WO2022113140A1

WO2022113140A1 - Formulation for treating cholesterol

Info

Publication number: WO2022113140A1
Application number: PCT/IT2021/050380
Authority: WO
Inventors: Giuseppe Giorgini; Claudio GARDANA; Antonio SCIALPI
Original assignee: Specchiasol S.R.L.
Priority date: 2020-11-24
Filing date: 2021-11-24
Publication date: 2022-06-02
Also published as: EP4251135A1

Abstract

A formulation for oral administration, in particular for treating cholesterol, is made as a differentiated-release multilayer formulation and comprises Monacolin K and Berberine.

Description

“FORMULATION FOR TREATING CHOLESTEROL”

FIELD OF THE INVENTION

Embodiments described here concern a formulation or composition with a differentiated release and comprising Monacolin K and Berberine. The formulation can be used to lower cholesterol, triglycerides and low-density proteins (LDL) in the blood, to increase those of high-density proteins (HDL) and reduce inflammatory states. The composition or formulation in accordance with the present description can be the pharmaceutical or food supplement or nutraceutical type.

BACKGROUND OF THE INVENTION

Atherosclerosis and coronary heart disease (CHD) have been the main causes of morbidity and mortality in Western countries for decades (Balbisi, 2006). It is widely recognized that lowering LDL-cholesterol (LDL-C) in the serum can interfere with the progression of atherosclerosis and reduce cardiovascular events in CHD suffering patients (Stamler 2000, Anderson 1987, Grundy 2004).

Among the compounds currently available that are able to modify plasma lipid levels, the most commonly prescribed are those able to inhibit 3 -hydroxy-3 - methylglutaryl coenzyme A (HMG-CoA) reductase, namely statins (Klotz 2003).

However, less than 20% of patients with a high risk of cardiovascular disease (CVD) have significant reductions in LDL and up to 60% of patients report some statin intolerance or side effects related to this treatment (Zhang 2013).

This constitutes the main reason for the interruption of and poor adherence to statin treatment, which remains a significant problem in the management of dyslipidemia (Banach 2016).

The increase in intolerance to statins entails the need to identify a non- pharmacological alternative, in particular for patients with moderate hypercholesterolemia, low cardiovascular risk or intolerance to traditional pharmacological treatment; it has led to the experimentation of many phytochemical compounds as nutraceuticals that have shown lipid lowering properties (Johnston 2017). Nutraceuticals are food components that can be used as therapeutic agents thanks to their beneficial effects on health (Kalra 2003).

Red yeast rice (RYR), a product obtained from the fermentation action of a specific strain of red yeast (Monascus purpureus), is a popular complementary medicine used to lower lipid levels in the blood (Liu 2006, Lu 2008). The main active component of the RYR extract is Monacolin K.

Another clinical choice, to increase the lipid-lowering effect, is a combination therapy of RYR with other compounds that are also able to alter the amount of plasma lipids. In addition, the advantages of combinatorial therapy are the reduced dosage of statins and the decreased risk of having adverse effects (Brown 2002, Davidson 2002).

Possible combinations currently available for treating hypercholesterolemia include statins combined with compounds able to sequester bile acids (Davidson 2002) or ezetimibe (Pearson 2005).

Cicero et al in Handbook of Nutraceuticals for Clinical Use (2018) lists several compounds, including Berberine and Monacolin K, indicating their uses and possible synergistic combinations and contraindications. This document teaches that Berberine and Monacolin K are used individually for treating hypercholesterolemia, and that these two compounds taken together have a synergistic effect in the same therapeutic treatment.

Similarly, EP-A-3064200 describes a composition comprising a mixture of Berberine, Monacolin K and Oleuropein to reduce the level of LDL-cholesterol in human blood, without affecting the level of HDL-cholesterol. These compounds are administered simultaneously. The formulation also includes some excipients such as antioxidants and vitamins.

US-A-2016/120882 describes a pharmaceutical composition comprising, as the active ingredient, a mixture of Monacolin K, lycopene and vitamin D3, in which the composition can also comprise Berberine as an additional active component. This composition is provided in a form suitable for oral administration and has positive effects on the cardiovascular system.

The Applicant has discovered that, unexpectedly, the association between Berberine and Monacolin K has some limits and disadvantages when these compounds are administered together, in particular connected to the fact that Berberine slows down the absorption of Monacolin K. This negative effect obtained from the co-administration of these two compounds is neither described nor mentioned in the state of the art. On the contrary, as described in the prior art documents referred to above, the combination of Berberine and Monacolin K is known to be very advantageous in particular in the treatment of hypercholesterolemia.

It is therefore necessary to implement specific measures intended to prevent Berberine from negatively affecting Monacolin K.

There is therefore a need to perfect a formulation or composition for treating cholesterol which can overcome at least one of the disadvantages of the state of the art.

In particular, one purpose of the present invention is to provide a formulation or composition, of a pharmaceutical or food supplement or nutraceutical type, for oral administration in order to reduce the plasma levels of total triglycerides, cholesterol and LDL and to increase those of HDL and reduce the inflammatory process.

The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

SUMMARY OF THE INVENTION

The present invention is set forth and characterized in the independent claims. The dependent claims describe other characteristics of the invention or variants to the main inventive idea.

In accordance with the above purposes, some embodiments concern a formulation or composition for oral administration. The formulation is multilayer with a differentiated release, in which a first layer comprises Monacolin K or an extract containing Monacolin K, and another layer comprises Berberine or an extract containing Berberine.

According to some embodiments, this first layer also comprises polyphenols.

According to some embodiments, the multilayer formulation comprises, or consists of, three differentiated-release layers, wherein the first layer and the other layer, or third layer, are separated by a second central layer.

According to some embodiments, the second central layer comprises vitamin K2 and/or coenzyme Q10.

According to some embodiments, the formulation described here is for reducing the plasma levels of cholesterol, triglycerides and LDL and for increasing those of HDL.

In some embodiments, the formulation comprises adjuvants and/or pharmacologically acceptable excipients, wherein the excipients comprise lipid excipients, in particular phospholipids and/or fatty acid esters and/or fatty acids. Preferentially, the lipid excipients are suitable for consumption, in particular they are of food grade or food supplement grade quality. In some embodiments, the fatty acid esters comprise, or consist of, glyceryl dibehenate.

In some embodiments, the lipid excipients are present in one or more of the layers of the formulation, in particular at least in the other layer, or third layer, containing Berberine or an extract containing Berberine.

In some embodiments, the formulation comprises vitamin K2 between 0.00001 and 0.05% weight/weight, in particular present in the second central layer.

In some embodiments, the formulation comprises coenzyme Q10 between 0.2 and 20% weight/weight, in particular present in the second central layer.

In some embodiments, in the formulation there is red yeast rice extract as a source of Monacolin K, in particular the Monacolin K is contained in the RYR, in the range 1-15% by weight.

In some embodiments, the formulation is in the form of a dose unit, ready for the administration of Berberine at a daily dose of 10 to 500 mg.

In some embodiments, the formulation is in the form of a dose unit, ready for the administration of Monacolin K at a daily dose of 1 to 15 mg, preferably from 3 to 10 mg.

In some embodiments, the formulation is in the form of a dose unit, ready for the administration of vitamin K2 at a daily dose of 0.1 to 500 meg, in particular from 50 to 100 meg.

In some embodiments, the formulation is in the form of a dose unit, ready for the administration of coenzyme Q10 at a daily dose of 2 to 200 mg, preferably from 10 to 30 mg. In some embodiments, the formulation is in the form of a dose unit, ready for the administration of polyphenols with antioxidant activity at a daily dose of 3 to 500 mg.

In some embodiments, the polyphenols contain in particular flavonoids as an active component, in particular from Bergamot, more in particular from natural Bergamot extract.

In some embodiments, the natural Bergamot extract is present in the formulation as a source of flavonoids, in particular the flavonoids are contained in the natural Bergamot extract in a range between 5 and 80% weight/weight.

Other embodiments concern a method to make a formulation for oral administration, the method comprising preparing a differentiated-release multilayer formulation, in which a first layer comprises Monacolin K or extract containing Monacolin K, and another layer comprises Berberine or an extract containing Berberine.

DESCRIPTION OF THE DRAWINGS

These and other aspects, characteristics and advantages of the present invention will become apparent from the following description of some embodiments, given as a non-restrictive example with reference to the attached drawings wherein:

- fig. 1 shows a graph showing the dissolution percentage of Monacolin K in the dissolution test carried out on a multilayer tablet product;

- fig. 2 shows a graph showing the dissolution percentage of vitamin K2 in the dissolution test carried out on a multilayer tablet product;

- fig. 3 shows a graph showing the dissolution percentage of Berberine in the dissolution test carried out on a multilayer tablet product;

- fig. 4 shows graphs of basolateral concentration profiles of Berberine after incubation of different Berberine-based formulations on Caco-2 cells (n=3);

- fig. 5 shows a graph showing the quantity of Berberine (pmol/L) detected in the basolateral solution after 180 minutes of incubation on Caco-2 cells;

- fig. 6 shows a graph showing the relative percentage of Berberine present in the basolateral solution after 180 minutes of incubation on Caco-2 cells;

- fig. 7 shows a graph showing the percentage increase, compared to the control, of Berberine permeated through the Caco-2 monolayer after 180 minutes of incubation.

DETAILED DESCRIPTION OF SOME EMBODIMENTS We will now refer in detail to the possible embodiments of the invention, of which one or more examples are shown in the attached drawings, by way of a non-limiting illustration. The phraseology and terminology used here is also for the purposes of providing non-limiting examples.

It is also understood that elements and characteristics of one embodiment can conveniently be combined or incorporated into other embodiments without further clarifications.

All measurements are carried out, unless otherwise indicated, at 25°C and at atmospheric pressure. All temperatures, unless otherwise indicated, are expressed in degrees Celsius.

All percentages and ratios indicated refer to the weight of the total composition (w/w), unless otherwise indicated.

All the percentage intervals reported here are provided with the provision that the sum with respect to the overall composition is 100%, unless otherwise indicated.

All the intervals reported here shall be understood to include the extremes, including those that report an interval “between” two values, unless otherwise indicated.

The present description also includes the intervals that derive from uniting or overlapping two or more intervals described, unless otherwise indicated.

The present description also includes the intervals that can derive from the combination of two or more values taken at different points, unless otherwise indicated.

Some embodiments described here concern a formulation or composition for oral administration, advantageously made as a multilayer formulation with a differentiated or prolonged release.

Favorably, the formulation described here is for treating cholesterol, in particular for reducing the plasma levels of total triglycerides, cholesterol and LDL and for increasing those of HDL.

In some embodiments, which can be combined with all the embodiments described here, the differentiated-release multilayer formulation comprises two differentiated-release layers and provides a first layer comprising Monacolin K or a Monacolin K extract, and another layer comprising Berberine or an extract containing Berberine.

According to some embodiments, the first layer also comprises polyphenols. According to some embodiments, the multilayer formulation comprises, or consists of, three differentiated-release layers, wherein the first layer and the other layer, or third layer, are separated by a second central layer.

In some embodiments, which can be combined with all the embodiments described here, the formulation described here can comprise, as active ingredients, a mixture of:

- Monacolin K or a Monacolin K extract;

- Berberine or an extract containing Berberine;

- polyphenols;

- coenzyme Q10 (ubidecarenone/ubiquinone) as an antioxidant to reduce inflammatory processes;

- vitamin K2, preferably MK-7, with the function of reducing calcium deposits in the veins and arteries.

In some embodiments, the formulation described here comprises Berberine, or an extract containing Berberine, between 1 and 50% weight/weight, preferably between 2 and 48%, more preferably between 3.5 and 45%.

Berberine (BBR) is a natural compound isolated from the rhizome of the traditional Chinese herb Coptidis, identified as a promising compound capable of lowering plasma cholesterol and with satisfactory clinical efficacy and safety. Berberine increases the expression of the LDLR gene in hepatocytes at the post- transcriptional level by stabilizing its mRNA, a mechanism distinct from that of statins. Due to its unique action and safety history, BBR is an ideal candidate for combination therapy with statins to treat hypercholesterolemia. However, the bioavailability of Berberine is generally very low, around 0.7%.

In some embodiments, the formulation described here comprises Monacolin K between 0.1 and 1.5% (weight/weight).

In some embodiments, the weight ratio between Monacolin K and Berberine in the formulation according to the present description is between 1 :500 and 1:1.

In some embodiments, the formulation comprises red yeast rice (RYR) or RYR extract as a source of Monacolin K. For example, the red yeast rice (RYR) extract comprises as an active component Monacolin K, preferably between 1 and 5% by weight of Monacolin K. In some embodiments, the red yeast rice (RYR) extract is present between 0.6 and 10% weight/weight in the formulation described here. Preferably, the red yeast rice extract used in the embodiments described here can be extracted naturally.

In some embodiments, the formulation described here comprises polyphenols between 0.3 and 50% (weight/ weight).

In some embodiments, the polyphenols present in the formulation contain in particular flavonoids as an active component. In possible embodiments, the flavonoids are derived from one or more of: Bergamot or Bergamot extract, or propolis or propolis extract, or extract of: berries (blueberry, bilberry, cranberry, currant, blackberry, raspberry, etc.), aronia (Chokeberry), mulberry, elderberry, hibiscus, orange, green tea, vitis vinifera, spices (rosemary, thyme, oregano, cumin, cinnamon or other), olives ( Olea europea ).

In the embodiments in which the flavonoids derive from Bergamot or Bergamot extract, the Bergamot extract is a natural Bergamot extract present in the formulation as a source of the flavonoids. In particular, the flavonoids are contained in the natural Bergamot extract in a range between 5 and 80% (weight/weight).

The Applicant has found that Bergamot juice ( Citrus bergamia ) and its flavonoids are able to reduce lipid serum levels, also improving atherosclerosis through the modulation of enzymatic activities, antioxidant and anti inflammatory mechanism and inhibition of the activation and proliferation of monocytes. Bergamot has a typical profile of flavonoids, such as neoeriocitrin, neoesperidin, naringin, rutin, neodesmin, rhoifolin and poncirin. Some of these flavonoids, such as naringin and its aglycone naringenin, have an antiatherogenic effect on animal models, while neoeriocitrin and rutin have been found to inhibit the oxidation of LDL. In addition, Bergamot juice is rich in 3-hydroxy-2- methylglutaryl neohesperidoside of hesperetin (brutieridin) and naringenin (melitidin), compounds capable of inhibiting HMG-CoA reductase. These compounds are likely to contribute to the substantial hypolipidemic effects that Bergamot juice has demonstrated in various animal studies. In fact, the evaluation of the effect of Bergamot on the lipid profile has shown a significant reduction in total cholesterol, triglycerides and LDL-C, and an increase in HDL- C after administration of Bergamot.

The Applicant has also found that the co-administration of Monacolin K from RYR with flavonoids represents a delivery system to improve the bioavailability of Monacolin K. In particular, it was surprisingly discovered that the co- administration of Monacolin K from RYR extract with some polyphenols/flavonoids extracted for example from Bergamot fruits is able to provide a synergistic effect between these molecules and Monacolin K from RYR extract, such as to increase the oral bioavailability of the latter.

In some embodiments, the formulation described here comprises coenzyme Q10 between 0.2 and 20% weight/weight.

Coenzyme Q10 (CoQlO) is an essential component of the human electron transport chain in mitochondria and an important liposoluble antioxidant that protects cell membranes and lipoproteins against oxidative damage. There is a clear relationship between plasma levels of total cholesterol or low density lipoproteins (LDL) and CoQlO. Dietary CoQlO intake is low, approximately 3-5 mg/day. Therefore, supplementation with CoQlO is recommended in cases of deficiency in order to at least restore the antioxidant capacity and to prevent oxidation of LDL. Unfortunately, the bioavailability of CoQlO is very low due to its chemical-physical characteristics, such as its high molecular weight, lipophilicity and poor aqueous solubility.

In some embodiments, the formulation described here comprises vitamin K2 between 0.00001 and 0.05% weight/weight.

Vitamin K is a generic term for compounds having 2-methyl- 1,4- naphthoquinone as a common nucleus and a variable alkyl substituent in 3 position. Vitamin K1 (phylloquinone) has a phytyl side chain and is of vegetable origin, while Vitamin K2, used in the formulation according to the present description, has bacterial origin and is represented by a group of analogues known as menaquinones-n (MK-n), where n represents the number of isoprenoid residues in the side chain. Regarding bioavailability, menaquinone-7 (MK-7) appears to be better absorbed than menaquinone-4 (MK-4) following oral administration.

In some embodiments, the weight ratio between vitamin K2 and Berberine in the formulation according to the present description is between 1:5x1 O ⁶ and 1:20.

Furthermore, the Applicant has found that, in contrast to the current belief that the reduction of cholesterol with statins reduces atherosclerosis, statins could, instead, cause the depletion of coenzyme Q10 with consequent calcium deposit in the coronary artery and in blood vessels, thus compromising their functionality. Furthermore, statins inhibit the synthesis of vitamin K2, the cofactor for the activation of the GLA protein, which in turn protects the arteries from calcification. Statins also inhibit the biosynthesis of selenoproteins, such as glutathione peroxidase which serves to reduce oxidative stress. A compromised biosynthesis of selenoproteins could be a factor in congestive heart failure. Thus, paradoxically, heart failure and atherosclerosis could be aggravated by using drugs such as statins.

Monacolin K has a mechanism of action very similar to that of statins. The Applicant has found that, even in the case of administration of Monacolin K from RYR, a patient could experience loss of vitamin K2 and consequent greater arterial calcification. The Applicant has therefore discovered that it is preferable that, when Monacolin K from RYR is taken, vitamin K2 is also administered.

In some embodiments, the formulation described here comprises one or more pharmacologically acceptable adjuvants and/or excipients. In particular, among the excipients there can be one or more of either diluents, absorbents, adsorbents, lubricants, binders, disintegrants, emulsifiers or surfactants, in particular surfactants, dyes, sweeteners, antioxidants or antimicrobials, polymers to modify the time or site of release, coating agents.

In specific embodiments, the formulation comprises one or more of such surfactant or emulsifying agents, in particular phospholipid emulsifiers. An example of a phospholipid emulsifier is lecithin, in particular soy lecithin or sunflower lecithin.

In other embodiments, among the excipients present in the formulation described here there are lipid excipients, in particular phospholipids and/or fatty acid esters and/or fatty acids. These lipid excipients are food grade or food supplement grade. Advantageously, the lipid excipients are present at a concentration comprised between 5 and 95% weight/weight with respect to the weight of the formulation, preferably between 10 and 90%, more preferably between 16 and 85%.

The fatty acid esters can be obtained from fatty acids esterified with, for example and not only, glycerin (mono-, di-, tri-glycerides), sugars (e.g. sucrose esters) of natural or artificial origin (e.g. sucralose and its esters), polyols (e.g. sorbitans). Another possible example of fatty acid esters is glyceryl dibehenate. In some embodiments, the glyceryl dibehenate is present between 5 and 50% (w/w) with respect to the weight of the formulation, preferably between 10 and 50%, more preferably between 16 and 50%.

In other embodiments, the glyceryl dibehenate is present between 5 and 80% (w/w) with respect to the weight of the formulation, preferably between 10 and

80%, more preferably between 16 and 80%.

In yet other embodiments, the glyceryl dibehenate is present between 5 and 95% weight/weight with respect to the weight of the formulation, preferably between 10 and 90%, more preferably between 16 and 85%. In some embodiments, the weight ratio between glyceryl dibehenate and Berberine in the formulation according to the present description is between 20:1 and 1:1.

The presence of lipid excipients allows to increase the bioavailability of Berberine, which allows to decrease its concentration in the formulation. For example, with this solution, it is possible to provide a concentration of Berberine lower than 45% weight/weight with respect to the weight of the formulation, even lower than 30%, for example comprised between 4 and 15%.

In the embodiments in which the formulation is multi-layer with a differentiated release, in particular with three layers, it is preferable that the layer comprising the Monacolin K, in particular supplied in the form of red yeast rice extract, and the polyphenols, in particular Bergamot extracts, is separated, both physically and in terms of timing of release, from the layer containing the Berberine.

In particular, in some embodiments a first layer can be provided which comprises the Monacolin K, in particular supplied in the form of red yeast rice (RYR) extract, and the polyphenols, in particular extracted from Bergamot, a second central, or intermediate, layer which comprises the vitamin K2 and/or coenzyme Q10 and a third layer which comprises the Berberine. Advantageously, the different layers release the respective active ingredients at different times. Specifically, the first layer releases between about 10 and 20 min, in particular in about 15 min, the second between about 1.5 and 2.5 h, in particular in about 2 h, the third between about 3.5 and 4.5 h, in particular in about 4 h. The distribution of the active ingredients in the different layers derives from the following considerations made by the Applicant.

Favorably, as better described below, the lipid excipients as above can be present at least in the second layer and in the third layer in order to improve the bioavailability of the active ingredients contained therein. In some embodiments, these lipid excipients can also be present in the first layer.

In particular, the lipid excipients described here can be present in the third layer. This solution considerably improves the bioavailability of the Berberine present in the third layer.

With regard to the first layer, the Applicant has found that after induction of Berberine for three days before the administration of a single oral dose of lovastatin (whose natural equivalent is Monacolin K), the pharmacokinetic parameter such as t_\n was significantly increased with the induction of Berberine, while the Area Under Curve (AUC, area under the concentration/time curve) and Cmax (maximum drug concentration at time tmax) parameters were significantly reduced compared to a single oral dose of Monacolin K from RYR extract. This observation revealed a potential drug-drug interaction between Berberine and Monacolin K from RYR extract. Therefore, Berberine could reduce the bioavailability of Monacolin K from RYR extract and, at the same time, increase its metabolization. Therefore, the Applicant has found that it is preferable that Berberine is not present in the first layer containing Monacolin K, in the form of RYR extract, and even more preferably that it is present in a separate, that is, not directly contiguous, layer.

Therefore, according to some embodiments, it is preferable that the first layer of the formulation described here contains RYR and Bergamot extract titrated respectively in Monacolin K and flavonoids, while the Berberine is contained in the third layer.

According to some embodiments, the central layer contains vitamin K2, preferably MK-7, and/or coenzyme Q10 and, according to other embodiments, also an appropriate quantity of the lipid excipients, more in particular phospholipids and/or fatty acids esters and/or fatty acids, in order to improve the bioavailability of the active ingredients.

In particular, the Applicant has found that the bioavailability of vitamin K2, preferably MK-7, and coenzyme Q10 is normally low due to their high molecular weight, high lipophilicity and therefore poor solubility in water. Furthermore, the quantity of phospholipids and or fatty acid esters and/or fatty acids in the central layer allows the release of these active components in a desired time, in particular between 1.5 h and 2.5 h, for example approximately 2 h, in order to counteract the potentially negative effects of Monacolin K from RYR extract.

According to some embodiments, the third layer contains Berberine and, according to other embodiments, also an appropriate quantity of the lipid excipients as above, in particular phospholipids and/or fatty acid esters and/or fatty acids, in order to improve the bioavailability of Berberine. In fact, Berberine is generally poorly absorbed, resulting in low bioavailability after oral administration. It appears that, due to its lipophilicity, its passage through the membranes of enterocytes is reduced. Furthermore, after oral administration, part of the Berberine remains intact through the gastrointestinal tract while the other part is metabolized through the small intestine.

In particular, the Applicant has devised an approach to increase the bioavailability of Berberine which, in some embodiments, contemplates the presence in the formulation described here, and in particular for example in the layer containing Berberine, also of the lipid excipients as above, in particular phospholipids and/or fatty acid esters and/or fatty acids, as agents to increase the bioavailability of Berberine. It has been found that, in this way, the liposolubility of Berberine, and therefore its bioavailability, is significantly increased. A particularly advantageous example of fatty acid esters is glyceryl dibehenate. The Applicant has noted that the complexation of Berberine with lipid excipients, in particular phospholipids and/or fatty acid esters and/or fatty acids described here, significantly increases its intestinal absorption.

Overall, according to some embodiments, the third layer therefore contains an appropriate quantity of lipid excipients, in particular phospholipids and/or fatty acid esters and/or fatty acids, which allows both to increase the bioavailability and also the release of Berberine over time.

In some embodiments, which can be combined with all the embodiments described here, the differentiated-release multilayer formulation provides a first layer comprising Monacolin K or a Monacolin K extract, a Bergamot extract or an extract rich in polyphenols, Vitamin K2 and coenzyme Q10 and another layer comprising Berberine or an extract containing Berberine

EXPERIMENTAL DATA Prolonged release of active compounds over time

The Applicant has developed a release strategy based on the production of a pharmaceutical form which would allow a modified release of the functional active components present. For example, the Monacolin K of the first layer, and the polyphenols if also present in this first layer, are released for example after 10-30 min, followed by other active ingredients present in the central layer, for example vitamin K2 and possible coenzyme Q10, and finally, after about 3-5 hours, the Berberine present in the third layer. Each release phase was advantageously treated with an approach aimed at increasing the bioavailability of the active ingredients present.

For example, the Applicant has adopted a multilayer tablet technology in order to allow differentiated releases in the desired time frame. A possible example provides that the first layer releases most of the active principle after about 15 minutes, the second layer after about 2 hours and the third layer after about 4 hours.

The Applicant has conducted, by way of example, a dissolution test carried out on a three-layer formulation, consisting of a first rapid layer (“fast”), a second normal layer, the intermediate one, and a third delayed-release layer (“retard”).

The data obtained, shown in figs. 1, 2 and 3, show that the Monacolin K, marker of the first “fast” layer, is completely dissolved after 15 min. Fig. 1 shows the release percentage of the Monacolin K present in the first layer of the three- layer tablet. Figs. 2 and 3 show the dissolution percentage of vitamin K2 and Berberine respectively. Figs. 2 and 3 show that vitamin K2 and Berberine are completely released from their respective layers, intermediate and third respectively, after 2 and 4 h.

Transport experiments in Caco-2 cell model The transport experiments in Caco-2 cell model allowed the Applicant to demonstrate that it is possible to increase the intestinal absorption of Berberine, improving its bioavailability. A system (BBR-ES) for delivery in emulsion (ES) for Berberine (BBR) was prepared and its effect on the absorption of BBR was evaluated by means of a Caco-2 cell transport method. The following formulations were tested, each with a total weight of 1000 mg.

(*) In Test 4 (T4) a hot preparation technology was used, in which the fatty acid esters were heated to about 70 °C and, subjected to stirring, Berberine was added. The mixture was stirred for 10 minutes, slowly cooled and then reduced to a fine powder.

Glyceryl dibehenate is a possible example of a fatty acid ester used in the experimental tests described here.

Transcellular transport of BBR was performed in accordance with a published study (Volpe 2011) with slight modifications. Each experiment was conducted in triplicate. The Caco-2 cells were supplied by the molecular biological laboratory of DeFENS, Universita degli Studi di Milano (I).

The cells were grown in T-75 culture flasks in a CO2.O2 atmosphere (5:95 %, v/v) at 37°C, and cultured in a Dulbecco culture medium also containing 10% (v/v) FBS, 90 U/mL penicillin, 90 pg/mL streptomycin, 1% non-essential amino acids and L-glutamine.

The Caco-2 model was tested by seeding the cells with 80% degree of confluence on Transwell® 24- well permeable supports at a density of 2xl0⁵ cells per well. The integrity of the monolayers was evaluated by means of a transepithelial electrical resistance method and experiments were conducted when the resistance values were above 300 W-cm².

In the experiments from apical (AP) to basolateral (BP) direction, 0.5 mL of test solution (5, 20 and 50 pg/mL of BBR) diluted in a pH 6.8 buffer was added to the AP side of the monolayer.

Aliquots (50 pL) were taken from the BP side at 30, 60, 90, 120 and 180 minutes and after centrifugation at 6000 x g, all samples were stored at -20 °C prior to Liquid Chromatography-High Resolution-Mass Spectrometry (LC-HR- MS) analysis.

At the end of the experiment, after 180 minutes, the cells were removed, counted and washed with pH 6.8 buffer, removing the adsorbed BBR. The washing solution was removed, and the cells treated with trypsin to separate the cells, which were lysed with Triton® lysis buffer. 50 pL of methanol was added to the lysate and the suspension centrifuged at 6000 x g for 1 min. The clear solution was transferred to a bottle, analyzed by means of LC-HR-MS and the quantity of Berberine expressed as ng/10⁶ cells.

The determination of the Berberine in all the samples was carried out by means of Acquity model Ultra Performance Liquid Chromatography (UPLC) (Waters, Milford, US) coupled to an E-Lambda model diode array detector (Waters) and to an Exactive model high resolution Fourier transform mass spectrometer (HR-FT-MS) (Thermo Scientific, San Jose, CA, USA) equipped with a HESI-II probe for electrospray ionization and a collision cell (HCD). The operating conditions were: spray voltage +4.0 kV, primary gas flow rate 55, auxiliary gas flow rate 10, capillary temperature 320°C, capillary +37.5 V, lens +10 V, skimmer +26 V, and probe temperature 130°C. 5 pL was injected onto a 1.7 pm Kinetex XB Cis column (100x2.1 mm, Phenomenex) maintained at 40°C and flow 0.7 ml/min. The eluents were 0.05% formic acid in MilliQ (solvent A) and acetonitrile (solvent B) treated water. The chromatographic separation was carried out by means of the following linear elution gradient: from 5 to 50% of solvent B for 5 min and then from 50 to 90% for 5 min at a flow rate of 0.7 mL/min.

The analyzes were carried out in full-scan mode in the range (m/z)⁺ 100-1000 u, using an isolation window of ±2 ppm. The AGC target, injection time, resolution and energy in the collision cell were lxlO⁶, 100 ms, 50 K and 30 V respectively. The MS data was processed using the software Xcalibur (Thermo Scientific). The identity of the peak was ascertained by evaluating the accurate mass of the fragments obtained in the collision cell. The calibration was in the range 0.01-10 pg/ml.

The results obtained seem to suggest that in particular the fatty acid esters (for example, glyceryl dibehenate) present in the formula could significantly change the transport of Berberine. In fact, the values of apical (AP) toward basolateral (BL) transport of the formulations 1, 2, 3 and 4 are larger than those of the respective controls, as shown in fig. 4.

As expected, and as shown in fig. 5, among the test formulations prepared in the same way, the largest quantity of Berberine in the basolateral solution was T1>T2>T3. On the other hand, among the formulations containing the same quantity of Berberine (T3 and T4), that is, 50 mg, the formulation prepared by heating the solution of fatty acid esters (T4) showed a better absorption of the active ingredient. Fig. 5 shows the concentration of Berberine in the basolateral solution after 180 minutes of incubation with Caco-2 cells.

It should be noted that the greater quantity of Berberine permeated through the Caco-2 cells, compared with the quantity initially present in the apical solution, was T4>T3>T2>T1, as seen in fig. 6. The formulations Test 1, Test 2, Test 3, Test 4 are as in Table 1.

Overall, the formulation containing fatty acid esters, for example glyceryl dibehenate, significantly increased the permeability of Berberine through the Caco-2 monolayer. In particular, a significant increase was measured in the percentage of Berberine, with respect to the control, absorbed by the Caco-2 cells, as the fatty acid esters:Berberine ratio increased from 1:1 to 20:1, as shown in fig. 7. Particularly interesting is the fact that, considering the absorption percentages determined in the in vitro tests, approximately 75 mg of the T3 and T4 formulations correspond to 500 mg of the control formulation Cl.

Some embodiments of formulations in accordance with the following Examples are described below, which have been developed on the basis of experimental data and are intended for illustrative and non-limiting purposes.

Example 1 - Three-layer prolonged-release tablet

An example of fatty acid esters is glyceryl dibehenate.

Some examples of pharmaceutically acceptable excipients and/or other ingredients that can be present in the embodiments of formulation described here are one or more of:

- cellulose, magnesium stearate, sodium bicarbonate, sodium silicate, silicon dioxide, talcum powder, bentonite, stearic acid, polyvinylpyrrolidone, polysorbates, polydimethylsiloxane, cross-linked sodium carboxymethyl cellulose, calcium phosphate, hydroxypropyl methylcellulose; - steviol glycosides, erythritol, maltodextrin, sucralose, neohesperidin, polyols

(for example xylitol, mannitol, sorbitol ...), sugars (for example fructose, glucose, sucrose);

- citric acid, lactic acid, tartaric acid;

- emulsifiers and thickeners (for example lecithin, gums, alginates, mono-, di-, tri-glycerides, fatty acids, phospholipids, fatty acid esters with sugars, polyols, etc.); - flavoring compounds (for example vanilla, strawberry, citrus, lemon, ...);

- dyes;

- vitamins;

- natural extracts that provide active compounds, such as antioxidants, vitamins, minerals or others;

- essential oils.

According to the present invention, it is possible, with a single formulation, to have the benefits of both Berberine and also Monacolin K, but without having the disadvantage obtained when they are administered together.

It is clear that modifications and/or additions of parts or steps may be made to the formulation as described heretofore, without departing from the field and scope of the present invention as defined by the claims.

REFERENCES

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Claims

1. Formulation for oral administration, wherein said formulation is a differentiated-release multilayer formulation, in which a first layer comprises Monacolin K or an extract containing Monacolin K, and another layer comprises Berberine or an extract containing Berberine.

2. Formulation as in claim 1, wherein said first layer comprises polyphenols.

3. Formulation as in claim 1 or 2, wherein said multilayer formulation comprises, or consists of, three differentiated-release layers, wherein said first layer and said other layer are separated by a second central layer.

4. Formulation as in claim 3, wherein said second central layer comprises vitamin K2 and/or coenzyme Q10.

5. Formulation as in any claim from 1 to 4, wherein said formulation is for reducing the plasma level of cholesterol, triglycerides and LDL and increasing that ofHDL.

6. Formulation as in any claim from 1 to 5, comprising pharmacologically acceptable adjuvants and/or excipients, wherein said excipients comprise lipid excipients, in particular fatty acid esters and/or fatty acids and/or phospholipids.

7. Formulation as in claim 6, wherein said lipid excipients are present in one or more of the layers of said formulation, in particular at least in said other layer containing Berberine or an extract containing Berberine.

8. Formulation as in claim 6 or 7, wherein said fatty acid esters comprise, or consist of, glyceryl dibehenate.

9. Formulation as in any claim from 1 to 8, comprising Berberine, or an extract containing Berberine, between 1 and 50% weight/weight.

10. Formulation as in any claim from 1 to 9, comprising Monacolin K between 0.1 and 1.5% weight/weight.

11. Formulation as in claim 2, or as in any claim from 3 to 10 when dependent on claim 2, comprising polyphenols between 0.3 and 50% weight/weight.

12. Formulation as in claim 4, or as in any claim from 5 to 11 when dependent on claim 4, comprising vitamin K2 between 0.00001 and 0.05% weight/weight.

13. Formulation as in claim 4, or as in any claim from 5 to 12 when dependent on claim 4, comprising coenzyme Q10 between 0.2 and 20% weight/weight.

14. Formulation as in any claim from 1 to 13, wherein red yeast rice extract is present as the source of said Monacolin K, in particular said Monacolin K is contained, in said red yeast rice extract, in the range 1-15% by weight.

15. Formulation as in any claim from 1 to 14, in the form of a dose unit, ready for the administration of Berberine at a daily dose of 10 to 500 mg.

16. Formulation as in any claim from 1 to 15, in the form of a dose unit, ready for the administration of Monacolin K at a daily dose of 1 to 15 mg, preferably from 3 to 10 mg.

17. Formulation as in claim 4 or as in any claim from 5 to 16 when dependent on claim 4, in the form of a dose unit, ready for the administration of vitamin K2 at a daily dose of 0.1 to 500 meg.

18. Formulation as in claim 4 or as in any claim from 5 to 17 when dependent on claim 4, in the form of a dose unit, ready for the administration of coenzyme Q10 at a daily dose of 2 to 200 mg, preferably from 10 to 30 mg.

19. Formulation as in claim 2 or as in any claim from 3 to 18 when dependent on claim 2, in the form of a dose unit, ready for the administration of polyphenols with antioxidant activity at a daily dose of 3 to 500 mg.

20. Formulation as in claim 2 or as in any claim from 3 to 19 when dependent on claim 2, wherein the polyphenols contain in particular flavonoids as an active component, in particular from Bergamot, more in particular from natural Bergamot extract.

21. Formulation as in claim 20, wherein said natural Bergamot extract is present in said formulation as a source of said flavonoids, in particular said flavonoids are contained in said natural Bergamot extract in a range between 5 and 80% weight/weight.

22. Method to make a formulation for oral administration, said method comprising preparing a differentiated-release multilayer formulation, in which a first layer comprises Monacolin K or an extract containing Monacolin K, and another layer comprises Berberine or an extract containing Berberine.