WO2023119167A1

WO2023119167A1 - Pharmaceutical composition based on glutathione thioester and oleuropein for use in the treatment of non-alcoholic steatohepatitis

Info

Publication number: WO2023119167A1
Application number: PCT/IB2022/062566
Authority: WO
Inventors: Andrea BIGOZZI; Oliviero Giusti; Gianfranco Liguri; Edoardo Mannucci; Fabio Marra; Massimo Stefani
Original assignee: Innbiotec Pharma Srl
Current assignee: Innbiotec Pharma Srl
Priority date: 2021-12-21
Filing date: 2022-12-21
Publication date: 2023-06-29
Anticipated expiration: 2024-06-21
Also published as: EP4452278A1; IT202100031985A1

Abstract

The present invention describes a pharmaceutical composition comprising a glutathione thioester and oleuropein for use in the treatment and/or prophylaxis of hepatic steatosis forms of metabolic origin such as NAFLD (non-alcoholic fatty liver disease), NASH (non- alcoholic steatohepatitis) and related diseases, such as their evolution into hepatic fibrosis, liver cirrhosis and liver cancer.

Description

PHARMACEUTICAL COMPOSITION BASED ON GLUTATHIONE THIOESTER AND OLEUROPEIN FOR USE IN THE TREATMENT OF NON-ALCOHOLIC STEATOHEPATITIS

FIELD OF THE INVENTION

The present invention refers to the field of pharmaceutical preparations comprising organic active ingredients, in particular it refers to the combination of Glutathione (GSH) thioester and Oleuropein (Ole) for use in the prevention and/or treatment of hepatic steatosis forms of metabolic origin such as NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis) and their evolution into hepatic fibrosis, liver cirrhosis and liver cancer.

STATE OF THE ART

NAFLD is a complex and severe degenerative liver disease characterized by oxidative stress, inflammation and injury following metabolic stress. NAFLD and its evolution into NASH involve a number of processes, including lipid accumulation, insulin resistance, inflammation and fibrogenesis, closely associated with complications such as obesity, diabetes and steatohepatitis with possible evolution into severe liver fibrosis and cancer. Recent attempts to establish effective therapies for NAFLD and NASH have identified potential micronutrients with antioxidant power that may reduce ROS accumulation and ultimately ameliorate the disease.

Glutathione or GSH is a tripeptide with antioxidant properties, consisting of cysteine and glycine, linked by a normal peptide bond, and glutamate, which is instead linked to cysteine with an isopeptide bond between the carboxy group of the glutamate side chain and the amino group of cysteine. In medicine, GSH is used as a direct and “fast” antidote in paracetamol poisoning.

Glutathione is only to a small extent bioavailable to humans; its introduction into the body using external sources is, in fact, poorly effective in increasing its plasma and/or intracellular concentration. The nature of glutathione which, being a peptide, is a substrate of the peptidases and proteases of the alimentary canal, and the absence of a specific glutathione carrier at the cell membrane level underlie its poor bioavailability. Recently, bioavailable derivatives of glutathione have been synthesized and studied, which have shown to be able to increase the intracellular levels of this peptide (A. Pensalfini et al. Free Radical Biology & Medicine 44 (2008) 1624-1636). Recent literature supports the involvement of glutathione (GSH) metabolism in the pathogenesis of steatohepatitis (Liver Research Volume 4, Issue 2, June 2020, Pages 64- 73; Antioxidants 2021 , 10, 364). It has been shown that low levels of GSH are found in liver tissue affected by steatosis (Molecular Aspects of Medicine Volume 30, Issues 1-2, February-April 2009, Pages 29-41 ). A study recently carried out on 30 patients diagnosed with NAFLD and orally treated with reduced glutathione, provided modest evidence of efficacy in reducing blood levels of alanine transaminase (ALT), while it was unable to significantly alter other markers such as g-glutamyl transpeptidase (gGT), aspartate transaminase (AST) and glycohemoglobin (HbA1c). (Honda et al. BMC Gastroenterology (2017) 17:96).

Oleuropein is the main secoiridoid polyphenol in the olive tree. Secoiridoids are structural derivatives of iridoids (where there is a cyclopentane ring most often fused with an oxygencontaining six-membered heterocycle ring) in which the cyclopentane ring is open upon oxidation and breaking of a carbon-carbon bond. Glycated oleuropein (methyl (4S,5E,6S)- 4-[2-[2-(3,4-dihydroxyphenyl)ethoxy]-2-oxoethyl]-5-ethylidene-6-[(2S,3R,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-pyran-3-carboxylate) is a well characterized chemical species with MW = 540.51 and chemical formula C25H32O13, whose glycated derivative is soluble in water while the aglycone has reduced polarity. In the human body, the oleuropein aglycone metabolism leads to the formation, inter alia, of hydroxytyrosol, a polyphenol with strong antioxidant power, and elenolic acid.

Studies carried out with oleuropein have shown many beneficial effects, particularly regarding normalization of lipid metabolism and protection against fat deposition in the liver following metabolic impairment. Data currently available from research on cellular and animal models are indicative of a potential pharmacological use of oleuropein in the prevention and treatment of NAFLD and NASH in Homo sapiens s. (Barbara B., et al., Int. J. Mol. Sci. 2,014, 15, 18508-18524; Porcu C., et al. Int. J. Mol. Sci. 2018, 19, 3948).

It is, however, apparent the continuous need to provide new pharmaceutical compositions suitable for counteracting the biochemical and morpho-functional alterations in liver cells affected by NASH.

DEFINITIONS AND ABBREVIATIONS

ALT: alanine aminotransferase

AST: aspartate aminotransferase yGT: Gamma-glutamyl transferase

GSH: glutathione

HbA1c: glycosylated hemoglobin

NASH: non-alcoholic steatohepatitis

NAFLD: non-alcoholic fatty liver disease

Ole: oleuropein

SAG: s-acetyl-glutathione

SLinG: s-linolenoyl-glutathione

EPA: eicosapentaenoic acid

DHA: docosahexaenoic acid

SUMMARY OF THE INVENTION

The present invention addresses the aforementioned problems by means of a pharmaceutical composition comprising an S-acyl-Glutathione and oleuropein for use in the treatment and/or prophylaxis of hepatic steatosis forms of metabolic origin such as NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis) and related diseases, such as their evolution into hepatic fibrosis, liver cirrhosis and liver cancer.

Surprisingly, oleuropein and S-acyl-glutathione have shown a synergistic effect in counteracting or reducing the appearance of adipose deposits of dysmetabolic origin in the liver and, possibly, counteracting the evolution of the disease into fibrosis and liver cancer. DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the present invention, S-acyl-glutathione is selected from bioavailable S-acyl-derivatives, such as for example S-acetyl-glutathione (SAG) or thioesters of omega- 3 fatty acids such as linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

For the purposes of the present invention, oleuropein is glycated oleuropein or in the form of an aglycone or metabolites thereof, such as for example hydroxytyrosol.

The composition according to the present invention is preferably in the form of formulations for oral administration, preferably capsules, tablets, or in another form which can be administered parenterally, inhaled or transcutaneously.

Preferably, the composition according to the present invention comprises 10-500 mg, more preferably 50-200 mg, of Ole and 30-1000 mg, more preferably 100-500 mg, of S-acyl- GSH. The composition of the present invention is preferably to be taken once or twice a day.

The composition of the present invention may obviously include excipients suitable for its formulation for oral, parenteral, inhalation or transcutaneous use.

The composition of the present invention may also comprise one or more other substances with hepatoprotective activity such as, for example, N-acetyl cysteine, choline, phosphatidylcholine, Moringa oleifera extract, Silybum marianum extract, and other hepatoprotectors known to those skilled in the art.

The present invention can be better understood in the light of the following embodiment examples.

EXPERIMENTAL PART

EXAMPLE 1

To test whether oleuropein and bioavailable glutathione supplementation reduced hepatic lipid accumulation in fatty liver disease animal models, 70 C57BL/6 mice were fed with a high-fat diet (steatogenic diet, SD) for eight weeks. At the end (t=0), 10 animals were sacrificed to evaluate the activity of liver enzymes (alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and Gamma-glutamyl transferase (yGT)), liver weight and to assess the degree of steatohepatitis. The 60 remaining animals were randomly divided into six groups of ten individuals, and treated for a further eight weeks as follows:

• the first group received a high fat diet (SD);

• the second group received a SD supplemented with oleuropein (Ole) at 2% w/w (SD + Ole);

• the third group received a SD supplemented with 3% w/w S-acetyl glutathione (SAG) (SD + SAG);

• the fourth group received a SD diet supplemented with 3% w/w S-linolenoyl glutathione (SD + SLinG).

• the fifth group received a SD diet supplemented with 3% w/w SAG and 2% w/w Ole (SD+Ole+SAG);

• the sixth group received a SD diet supplemented with 3% w/w SLinG and 2% w/w Ole (SD+Ole+SLinG). At week 8 (t=56) blood samples were taken from the tail and blood values for liver enzyme activity were determined. Mice were then sacrificed, and histological analysis was performed on liver tissue sections stained with hematoxylin-eosin and viewed at 40x magnification. The experiment complied with all experimental procedures and protocols approved by the National Committee for the Protection of Animals Used for Scientific Purposes.

Table 1 shows the values of the parameters measured at the beginning and at the end of the study.

No adverse events were observed in treated individuals throughout the study period.

Table 1

‘evaluated as absent (0), mild (1), moderate (2) and severe (3) based on the area percentage of the liver parenchyma visually affected by steatosis. ** p<0.05. *** p<0.01 .

Values are expressed as means. The results show a surprising synergistic effect between Oleuropein and Glutathione thioester.

Effectively, the animals studied after 56 days of treatment with both substances showed a surprising reduction of lipid deposits in the liver together with strongly improved histopathological characteristics. This protection resulted to be significantly improved compared to that observed in mice treated with glutathione thioester alone (both SAG and SLinG) or with Ole alone.

In addition, a reduction in body and liver weight was also observed in mice fed with an oleuropein-enriched diet. EXAMPLE 2

Eight subjects of Homo s. adults, four males and four females aged between 36 and 68, all with evidence of NAFLD/NASH and impaired fasting glycemia, voluntarily took 120 mg/day of oleuropein for 6 consecutive months and 200 mg/day of S-acetyl-glutathione for the following six months without showing significant improvements in diagnostic parameters with the exception of a modest (less than 10%) reduction in HbA1c after treatment with oleuropein.

In the following six months, the same subjects voluntarily took 120 mg/day of oleuropein and 200 mg/day of S-acetyl-glutathione for 6 consecutive months. Abdominal ultrasound tests, hematological analysis (ALT, AST, yGT and glycosylated haemoglobin (HbA1c)) and the detection of anthropometric parameters were performed at the beginning and at the end of the treatment (Table 2).

Table 2

The results show that in almost all the subjects studied, the three blood chemistry parameters measured at the end of the six months of treatment were reduced with respect to the initial level. Furthermore, in all eight subjects, a clear reduction in the “bright” ultrasound signal was observed at the end of the treatment, while the effect with regard to body weight was negligible.

Claims

1. A pharmaceutical composition comprising an S-acyl-Glutathione and oleuropein (Ole) for use in the treatment and/or prophylaxis of hepatic steatosis forms of metabolic origin such as NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis) and related diseases, such as their evolution into hepatic fibrosis, liver cirrhosis and liver carcinoma; wherein said S-acyl-Glutathione is selected from the group consisting of S-acetyl-Glutathione and thioesters of Glutathione with an omega-3 fatty acid.

2. The composition according to claim 1 , wherein the omega-3 fatty acid is selected from the group consisting of linolenic acid, EPA and DHA

3. The composition according to any one of claims 1 -2, wherein Ole is in a glycated or aglycone form, or it is the hydroxytyrosol metabolite.

4. The composition according to any one of claims 1-3 in the form of a formulation suitable for oral or parenteral administration.

5. The composition according to any one of claims 1 -4 comprising 10-500 mg of Ole and 30-1000 mg of S-acyl-GSH.

6. The composition according to claim 5 comprising 50-200 mg of Ole and 100- 500 mg of S-acyl-GSH.

7. The composition according to any one of claims 1-6 further comprising one or more other substances with hepatoprotective activity.

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