The present invention relates to compositions containing berberine in association with extracts or compounds of plant origin which enhance its bioavailability.

Prior art Cholesterol is a molecule which plays an essential physiological role: it prevents excessive fluidity of the cell membranes, and is an important precursor in the synthesis of hormones, Vitamin D and bile salts.

In the blood, cholesterol is bound to proteolipid substrates with carrier functions. These carriers are distinguished according to their density. On the basis of this subdivision, cholesterol can be found in the form of kilomicrons, HDL (High-Density Lipoproteins), LDL (Low-Density Lipoproteins), VLDL and IDL (Very Low and Intermediate Density Lipoproteins respectively). Cholesterol, especially in the form of LDL, can undergo an oxidation process which endangers the arteries. Oxidised cholesterol tends to accumulate in the cells (in this case called Foam Cells) which constitute the inner wall of the arteries. The resulting thickening, which narrows the lumen of the artery, is called an atheromatous plaque. Said narrowing considerably limits the flow of oxygen-rich arterial blood, leading to atherosclerosis and its cardiovascular complications (heart attack, stroke, etc.). Hypercholesterolemia is often accompanied by a marked increase in blood triglycerides. Conditions characterised by plasma cholesterol levels exceeding 180-200mg/dl, and triglyceride levels exceeding 200 mg/dl, are therefore now more correctly described as dyslipidaemia. A deterioration in the lipid balance can result from serious dietary disorders, liver metabolism disorders, and other diseases.

Conditions predisposing to an increase in LDL and triglycerides include diabetes mellitus, hypothyroidism, obesity, alcoholism, kidney disease, the use of oral contraceptives, liver disease and genetic disorders.

Two main classes of drugs are used to correct the altered lipid balance: statins and fibrates. Statins (e.g. atorvastatin, fluvastatin, pravastatin and simvastatin) act by inhibiting the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) involved in the biosynthesis of cholesterol. The use of statins has reduced mortality due to coronary disease: by normalising the blood cholesterol and consequently reducing the development of atherosclerotic plaques, cardiovascular events and mortality are also reduced. Fibrates are used in forms of dyslipidaemia in which triglycerides prevail. In addition to reducing the circulating levels of triglycerides, these substances increase the HDL fraction, modify the LDLs, making them less atherogenic, and reduce the fibrinogen levels. In order to thin the blood and prevent clot formation the treatment is often associated with platelet aggregation inhibitors such as low doses of acetylsalicylic acid, especially as secondary prevention after a thrombotic event, such as a myocardial infarction or stroke. Other drugs used to treat dyslipidaemia include probucol, nicotinic acid, niacin and derivatives thereof.

In addition to the established treatments, there are many compounds of natural origin which can supplement or replace the classic drugs listed above.

For example, red yeast rice {Monascus purpureus) inhibits the enzyme HMG-CoA-reductase, consequently acting as a natural statin.

Garlic, used in the form of standardised dried extract or essential oil, is useful in cases of hypercholesterolaemia due to its platelet aggregation inhibiting and fibrinolytic properties and its ability to improve the HDL/LDL ratio and reduce the triglyceride level.

Caigua is a plant native to South America which has been used to treat hypercholesterolaemia for centuries. Recent studies have demonstrated the ability of this plant to reduce LDL levels and increase HDL levels. Chitosan is a polymer obtained from chitin, a substance extracted from the exoskeleton of shellfish. Its use reduces the absorption of dietary lipids by up to 30%.

Gugulipid is a resin obtained from an Indian tree, Commiphora mukul, used in ayurvedic medicine to treat hypercholesterolaemia by rebalancing the ratio between HDL and LDL and reducing the viscosity of the blood.

Policosanols, extracted from sugar cane or rice, effectively reduce cholesterol and triglycerides.

Artichoke {Cynara scolymus) leaf extract is a preparation which has been studied for its cholesterol-reducing properties. The active ingredient contained in the extract is believed to be luteolin-O-glucoside, a probable inhibitor of the reductase responsible for liver cholesterol synthesis.

It has been known for nearly 50 years that plant sterols and stanols, also called phytosterols and phytostanols, reduce the cholesterol level by partly inhibiting the absorption of cholesterol in the intestine. Sterols are found in nature in small amounts in many types of fruit, vegetables, nuts, seeds, pulses and vegetable oils. Finally, the polyunsaturated acids in the omega-6 series appear to reduce the blood cholesterol level by reducing the plasma LDL levels. However, this benefit seems to be partly attenuated by the fact that the same fatty acids simultaneously reduce HDL cholesterol. Omega-3 polyunsaturated fatty acids basically reduce the plasma levels of triglycerides, by interfering with their incorporation in the VLDLs in the liver. They consequently seem to possess an important antithrombotic action, as high levels of triglycerides in the blood reduce the fibrinolysis process. Therapeutic approaches designed to improve the altered lipid balance have so far either proved partly ineffective or caused sometimes serious side effects.

Some authors recently analysed the efficacy of an alkaloid known as berberine obtainable from various medicinal plants used in Chinese medicine. Berberine is an isoquinoline alkaloid which, in the form of the sulphate and phosphate, has been used to treat malaria, bleeding and fever. According to more recent studies, this compound reduces the cholesterol level. Berberine is mainly extracted from Copt is chinensis and Berber is aristata. When taken orally for 3 months by 32 patients with high blood cholesterol levels it reduced plasma cholesterol by 29%, triglycerides by 35%, and LDL cholesterol by 25%. Researchers from the Chinese Academy of Medical Sciences in Beijing, using human hepatoma cells, have demonstrated that berberine increases the expression of LDLR (low-density lipoprotein receptor). Increased expression of LDLR by berberine takes place via a post-transcription mechanism which stabilises the messenger RNA. Berberine therefore potentially seems to be a new blood-lipid reducing agent with an action mechanism different from those of the statins and other conventional therapeutic compounds. In view of its high safety profile and tolerability, demonstrated clinically and by very long use in traditional Chinese medicine, berberine presents as an elective molecule in the treatment of hypercholesterolaemia. Moreover, in view of its blood-sugar reducing efficacy in diabetics, and the increasingly common concomitant alteration of blood sugar and lipid levels in patients suffering from obesity and/or type II diabetes, berberine is also an elective compound in the treatment of carbohydrate disorders, not necessarily accompanied by alterations in the lipid balance. The use of berberine to treat alterations of the lipid balance is described in WO 2007/090289A 1 and WO 2007/1 13748. Compositions containing berberine, silymarin and a number of other active ingredients are disclosed in CN 101049349 and CN 1362216. WO 2007/035515 discloses anticancer/antiviral compositions comprising berberine in combination with S. marianum. However, the oral bioavailability of berberine is low, which limits its efficacy. Its pharmacokinetic characteristics are not yet fully understood. After intravenous administration, berberine presents linear kinetics with an AUC value that increases in proportion to the dose injected. From the blood, it is transferred to the liver, where it is processed and secreted through the hepatobiliary tract. In the liver, berberine is metabolised by a first demethylation stage followed by a second glucuronidation stage. The proportion of berberine detectable in the plasma after oral administration is very low. In the blood, berberine is detectable unchanged or in glucuronidated form. Its main metabolites are berberrubine, talifendine, dimethylene berberine and jatrorrhizine, detectable in the blood in free or glucuronidated form.

Description of the invention

It has now been found that the oral bioavailability of berberine and the analogues, derivatives or metabolites thereof can be considerably increased by associating it with some phytotherapeutic preparations, especially the flavonolignan fraction obtained by extraction from milk thistle (silymarin).

The oral bioavailability of the compound can increase from 2 to up 6-8 fold.

As glycoprotein P is abundantly expressed in the intestinal cells, and in view of the evident effects of the first stage associated with protein P-450, the oral bioavailability and absorption of berberine are presumably influenced by co-administration of inhibitors of those protein systems, as demonstrated by the data in our possession. From the kinetic standpoint, however, the most striking finding was obtained by co-administering silymarin. In view of the known hepatoprotective action of said compound and the crucial role played by the liver in lipid and carbohydrate disorders, silymarin can be considered an ideal candidate for the treatment of lipid/carbohydrate disorders with any etiology, as its use is designed to increase the oral bioavailability of berberine, its analogues and extracts containing it/them, and to contribute to their hypocholesterolaemic and hypoglycaemic effect. However, the validity of the invention does not depend in any way on the verification of that hypothesis.

As a matter of fact, the clinical evaluation of the two actives given separately has given the following results. Berberine alone at 1000 mg/day for 30 days did not modify at all total cholesterol (CT) or LDL; the same results were obtained after 60 days; differently, after 90 days, 1000 mg/day of berberine lowered CT and LDL to about 25-29%. Silymarin, even at 420 mg/day, did not modify CT and LDL in a significant way: at 30 days the two parameters were respectively -2 and -3% and, at 60 days, -5 and -5%; after 90 days the two parameters were -7 and -5%. Instead, the administration of berberine ( 1000 mg/day) together with silymarin (210 mg/day) already after 30 days modified the two parameters to -25 and -27%; after 60 days, the values were -30 and -32% and after 90 days the values were respectively -40 and -38%. Moreover, the administration of 50% of the dose (500 mg of berberine plus 105 mg of silymarin) decreased after 90 days the two parameters to-28 and -25%, respectively.

Anyway, both the presence of silymarin and the following bioavailability increase of berberine do not justify the results. Actually, even the administration of 3000 mg/die of berberine alone did not allow to reduce the two parameters as they have been reduced by administering 1000 mg of berberine together with 210 mg of silymarin.

The same results were obtained in diabetic patients. Berberine alone, given at 1500 mg/day, modifies glycate haemoglobin (gh) of 1 - 1.5% after 90 days. No evidence of activity was found at 30 or 60 days. Silymarin alone did not alter the same parameter at any dosage and at any time. The administration of the two compounds at 1000 mg/day (berberine) and at 210 mg/day (silymarin), decreased gh of 2% already after 30 days. These results could not even be obtained by administering 5000 mg/day of berberine (for 30 days).

These results again cannot be attributed only to the expected bioavailability increase of berberine since they were not obtained even administering berberine dosages from 3 to 5 times higher. It has in fact been found that AUC is increased by 6-8 times only in 20% of the subjects administered with berberine plus silymarin. In 80% of them, the concomitant administration of silymarin only doubles the berberine AUC.

These unexpected clinical synergistic results have been obtained administering berberine and silymarin at weight ratios berberine:silymarin from 5 : 1 to 5: 1.5, preferably 5 : 1.05. Said ratio appears to be critical. This was evaluated both increasing or decreasing the amount of berberine vs. silymarin ( 1000 mg vs 105 or 500 vs 210), respectively. The same negative result was also obtained by administering the two compounds in 1 : 1 ratio. The invention therefore relates to compositions containing: a) berberine or analogues thereof or extracts containing them; b) an extract of Silybum marianum, silymarin or one of its constituents, namely silybin, isosilybin, silychristin and silydianin in a weight ratio effective for increasing the berberine bioavailabilty up to 2 fold, preferably up to 4 to 6.8 fold. The compositions of the invention may also include other ingredients, preferably catechins of Camellia sinensis, ginsenosides obtained from Panax ginseng, quercetin, kaempferol and flavones obtained from Ginkgo biloba, curcumin from Curcuma longa, and hyperforin and/or hypericin from Hypericum perforatum.

Berberine may be present in the compositions according to the invention as such or in the form of an extract containing it, such as an extract of Berber is aristata. The quantity of berberine per dose unit is preferably between 500 and

2000 mg, while ingredient b) is preferably present in quantities of between 105 and 420 mg per unit dose.

The compositions of the invention preferably contain the component a) and b) as the sole active ingredients. The compositions according to the invention can be prepared with conventional techniques and excipients. Examples of dosage forms include tablets (film-coated, gastroprotected, time- or pH-dependent controlled-release, multi-layer), capsules, sachets, granulates, chewable gum discs, and orodispersible forms. The phytotherapeutic ingredients of the compositions according to the invention, and in particular the extracts of Silybum marianum (in this text referred simply also as "Silymarin"), may be present in phytosome (complexed with phospholipids), liposome, co-ground or ter-clathrate form.

The compositions according to the invention may also contain antioxidants such as vitamins (C and E), extracted polyphenols (OPC from Vitis vinifera, anthocyanosides and proanthocyanidins from bilberry), cysteine, cystine, methionine, glutathione, lipoic acid and ubidecarenone.

A further aspect of the invention relates to the use of berberine or analogues thereof or extracts containing them in association with silymarin or of extracts of Silybum marianum for the preparation of hypocholesterolaemic and hypoglycaemic compositions.

The following examples illustrate the invention in greater detail. EXAMPLE 1 - Film-coated tablets

Ingredient Quantity

Berberis aristata (85% as berberine) 588 mg

Silybum marianum dried extract (Silymarin) 210 mg

Microcel 200 mg

Dicafos 200 mg

Vegetable magnesium stearate 40 mg

PVP CL 40 mg

Silicon dioxide 20 mg

Shellac 50 mg

EXAMPLE 2 - "0" format capsules

Ingredient Quantity

Berberis aristata (97% as berberine) 515 mg

Silybum marianum dried extract (Silymarin) 210 mg

Talc 10 mg

Microcel 101 50 mg

EXAMPLE 3 - Sachets

Ingredient Quantity

Berberis aristata (97% as berberine) 1545 mg

Silybum marianum dried extract (Silymarin) 420 mg

Fructose 1000 mg

Methocel E5 20 mg

Aerosol 50 mg

Acesulfame K sweetener 10 mg

EI lO 2 mg

Citrus fruit flavouring 150 mg EXAMPLE 4 - Sachets

Ingredient Quantity

Berberis aristata (97% as berberine) 515 mg

Silybum marianum dried extract Silymarin) 105 mg

Saccharose 2265 mg

Citric acid 50 mg

Silicon dioxide 20 mg

Flavouring 150 mg

Acesulfame K 15 mg

EXAMPLE 5 - Programmed-release two-layer tablets

Ingredient Quantity

NORMAL-RELEASE LAYER

Berberis aristata (as berberine 97%) 515 mg

Silybum marianum dried extract (Silymarin) 105 mg

Dicafos 304 mg

Aerosil 3 mg

Vegetable magnesium stearate 6 mg

Colouring 1 mg

SLOW-RELEASE LAYER

Berberis aristata (as berberine 97%) 515 mg

Silybum marianum dried extract (Silymarin) 105 mg

Metholose 80 mg

Aerosil 2 mg

Vegetable magnesium stearate 3 mg

Microcel 80 mg

Dicafos 164 mg EXAMPLE 6 - Orodispersible formulation

Ingredient Quantity

Berberis aristata (as berberine 85%) 588 mg

Silybum marianum dried extract (Silymarin) 105 mg

Sorbitol 160 mg

Orange flavouring 20 mg

Mandarin flavouring 5 mg

Acesulfame K 2 mg

Aerosil 5 mg

Fructose 1566 mg

EXAMPLE 7 - Gum disc

Ingredient Quantity

Berberis aristata (as berberine 97%) 515 mg

Silybum marianum dried extract (Silymarin) 105 mg

Gum base 800 mg

Aspartame 2 mg

Menthol 2 mg

Acesulfame 1 mg

Levilite 20 mg

Talc F.U. 20 mg

Vegetable magnesium stearate 18 mg

Shellac 12 mg

Xylitol 250 mg

Gum arabic 6 mg

Titanium dioxide 6 mg

Carnauba wax 0.2 mg