The present invention relates to new compositions for the oral administration of apolipoproteins in the dimeric or multimeric form, with specific dosage regimens for use in the treatment of atherosclerosis.

PRIOR ART

Myocardial infarction, stroke, unstable angina and sudden cardiac death collectively represent the main cause of death worldwide, with an ever-increasing incidence. The most common cause of these CVDs is atherosclerosis, a slowly progressing disease in which lesions, called plaques or atheromas, are formed in large- and medium-sized arteries. The atherogenic process is triggered by subendothelial retention of cholesterol-containing plasma lipoproteins in focal areas of arteries, particularly in anatomical districts (such as bifurcations, curvatures, etc.) where blood flow is characterized by turbulences.

High cholesterol levels in blood are associated with a disorder referred to as dyslipidemia, representing, both in a hereditary form and caused by secondary conditions not of genetic origin, a relevant risk factor for atherosclerosis. The increase of total cholesterol (TC) and LDL cholesterol (LDL-C, Low-Density Lipoprotein Cholesterol) in blood, as well as the so-called atherogenic lipid triad given by an increase of very low-density lipoproteins (VDLD), of small dense low-density lipoproteins and the reduction of HDL cholesterol (HDL-C, High-Density Lipoprotein Cholesterol), seem to be relevant factors for cardiovascular diseases. Early identification and management of modifiable risk factors, and above all of those factors contributing to dyslipidemias, constitute the first line of prevention for cardiovascular diseases.

The promising therapeutic potential of ApoA1 protein (Apolipoprotein A1) is known in the literature. The protein is found strongly associated with phospholipids, to form complexes and promote cholesterol outflow from cholesterol-rich cells. Maintaining an adequate plasmatic level of ApoA1, to mitigate or prevent atherosclerosis symptoms, is not easy to carry out.

The reverse correlation found between a high plasmatic level of HDL cholesterol and cardiovascular diseases is ascribed to the role that HDL and its major component, ApoA1 protein, have in the Reverse Cholesterol Transport—RCT phenomenon. RCT efficiency depends on ApoA1 protein ability to promote cholesterol outflow from cells, bind lipids, activate lecithin-cholesterol acyltransferase (LCAT) and form mature HDL which interacts with specific receptors and transfer proteins eliminating it from plasma flow through the liver.

It has been demonstrated that the infusion of proteins such as ApoA1 or ApoE, both recombinant and extraction ones, determine a reduction of the atherosclerotic burden in animal experimental models of atherosclerotic disease. Although various studies on animal models of atherosclerosis have demonstrated the potential of HDL use as a therapeutic strategy, clinical studies did not substantially succeed to demonstrate a significant reduction in atheroma volume, an important effectiveness parameter, when recombinant HDL molecules or the related HDL-mimetic peptides have been administered by infusion [A. M. Fogelman, Trying to harness the potential of HDL: wishful thinking or sound strategy? Eur Heart J. 35 (2014) 3248-3249.]. The therapies targeting HDL cholesterol for atherosclerosis are based on normalization of HDL-C plasma levels by acting on different sides of reverse cholesterol transport metabolism. Among these, statins are used to reduce endogenous cholesterol synthesis in the liver, thereby reducing LDL-C levels. On the other hand, CETP inhibitors increase HDL-C levels. Other therapeutic approaches are based on the infusion of reconstituted HDL and ApoA1 mimetics in the bloodstream. With this type of molecules, the aim is to increase the amount of cholesterol in HDL particles, and thereby improve the functionality of these molecules. These strategies yielded relevant results in experimental models, which however did not lead to a clear advantage in clinical studies.

Regardless of the actual clinical application, and despite the high therapeutic potential of ApoA1 lipoproteins, their therapeutic use in disorders associated with dysfunctions or anomalies of lipid metabolism (such as familial hypercholesterolemia, atherosclerosis, and others) has not been satisfactorily exploited yet. There are two main limitations accounting for the lack of success of ApoA1-based therapies, i.e. the very low efficiency of methods for the production and/or purification of these molecules, and the limits in their administration to patients in need thereof.

An integral part of therapies currently in use for the treatment of atherosclerosis is the substantial modification of the patient's dietary habits. Such a modification, since often required for persons hardly able to control their dietary habits, is oft-times a failure and seriously undermines the effectiveness and results of pharmacological therapies used on patients. The relevance of diet in the treatment of the above-indicated pathologies is well-known in the literature (e.g. Connor W E, Connor S L “Importance of diet in the treatment of familial hypercholesterolemia” Am J Cardiol. 1993 Sep. 30; 72(10):42D-53D; Connor W E, Connor S L “Diet, atherosclerosis, and fish oil” Adv Intern Med. 1990; 35:139-71; Chapman M J, Blankenberg S, Landmesser U (2016) The year in cardiology 2015: prevention. Eur Heart J 37:ehv721-519. doi: 10.1093/eurheartj/ehv721).

Patent Application WO2008/01790 describes the production in plant seeds of muteins of ApoA1 in the dimeric or multimeric form and assumes, without however demonstrating it, not only an intravenous administration of these apolipoproteins, but also an oral or a rectal one. In the oral administration, an administration is assumed directly by administering milk extracted from seeds of plants described in the patent application, or from flours thereof, etc.

Moreover, the document teaches a daily dosage of Apolipoproteins A1 by pharmaceutic compositions comprising one or more dimers and/or oligomers of the muteins described in pharmaceutically effective concentrations, such as, e.g., 10 mg/Kg of body weight, or 50 mg/Kg of body weight.

SUMMARY OF THE INVENTION

The Authors of the present invention have verified the possibility of effectively using an oral administration, by milk extracted from seeds of plants as described in WO2008/017906, and by assessing the activity thereof in various cell and animal models.

However, the Authors of the present invention have surprisingly discovered that the administration of milk extracted from seeds of plants as described in WO2008/017906 was effective, in vivo, at dosages 10-100-fold lower than those suggested in the above-indicated Patent Application, and that the anti-atherosclerotic therapeutic effect was observable also in animals subjected, during treatment with the assayed oral composition, to a hypercholesterolizing diet.

The Authors have also advantageously discovered that milk extracted from the seeds of plants as described in WO2008/017906 retained its effectiveness also in lyophilized form, after storage of the frozen lyophilizate (e.g., −20° C.), or at room temperature and after resuspension thereof with an appropriate diluent.

In in vivo experiments on mice treated with a hypercholesterolemic diet, said composition proved surprisingly capable of preventing the progression of atherosclerotic plaques, but also of significantly reducing their area, also in animals subjected, before and after treatment, to a hypercholesterolizing diet.

In the literature, there are no experimental data on oral administration of apolipoprotein A1 (ApoA1) or muteins thereof, data that would therefore enable to assume correct dosage regimens; on top of that, existing data on intravenous administration of said molecules (see Ibanez et al, Atherosclerosis 2012, and Cimmino et al, J Cell Mol Med 2009, reporting the administration of 75 mg/Kg of APOA1Milano to hypercholesterolemic and atherosclerotic rabbits, in two IV infusions 4 days apart, for a total of 150 mg/Kg in a week) show, at dosages much higher than those described and claimed herein for oral administration, a <5% reduction of the atherosclerotic plaque.

In data reported in the present description, a plaque reduction of about 50% at the level of the aortic sinus, and a plate area reduction 40% in the aortic arch are demonstrated.

Such data were moreover obtained on mice fed with a hypercholesterolemic diet, reflecting much more realistically a possible dietary regimen of patients not especially disciplined at the dietary level.

Therefore, object of the invention is a composition comprising one or more muteins of apolipoprotein A1 (ApoA-1) in the dimeric and/or multimeric form, wherein said muteins are produced in the dimeric and/or multimeric form in plant seeds, said composition comprising milk extracted from seeds of said plants and/or derivatives thereof, and wherein said muteins are administered orally to patients in a daily dosage of from 0.2 to 4 mg/kg of body weight for use in the treatment of atherosclerosis.

Object of the invention is also a method for the treatment of atherosclerosis by oral administration, to a patient in need thereof, of a composition comprising one or more muteins of apolipoprotein A1 (ApoA1) in the dimeric and/or multimeric form, wherein said muteins are produced in the dimeric and/or multimeric form in plant seeds, said composition comprising milk extracted from seeds of said plants and/or derivatives thereof, and wherein said muteins are administered in a daily dosage of from 0.2 to 4 mg/kg of body weight.

