WO2014147119A1

WO2014147119A1 - Silybin for the treatment of metabolic oxidative alterations in patients with non-alcoholic steatohepatitis (nash)

Info

Publication number: WO2014147119A1
Application number: PCT/EP2014/055499
Authority: WO
Inventors: Carmelina Loguercio; Alessandro FEDERICO; Paola STIUSO
Original assignee: Istituto Biochimico Italiano Giovanni Lorenzini S.P.A.
Priority date: 2013-03-20
Filing date: 2014-03-19
Publication date: 2014-09-25
Also published as: EP2976075B1; JP2016515540A; BR112015024181A2; RU2694896C2; ES2749192T3; PT2976075T; EP2976075A1; ITMI20130425A1; MX2015013409A; PL2976075T3; HUE045695T2; RU2015144870A; MX370252B; DK2976075T3

Abstract

The invention relates to a composition for use in the treatment of metabolic oxidative alterations in non-alcoholic steatohepatitis (NASH), wherein said composition comprises silybin and wherein the treatment is administered to patients having an altered lipidomic profile. Patients having altered lipidomic profile show an high TBARS value in TBARS test (lipid peroxidation marker) with respect to healthy subjects.

Description

SILYBIN FOR THE TREATMENT OF METABOLIC OXIDATIVE ALTERATIONS IN PATIENTS WITH NON-ALCOHOLIC STEATOHEPATITIS (NASH)

DESCRIPTION FIELD OF THE INVENTION

The present invention relates to a composition comprising silybin for the treatment of metabolic oxidative alterations in non-alcoholic steatohepatitis (NASH).

BACKGROUND OF THE ART

Oxidative stress, lipid peroxidation, antioxidant therapy, these terms are very common in the literature and in everyday clinical practice in recent years. In fact, in the pathogenesis of various diseases, including cancer, considerable emphasis is often given to the formation of free radicals and their entrapment by antioxidant substances.

In humans, evidence has clearly been provided that an increase in circulating oxidative damage markers is an expression of organ pathology.

Among the oxidative stress markers considered expression of pathology of various organs and systems, the increase of products from lipid peroxidation plays a prominent role. Lipid peroxidation markers in the circulation are present in many pathological conditions as an expression of peroxidation of cell membranes. Among various diseases, surely the metabolic syndrome, but also the chronic liver disease and the advanced stages of many cancers, represent pathognomonic situations of chronic diseases, in which the oxidative stress plays a key role.

Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome. Non-alcoholic steatohepatitis (NASH) is a type of chronic damage to the liver belonging to metabolic diseases and represents the degenerative inflammatory progression of the non-alcoholic fatty liver disease (NAFLD).

The presence of the metabolic syndrome or of one or of more components of this syndrome (i. e. visceral obesity, hypertension, hypertriglyceridemia, fasting hyperglycemia) greatly increases the risk of progression of non-alcoholic hepatic steatosis to steatohepatitis (NASH). NASH is in fact almost always present in patients with metabolic syndrome and affects their morbidity and mortality.

It is established through several steps which involve the modulation of a large number of genes and proteins, starting from an altered trafficking of lipids between the periphery (adipose tissue) and liver, with final accumulation of fatty acids in the liver, peroxidation of both circulating and intrahepatic lipids, which induce, through oxidative stress, hepatocyte inflammation and death, up to triggering of reparative phenomena, such as fibrosis and cirrhosis. At present, no pharmacological agents have been approved for the treatment of metabolic syndrome, in particular for the treatment of NASH. In the latter case, a long-term treatment was attempted with insulin sensitizing agents or with high doses of vitamin E, without definitive results.

While the use of natural products is increasing throughout the world, the fact that their use is not always safe and effective is, however, also known. Patients with non-alcoholic fatty liver disease (NAFLD) have been shown to respond positively when subjected to chronic administration of silybin. In particular, treatment of patients with NAFLD with silybin has been shown to improve the levels of transaminases, insulin resistance and liver histology. NASH is characterized, histologically, by a chronic inflammation and early fibrosis, as compared to other chronic liver diseases included in NAFLD.

Object of the present invention is to provide a treatment of the metabolic syndrome, when associated to NASH

SUMMARY OF THE INVENTION

The inventors of the present invention have identified among the patients with metabolic syndrome, when associated to NASH, a group of patients presenting a lipidomic profile alteration associated with high values of TBARS in serum, and have surprisingly discovered that treatment with a composition including silybin allows normalization of said lipidomic profile, thus having effects in the treatment of non-alcoholic steatohepatitis.

The invention thus relates to a composition for use in the treatment of metabolic oxidative alterations in patients with non-alcoholic steatohepatitis (NASH), wherein said composition comprises silybin, and wherein the treatment is administered to patients having an altered lipidomic profile.

In the present invention, when the definition "altered lipidomic profile" is used, it is meant the lipidomic profile of a patient with metabolic syndrome, preferably also with NASH, who is positive to the TBARS assay, who specifically show high values of TBARS in the TBARS assay ((2-ThioBarbituric Acid Reactive Substances (TBARS)). The TBARS assay measures in a serum sample of a patient the TBARS, i.e. the by-products generated by a process of lipid peroxidation, through the use of barbituric acid (Armstrong, D., and Browne, R. The analysis of free radicals, lipid peroxides, antioxidant enzymes and compounds to oxidative stress as applied to the clinical chemistry laboratory. Free Radicals in Diagnostic Medicine 366, 43-58 1994). In the present invention, when it is stated that high levels of TBARS or low levels of TBARS are present, it is meant that the values found in the TBARS assay are higher or lower than those observed in healthy subjects. In the present invention, while referring to average values of TBARS in healthy subjects average values of approximately 0.01 ±0.002 M/ gprot (μΜ of TBARS with respect to g of plasma proteins).

The inventors of the present invention have therefore identified a specific group of patients with the metabolic syndrome and with also NASH, who have an alteration of serum lipidomics and who are responsive to treatment with a composition comprising silybin. The group of patients identified by the inventors in the group of patients with metabolic syndrome with also NASH, have a lipidomic profile associated with altered lipid peroxidation (high values of TBARS) that distinguishes it from the group of patients with metabolic syndrome with also NASH, who, in basic conditions, do not show high levels of circulating lipid peroxidation markers (TBARS). The two groups of patients are in no way overlapping on the basis of the values of TBARS determined by the TBARS assay, as will be evident from the experimental section below. Upon completion of the treatment with the composition including silybin, the group of patients of the invention showed a normalization of the serum lipidomic profile, with a corresponding lowering of the values of TBARS in the TBARS assay, confirming the treatment of oxidative stress typical of NASH pathology.

The invention will now be described in detail and with reference to the following figures.

DESCRIPTION OF THE FIGURES

Figure 1 includes graphs of mass spectroscopy of a pool of serum samples of the control group (CTR) (a)), the first group of NASH patients at time zero (TO, Group 1 ) (b)) and the second group of NASH patients at time zero (TO, Gruppo 2) (c)). Figure 2 includes the graphs of mass spectroscopy of a pool of serum samples of the first group of NASH patients after 12 months of treatment (T12) with the composition of the invention (Group 1 ) (b)) and the second group of NASH patients after 12 months of treatment (T12) with the composition of the invention (Group 2) (c)).

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore relates to a composition for use in the treatment of metabolic oxidative alterations in non-alcoholic steatohepatitis (NASH), and wherein said composition comprises silybin, and wherein the treatment is administered to patients having an altered lipidomic profile.

The group of patients having the "altered lipid profile" show an high TBARS value in TBARS assay with respect to healthy subjects. The TBARS assay detects the substances reactive to barbituric acid, which are formed as by-products of lipid peroxidation. Therefore, TBARS levels represent a marker of lipid peroxidation. A patient having the metabolic syndrome with also NASH, who is positive to the TBARS assay, show high values of TBARS in the TBARS assay, i.e. a large number of lipidic peroxidation by-products of the cell membranes is present in the serum of the patient.

The inventors of the present invention have therefore identified a sub-group of patients with the metabolic syndrome with also NASH, who have an alteration of lipidomics and who are responsive to treatment with a composition comprising silybin.

The composition of the invention comprises silybin, preferably complexed with phosphatidylcholine. Silybin used in the invention can be in the form of an extract of Silybum marianum, i.e. Milk thistle. It is known that extracts of Milk thistle can be titrated with silybin or silymarin, in the invention all these extracts can be used. Extracts of Milk thistle are preferred, titrated with silybin in the range of 20 to 90%, more preferably of 30 to 70%. Even more preferably, the composition of the invention comprises an extract of Silibum marianum titrated with 33% silybin.

According to the invention, the composition comprises preferably vitamin E. Advantageously, the composition of the invention includes an extract of Milk thistle, complexed with phosphatidylcholine and vitamin E acetate.

The composition of the invention comprises also excipients suitable for the preparation of a pharmaceutical formulation.

Preferably, according to the invention, the daily dose of silybin is in the range of 150 to 300 mg/day, being even more preferable a dose of about 200 mg/day of silybin.

The group of patients with the metabolic syndrome with also NASH, and identified in the treatment according to the invention have therefore an altered lipidomic profile, which are normalized with the composition of the invention, as will be evident from the experimental part that follows.

Example 1

Preparation of the composition used in the invention

Silybin in the form of an extract of Milk thistle complexed with phosphatidylcholine, was added to vitamin E acetate in the amounts indicated in Table 1 :

Table 1

^* equal to 15 mg Vit E

The composition so obtained was administered to the patients in the form of sachets of powder composition.

Example 2:

Identification of the two groups of patients

We enrolled 20 patients affected by metabolic syndrome and non-alcoholic steatohepatitis (NASH). We also enrolled 20 healthy subjects, who formed the control group. The serum of 20 patients was assessed at basal conditions and after 12 months of treatment with the composition of the invention, by performing the TBARS assay. Then, the level of TBARS, i.e. the level of reactive substances as lipidic peroxidation by-products of the cell membranes in the serum of all 40 subjects was evaluated.

The analysis performed on the serum of these patients showed the presence of two distinct groups of patients (Tables 2 and 3). 1 1 of the 20 patients involved in the study were in group 1 and 9 belonged to group 2, based on the values of TBARS found and reported in Table 2 already divided into two groups.

Table 2 also shows the values of TBARS measured in patients not only at time zero (TO), but even after 12 months of treatment (T12).

Table 2. Values of TBARS (MM/ gprot.) measured in the serum of patients with metabolic syndrome and NASH belonging to group 1 (n=11) and of group 2 (n=9). TBARS = substances reactive to thiobarbituric acid.

Table 3 shows the average of the levels of TBARS measured at time zero (TO) and after 12 months of treatment for groups 1 and 2 of the patients and the average of the values of TBARS in the control group (CTR). At time TO, group 1 showed TBARS values lower than the values of the control group, in contrast to what was found in group 2, in which patients showed TBARS values higher than the control group.

Table 3. Serum values of TBARS (MM/ gprot.) in the two groups of NASH patients.

Group 1 Group 1 Group 2 Group 2 CTR

Parameters TO T12 TO T12

TBARS 0.004410.0003 0.02110.001 0.07410.006 0.04710.004 0.0110.002 The first group of patients (Group 1 ) in basal conditions showed a value of substances reactive to thiobarbituric acid (TBARS) lower compared to the control values. In this group, after the start of the treatment with the composition of the invention, TBARS levels increased immediately, but these values returned to normal values at the end of the treatment, after 12 months.

The second group (Group 2) showed a very high value of TBARS in basal conditions, compared to the average value of the control. In these patients, the treatment with the composition of the invention significantly reduced the average level of TBARS (p <0.01 ). Within the group of 20 patients with metabolic syndrome and NASH, two groups of patients were actually present, which differed in the levels of TBARS at the basal condition, group 1 had low values at time 0 and the second on the contrary had high values (compared to the control group of healthy subjects), getting closer to the values of the control group after 12 months of treatment.

The two groups of patients were then further characterized both at time zero and after 12 months of treatment with the composition of the invention (Table 4). All patients in group 1 had a body mass index (BMI), insulin and HOMA index (Homeostasis Model Assessment - an indicator of insulin resistance) which did not change after 12 months of treatment, only the glycaemia in the blood of group 1 decreased compared to basal value (p<0. 05). On the contrary, in group 2, BMI, insulinemia and HOMA index were significantly reduced (p<0.0001 ) after the treatment, while glycaemia in the blood remained normal after 12 months, as at the time of basal condition (TO).

Table 4. Average values of metabolic parameters for the two groups of patients at basal condition (TO) and after 12 months of treatment with the composition of the invention (T12). Group 1 Group 2

Parameters TO T12 TO T12

BMI 30±1.86 30±1.8 28±0,50 26±0,50***

Glycaemia 116±10 105+8,2* 99±2.15 99±2

Insulin 18±2.26 17±3.77 23+4.34 14±1.9***

HOMA 5±1 4.5+1.07 5.97±0.6 3.43±0.5***

^* p<0.05, ^** p<0.005; ^*** p<0.0001 Therefore, patients in Group 1 who at the time of basal condition had lower TBARS values as compared to the control group, resulted not to show changes after treatment with the composition of the invention, in contrast to patients in Group 2 having high TBARS values as compared to the controls, who responded to the treatment with silybin in the composition.

The use of the composition of the invention therefore reduced the body mass index, insulin and insulin resistance (HOMA) of the sole Group 2, determining thus a treatment of the metabolic syndrome and NASH only for this group of patients. Example 3

Normalization of the lipidomic profile of Group 2

The 20 patients with metabolic syndrome and non-alcoholic steatohepatitis (NASH), divided into two groups as shown in Example 2 were treated with the composition of the invention for 12 months at a dose of 4 sachets/day.

On the serum of 20 patients with metabolic syndrome and NASH and 20 healthy subjects in the control group, lipidomics analysis was performed by MALDI-TOF mass spectroscopy. Lipidomic species studied in the analysis, corresponding to the main peaks were:

Lyso: lysophosphatidylcholine, which can have different combinations of fatty acids with varying length and saturation bound at position C-1 .

lyso PC 16:0: lysophosphatidylcholine with palmitic acid

lyso PC 18:2 lysophosphatidylcholine with linoleic acid

lyso PC 18:1 lysophosphatidylcholine with oleic acid

lyso PC 18:0 lysophosphatidylcholine with stearic acid

SM: sphingomyelin PC: phosphatidylcholine, which can have different combinations of fatty acids with varying length and saturation bound at positions C-1 and C2.

PC 16:0/18:2 phosphatidylcholine, which has a chain of palmitic acid in position C- 1 and a chain of linoleic acid in position C2

PC 16:0/18:1 phosphatidylcholine, which has a chain of palmitic acid in position C- 1 and a chain of oleic acid in position C2

PC 18:1 /18:2 phosphatidylcholine, which has a chain of oleic acid in position C-1 and a chain of linoleic acid in position C2

PC 18:0/18:2 phosphatidylcholine, which has a chain of stearic acid in position C- 1 and a chain of linoleic acid in position C2

PC 18:1 /18:1 phosphatidylcholine, which has a chain of oleic acid in position C-1 and a chain of oleic acid in position C2

PC 18:2/18:2 phosphatidylcholine, which has a chain of oleic acid in position C-1 and a chain of linoleic acid in position C2

PC 18:1 /20:4 phosphatidylcholine, which has a chain of oleic acid in position C-1 and a chain of arachidonic acid in position C2

PC 18:0/20:4 phosphatidylcholine, which has a chain of stearic acid in position C- 1 and a chain of arachidonic acid in position C2

PC 18:0/20:3 phosphatidylcholine, which has a chain of stearic acid in position C- 1 and a chain of homo-g-linolenic acid in position C2.

A pool of the patients' serum was then made, i.e. a pool of the serum of patients belonging to group 1 and a pool of the serum of patients in group 2. Also a pool of the serum was made for the subjects belonging to the control group. The pools were collected and analyzed before treatment and after the treatment. For each pool graphs were drafted of MALDI-TOF mass spectrometry of organic extracts obtained from the sera of the patients themselves.

Figure 1 shows the following graphs: spectrogram a. reports the lipidomic profile of the pool of the normal subjects (control group), the spectrogram b. shows the lipidomic profile of the pool of patients belonging to group 1 and the spectrogram c. shows the lipidomic profile of the pool of patients belonging to group 2 (at time As it is evident from Figure 1 , both patients of Group 1 and Group 2 had a lipidic profile different from the profile of the control group. Specifically, all tested species were present in the serum of patients in amounts lower than the amounts of the control subjects (the experiments were repeated three times).

The two groups of patients were then subjected to a treatment with the composition of the invention. After 12 months of treatment with 4 sachets/day, the pools of patient sera, for which the mass spectrum was taken again, were analyzed. Figure 2 shows the mass spectrum of the pool of the same patients in group 1 and group 2 of Figure 1 , but after 12 months of treatment with silybin.

As it is evident from Figure 2, the spectrum of the pool of patients in Group 1 showed that the amount of detected lipidic species were still low, as at time zero, and indeed for some species the amount was more decreased.

Conversely, lipidomic profile in Group 2 at T12 (shown in Figure e)) resulted more similar to that one of the control group, thus demonstrating that a normalization had occurred for the lipidomic profile, as tested after the treatment with the composition of the invention.

The intensity of the values from the mass spectrometry was then normalized, and the values are shown in Table 5 below:

Table 5

Grou 1 Group 2

496,36 lyso PC 16:0 100 100 100 100 100

369,37 FC (-H₂0) (H⁺) 68 35 16 15 41

520,4 lyso PC 18:2 37 12 2 11 9

522,41 lyso PC 18:1 30 18 17 17 15

524,37 lyso PC 18:0 31 32 30 30 59

703,5 SM 16:0 20 5 1 8 18

758,65 PC 16:0/18:2 104 21 8 19 64

760,51 PC 16:0/18:1 (H⁺) 42 24 13 8 27

784,66 PC 18:1/18:2 43 13 5 9 34

786,53 * PC 18:0/18:2 (H⁺)/ PC 18:1/18:1 (H⁺) 40 9 4 6 18

804,52 PC 18:2/18:2 (Na⁺) 9 2 1 2 10

808,55 * PC 18:0/18:2 (Na⁺)/ PC 18:1/20:4 (H⁺) 19 3 9 5 19

810,55 PC 18:0/20:4 25 5 3 5 18

812,62 PC 18:0/20:3 12 5 22 3 16 # Identification is not unique, molecular weight can be attributed to two possible identities FC= free cholesterol

lyso PC = lysophosphatidylcholine

PC= phosphatidylcholine

SM= sphingomyelin

Therefore, normalized data confirmed what could be seen from the figures. In NASH patients of group 2, increased circulating levels of free cholesterol, sphingomyelin and phosphatidylcholine could be found after 12 months of treatment. Intensities of these species were low in basal conditions, and returned to control values after the treatment with the composition including silybin. Furthermore, the lipidomic analysis data show that in the group of patients with high TBARS values (Group 2) treatment with the composition including silybin induced an improvement in the ratio phosphatidylcholine/arachidonic acid. As it is well known, sphingomyelin is synthesized in the lumen of the Golgi apparatus, and is then transported to the cell membrane level by specific carriers. Sphingomyelin has a high affinity for cholesterol, with which it binds at the cell membrane level. Without being bound to any theory, based on the above data, the inventors of the present invention assume therefore that the treatment according to the invention catalyzes the release of free circulating sphingomyelin and cholesterol, through an increase in the lyso-phosphatidylcholine ratio, which has a "detergent-like" action on the membranes.

In order to reduce the experimental error, lipid extraction and analysis of lipidomic profile with mass spectrometry were carried out several times for the pool of patients of group 1 , group 2, and the control group, thus obtaining results which could be superimposed to those reported above, thus confirming that the lipidomic profile of the patients in group 2 was normalized.

Therefore, the results obtained from the lipidomic analysis, when added to the reductions in the insulinemia and insulin resistance (see Table 3 of Example 2), show that only for patients with metabolic syndrome and NASH of Group 2, the composition including silybin interfered with the cell intermediate metabolism concerning the oxidation of lipids, decreasing the oxidative stress typical of the metabolic syndrome and NASH, and, therefore, having the effects of the treatment on the same only for this specific group of patients.

Claims

1 . A composition for use in the treatment of treatment of metabolic oxidative alterations in non-alcoholic steatohepatitis (NASH), wherein said composition comprises silybin and the treatment is of patients having an altered lipidomic profile and wherein the patients having the altered lipidomic profile show an high TBARS value in TBARS test with respect to healthy subjects.

2. The composition according to claim 1 , wherein the treatment consists in the normalization of the lipidomic profile of patients having an altered lipidomic profile.

3. The composition according claim 1 or claim 2, wherein the composition includes silybin complexed with phosphatidylcholine.

4. The composition according to any of claims 1 to 3, wherein silybin is in the form of an extract of Silybum marianum, i.e. Milk thistle.

5. The composition according to claim 4, wherein silybin titre of the Milk thistle extract is in the range of 20 to 90%, more preferably 30 to 70%.

6. The composition according to claim 5, wherein the composition of the invention includes an extract of Milk thistle with a silybin titre of 33%.

7. The composition according to any of claims 1 to 6, wherein the composition includes vitamin E.

8. The composition according to any of claims 1 to 7, wherein patients having an altered lipidomic profile after the treatment have values of BMI, HOMA and insulin lower than the basal condition.