognized by those skilled in the art, for strongly hydrophobic pharmaceutical active ingredients such as pentacyclic triterpenoids, lipid-based compositions are reasonably envisioned as suitable drug (nano)carriers. However, various polymer-based (nano)carriers also have the capacity to carry hydrophobic compounds

[74] .

[0056] Lipid-based colloidal nanocarriers can be classified into (1) liposomes, (2) solid lipid nanoparticles (SLNs), (3) nanostructured lipid carriers (NLCs), and (4) nanoemulsions [74,75].

[0057] Historically, liposomes, which are core-shell lipid-based (i.e., aqueous core contained within one or more lipid bilayers) carriers, have been the longest studied in this field since their invention in the 1960s. Nano-sized liposomes are classified as nanocarriers, although much larger liposomes (up to >10 μm) also exist.

[0058] In the mid-1990s, solid lipid nanoparticles (SLNs) and, somewhat later, nanostructured lipid carriers (NLCs) were developed to overcome some of the drawbacks of liposomes.

[74] SLNs have a hydrophobic core of solid lipids, such as triglycerides, waxes, or fatty acids, stabilized by a phospholipid monolayer and / or emulsifiers, such as Tween or bile salts, which act as coatings and surfactants. As a next step, NLCs were developed by replacing part of the solid lipid core of SLNs with liquid lipids. This creates space for further drug loading of hydrophobic active pharmaceutical ingredients (APIs) and improves long-term stability by preventing API repulsion from the lipid core.

[0059] Water-in-oil (O / W) nanoemulsions are quite similar to SLNs and NLCs, but are composed entirely of liquid lipids and surrounded by an emulsified monolayer of phospholipids. Regarding drug loading properties, liposomes are primarily suitable for the delivery of hydrophilic APIs (in the aqueous core); liposomes can also contain hydrophobic APIs in their lipid bilayer, although less efficiently

[74] . The properties of SLNs, NLCs, and O / W nanoemulsions make them uniquely suitable for the delivery of lipophilic bioactive compounds.

[0060] The intense attention in the drug delivery field over the past two decades towards small nano-sized drug carriers is thought to be somewhat overrated [76-78], especially since micrometer-sized delivery systems have certain advantages in some ways and depending on the intended use. Importantly, the loading capacity of nano-sized particles is significantly lower than that of larger carriers (such as large liposomes or (macro)emulsions) due to the small particle volume

[79] .

[0061] Indeed, it has been shown that betulinic acid can be incorporated at a much higher payload in large liposomes compared to small nano-sized liposomes for triterpenoid delivery [9]. The most important betulinic acid formulations reported in the literature are summarized in Table 1.

[0062] The current biased emphasis on nanosized delivery systems may therefore hinder the discovery of optimal drug formulation systems for the (oral) delivery of pentacyclic triterpenoids. The limited availability of technical expertise in industry and academic research groups is known to introduce another bias that hinders the discovery of optimal formulation approaches

[80] .

[0063] The present invention is the result of a thorough analysis of the formulation problem to be solved, particularly with regard to the chemical properties of the triterpenoids of interest, combined with extensive experimental research efforts.

[0064] The milk fat globule-triterpenoid drug carriers of the present invention are capable of carrying very high payloads of triterpenoids, which can be explained by the unique combination of the properties of milk fat globules as excipients on the one hand and the properties of triterpenoids on the other hand, with the particularly preferred triterpenoid being betulinic acid or betulin.

[0065] As shown in the experimental section: Betulinic acid and betulin can be efficiently solubilized and incorporated into milk fat globule-betulinic acid (or betulin) formulations by using ultrasonication techniques as described and shown in Examples 1 and 2. The milk fat globule-betulinic acid (or betulin) formulation of the present invention contains a high payload of betulinic acid (or betulin), as shown in Example 2. The incorporation of betulinic acid (or betulin) alters the properties of milk fat globule compositions, as shown in Examples 3 and 4. Furthermore, the milk fat globule-betulinic acid formulation has high stability, as shown by the data in Example 5.

[0066] 3.6 Triterpenoid and milk fat globule characteristics that synergize to high drug loading levels The special properties of milk fat globules, on the one hand, and the bioactive pentacyclic triterpenoids of the present invention (preferably betulinic acid and betulin), on the other hand, jointly support the favorable properties of milk fat globule-triterpenoid formulations. In general, milk fat globules are known to be equipped to carry lipophilic bioactive substances [81-83].

[0067] As disclosed by the present invention, drug loading of pentacyclic triterpenoids appears to be highly efficient in milk fat globules, especially when performed by appropriate loading methods.

[0068] The relatively large size of (bovine) milk fat globules (4 μm on average, but the average size may be smaller after a possible prior homogenization process) provides a relatively large volumetric content for lipophilic drug loading in the triglyceride core. Furthermore, the size and composition of the milk fat globule membrane (a complex three-layer membrane composed of different phospholipids and proteins, with very good emulsifying properties) also makes it well suited to contain high amounts of hydrophobic bioactive components.

[0069] The unexpected efficiency and high triterpenoid loading capacity of the composition of the present invention reveal that the properties of milk fat globules are completely consistent with those of pentacyclic triterpenoids. It can be theorized, and is almost certain, that some special features of triterpenoids, particularly betulinic acid or betulin, contribute to the apparently efficient physicochemical interactions between milk fat globules and triterpenoids, as detailed herein.

[0070] First, lupane-type pentacyclic triterpenoids are known to be miscible with the major structural outer leaflet membrane phospholipids (glycerophosphatidylcholine and sphingomyelin) in various molar ratios, as shown in the literature for betulinic acid and lupeol

[84] .

[0071] Second, and consistent with this finding, betulinic acid is known to function like cholesterol as a structural element in the lipid bilayer of liposomes [9], and indeed there is strong indication of a direct interaction between phospholipids and pentacyclic triterpenoids [84-86].

[0072] Third, betulinic acid can be solubilized in nanoemulsions containing triglycerides

[48] .

[0073] Therefore, these properties of pentacyclic triterpenoids, especially betulinic acid, very likely underlie the highly efficient assembly of milk fat globule-triterpenoid particles and the incorporation of betulinic acid into milk fat globules, where it is present in both the membrane and the core at high concentrations.

[0074] Drug loading of triterpenoids in the milk fat globule microvesicle structure can therefore occur in both the lipid core and the trilayer membrane, as shown in Figure 2 .

[0075] Importantly, and despite the specific advantages of nanosized carrier systems (as mentioned above), the most important requirements for effective oral administration of triterpenoids with high bioavailability are better addressed by microsized milk fat globule carrier systems, since, as shown in Figure 2, a large difference exists between the loading capacity of milk fat globules for hydrophobic compounds on the one hand, and that of nanosized carrier systems, especially (nanosized) liposomes with a water-soluble core that cannot hold lipophilic bioactive compounds on the other hand.

[0076] 3.7 Additional Advantageous Properties of Triterpenoid-Milk Fat Globule Compositions In addition to optimal drug loading properties, milk fat globules have additional properties that make them advantageous for the oral administration of pharmaceuticals and functional foods.

[0077] The bioavailability of triterpenoids after oral administration is predicted to be good when included in milk fat globule delivery systems (see Table 1). In general, milk and its components are increasingly being considered as excipients for drug delivery systems

[87] .

[0078] Although the exact digestion of milk fat globules in the gastrointestinal tract is not yet fully understood, it is well known that all nutritional components of milk, including milk fat globules, are highly digested and absorbed after oral ingestion

[88] .

[0079] As experts understand, any bioactive compounds incorporated into milk fat globules can only be released, absorbed, and become bioavailable after intestinal digestion and absorption of the milk fat globules. There are strong indications that milk fat globule membrane components can enhance the digestion of triglycerides

[89] and improve the bioavailability of bioactive compounds

[90] .

[0080] Furthermore, the formulation of the present invention was found to be stable, which is in agreement with the literature, since milk fat globule membrane material has the ability to stabilize emulsions

[91] .

[0081] Apart from the advantageous properties of milk fat globule membrane components in digestion and stability, the milk fat globule membrane components (polar lipids, proteins, and glycoproteins) themselves have also been widely demonstrated to confer beneficial health effects

[92] . Naturally, being natural food components, milk fat globules do not induce any adverse reactions when used as a carrier system. Finally, being based on common and widely available milk components, the milk fat globule-triterpenoid formulation of the present invention can be produced at a relatively low cost. DETAILED DESCRIPTION OF THE INVENTION

[0082] 4. Detailed Description of the Invention The present invention relates to a composition comprising (at least one) triterpenoid compound, preferably a pentacyclic triterpenoid compound, wherein the pentacyclic triterpenoid compound is incorporated into milk fat globules or lipid vesicles having a membrane, the membrane comprising: -phospholipid monolayer; and -phospholipid bilayer It has a three-layer structure including: Both the triglyceride core and the three-layer membrane of the milk fat globule or lipid vesicle can function as lipid compartments that can assemble with and incorporate bioactive pentacyclic triterpenoids.

[0083] Milk fat globules or lipid vesicles are surrounded by a three-layer membrane, which therefore a phospholipid monolayer, i.e., a lipid monolayer having the composition normally occurring in the triple membrane of a milk fat globule; and / or -A bilayer of phospholipids, i.e., a lipid bilayer Includes:

[0084] In one embodiment, one pentacyclic triterpenoid compound or a combination of two or more different pentacyclic triterpenoid compounds is contained in the plurality of milk fat globules.

[0085] The (spherical) lipid vesicles or globules according to the present invention have a core comprising triglycerides. The core can be considered as the part of the lipid vesicle or globule enclosed or surrounded by the (outer) trilayer or trilamellar membrane. The core can, for example, comprise at least 5% by weight of triglycerides relative to the weight of the lipid vesicle or globule.

[0086] The core is thus surrounded or enclosed by an (inner) monolayer comprising (polar) phospholipids and / or proteins, and / or an (outer) bilayer of (polar) phospholipids and / or proteins. The proteins may in particular be partially glycosylated and partially non-glycosylated, and / or the lipids may be glycerophospholipids and / or sphingolipids. The monolayer and / or bilayer may also comprise cholesterol, such as at least 1% cholesterol by weight relative to the weight of the particle or globule.

[0087] Additionally, an interstitial layer of protein may be present between the monolayer and bilayer. The (spherical) trilayer membrane, i.e., the monolayer, bilayer, and optional interstitial layer, is 10-50 nm thick. This can be considered the outer shell or membrane of the lipid vesicle or globule.

[0088] The lipid vesicles or globules of the present invention are preferably milk fat globules. The present invention therefore preferably relates to milk fat globules comprising at least one triterpenoid compound.

[0089] Thus, the present invention basically relates to compositions comprising pentacyclic triterpenoid compounds, which are incorporated into milk fat globules or into lipid vesicles having a membrane, the membrane being three-layered, including a phospholipid monolayer and a phospholipid bilayer.

[0090] The milk fat globules, preferably of bovine origin, used in compositions according to the present disclosure can be obtained by using fresh (raw) non-skimmed bovine milk as a raw material, still containing its full fat content (i.e., cream). Conventional dairy processing procedures can separate the fat fraction of the milk, including the milk fat globules. Essentially, the density difference between the milk fat and the (non-fat) skim milk fraction causes the passive separation of the lighter milk fat fraction, including the milk fat globules, which in turn rises to the surface when the whole milk (full fat content) is allowed to stand for a period of time (e.g., 24 hours). Common dairy technology can actively accelerate the separation of the cream by applying centrifugal force using a centrifuge (also called a (milk) separator). After separation, the cream fraction can be washed with water or a salt solution, if desired, to (further) remove casein and whey proteins.

[0091] Instead of raw milk as a source of milk fat globules, homogenized milk can also be used. Homogenized milk contains milk fat globules of reduced size on average, achieved by the homogenization process applied to (raw) milk to avoid cream separation (i.e., the rise of fat globules to the surface due to the aforementioned density difference). Thus, the milk fat globules obtained from homogenized milk have a significantly reduced average diameter: the size of the milk fat globules is reduced from an average of 2-12 μm to an average of <2 μm. The exact fat content in the collected cream fraction, and therefore the milk fat globule fraction, can be analyzed using routine dairy techniques, such as the Gerber fat determination method.

[0092] Dairy technology is constantly evolving, and sophisticated isolation of milk fat globule membranes is now possible

[93] . Therefore, the compositions of the present invention can be prepared using isolated milk fat globule membrane fractions as the base component, in combination with triglycerides, which provide the fat core of the lipid vesicle carrier, and the pentacyclic triterpenoids of the present invention. An obvious potential disadvantage of such artificially synthesized triterpenoid-containing lipid vesicles with a triplymembrane structure is their more laborious preparation and the (significantly) higher costs involved compared to using natural isolated milk fat globules as the base material for the vesicles of the present invention.

[0093] Thus, preferred compositions comprising pentacyclic triterpenoid compounds according to the present disclosure comprise one pentacyclic triterpenoid compound or a combination of two or more different pentacyclic triterpenoid compounds contained in multiple (natural) milk fat globules.

[0094] The lipid vesicles or globules, preferably milk fat globules, contained in the compositions of the present disclosure have a (median or mean) diameter, as measured by laser diffraction, of 0.1 to 15 μm, preferably 1 to 10 μm, more preferably 2 to 6 μm. In the present disclosure, the median particle size is preferably measured, for example, by laser diffraction measurement of each lipid vesicle or globule contained in the composition (or a sample thereof), and the D50 percentile value of such measurement represents the median particle size as described above. The D50 value may be described as the value of the particle size at 50% in the cumulative volume-weighted particle size distribution as measured by laser diffraction. Alternatively, the mean diameter (i.e., average diameter) may be measured, for example, by laser diffraction measurement of each lipid vesicle or globule contained in the composition (or a sample thereof), preferably according to the formula D[4,3]=(Σn i ·d i 4 ) / (Σn i ·d i 3 ) by calculating the volume-weighted average (i.e., mean) particle diameter (often denoted as D[4,3], also called the De Brouckere mean diameter). In other words, the mean diameter herein is preferably the sum of the fourth power of all measured particle diameters of the molecules (1-n particles, d i 4 indicates the fourth power of the diameter of the i-th particle) is the sum of the cubes of all the measured particle diameters in the denominator (1-n particles, d i 3(where σ denotes the cube of the diameter of the i-th particle). Laser diffraction data is inherently volume-weighted because the diffraction of light is proportional to the volume of the particle. Thus, laser diffraction provides volume-weighted results. Those skilled in the art are familiar with laser diffraction measurements to measure average or mean particle size. For example, this can be performed using a Bettersizer S3 Plus instrument (Bettersize Instruments Ltd, Dandong, China) or a Mastersizer™ 3000 instrument (Malvern Panalytical) according to the manufacturer's instructions. In such measurements, a laser beam is passed through the sample and the scattered light is analyzed to determine the particle diameter distribution. The diffraction pattern provides information about the diameters of the particles present, allowing the calculation of the average or mean diameter.

[0095] A slightly reduced milk fat globule size and narrower size distribution may have the advantage of better maintaining the stability of the formulation. On the other hand, a larger average size of the milk fat globules (or milk fat globule-based vesicles) advantageously adds to the final triterpenoid payload in the formulation. Thus, a trade-off likely exists between large milk fat globules (or their derived vesicles) on the one hand and small milk fat globules on the other. Depending on the specific application and the associated end product, a preferred choice may be made between large milk fat globules (with a broad particle size distribution) or those with an average reduced size (such as those obtained by the homogenization procedure described above). Milk fat globules with a slightly smaller size and narrower size distribution are expected to have a more favorable overall combination of properties.

[0096] The pentacyclic triterpenoid compound can be a triterpenoid selected from the lupane-type pentacyclic triterpenoid group, the oleanane-type pentacyclic triterpenoid group, or the ursane-type pentacyclic triterpenoid group.

[0097] Preferably, the pentacyclic triterpenoid compound according to the present disclosure is betulinic acid, and / or betulin, and / or lupeol, and / or oleanolic acid, and / or ursolic acid. More preferably, the pentacyclic triterpenoid compound is betulinic acid and / or betulin, and most preferably, the compound is betulinic acid.

[0098] The support for the compositions according to the present disclosure provided herein is mainly achieved through experiments using betulinic acid. However, since other preferred five-ring triterpenoid compounds of interest have very similar chemical structures, the milk fat globule drug composition can almost certainly also be applied as a preferred drug carrier system for these compounds. Therefore, experimental support for the latter compounds (betulin, lupeol, oleanolic acid, and ursolic acid) is very likely to be obtained through further elaboration of experiments.

[0099] The compositions of the present invention comprising a triterpenoid compound and lipid vesicles or globules, preferably milk fat globules, as a carrier system preferably contain a number of said lipid vesicles, globules or milk fat globules, e.g., at least 10 per ml of the formulation. 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 It is contained in a liquid emulsion formulation containing

[0100] Liquid emulsion formulations according to the present disclosure preferably contain at least 10, 15, 20, 25, 30, 35, 40, 50, 60, 70 mg / ml (pentacyclic) triterpenoid compounds, of which preferably at least 50, 60, 70, 80, 90 wt. % (based on the total weight of triterpenoid compounds in the formulation) is surrounded by a triphasic membrane or incorporated into milk fat globules or lipid vesicles.

[0101] By using milk fat globules obtained from the cream fraction of milk as the source of excipients for preparation, it is clear that the initial composition according to the present disclosure will have liquid properties. The viscosity of this liquid formulation is increased by the incorporation of triterpenoid compounds, as detailed in Example 4 and shown in Figure 7. This is believed to be an advantageous effect, as it improves the stability of the formulation. In part, viscosity may also be affected by the preparation method, since sonication affects the size and possibly membrane composition of the milk fat globules.

[0102] Alternatively, in another preferred embodiment, the composition according to the present disclosure comprising a (pentacyclic) triterpenoid compound incorporated into lipid vesicles, preferably milk fat globules, may be in the form of a powder formulation. The composition comprising a (pentacyclic) triterpenoid compound in powder form is preferably obtained by the spray drying or freeze drying process applied to a liquid formulation, as described above.

[0103] Spray drying involves the conversion of a fluid substance into dry particles using a gaseous, hot drying medium. This method is routinely used for the preparation of dairy products.

[0104] Freeze-drying (or lyophilization) is an alternative method of drying in which water is removed from a liquid material by freezing it, removing the ice by placing the product under vacuum and converting the ice directly from a solid to a vapor. Both drying methods have relative advantages and disadvantages, particularly with regard to product quality (including possible structural changes that may occur), process complexity, and equipment costs.

[0105] After drying, the triterpenoid compositions of the present invention in powder form can be used for oral ingestion (i.e., oral administration) after their solubilization (rehydration) in beverages or in pharmaceutical liquid or syrup products. Alternatively, powdered embodiments of the compositions of the present invention can be used in the preparation of edible (food) products and / or dietary supplements, for example, in the form of energy, protein, and granola bars, or baked goods such as cookies, cakes, pastries, sauces, dips, etc. It is also conceivable to orally administer powdered embodiments of the present invention after formulating the compositions into tablets or capsules, although this option is less preferred from the standpoint of absorption in the gastrointestinal system.

[0106] In general, triterpenoid compositions according to the present disclosure in powder form may have the advantage of extended shelf life, less restrictive storage conditions, and / or easier transportation conditions than liquid triterpenoid formulations.

[0107] In a preferred embodiment, the compositions comprising the triterpenoid compounds according to the present invention, e.g., in triphasic lipid vesicles or milk fat globules, are for oral administration and / or are comprised in a food composition, beverage composition, dietary supplement composition, dietary composition, functional food composition, or pharmaceutical composition.

[0108] Thus, the formulation of the present invention may be used in the form of a beverage for direct oral administration and ingestion, it may also be mixed with other beverages before oral administration, or it may be used in the preparation of baked or other manufactured foods that function as dietary supplements or functional foods.

[0109] Alternatively, in another preferred embodiment, a formulation comprising a triterpenoid compound contained in triphallama lipid vesicles or milk fat globules according to the present disclosure can be applied in the form of an ointment or cream suitable for topical administration to the skin, for example, to treat melanoma or skin diseases, or for cosmetic purposes. Administration of one embodiment of a triterpenoid composition according to the present disclosure in the form of an eye ointment or eye cream to the eyelid can be used for application to the eye.

[0110] Because the excipients of the present invention are naturally and food-derived, and because they are known for their optimal gastrointestinal uptake and absorption, the triterpenoid formulations of the present invention will almost certainly provide favorable bioavailability of the triterpenoid compounds when administered orally.

[0111] Due to the nature of the excipients, topical application of the above triterpenoid-milk fat globule formulations of the present invention is also expected to provide sufficient transdermal bioavailability of the triterpenoid compounds, enabling beneficial bioactive effects on skin-related diseases and / or improving skin conditions.

[0112] As detailed in the preceding sections of this application, the subject pentacyclic triterpenoids, particularly betulinic acid, betulin, lupeol, oleanolic acid, and / or ursolic acid, have known biological effects that make them suitable for therapeutic use, either alone or in combination with other chemotherapeutic compounds, particularly for the treatment of cancer. Thus, pentacyclic triterpenoids, preferably betulinic acid, can be used therapeutically to inhibit tumor growth and ameliorate cancer-related health conditions, and / or can be used as a priming active pharmaceutical ingredient to enhance the effects of other cancer chemotherapeutic agents, for example, by inhibiting (limiting) drug resistance. Many other beneficial effects are known for the triterpenoid compounds described herein, as detailed above. Thus, other disease states likely would also benefit from treatment with any of the pentacyclic triterpenoids described herein, including, in particular, betulinic acid, contained in the compositions of the present disclosure. The known effect of betulinic acid, in particular, on cancer cells to selectively inhibit their proliferation also makes the pentacyclic triterpenoid compositions of the present invention highly suitable for use as a (chemo)preventive measure to prevent the onset of cancer. Such use can be envisioned in the form of dietary (food) supplements or functional foods.The use of the compositions described herein as dietary supplements is also expected to be useful for improving health in general terms (not necessarily disease).For example, as documented, the compositions of the present invention may be useful for therapeutic purposes such as suppressing obesity, but also as inferred from preclinical in vitro and in vivo techniques, it is expected to be useful for preventing, improving, or treating one or more of the following health-affecting symptoms and conditions: type 2 diabetes, cardiovascular disease and / or its precursors, skin diseases, chronic inflammation, and movement disorders associated with Parkinson's disease.The demonstrated efficacy of preferably betulinic acid, and of the other pentacyclic triterpenoids described herein, against multiple cancer types, including the most prevalent cancers in men and women (lung, colorectal, breast, prostate, cervical or melanoma), both in vitro and in preclinical animal studies, makes the application of the compositions described herein as pharmaceuticals or dietary supplements (or functional foods) against or to prevent cancer most favorable.

[0113] Thus, compositions comprising the pentacyclic triterpenoid compounds according to the present invention are provided for therapeutic use, for example in the treatment or prevention of cancer, type 2 diabetes, obesity, metabolic syndrome, cardiovascular disease, skin disease, (chronic) inflammation, or Parkinson's disease and / or related movement disorders, preferably for use in the treatment or prevention of cancer, which may be lung cancer, colorectal cancer, breast cancer, prostate cancer, cervical cancer, or melanoma.

[0114] In the comprehensive research leading to the present invention, it was surprisingly found in accordance with the present invention that the use of high energy emulsion preparation techniques, particularly ultrasonication in combination with high shear homogenization, unexpectedly enables a drug carrier system consisting of milk fat globules (which are natural lipid vesicles with a triple layer membrane and a triglyceride core) to entrap, incorporate and encapsulate a high payload of triterpenoid compounds, particularly compared to other known (lipid-based) drug carrier formulations of the present invention (as detailed in Table 1).

[0115] In addition to the composition itself, the present invention further provides a method for the preparation of a (pentacyclic) triterpenoid compound composition, i.e., a composition contained in a (milk) fat globule or lipid vesicle according to the present invention, which method comprises the steps of: a) providing a composition comprising lipid vesicles having a triple-layer membrane comprising a single layer of phospholipids together with a bilayer of phospholipids, preferably milk fat globules themselves; b) mixing the composition provided in a) with one or more different (5-ring) triterpenoid compounds, preferably by applying high shear mixing and / or ultrasonication and / or high pressure homogenization techniques, to provide a (5-ring) triterpenoid composition of the present invention comprising one or more (5-ring) triterpenoid compounds incorporated into lipid vesicles, preferably milk fat globules, thereby achieving incorporation of the triterpenoid compounds into lipid vesicles, preferably milk fat globules, and assembly of milk fat globules or lipid vesicles incorporating the triterpenoid compounds.

[0116] In a preferred embodiment, in step b), the method of preparation therefore comprises one, or a combination of two or three high energy emulsification and homogenization methods such as sonication, high shear mixing, high pressure homogenization, most preferably sonication.

[0117] These techniques, separately or in combination, by mixing and stirring the particles and / or triterpenoid compounds, greatly facilitate the efficient and rapid incorporation of high payloads of triterpenoid compounds into the lipid vesicles (preferably milk fat globules) of the present invention, and are believed to be ideal for complete integration of triterpenoid compounds into lipid vesicles (preferably milk fat globules).

[0118] The high shear mixer referred to herein is a high-performance dispersing device that applies shear forces based on the rotor-stator principle. The high circumferential rotation speed of the rotor (e.g., in the range of 3,000 rpm to 35,000 rpm) in combination with the stator generates the necessary shear forces to break down and mix the ingredients being mixed, thereby, for example, emulsifying a liquid and / or dispersing powders or other materials in a liquid.

[0119] Thus, as used herein, high shear mixing may refer to mixing using a rotor mixing device, which typically consists of a rotor and a stator, the rotor being rotatable to generate shear force. As mentioned above, rotor speeds ranging from, for example, 3,000 rpm to 35,000 rpm may be used.

[0120] High shear mixing is particularly useful and therefore particularly used as an initial dispersion step in combination with subsequent preparation steps including sonication and / or high pressure homogenization (preferably sonication).

[0121] In the (ultra)sonication process, preferably as applied in step b, an ultrasonic transducer is used to generate ultrasonic vibrations. Sonication is a process of disrupting and mixing ingredients using sound waves. This process is usually carried out using an ultrasonic generator, which is a device that generates sound waves and directs them at the ingredients to be mixed. There are different types of ultrasonic transducers, such as horn (also called probe) transducers, piezoelectric plates, and Langevin transducers

[94] . In the present invention, preferably a horn (probe) ultrasonic generator is used, where ultrasonically induced pressure cycles cause the formation of tiny vacuum bubbles in the solution. These bubbles continuously collapse in a process known as cavitation, which generates powerful vibrational waves with high energy, exerting various physicochemical effects on particles in the emulsion and leading to enhanced and accelerated incorporation of triterpenoids into milk fat globules or milk fat globule-based vesicles of the inventive composition.

[0122] Additionally or alternatively, step b) may involve the use of high-pressure homogenization techniques, which means applying a pressure of at least 800 bar and up to 4,000 bar to force the (mixed) composition through a nozzle-shaped orifice (with a diameter of 10 to 10,000 nm) or using an associated high-pressure homogenization device.

[0123] Additionally or alternatively, step b) may comprise heating the (mixed) composition to a temperature of 30 to 90°C and / or step b) may be carried out at such a temperature.

[0124] At least one triterpenoid compound of the present invention is advantageously incorporated into milk fat globules in combination with casein micelles and / or whey proteins (derived from natural milk), which can be attached to the milk fat globules, thereby forming a more homogeneous and stable emulsion.

[0125] The addition of such native milk proteins to milk fat globules or to lipid vesicles based on milk fat globules may preferably be carried out before the preparation of the compositions of the present invention, but may alternatively also be achieved after preparation. Depending on the processing of the milk fat globule raw material used, as explained earlier in this specification, milk proteins may still be present in the milk fat globule emulsion used to prepare the composition. As explained in detail in previous sections of this application, both casein and whey proteins, of which a wide variety of variants exist in milk, are known in the literature for their ability to improve the emulsification process and may therefore advantageously help to prepare and stabilize formulations.

[0126] Alternatively, and also in order to further improve the stability of the compositions of the invention, a non-dairy derived stabilizer, preferably carrageenan (which is a mixture of natural sulfated polysaccharides extracted from red edible seaweed, which is approved for food applications and has European food additive number E407), may be beneficially added together with or alone to the dairy protein either before or after preparation of the composition.

[0127] In this specification and its claims, the verb "comprise" and its conjugations are used in an open-ended sense to mean that the items that follow are included, and items not specifically mentioned are not excluded. Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the presence of more than one of that element unless the context clearly requires that only one of that element is present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0128] The following examples illustrate different embodiments of the present invention. [Brief explanation of the drawings]

[0129] [Figure 1] Chemical structures of five pentacyclic triterpenoids addressed in the present invention.Five pentacyclic triterpenoids with strong pharmacological properties, the pharmaceutical formulations of which are addressed in this patent application.

[0130] Betulinic acid is derived from the lupane subfamily and its IUPAC name is (3β)-3-hydroxylup-20(29)-en-28-oic acid, with the chemical formula C 30 H 48 Betulin is from the lupane subfamily and its IUPAC name is (3β)-lup-20(29)-ene-3,28-diol, with the chemical formula C 30 H 50 Lupeol also has a lupane skeleton, its IUPAC name is (3β)-lupe-20(29)-en-3-ol, and its chemical formula is C 30 H 50 O. Oleanolic acid has an oleanane skeleton and its IUPAC name is (3β)-3-hydroxyolean-12-en-28-oic acid, and has the chemical formula C 30 H 48 Ursolic acid is from the ursane subfamily and its IUPAC name is (3β)-3-hydroxyurs-12-en-28-oic acid, with the chemical formula C 30 H 48 Has O3. [Figure 2]Principle of efficient assembly of milk fat globules (MFG) containing pentacyclic triterpenoids and incorporation of triterpenoids at high payloads. The membrane thickness is not to scale in Figure 2. The bottom half of the MFG contains no triterpenoids. Incorporation of hydrophobic triterpenoid drugs almost certainly occurs in both the trilayer membrane of the MFG and the triglyceride MFG core. Compared to smaller carrier systems, such as large or nanosized liposomes, shown as examples, there is ample space for incorporation of bioactive triterpenoids in the MFG carrier. The membrane surface area of ​​a 200 nm diameter nanoparticle is 400 times smaller than that of a 4 μm diameter MFG vesicle, and the core content of such MFG is 8,000 times greater than that of a 200 nm diameter nanoparticle. Furthermore, the aqueous core of the liposomal vesicle is unavailable for loading of hydrophobic compounds.

[0131] Depending on the relative contributions of the membrane and core to triterpenoid loading in MFG (which is precisely unknown), this would result in a 20-75% distribution of triterpenoid compounds loaded in the MFG core, making the total drug loading capacity of a single nanoliposome vesicle (200 nm diameter) 500-1,600 times lower than that of a single MFG vesicle (4 μm diameter). The extremely high drug loading capacity of MFG is its primary advantage as an (oral) drug delivery system. When included in an emulsion, even a much higher density of nanoparticles (per volume unit) is not expected to significantly compensate for the significantly lower drug loading capacity per nanoparticle. [Figure 3]RP-HPLC analysis of pentacyclic triterpenoid milk fat globule (MFG) formulations according to the present invention. Panel A: MFG-betulinic acid formulation. Panel B: MFG-betulin formulation. Top panels (both A and B): Representative RP-HPLC elution profiles of samples taken from betulinic acid or betulin-milk fat globule formulations, with increasing triterpenoid payloads of 10, 20, 30, and 50 mg / ml, demonstrating highly efficient incorporation of triterpenoid compounds across this concentration range. Bottom panels (both A and B): Peak area analysis by linear regression of the chromatographic elution profiles of four samples (n=4; standard deviation, SD, error bars shown at each payload concentration) of formulations with increasing triterpenoid concentrations, as shown. Strong linearity of drug incorporation was observed across this range of payloads. Four samples (n=4) were taken from different positions in the formulation without prior mixing to test for possible heterogeneous triterpenoid incorporation (or distribution) within the formulation during preparation and / or ongoing instability phenomena during storage, such as aggregation and / or phase separation (demulsification). [Figure 4] Microstructure of betulinic acid-milk fat globule formulations according to the present disclosure compared with a control formulation by optical microscopy. Bright-field microscopy images at 100x magnification of (A) a control formulation of betulinic acid dispersed in bovine skim milk without milk fat globules, and (B) a composition according to the present invention containing betulinic acid incorporated into milk fat globules at a payload of 32 mg / ml. The dispersion and emulsification procedures were completely identical for the preparation of (A) and (B). In [A], undissolved rod-like precipitates of betulinic acid are clearly observed, which are completely absent in the composition of betulinic acid contained in milk fat globules. [Figure 5]Particle size distribution analysis of triterpenoid-milk fat globule compositions of the present invention, measured by laser diffraction. Laser diffraction experiments were performed using a Bettersizer S3 Plus particle size analyzer from Bettersize Instruments Ltd (Dandong, China). Formulations containing no triterpenoids (control) or no triterpenoids (betulinic acid or betulin) were compared. Panel A: Left, a betulinic acid-milk fat globule composition (betulinic acid payload, 32 mg / ml) was compared with a control milk fat globule formulation containing no betulinic acid but undergoing the same preparation procedure (high-shear mixing and sonication); Right, a composition of betulin incorporated into milk fat globules (betulin payload, 20 mg / ml) was compared with a control milk fat globule formulation containing no betulin but undergoing the same preparation procedure (high-shear mixing and sonication). Panel B: The same experiment as panel [A], represented as a pie chart to visually clarify the differences in particle size distribution. The percentage of particles per size category is shown. [Figure 6] Characteristics of triterpenoid-milk fat globule formulations analyzed by acoustic spectroscopy. The figures show acoustic spectroscopy experimental results for a control milk fat globule formulation without betulinic acid and a composition of the present invention containing betulinic acid incorporated into milk fat globules (MFG). The control milk fat globule formulation does not contain betulinic acid, but it underwent the same preparation procedure (high shear mixing and sonication) as the MFG-betulinic acid formulation. Measurements were performed under controlled temperature. A significant difference in the attenuation spectra was observed when betulinic acid was incorporated into the MFG carrier. [Figure 7] Viscosity profiles of betulinic acid-milk fat globule formulations at defined temperatures. The viscosities of three betulinic acid-milk fat globule compositions with increasing betulinic acid payloads were tested along with a control formulation of unloaded milk fat globules (empty MFG). Viscosity was measured under strictly controlled temperature conditions due to the strong interdependence of viscosity and temperature. The betulinic acid payloads of the formulations are shown in the graph (15, 25, and 35 mg / ml). Panel A shows all three betulinic acid formulations along with the control. Panel B shows only the 15 mg / ml formulation along with the "empty" control to better visualize the dynamic effect of betulinic acid incorporation and the viscosity increase observed even at low concentrations of betulinic acid. [Figure 8] Stability of betulinic acid-milk fat globule formulations after centrifugation and long-term storage. Panel A: Effect of centrifugation on emulsion stability by measuring centrifugation-induced collapse of betulinic acid-milk fat globule compositions compared to control emulsions. A composition containing betulinic acid at a payload of 35 mg / ml, assembled in milk fat globules according to the present disclosure, was centrifuged at 3,400 g for 50 minutes (Tube 1). Control emulsions included unprocessed O / W emulsions from either milk fat globules derived from similarly centrifuged and homogenized milk fat globules (with a particle size distribution of 0.2-2 μm) (Tube 2) or milk fat globules derived from raw, unhomogenized dairy cream containing significantly larger milk fat globules (with a particle size distribution of 1-10 μm) (Tube 3). Panel B: Shelf-life stability of the inventive milk fat globule-betulinic acid formulations after 3 months of storage at 4° C. Different variants of the inventive betulinic acid compositions incorporated into milk fat globules (MFG) are shown. Tube 1: MFG from homogenized milk with a betulinic acid payload of 25 mg / ml; Tube 2: MFG from homogenized milk with a betulinic acid payload of 40 mg / ml; Tube 3: MFG from raw milk with a betulinic acid payload of 25 mg / ml.

[0132] Experimental Section Example 1 Preparation of triterpenoid milk fat globule compositions according to the present invention Milk fat globule source The compositions according to the invention were prepared using a basic protocol, several variations of which were carried out with regard to the exact source and content of excipients used, and the details of the preparation protocol. Essentially, the milk fat globules used as raw material for the compositions were either derived from homogenized milk cream commercially supplied by various dairy companies, or milk fat globules collected from raw milk (fresh milk) supplied, for example, by (biological) dairy farms. The raw milk cream was collected (or "skimmed") according to the basic dairy manufacturing procedure, as previously outlined, to obtain the milk fat globules contained in the cream fraction.

[0133] Both of these milk fat globule sources can then be used to prepare compositions according to the present invention, thereby taking into account the shelf life of the source material. As previously outlined, the compositions prepared using milk fat globules from these two sources have different mean globule diameters, but are similar in terms of the payload of pentacyclic triterpenoid incorporation into the milk fat globule drug carrier system.

[0134] Formulation preparation, methods and materials Heavy cream preparations containing high-density (at least 30% milk fat, v / v) milk fat globules in volumes of 20 to 100 ml were used as the excipient source for the compositions of the present invention. Highly purified (>98%, as confirmed by mass spectrometry) isolated pentacyclic triterpenoid compounds were added in powder form at various concentrations (expressed as mg triterpenoid compound per ml excipient, e.g., 10, 20, 30, 40, or 50 mg / ml). No triterpenoid compounds were added to the control compositions, but the preparation protocol followed was identical to that used for the compositions containing triterpenoid compounds (compared paired compositions).

[0135] For most preparations, an initial dispersion step involving high-shear mixing (apparatus: Ultra Turrax dispensing device, model T25, IKA) at 15,000-20,000 RPM for 2-5 minutes was applied to achieve an initial coarse dispersion of the triterpenoid powder throughout the entire volume of excipients used. However, in some other preparations, the initial high-shear mixing step was omitted because it was deemed unnecessary if the second sonication step was performed according to the most preferred steps and parameter settings.

[0136] During the second preparation stage, a sonication procedure was subsequently performed, which was deemed essential to ensure proper and complete encapsulation and incorporation of the pentacyclic triterpenoids in the milk fat globules. Therefore, sonication was used in the preparation of all formulations according to the present invention; otherwise, it would be impossible to achieve complete triterpenoid incorporation at high payloads. Sonication was performed using an ultrasonic probe-type processor (also known as a tip, horn, or pole-type sonicator). The equipment used was an ultrasonic processor model FS150-N (Zhengzhou TCH Instrument Co, Zhengzhou, China) for small-volume preparations and an ultrasonic generator model FS1800-N (same as above) for large-volume preparations, with adjustable maximum powers of 80 watts and 1,800 watts, respectively, and a frequency of 20 kHz. The following sonication cycle sequence and parameter settings were used for the 80-watt ultrasonic generator (FS150-N) with an 8 mm diameter probe: Five sonication cycles were applied, using formulation volumes of 25 ml to 40 ml. Each cycle used increasing power usage, starting at 35% power intensity, followed by subsequent cycles at 70%, 80%, 85%, and 90% power intensity. Each cycle had a 5-minute duration, and within each cycle, sonication alternated between 10-second sonication pulses with 5-second rest periods between pulses (a total of 20 10-second pulses per 5-minute cycle). The primary reason for using this setting was to prevent the temperature of the composition being prepared from excessively increasing above 60°C. While increasing the temperature can aid in the emulsification process and encapsulation of triterpenoid compounds into milk fat globules, excessively high temperatures can undesirably cause denaturation of proteins in the milk fat globule membrane. Therefore, it was chosen to stay within safe temperature limits. The temperature was monitored between and within cycles during sonication. The applied cycle and pulse settings ensured that the formulation temperature never exceeded 60°C. The immersion depth of the applied sonication probe was approximately 25% below the surface of the total formulation height. After the sonication procedure, the formulation was allowed to come to room temperature and then stored at 6-8°C until use.

[0137] As will be appreciated by those skilled in the art, it is anticipated that the use of high-pressure emulsification techniques in place of sonication may similarly effect complete incorporation of pentacyclic triterpenoid compounds in milk fat globules. Similarly, in some embodiments of the compositions of the present invention, the combination of sonication and high-pressure emulsification techniques during preparation may advantageously aid in the scale-up process required for the manufacture of high-volume formulations

[94] .

[0138] It is noteworthy regarding the preparation method applied in this invention that in none of the published literature in which milk fat globules were used to incorporate active biological compounds, the encapsulation method involved the use of sonication or other high-energy emulsification techniques such as high-pressure emulsification or high-shear mixing. Instead, only simple co-incubation (i.e., stirring at 25 °C) was applied

[83] , or organic solvents were used to aid the incorporation process [81, 82].

[0139] As shown in Example 2, the preparation method described herein successfully incorporated triterpenoids, preferably betulinic acid and betulin, at high concentrations in milk fat globules when proceeding according to the construction principle shown in Figure 2. Emulsification and incorporation of triterpenoid compounds into milk fat globules using several tested low-energy emulsification methods (e.g., stirring at 40°C) was unsuccessful, again demonstrating the importance of using the applied high-energy emulsification techniques described herein.

[0140] Example 2 Efficient incorporation of triterpenoid compounds in milk fat globules revealed by RP-HPLC and microscopy Sample Preparation Method for Triterpenoid-Milk Fat Globule Composition A two-phase liquid-liquid extraction (LLE) method using methanol (MeOH) and isooctane as immiscible organic solvents was developed to separate pentacyclic triterpenoid compounds from their sample matrix, which is milk fat globules. Briefly, samples of triterpenoid-milk fat globule preparations were collected using a sampling spatula at at least four different locations within one preparation (contained in a 50 ml storage vial), and the samples were weighed. The two-phase liquid extraction system consisted of MeOH to dissolve the triterpenoids (betulinic acid or betulin) and isooctane to dissolve the matrix lipids from the milk fat globules. For this purpose, equal amounts (7 ml) of MeOH and isooctane were collected, and 3% by volume of water was added to favorably adjust the liquid-liquid equilibrium between the two organic solvents. The sample collected from the preparation was added to the two-phase LLE system. The preparation sample was selected to be approximately 200 mg. A series of samples, primarily of slightly varying weights, whose triterpenoid content needed to be quantitatively compared by HPLC were collected. The volumes of MeOH and isooctane were adjusted, as needed, to obtain equal mg of formulation per ml of the two-phase LLE organic solvent system. The samples were vigorously mixed in the LLE system using vortexing techniques. The LLE system was then heated to 40 °C and left for 30 minutes to aid in sample dissolution. To further improve sample dissolution, the LLE system containing the samples was then vigorously mixed in a high-shear mixer (Ultra Turrax, Model T25, high-shear mixer, operating on the rotor-stator principle) at 10,000 RPM and then left at 40 °C for 2 hours to re-perform phase separation between the MeOH and isooctane solvent layers.

[0141] A 1.2 ml sample was taken from the lower MeOH layer containing the dissolved triterpenoids and centrifuged at 10,500 RPM for 5 minutes to remove any debris, after which a 300 μl sample was taken for HPLC analysis. Quantitative RP-HPLC analysis of the HPLC-prepared samples compared to a concentration series of triterpenoid (betulinic acid or betulin) standards, combined with standard addition experiments, revealed that the sample preparation procedure described here resulted in an average loss of 30% of the triterpenoid content (data not shown).

[0142] RP-HPLC analysis and methods for triterpenoid compounds RP-HPLC analysis was performed using a chromatography system consisting of a Waters Alliance 2695 separation module coupled to a Waters 996 photodiode array detector. The stationary phase was a C-18 column (Waters Symmetry® C18, 5 μm, 4.6 × 250 mm). The injection volume was 20 μl, and the flow rate was 1.5 ml / min at a controlled temperature of 30 °C. For separation, a mobile phase consisting of acetonitrile-water in a ratio of 91:9 (v / v) was used for elution in isocratic mode. The detection wavelength of the PDA detector was set at 210 nm. Chromatographic peaks of betulinic acid and / or betulin were confirmed by comparing their retention times with those of betulinic acid and betulin standards (Sigma-Aldrich) with purity >98% dissolved in methanol.

[0143] RP-HPLC analysis and results of triterpenoid-milk fat globule preparations HPLC chromatography of samples prepared from the compositions of betulinic acid in milk fat globules and betulin in milk fat globules efficiently detected the triterpenoid compounds in the formulations (Figure 3A and B, top panel, showing a representative elution profile; note the difference in retention time between betulinic acid and betulin) and demonstrated linearity of triterpenoid incorporation, even at high payloads (Figure 3A and B, bottom panel).

[0144] The complete incorporation of betulinic acid and betulin into milk fat globules was inferred from different findings. Both emulsion formulations containing betulinic acid or betulin in milk fat globules were extremely stable (Figure 8A and B), and betulinic acid (or betulin) was homogeneously emulsified, as indicated by the small standard deviations between samples prepared from four different positions in the emulsion, as shown in Figure 3A and B. Note: Random sampling at different positions in the emulsion was performed after at least one week of standing, and there was no premixing (dispersion) of the emulsion.

[0145] Furthermore, when samples were collected from the upper lipid fraction of the emulsion by forced centrifugation (see centrifugation as described in Example 5 and tube 1 in Figure 8.A), results were similar: nearly identical triterpenoid concentrations were observed in samples collected at different locations throughout the lipid fraction of the emulsion, again indicating a stable and homogeneous distribution of triterpenoids throughout the formulation (data not shown). However, as expected, higher concentrations of triterpenoids were detected in the upper lipid fraction of the HPLC samples compared to the uncentrifuged emulsions, because the aqueous lower fraction separated (and analyzed) after centrifugation (tube 1 in Figure 8.A) does not contain triterpenoids (data not shown).

[0146] Furthermore, microscopic observation of the triterpenoid-milk fat globule (MFG) compositions and their comparison with a triterpenoid control formulation without MFG provides strong additional indication that the amount of triterpenoid added initially during the preparation of the formulation according to the invention was fully incorporated into the MFG.

[0147] Bright-field microscopy of triterpenoid-milk fat globule composition The bright-field optical microscope (100x magnification, Kern OBL137C832 microscope, Kern & Sohn GmbH, Germany) image in Figure 4B of the betulinic acid-milk fat globule formulation (with a payload of 32 mg / ml betulinic acid) shows the normal appearance of the milk fat globules without any visible signs of crystalline or amorphous betulinic acid deposits. On the other hand, the control formulation of betulinic acid dispersed in skim milk by the same emulsification procedure (Figure 4A) is largely devoid of milk fat globules, and betulinic acid precipitates are abundantly and clearly observed as rod-like structures.

[0148] The importance of this type of particle characterization by microscopy is that it is best achieved through direct observation, without the often complex interpretation problems of, for example, laser diffraction or acoustic spectroscopy results of complex emulsions, which often confound the results and prevent clear conclusions.

[0149] Example 3 Particle size characterization of triterpenoid-milk fat globule compositions Particle characterization, including particle size distribution, measured by laser diffraction The particle characteristics of the milk fat globule (MFG) control formulation and the inventive MFG formulation containing bioactive pentacyclic triterpenoids were analyzed by laser diffraction using a Bettersizer S3 Plus particle size analyzer (Bettersize Instruments Ltd, Dandong, China). The control formulation did not contain triterpenoid molecules, but the preparation procedure, including high-shear mixing and sonication, was carried out in the same manner as the preparation protocol for the formulation containing triterpenoids.

[0150] Laser diffraction measurements can be difficult to interpret reliably under certain conditions. The experiment shown in Figure 5 (Panels A and B show the same data but in different formats for clarity and interpretation) strongly suggests that the particle size of MFG is enlarged by the incorporation of bioactive triterpenoids. This is because, in the control formulation, no MFG particles larger than 5 μm in size were observed, whereas this size category was prominent after the incorporation of pentacyclic triterpenoids (betulinic acid or betulin). However, it cannot be completely ruled out that differences between the control and triterpenoid formulations other than differences in MFG particle size, particularly the occurrence of MFG particle aggregation, are responsible in whole or in part for the observed effect. However, all samples were identically pretreated, including the aforementioned short sonication step to break down aggregates immediately before laser diffraction measurements, making the latter explanation less likely.

[0151] Additionally, the volume-weighted mean (i.e., average) particle size and median particle size values ​​obtained by laser diffraction were significantly increased in the MFG formulations containing betulinic acid compared to the formulations containing excipients only, which were similarly treated but did not contain triterpenoids (data not shown). This is consistent with the results in Figure 5, demonstrating the particle-enlarging effect of triterpenoids. The volume-weighted mean (i.e., average) particle diameter (designated D[4,3]) was:

number

[0152] Particle characterization measured by acoustic spectroscopy The principle of acoustic attenuation for analyzing particles in concentrated emulsions requires an acoustic sensor to measure the attenuation of ultrasound passing through the emulsion in the frequency range of 1–100 MHz at various transmitter–detector distances. For this purpose, an acoustic and electroacoustic spectrometer DT-1202 (Dispersion Technology Inc., USA) was used.

[0153] Figure 6 shows a clear reciprocal effect, with the acoustic attenuation of the betulinic acid-milk fat globule pharmaceutical formulation being lower than the "empty" control milk fat globule formulation at acoustic frequencies below 35 MHz, whereas an opposite effect is observed at frequencies above 35 MHz. The significant difference in acoustic attenuation profiles between milk fat globules with and without the pentacyclic triterpenoid compound is necessarily caused by the incorporation of the compound, since the preparation procedures were similar.

[0154] The detailed interpretation of these results is complicated

[95] because, in addition to particle size, viscosity and particle composition can also affect the attenuation spectrum, and the latter two parameters can also be modified by the incorporation of bioactive triterpenoid compounds. In general, in the low acoustic frequency range, lower acoustic attenuation correlates with larger milk fat globules

[95] , suggesting an enlargement of milk fat globules upon the incorporation of triterpenoid compounds, which is also consistent with the results observed using laser diffraction (Figure 5). However, changes in viscosity (see Figure 7) could also explain, at least in part, the observed effect.

[0155] Example 4 Incorporation of betulinic acid increases the viscosity of the formulation Viscosity measurements of betulinic acid-milk fat globule formulations were performed using a sinusoidal oscillation viscometer SV-10 from A&D Company Ltd. (Tokyo, Japan), equipped with a temperature sensor and a viscosity measurement limit of 0.3 to 10,000 mPa·s (milliPascal·seconds). This instrument is based on the sinusoidal oscillation method. Kinematic viscosity was measured continuously under strictly temperature-controlled conditions. The formulation to be tested was placed in a sample cup in a water jacket with circulating water, which allowed for precisely controlled formulation temperature. The refrigerated bath circulator used for this purpose was a Thermo Scientific Neslab RTE-201 model.

[0156] The results shown in Figure 7 indicate that viscosity is significantly increased by the incorporation of betulinic acid, including a strong positive correlation between the payload of betulinic acid in the formulation and the level of viscosity enhancement. The increased viscosity most likely results in improved formulation stability, which is an additional advantageous property of the triterpenoid compounds of the present disclosure. The increased viscosity is likely caused by the incorporation of betulinic acid, which mimics cholesterol, in the triple-layer membrane of milk fat globules. Cholesterol is known to make the membrane more robust, thereby increasing viscosity

[96] .

[0157] Example 5 The compositions according to the present invention are stable For purposes of oral or topical administration, and as one skilled in the art will appreciate, it is important that the administered formulation be stable, have a homogeneous composition, and be stable over time. Stability was tested using two methods: centrifugation and a (simple) shelf-life test.

[0158] Accelerated stability testing was performed using high-gravity centrifugation. For this purpose, betulinic acid-milk fat globule (MFG) formulations were centrifuged at 3,400 g for 50 minutes. A significant effect of incorporating betulinic acid into the MFG composition, along with its preparation method, was observed. The betulinic acid-MFG composition exhibited two-phase separation after centrifugation, with an aqueous layer at the bottom of the tube and a homogenous lipid layer at the top (Figure 8.A, Tube 1). In contrast, the unprocessed milk fat globule "source composition" obtained from homogenized milk or raw milk (containing relatively small or large MFGs, respectively, as previously described), exhibited a significantly different phase separation after centrifugation, with a three-phase separation with a distinct oil layer at the top (Figure 8.A, Tubes 2 and 3). Notably, in Tube 2 (containing an O / W dairy emulsion of homogenized small MFGs), the second phase separation was only detectable under transmitted light. These results suggest that the incorporation of betulinic acid into milk fat globules, in conjunction with the preparation procedure (best of all, sonication), achieves increased lipid particle stability in the formulation, since no oil phase containing "liberated" fat from the core of the milk fat globules appeared. This is explained by the incorporation of betulinic acid into the MFG membrane, thereby increasing the membrane's rigidity and its resistance to centrifugation-induced membrane disruption.

[0159] Storage stability was tested after various storage periods. Figure 8.B shows the results of three betulinic acid-milk fat globule formulations with different betulinic acid payloads stored at 4°C for three months. In all cases tested, the compositions disclosed herein maintained high stability without phase separation. Phase separation, if present, would indicate undesirable aggregation or fusion within the emulsion. A storage temperature of approximately 4°C is a commonly accepted and required condition for dairy-based (food) products, and the apparent undisturbed emulsion stability after three months at this temperature can be considered an advantageous property of the triterpenoid-MFG formulations of the present invention.

[0160] Furthermore, in room temperature storage experiments, similar stability was observed after 1 month, and bacterial growth was also absent in the majority of cases tested (8 of 9 formulations tested (>85%)) (data not shown). This apparent beneficial antibacterial effect can be explained by the synergistic action of sonication, which has a known bacterial growth inhibitory effect, and the incorporation of betulinic acid, a compound with proven antibacterial activity.

[0161] Example 6 Evaluation of the betulinic acid-milk fat globule composition of the present invention compared to betulinic acid formulations reported in the art A comprehensive evaluation was performed on the most representative reported betulinic acid formulations with pharmaceutical potential, as shown in Table 1. In this overview, betulinic acid was selected because of its chemical structure, which is strongly similar to other triterpenoids addressed in this invention, resulting in their strong hydrophobicity, and because most published formulation research efforts have been directed towards betulinic acid.

[0162] The drug loading capacity (payload) and the effect of formulation excipients were evaluated. Other characteristics mentioned were (1) preparation difficulty and associated cost-effective manufacturing, (2) potential toxicity and associated potential regulatory constraints, (3) assessment or estimation of formulation stability, and (4) potential suitability for oral administration and nutraceutical development, which were also evaluated.

[0163] A comprehensive evaluation of these different betulinic acid formulations compared to the composition of the present invention, which comprises a triterpenoid compound (preferably betulinic acid and / or betulin) preferably incorporated into milk fat globules, makes the composition of the present invention the most promising by possessing advantageous properties compared to the other formulations in all respects listed in Table 1. [Table 1] TIFF2026504193000003.tif24264

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Claims

1. 1. A composition comprising a pentacyclic triterpenoid compound, wherein the pentacyclic triterpenoid compound is incorporated into milk fat globules or lipid vesicles having a membrane, the membrane comprising: - a phospholipid monolayer; and -phospholipid bilayer The composition is three layers comprising:

2. 2. A composition comprising the pentacyclic triterpenoid compound of claim 1, wherein one pentacyclic triterpenoid compound or a combination of two or more different pentacyclic triterpenoid compounds is contained in multiple milk fat globules.

3. 3. A composition comprising a pentacyclic triterpenoid compound according to claim 1 or 2, wherein the milk fat globules or lipid vesicles have an average diameter of 0.1 to 15 μm, preferably 1 to 10 μm, more preferably 2 to 6 μm, as measured by laser diffraction.

4. A composition comprising the pentacyclic triterpenoid compound according to any one of claims 1 to 3, wherein the pentacyclic triterpenoid compound is betulinic acid, and / or betulin, and / or lupeol, and / or oleanolic acid, and / or ursolic acid.

5. 5. A composition comprising the pentacyclic triterpenoid compound according to any one of claims 1 to 4, wherein the pentacyclic triterpenoid compound is betulinic acid and / or betulin.

6. 6. A composition comprising the pentacyclic triterpenoid compound according to any one of claims 1 to 5, wherein the pentacyclic triterpenoid compound is betulinic acid.

7. 7. A composition comprising a pentacyclic triterpenoid compound according to any one of claims 1 to 6, wherein the pentacyclic triterpenoid compound is contained in a liquid emulsion formulation, preferably the liquid emulsion formulation contains at least 10, 15, 20, 25, 30, 35, 40, 50, 60, 70 mg / ml of the pentacyclic triterpenoid compound, preferably at least 50, 60, 70, 80, 90% by weight of which is incorporated into milk fat globules or lipid vesicles.

8. A composition comprising a pentacyclic triterpenoid compound according to any one of claims 1 to 6, wherein the pentacyclic triterpenoid compound is contained in a powder formulation obtained by spray drying or freeze drying techniques, preferably as applied to the liquid formulation according to claim 7.

9. 9. A composition comprising the pentacyclic triterpenoid compound according to any one of claims 1 to 8, wherein the pentacyclic triterpenoid compound is for oral administration and / or is contained in a food composition, beverage composition, nutritional supplement composition, health supplement composition, functional food composition, or pharmaceutical composition for oral administration.

10. 9. A composition comprising a pentacyclic triterpenoid compound according to any one of claims 1 to 8, wherein the pentacyclic triterpenoid compound is for topical administration to the skin, preferably as a medicinal or cosmetic ointment or cream.

11. A composition comprising a pentacyclic triterpenoid compound according to any one of claims 1 to 10 for therapeutic use.

12. 12. A composition comprising a pentacyclic triterpenoid compound according to any one of claims 1 to 11 for use in the treatment or prevention of cancer, type 2 diabetes, obesity, metabolic syndrome, cardiovascular disease, skin disease, (chronic) inflammation, or Parkinson's disease and / or related movement disorders, preferably for use in the treatment or prevention of cancer.

13. 13. A composition comprising the pentacyclic triterpenoid compound of any one of claims 1 to 12 for use in treating or preventing cancer, wherein the cancer is lung cancer, colorectal cancer, breast cancer, prostate cancer, cervical cancer or melanoma.

14. A method for preparing a composition comprising a pentacyclic triterpenoid compound according to any one of claims 1 to 13, comprising: a) providing a composition comprising lipid vesicles, preferably milk fat globules, having a triple-layer membrane comprising a phospholipid monolayer and a phospholipid bilayer; b) providing a pentacyclic triterpenoid composition according to any one of claims 1 to 13 by mixing the composition provided in a) with one or more different pentacyclic triterpenoid compounds, preferably by applying high shear mixing and / or sonication and / or high pressure homogenization techniques, thereby achieving incorporation of said triterpenoid compounds into said lipid vesicles, preferably milk fat globules, and assembly of milk fat globules or lipid vesicles incorporating said triterpenoid compounds, A method comprising:

15. 15. The method of claim 14, wherein in step b) ultrasonic treatment is applied to agitate the milk fat globules, or lipid vesicles, together with the pentacyclic triterpenoid compound, thereby resulting in dispersion of the pentacyclic triterpenoid compound in the emulsion comprising the milk fat globules or lipid vesicles, and to achieve incorporation of the pentacyclic triterpenoid compound into the milk fat globules or lipid vesicles.

16. 16. The method according to claim 14 or 15, wherein in step a) or b) milk proteins, preferably casein proteins, casein micelles and / or whey proteins, are added.

17. A composition comprising a pentacyclic triterpenoid compound according to any one of claims 1 to 13 in combination with milk proteins, preferably casein proteins, casein micelles and / or whey proteins.

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