Compositions for use in preventing or treating renal failure and associated disorders

Abstract

The present disclosure relates to a composition for use in preventing and / or treating renal failure, such as uremia, in a subject. Said composition is a composition for oral consumption and comprises i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root. Said composition is also useful for preventing or reducing a gain of body weight, or for reducing body weight, in a subject in need thereof.

Description

[0001] COMPOSITIONS FOR USE IN PREVENTING OR TREATING RENAL FAILURE

[0002] AND ASSOCIATED DISORDERS

[0003] TECHNICAL FIELD OF THE DISCLOSURE

[0004] The present disclosure relates to the field of lifestyle diseases and disorders commonly occurring in the industrialized world due to dietary habits and exercise patterns. Individuals in the industrialized world are often more prone to experience overweight and obesity as well as kidney malfunctions caused by the diet and non-adequate physical exercise. Herein it is disclosed a composition for reducing or preventing the progress of such disorders or malfunctions before they have entered a severe disease stage. More specifically, the present disclosure relates to a composition for use in preventing and / or treating renal failure, such as uremia, in a subject. The present disclosure also relates to a composition for use in preventing and / or reducing gain of body weight.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] Cardiovascular and metabolic disease, including hypertension, cardiac and renal diseases, lipid and glucose metabolism linked to lifestyle in the industrialized world are major health problems. Lifestyle and dietary habits in the industrialized world contribute to certain major health problems which are associated with weight gain and obesity, leading to metabolic and cardiovascular aberrations, which are related to development of disease manifestations in such individuals.

[0007] Metabolic aberrations related to obesity are accelerated depositions of lipids in various tissues and hyperlipidemia, glucose intolerance, may occur as well as insulin resistance, simulating type 2 diabetes mellitus. Mitochondrial function on a cellular level may also become affected and allow for a disturbed oxygenation of several tissues and organs. Secondary renal manifestations with deteriorating renal function, proteinuria, disturbed handling of water, salt, blood pressure regulation and accumulation of uremic toxins, which may affect most tissues in the body may also occur.

[0008] The cause of renal impairment is dependent upon both the above metabolic deviations and on vascular changes described below. An induced hyperfiltration will also contribute to renal damages. Vascular deterioration will occur with endothelial cell dysfunction and disturbed ability to dilate an artery, which will subsequently contribute to hypertension and disturbances in microcirculation. At a later stage an atherosclerotic phenotype will emerge with stiffening of the vascular wall due to secondary calcifications. Hypertension will occur due to both these direct vascular effects and because of renal dysfunction which leads to disturbed water and salt metabolism and accelerated secretion of blood pressure regulating hormones from the kidney. Subsequently hypertension, atherosclerosis, hyperlipidemia, and the excretion of uremic toxins will also lead to development of cardiac stress and a state with apoptosis of cardiomyocytes and eventually a manifest heart failure. Other related organ and tissue damages also occur in the context of these disease states.

[0009] GLP-1 (Glucagon-like peptide-1) is a gut hormone that is synthesized and secreted from the gut in response to food intake. GLP-1 is an incretin and can decrease blood sugar levels in a glucose-dependent manner by enhancing the secretion of insulin. This hormone has been a long-term target for the development of medications against obesity and associated conditions, such as diabetes type 2. In this regard, there are a few GLP-1 agonists on the market today, e.g., Ozempic®, Victoza®, and Trulicity®. These assist in regulating the appetite and the feeling of satiety in an individual and has a positive effect on diabetes 2. However, there is often a lack of supply of these medicaments, they are expensive for the consumer and there have been some reported side effects.

[0010] There is still a need in the art to identify new measures to limit the consequences of modern lifestyle diets and the progress of overweight, obesity and therewith associated disease manifestations. There is a need in the art to identify alternative measures for the treatment or prevention of overweight or obesity as compared to the treatments available today.

[0011] There is a need in the art to treat or prevent renal manifestations due to a western lifestyle and dietary habits, such as manifestations eventually resulting in renal failure. Such manifestations often progress in connection with overweight, obesity and / or hyperfiltration in the kidneys because of such a lifestyle.

[0012] SUMMARY OF THE DISCLOSURE

[0013] The above-mentioned problem has now been solved or at least mitigated by the provision herein of a composition comprising: i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root.

[0014] Herein, such a composition was surprisingly shown to be useful for use in preventing or reducing gain of body weight, for reducing body weight and / or for preventing and / or treating renal failure, such as uremia in a subject. Accordingly, said composition is intended for use in preventing or reducing gain of body weight, or for reducing body weight, in a subject, particularly an obese subject, i.e. where the condition is a medical condition. Secondly, the composition is intended for use in preventing and / or treating renal failure, such as uremia, in a subject.

[0015] Use is also intended of the composition for preventing or reducing a gain of bodyweight, or for reducing body weight, in a subject. Such a use is hence also envisaged in a non-medical or non-therapeutical context as clearly set out herein.

[0016] Herein, the composition is referred to interchangeably as a composition or an oral composition. The composition is intended to be used for oral consumption.

[0017] The present composition, that has a background as a general health and well-being improving composition assisting in vitality and in the relief of muscle aches, of “above 40” individuals, was in the present context surprisingly shown to have direct effects on the prevention or reduction of weight gain and in the progress and development of conditions associated with renal failure, such as uremia. The present composition is also intended for reducing body weight in a subject, wherein said effect may be medical or non-therapeutical depending on the subject that is provided with the composition.

[0018] These effects were identified by the inventors’ development of an animal model that included exposure of the animals to a western diet high in salt and sugar, i.e., a diet that would generally result in the animals gaining weight. The same animals that were exposed to the diet had also been physically compromised through a nephron reduction (UNX, unilateral nephrectomy) mirroring a uremic condition. Presenting the animals with a composition of the present disclosure during a treatment period of 12 weeks, even in combination with a western diet, surprisingly prevented or at least reduced weight gain and resulted in a redistribution and reduction of tissue fat in the animals. In addition, the treatment had beneficial effects on renal failure including the uremic conditions induced in the animals. In turn, this resulted in other physical improvements of the health of the animals, which are further described and disclosed elsewhere herein.

[0019] These effects present implications and uses of the composition of the present disclosure in disease states or disorders that are a direct consequence of, or linked to, overweight, obesity and / or to renal failure, such as uremia, e.g., diseases commonly associated with a western lifestyle high in fat, salt and / or carbohydrates.

[0020] In addition, there is a continuous need in the art to identify new treatments that have a reducing effect on body weight perse, or that at least influence further gain of body weight. This can be for medical reasons but also mainly for cosmetic or non-therapeutic purposes.

[0021] The composition of the present disclosure can also meet this need.

[0022] As for the composition of the present disclosure, this composition may also comprise one or more additional ingredients such as one or more of a stabilizing agent, an anti-caking agent, a preservative and an emulsifier. These are all ingredients useful for presenting the composition in a suitable formulation for consumption. As for the composition of the present disclosure, the composition may also comprise magnesium citrate and / or vitamin C (L- ascorbic acid).

[0023] There is also provided herein the use of a composition comprising i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root, for preventing or reducing gain of body weight, or for reducing body weight, in a subject.

[0024] Hence, such a use may be described as a non-medical or non-therapeutic use providing an effect on the general well-being on the subject in question wherein said subject does not suffer from overweight by a medical definition.

[0025] There is also provided a method for preventing or reducing gain of body weight, for reducing body weight and / or for preventing and / or treating renal failure, such as uremia, in a subject, said method comprising administering a pharmaceutically effective amount of a composition comprising i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root, to a subject in need thereof.

[0026] There is also provided a method for preventing a gain of body weight and / or inducing a loss of bodyweight in a subject by providing to said subject a composition of the present disclosure but wherein said subject may not suffer from overweight by a medical definition.

[0027] Additional aspects and alternatives of the present disclosure will be evident from the disclosure as a whole.

[0028] BRIEF DESCRIPTION OF THE FIGURES

[0029] Figure 1 shows the experimental design of the animal model including unilateral nephrectomy (referred to as “UNX”) followed by feeding mice on a western junk diet consisting of lipids, carbohydrates and salt (WD) that was given to the animals for 3 months (12 weeks), followed by sacrificing the subjects. There was one sham or control group (referred to as “SHAM”), one experimental group subject to UNX + WD and a third group which was given the composition of the present disclosure, FLX (this group is referred to as “UNX-WD-FLX”). Groups: 1: SHAM: Control water, (n= 19); 2: (UNX + WD) + Control water (n= 18); 3: (UNX + WD + FLX), (n= 8). Animals: Males, C57BL / 6J, 4 weeks old, BW = body weight = 21.23 ± 1.03.

[0030] Diet information: NsD06111702-1.5Vii, RD Western Diet, 4% Sodium Chloride, 1.5X Vitamin Mix. IPGTT = IP Glucose Tolerance Test; DEXA = Dual-energy X-ray absorptiometry; BP = Blood Pressure.

[0031] Figure 2 shows effects of the composition FLX on body weight gain (BW gain) in all participating groups. Figure 2a shows BW gain (g), Figure 2b shows BW Gain AUC.

[0032] Figure 3 shows effects of the composition FLX on fat mass and lean body mass in the body using DEXA (Dual-energy X-ray absorptiometry). Figure 3a shows Fat Mass (g), Figure 3b shows Lean Mass (g) and Figure 3c shows Fat / Lean Mass ratio, on the y-axes, respectively.

[0033] Figure 4 shows effects of the composition FLX on adipocyte area in the subjects of the respective treatment groups.

[0034] Figure 5 shows effects on glucose levels of the respective treatment groups. Figure 5a shows fasting glucose levels, Figure 5b shows non-fasting glucose levels, Figure 5c shows blood glucose 15 minutes after insulin injection, Figure 5d shows a glucose tolerance test and Figure 5e shows a IP Glucose Tolerance Test (IPGTT).

[0035] Figure 6 shows effects of the composition FLX on blood pressure measured as Mean, MAP (Figure 6a), Systolic, SAP, mmHg (Figure 6b), Diastolic, DAP, mmHg (Figure 6c), Heart Rate, HR, bpm (Figure 6d), by tail cuff instrument (MAP = Mean Arterial Pressure; SAP = Systolic Arterial Pressure, DAP = Diastolic Arterial Pressure). Results for all groups are shown.

[0036] Figure 7 shows effects of the composition FLX on endothelial dependent vasodilation in relation to Acetylcholine concentration on arterial rings in a myograph ex vivo. Results for all groups are shown. Figure 7a shows contraction (% of KPSS plateau) and Figure 7b shows Dilatation (% of PE plateau).

[0037] Figure 8 shows effects on renal function measured as Glomerular Filtration Rate (GFR) by inulin infusion (FITC). Results for all groups are shown. Figure 8a shows GFR (pl / min), Figure 8b shows GFR / BW (Body Weight) (pl / min / g) and Figure 8c shows GFR Percentage (SHAM 100%).

[0038] Figure 9 shows effects on renal glomerular injury as judged by degree of mesangial cell proliferation in renal tissue. Results for all groups are shown. Figure 9a shows glomerular area (pm2), Figure 9b shows glomerular injury (%), Figure 9d shows a tubular injury score and Figures 9c and 9e show a Hematoxylin & Eosin stainings of the kidney. Figure 10 shows a correlation between Glomerular Injury I GFR (Glomerular Filtration Rate).

[0039] Results for all groups are shown.

[0040] Figure 11 shows a correlation between Tubular Injury I GFR (Glomerular Filtration Rate). Results for all groups are shown.

[0041] Figure 12 shows effects of the composition FLX on urinary excretion of albumin. Results for all groups are shown. Figure 12a shows urinary albumin / density, Figure 12b shows Urinary Urea Nitrogen / Density, Figure 12c shows Urinary Creatinine I Density and Figure 12d shows Urinary Albumin I Density.

[0042] Figure 13 shows effects of the composition FLX on mitochondrial function measured as P / O ratio (Produced ATP / consumed oxygen / Mitochondrial oxygen efficiency). Results for all groups are shown.

[0043] Figure 14 shows effects on plasma Troponin I values over time. Results for all groups are shown.

[0044] Figure 15 shows effects on IL-6 levels (pg / ml) when mice of the different groups were treated with FLX (FLEXO).

[0045] DETAILED DESCRIPTION OF THE DISCLOSURE

[0046] Definitions

[0047] In the description, the singular forms also include the plural unless the context clearly dictates otherwise.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0049] Compositions “comprising” one or more recited ingredients or elements may also include other ingredients or elements not specifically recited.

[0050] Herein, wt% (weight %), % of the total weight of the composition, or wt% based on the total weight of the composition refers to the dry weight of the ingredients of the composition, meaning that the moisture content of the composition in total is very low but also meaning that there may be some moisture present in the composition.

[0051] The term “oral composition” is used herein to define a composition which is suitable to be consumed orally by the subject. A composition is suitable e.g., if it has a texture, odour, taste and content to make it suitable for this purpose. Herein, the terms “subject”, “individual” or “patient” are terms that may be used interchangeably herein and refers to someone, a mammal, usually a human, who would benefit from the composition of the present disclosure.

[0052] An “extract of Rhodiola rosea" root (family Crassulaceae) as referred to herein, comprises a standard preparation of a stem (rhiozome) and / or a root (radix) part (i.e., a below ground part) of Rhodiola rosea or Rhodiola Rosea L,. An “extract of a Rhodiola rosea root” may include a part of the root and / or the stem. Preparations or compositions of Rhodiola Rosea are usually made from dried extracts of the root that have been prepared by extraction techniques comprising ethanol or similarly to obtain a composition that is suitable for oral ingestion. Such techniques are known in the art. An extract of Rhodiola rosea is usually substantially pure meaning that it contains close to 100 wt%, such as 98 wt% or 99 wt% of material from the root and / or the stem of the plant based on the total weight of the extract of Rhodiola rosea root.

[0053] An extract of Rhodiola rosea root comprises the substance rosavin (a cinnamyl alcohol glycoside, formula C20H28O10) or rosavins (in the plural), wherein rosavins in an extract of Rhodiola rosea root herein may comprise rosarin, rosavin and / or rosin (small variants). The presence and content of such rosavin or rosavins in a dried extract of Rhodiola rosea root may be used as a characterization of an extract suitable for use in the present disclosure. The presence of such an amount of rosavin or rosavins may be interpreted as to provide a certain quality to said extract.

[0054] An extract of Rhodiola rosea root for use in the present disclosure normally contains from about 1 wt% to about 5 wt% of rosavin or rosavins, such as about 1wt%, 2 wt%, 3 wt%, 4 wt% or 5 wt% rosavin or rosavins based on the total weight of the extract of Rhodiola rosea root, which extract of Rhodiola rosea root forms a part of a composition of the present disclosure. Usually, when only rosavin is considered, the content of rosavin is from about 1 wt% to 2 wt% of the total weight of the Rhodiola rosea root extract. Usually when rosavins are considered, the content is from about 3 wt% to about 5 wt% of the total weight of the Rhodiola rosea root extract. These numbers are however not limiting. Further examples are provided elsewhere herein.

[0055] An “extract of beet root” (Beta vulgaris, family Amaranthaceae) as referred to herein, is a standard preparation of a beet root. Preparations or compositions of beet root are usually made from dried extracts that have been prepared by extraction techniques comprising ethanol or similarly to obtain a composition that is suitable for oral ingestion. An extract of beet root is commercially available and can be bought over the counter. Similarly, the skilled person can easily prepare extracts of beet roots from its natural source. An extract of beet root is usually substantially pure meaning that it contains close to 100 wt%, such as about 98 wt% or about 99 wt% of material from the root, based on the total weight of the extract of beet root. An extract of beet root may be characterized by its nitrate content, which usually is from about 1 wt% to about 4 wt%, such as about 1 wt%, 2 wt%, 3 wt% or 4 wt% based on the total weight of the extract of beet root which forms a part of a composition of the present disclosure. These numbers are, however, not limiting.

[0056] L-arginine is an amino acid that is naturally found e.g., in red meat, poultry, fish, and dairy. It is required for making proteins and is commonly used for improving blood circulation. L- arginine is converted into nitric oxide in the body. In a composition of the present disclosure, substantially pure L-arginine may be used as a source for preparing the L-arginine part of the composition meaning that the L-arginine part, or L-arginine composition usually contains close to 100 wt%, such as 98 wt% or 99 wt% of the amino acid L-arginine, alone or in a salt form based on the total weight of the L-arginine part of the composition. L-arginine is commercially available and can be bought over the counter. The amino acid L-arginine may be provided from a suitable source in a suitable format, such as in powder format.

[0057] Sometimes the L-arginine part is provided in the salt form L-arginine hydrochloride. Herein it may also be referred to a L-arginine composition, or a composition comprising L-arginine, whenever the L-arginine part of the composition is discussed.

[0058] L-citrulline is an amino acid that can be produced by the body and that can also be found in external sources such as e.g., in watermelon. L-citrulline can be metabolized into L-arginine. In a composition of the present disclosure, substantially pure L-citrulline may be used as a source for preparing the L-citrulline part of the composition meaning that the L-citrulline part, or L-citrulline composition usually contains close to 100 wt%, such as 98 wt% or 99 wt% of the amino acid L-citrulline, alone or in a salt form based on the total weight of the L-citrulline part of the composition. L-citrulline is commercially available and can be bought over the counter. L-citrulline may be provided from a suitable source in a suitable format, such as in powder format. Sometimes L-citrulline is provided in the form L-citrulline DL-malate. Herein it may also be referred to a L-citrulline composition, or a composition comprising L-citrulline, when the L- citrulline part of the composition is discussed.

[0059] Herein when “preventing or reducing a gain of body weight” and “reducing body weight” is referred to this means that there is an effect on the body weight of a subject in the manner that the body weight of the subject will not, or in essence will not (meaning that minor fluctuations can occur) increase as compared to an initial body weight, i.e., the body weight of the subject when the composition of the present disclosure was first taken by the subject. It may also be so that the body weight of the subject may decrease after some time when the composition of the disclosure has been ingested on a regular basis. Hence, sometimes the body weight of the subject may stay the same, or approximately the same as when the composition was first ingested. Sometimes ingesting the composition will reduce the body weight of the subject from the initial body weight when the composition was first ingested. Without wishing to be bound by theory, the reduction of body weight or the prevention of further gain of body weight may be due to a reduced appetite of said subject and / or it may be due to a reduction of fat depositions, possibly because of enhanced metabolism in said subject.

[0060] Herein “preventing or reducing a gain of body weight” and “reducing body weight” can be referred to either in a medical context or alternatively in a non-medical or non-therapeutic context, where the effect is mostly cosmetic and not intended to treat or cure a medical condition. It can be that gaining weight is prevented in a subject that is not overweight or obese when the composition of the present disclosure is ingested for the first time, i.e., said subject is a healthy human subject. It can also be so that gaining weight is prevented in a subject that already suffers from overweight or obesity. Hence it is envisaged that different subject groups may benefit from the composition of the present disclosure, healthy subjects or subjects that are suffering from a medical condition. In general, obesity can be seen as a medical condition that needs to be treated as the subject suffering from obesity very often suffers from other medical conditions or health issues due to the obesity. Overweight is usually related to health issues and can be severe, but it can also be mild. Herein, it is intended for the composition of the present disclosure to find uses either in a non-medical or in a medical context depending on the subject that is being treated.

[0061] “Renal failure” as referred to herein, is a medical condition characterized by the inability of the kidneys to perform excretory function leading to retention of nitrogenous waste products from the blood. A ’’uremic” condition or “uremia” may be a result of renal failure.

[0062] A ’’uremic” condition, or “uremia”, as referred to interchangeably herein, is a medical condition characterized by a buildup in the blood circulation of urea and other metabolic waste products which are normally excreted by the kidneys and occurs when the kidneys capacity of filtering waste out from the body through the urine is compromised. Uremia is associated with fluid, electrolyte and hormone imbalances as well as metabolic and cardiovascular abnormalities that will develop in parallel with deterioration of kidney function. Uremia is often a sign of end-stage renal disease and if it is untreated it can lead to severe health problems or death.

[0063] A ’’western diet” (WD) as referred to herein, is a diet that is characterized by being high in fat and / or in carbohydrates and / or in salt. The result of being exposed to such a diet for a longer period of time is usually a gain of weight, with a risk of leading to obesity, which in turn may lead to other health problems as exemplified herein. An individual in the context of the present disclosure, and in need of a composition of the present disclosure, has most often been exposed to, or is consistently exposed to, a western diet.

[0064] Detailed description

[0065] The present disclosure concerns a composition for use in reducing, treating and / or preventing renal failure, particularly uremia, in a subject. The composition is an oral composition intended for ingestion on a regular basis. The present disclosure also concerns a composition for use in preventing or reducing a gain of body weight, or for reducing body weight in a subject.

[0066] Initially, the aim of the present inventors was to develop an animal model based on the intake of a western junk diet rich in fat, carbohydrates and salt in which manifestations of the corresponding disease phenotype would occur, allowing for tests using compounds that may have beneficial effects on dietary and obesity driven disease manifestations as mentioned above. The same animal model also allowed for trials of compounds that would have an effect in subjects with a diminished kidney function, as the animals of the study had only one functional kidney after a surgical excision of one kidney (UNX). The experiments are illustrated in Figure 1.

[0067] When developing such a model, some surprising findings were made in relation to a composition, the composition of the present disclosure, which had previously only been used as a health supplement for muscle aches and to improve blood circulation in general, as discussed elsewhere herein. The components of the composition of the present disclosure had also been described for other various uses, but no effects have to the inventor’s best knowledge until now been confirmed or manifested in the conditions as shown herein.

[0068] The composition of the present disclosure was found to have several beneficial effects and mechanisms of actions, of which some are described and put into its context herein.

[0069] In this regard, it was surprisingly shown that a composition of the present disclosure that was given to a subject could prevent a gain of body weight in the same subject while said subject was ingesting a western diet high in carbohydrates, salt and fat. In subjects not receiving the composition, there was an increase in weight due to the intake of this diet. As the subjects were also suffering from decreased kidney function, mirroring uremia, it was possible to identify further beneficial effects of this treatment that would be useful for the treatment of uremic subjects. It was also particularly surprising that the present composition had such significant effects on cardiac troponin leakage thereby supporting the ability of the present composition to prevent cardiac stress that may lead to cardio myocyte damage and associated disorders.

[0070] In view of the composition of the present disclosure having effects on body weight, and further in view of all the results presented herein, it is believed that the present composition exerts effects similar to GLP-1 (Glucagon-like peptide-1) agonists possibly accelerating the metabolism of deposited lipids in various tissues. This belief is made without wishing to be bound by theory, but it is considered that the finding opens the door to a new and very useful application of the present composition, independently of its exact mechanism of action. Hence, it is envisaged herein a composition of the present disclosure for use as a GLP-1 agonist.

[0071] Referring to the above, the composition finds use in conditions that are initiated or triggered by weight gain and / or by renal failure, such as uremia, i.e., conditions that will arise as a result thereof wherein the initial effect of the composition will be on the weight gain, i.e., the preservation or reduction of the weight and / or on a condition comprising renal failure, such as uremia. This will hence make it possible to prevent or at least mitigate the development of any further conditions that usually follow because of these conditions.

[0072] In summary, the findings made by the inventors open a new field of use for the composition of the present disclosure. The composition will now be described in more detail.

[0073] Hence, herein there is in a first aspect provided a composition comprising: i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root, for use in preventing and / or treating renal failure, such as uremia, in a subject.

[0074] There is also provided said composition for use in preventing or reducing gain of body weight, and / or for reducing body weight as set out elsewhere herein, particularly in an obese subject.

[0075] There is also provided a composition for use wherein said subject is suffering from overweight, particularly obesity, and further wherein said composition prevents weight gain and / or reduces body weight in said subject.

[0076] An extract of Rhodiola rosea root for use in the present disclosure usually contains from about 1 wt% to about 5 wt% of rosavin or rosavins, such about 1wt%, 2 wt%, 3 wt%, 4 wt% or 5 wt% rosavin or rosavins based on the total weight of the extract of Rhodiola rosea root which forms a part of a composition of the present disclosure.

[0077] As explained elsewhere herein, when it is referred to rosavins (in the plural), these may include rosarin, rosavin and / or rosin. More specifically, an extract of Rhodiola rosea root may comprise from about 1 wt% to 3 wt% rosavin, such as 1wt%, 2 wt%, or 3 wt% of rosavin based on the total weight of the Rhodiola rosea root extract. An extract of Rhodiola rosea root may also comprise about 3 wt% to about 5 wt% of rosavins, such as 3 wt%, 4 wt% or 5 wt% of rosavins based on the total weight of the Rhodiola rosea root extract.

[0078] A specific example of an extract of Rhodiola rosea root comprises 3 wt% rosavins, wherein rosarin constitutes from about 0.6 wt% to about 1.5%, rosavin constitutes from about 1.6 wt% to about 2.3 wt% and rosin constitutes from about 0.2 wt% to about 0.7 wt%, of the total weight of the extract of Rhodiola rosea root.

[0079] Said composition may further comprise magnesium citrate and / or vitamin C.

[0080] In a similar manner, there is also provided a method for preventing and / or treating renal failure, such as uremia, in a subject, said method comprising administering a pharmaceutically effective amount of a composition comprising i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root, to a subject in need thereof

[0081] In yet another similar manner, there is also provided a method for preventing or reducing gain of body weight, for reducing body weight, and / or for preventing and / or treating renal failure, such as uremia, in a subject, said method comprising administering a pharmaceutically effective amount of a composition comprising i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root, to a subject in need thereof. Said method for preventing or reducing gain of body weight, for reducing body weight may be intended for an obese subject, also described as a subject that is suffering from overweight as a medical condition.

[0082] The skilled person will know how to determine a pharmaceutically effective amount of said composition in addition to the examples of such amounts that are provided elsewhere herein. Such a pharmaceutically effective amount may be determined by the skilled practitioner based on the specific medical condition or health of the subject, the subject’s weight as well as other parameters relevant in the present context.

[0083] Whenever it is referred to herein a composition for use in its various contexts, this also includes a method of using said composition, wherein said method may comprise administering a pharmaceutically effective amount of said composition to a subject in need thereof. It also includes use of a composition as disclosed herein in the manufacture of a medicament for preventing or reducing gain of body weight and / or for preventing and / or treating renal failure, such as uremia in a subject.

[0084] Herein, said composition may further comprise vitamin C and / or magnesium citrate.

[0085] Herein, said composition may further comprise one or more of a stabilizing agent, an anticaking agent, a preservative and an emulsifier. Examples of such ingredients are further described herein.

[0086] There is furthermore disclosed herein a composition for use, wherein said composition comprises i) from about 10 wt% to about 20 wt% of an extract of Rhodiola rosea root, such as from about 12 wt% to about 18 wt%, such as about 15.5 wt% of an extract of Rhodiola rosea root (or ±1 , 2, 3, 4 or 5 wt%), based on the total weight of the composition; ii) from about 22 wt% to about 32 wt% of L-arginine, such as from about 24 wt% to about 29 wt%, such as about 27 wt% of L-arginine (or ±1, 2, 3, 4 or 5 wt%), based on the total weight of the composition; iii) from about 12 wt% to about 25 wt% of L-citrulline, such as from about 16 wt% to about 22 wt%, such as about 19 wt% L-citrulline (or ±1, 2, 3, 4 or 5 wt%), based on the total weight of the composition; and iv) from about 10 wt% to about 20 wt% of an extract of beet root, such as from about 12 wt% to about 18 wt%, such as about 15.5 wt% of an extract of beet root (or ±1, 2, 3, 4 or 5 wt%), based on the total weight of the composition, and optionally; v) about 4 wt%, such as about 3.8 wt% or 3.9 wt% of magnesium citrate and / or vitamin c, based on the total weight of the composition, and optionally vi) from about 10 to about 20 wt% of one or more additional ingredients, such as 15 wt% of one or more additional ingredients (or ±1, 2, 3, 4 or 5 wt%), such as one or more of a stabilizing agent, an anti-caking agent, a preservative and an emulsifier, based on the total weight of the composition.

[0087] Accordingly, in general, the four main ingredients of a composition of the present disclosure, an extract of Rhodiola rosea root, L-arginine, L-citrulline and an extract of beet root may constitute in total from about 60 wt% to 80 wt%, such as from about 70 wt% to about 80 wt%, such as about 77 wt% (or ±1 , 2, 3, 4 or 5 wt%) of the total weight of the composition.

[0088] Magnesium citrate and vitamin C may be present in said composition from about 2 wt% to about 6 wt% each, such as about 4 wt% each based on the total weight of the composition (such as 3.8 wt% or 3.9 wt% of the total weight of the composition, each). The remaining from about 10 wt% to about 20 wt%, such as about 15 wt% of the total weight of the composition may constitute additional ingredients such as one or more stabilizing agents, emulsifiers, anticaking agents.

[0089] A composition of the present disclosure may comprise about 77 wt% of the four main ingredients, about 4 wt% of each of magnesium citrate and vitamin C and the remaining part, about 15 wt% of additional ingredients as defined elsewhere herein, based on the total weight of the composition.

[0090] There is also provided a composition herein, wherein the active ingredients; an extract of Rhodiola rosea root, L-arginine, L-citrulline and an extract of beet constitute about 75 wt% to about 90 wt% of the total weight of the composition.

[0091] More particularly, a composition may comprise about 10.5 wt%, 11 wt%, 11.5 wt%, 12 wt%, 12.5 wt%, 13 wt%, 13.5 wt%,14 wt%, 14.5 wt%, 15 wt%, 15.5 wt%, 16 wt%, 16.5 wt%, 17 wt%, 17.5 wt%, 18 wt%, 18.5 wt%, 19wt%, 19.5 wt%, 20 wt% or 20.5wt% of an extract of Rhodiola rosea root based on the total weight of the composition.

[0092] The composition may also comprise about 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, or 32 wt% of L-arginine based on the total weight of the composition.

[0093] The composition may also comprise about 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, or 24 wt% of L-citrulline based on the total weight of the composition.

[0094] The composition may comprise about 10.5 wt%, 11 wt%, 11.5 wt%, 12 wt%, 12.5 wt%, 13 wt%, 13.5 wt%,14 wt%, 14.5 wt%, 15 wt%, 15.5 wt%, 16 wt%, 16.5 wt%, 17 wt%, 17.5 wt%, 18 wt%, 18.5 wt%, 19wt%, 19.5 wt%, 20 wt% or 20.5 wt% of beet root based on the total weight of the composition.

[0095] It is also encompassed any combinations of the herein mentioned wt% of the ingredients in a composition of the present disclosure.

[0096] As an example, there is provided herein a composition for use, said composition comprising about: i) from about 10 wt% to about 20 wt% of an extract of Rhodiola rosea root, such as from about 12 wt% to about 18 wt%, such as about 15.5 wt% of an extract of Rhodiola rosea root, based on the total weight of the composition; ii) from about 22 wt% to about 32 wt% of L-arginine, such as about 24 wt% to about 29 wt%, such as about 27 wt% of L-arginine, based on the total weight of the composition; iii) from about 12 wt% to about 25 wt% of L-citrulline, such as from about 16 wt% to about 22 wt%, such as about 19 wt% L-citrulline, based on the total weight of the composition; and iv) from about 10 wt% to about 20 wt% of an extract of beet root, such as from about 12 wt% to about 18 wt%, such as about 15.5 wt% of an extract of beet root, based on the total weight of the composition; and optionally one or more additional ingredients as mentioned elsewhere herein.

[0097] The composition may also further comprise magnesium citrate and / or vitamin C. These may be present in said composition from about 1 wt% to about 8 wt%, such as about 2 wt % to about 6 wt%, such as about 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt, 6 wt%, 7 wt%, 8 wt% based on the total weight of the composition.

[0098] The composition may also comprise one or more further components in addition to the active ingredients. These additional components may constitute from about 10 wt% to about 20 wt%, such as about 15 wt% of the total weight of the composition, such as about 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt% based on the total weight of the composition.

[0099] Regarding the content of active components in the formulation, it is illustrated in table 2 the contents thereof in the formulation and their relative proportions. Hence, there may also be other formulations of the composition of the present disclosure where the proportion of active ingredients in relation to additional components differ, but where the proportions in between the active ingredients are as in essence as illustrated in table 2.

[0100] Naturally, when modifications are made to the percentages of the respective ingredients of the composition, the total adds up to 100 wt%.

[0101] A product FLX according to the present disclosure is presented in Table 1.

[0102] More specifically, one dose, such as in the form of a tablet, pill or any other formulation, preferably suitable for oral consumption may contain the following amounts of the main active ingredients, when only the active ingredients are taken into account: Table 2: Main active ingredients of a serving (one dose) of FLX (FLEXO), a product according to the present disclosure.

[0103] Table 2 is meant to illustrate proportions of active ingredients of a composition of the present disclosure, usually such a composition also contains other ingredients as mentioned elsewhere herein. One serving of a product according to the present disclosure may comprise 647 mg, i.e., 147 mg constitutes other ingredients than the four main ingredients mentioned above. Examples of these ingredients are exemplified elsewhere herein.

[0104] Additional ingredients that may form part of a composition of the present disclosure comprises one or more of the following ingredients: calcium carbonate, silicon dioxide, sodium carboxy metylcellulose, microcrystalline cellulose (stabilizing agent), magnesium salts of fatty acids, hydroxypropyl metylcellulose, glycerol, modified potato starch, and silica. Some of these ingredients may be used interchangeably, or ingredients similar to the ingredients may be used that will provide the same function, i.e., a stabilizing function, a moisture-reducing function or an antioxidant function.

[0105] The composition of the present disclosure may include other constituents and / or excipients, as deemed suitable.

[0106] The present composition is an oral composition, i.e., the composition is intended for oral use, ingestion, to be swallowed, chewed, or sucked to finally dissolve when ingested by the subject.

[0107] The oral composition may be in the form of a tablet or a pill, such as a tablet or pill that is intended to be swallowed. It may also be in the form of a tablet that will dissolve when put in a glass of water, or that is in the form of a powder packaged into a small bag or similarly to be mixed with a glass of water where it will dissolve and be consumed by the subject in question.

[0108] Depending on which formulation is used for providing the active ingredients to the subject in question, various additional ingredients may be added to the composition to ensure that it will provide its function in an optimal way independently of the specific formulation. For a tablet that is to be swallowed, it is for example important that the outside of the tablet makes it suitable for and easy to swallow for the subject.

[0109] Herein, the subject for which the composition is intended, may be a subject suffering from overweight, which when severe may be categorized as obesity. Hence, the overweight may be mild, mid severe or severe, or similarly, and it will therefore, depending on how severe the overweight is, usually result in one or several additional health problems.

[0110] As shown in figure 2 and 3, the composition showed positive effects on body weight gain and fat mass / lean body mass in the subjects treated with the composition of the present disclosure. It is also envisaged that the composition of the disclosure may be useful for preserving a normal glucose metabolism and / or for reducing hyperglycemia in a subject. Accordingly, a composition of the present disclosure is useful for the treatment and / or prevention of hyperglycemia in a subject. As shown in Figure 5, there was a beneficial effect in this regard in the subjects that had been treated with the composition of the present disclosure. Hence it is envisaged that the composition may be used as a complement to a normal diet to keep or maintain the glucose levels at a normal level and to avoid “highs-and-lows” of the glucose levels in the body.

[0111] A subject to which a composition is provided may be a subject that is or has been exposed to a diet high in fat and / or carbohydrates and / or a diet high in salt for a shorter or longer period. It is envisaged the use of the composition as a complement to the diet to individuals that consume food with a high content of salt, sugar, and / or fat. Even if it would be preferable for any individual to also reduce the amount of salt, sugar, and / or fat in their diet, using the composition of the present disclosure could be beneficial for subjects that are struggling in this regard and could provide a springboard for gaining a healthier lifestyle if further weight gain is prevented at an early stage before overweight progresses into obesity.

[0112] There is also provided a composition for use as disclosed herein, wherein said composition attenuates and / or prevents progressive renal failure and / or progressive renal damage in a subject. Accordingly, said composition is for use in the treatment or prevention of progressive renal failure and / or progressive renal damage in a subject. There is also provided a composition for use as disclosed herein, wherein said composition prevents development of uremia- and / or overweight- or obesity related renal structural or functional deterioration.

[0113] There is also provided a composition for use as disclosed herein, wherein said subject suffers from organ and / or tissue damage and wherein said composition further prevents and / or treats said organ and / or tissue damage. Hence, said composition may be used for the treatment and / or prevention of organ and / or tissue damage in a subject.

[0114] There is also provided a composition for use as disclosed herein, wherein said composition preserves endothelium-dependent vascular relaxation and / or prevents subsequent development of hypertension in said subject. Particularly, said composition is for use in the treatment and / or prevention of hypertension in a subject. Figures 7 to 14 support the effect of the present composition in these states.

[0115] In this regard, without wishing to be bound by theory, it is believed that the composition of the present disclosure can act as a weak PDE5i (phosphodiesterase type 5 inhibitor). This ability is believed to make it possible to preserve endothelial driven vasodilation, for which supportive data is provided herein (see “Vasodilation", Figure 7). In general, a PDE5i is a vasodilating substance that blocks the degradative action of cGMP-specific phosphodiesterase type 5 (PDE5) (cyclic guanosine monophosphate-specific phosphodiesterase-specific phosphodiesterase type 5) on cyclic GMP in the smooth muscle cells lining the blood vessels supplying various tissues. As an example, such substances are used today for the treatment of erectile dysfunction. Hence, the composition of the present disclosure is also envisaged for use as a PDE5L Even it is known from the prior art that L- arginine may exert a limited such effect, it was now shown that the combination of the ingredients of a composition of the present disclosure could be even more beneficial in this regard (data not shown) potentially supporting that the effect is not due to L-arginine alone but the specific combination of ingredients including an extract of Rhodiola rosea root.

[0116] Furthermore, there is also provided a composition for use as disclosed herein, wherein said composition preserves a normal blood pressure in said subject. As shown in Figure 6, there was an increase in blood pressure in the compromised group treated with the western diet (LINX-WD), but a significantly lower blood pressure, SBP, DBP and MAP in the group that was given the composition of the present disclosure (UNX-WD-FLX). However, there was no influence on the heart rate.

[0117] There is also provided a composition for use as disclosed herein, wherein said composition prevents renal failure-, such as uremia- and / or overweight- or obesity related heart disease in said subject. Hence, there is also provided a composition for use, wherein said composition further prevents and / or treats heart disease or is for use in the treatment and / or prevention of heart disease, related to renal failure or uremia in said subject.

[0118] In figure 14, the composition had effects on Troponin I values over time. Troponin I is a protein used as an indicator of acute damage of cardiomyocytes in the heart. The fact that the composition influenced the levels of Troponin I in the blood indicates that there is not only a preventive effect of further damage to the heart but also, surprisingly, a regression of the damage that the diet and the loss in kidney function has caused to the heart. Hence, the present composition as disclosed herein finds use in the treatment or prevention of a condition or disorder that damages the heart.

[0119] Accordingly, there is also provided herein a composition for use in the treatment and / or prevention of a heart disorder or disease. There is also provided herein the composition for use in the treatment and / or prevention of cardiac stress and injury. More specifically, there is also provided a composition for use in the treatment and / or prevention of myocardial injury.

[0120] There is also provided a composition for use as disclosed herein, wherein said subject is suffering from glomerular damage (mesangial hypercellularity and hypertrophy) and renal injury in parallel with reduced renal function, and wherein said subject would benefit from being provided with said composition to at least prevent further damage in the subject as a result of this condition.

[0121] There is also provided a composition for use as disclosed herein, wherein said composition prevents or reduces renal failure-, such as uremia-, and / or overweight- or obesity related systemic inflammation. Herein, a composition of the present disclosure was shown to have an effect on IL-6 levels (see Figure 15) when provided to subjects suffering from a lack of kidney function and exposed to a western diet. Accordingly, a composition as disclosed herein is further intended for use in the treatment and / or prevention of systemic inflammation in a subject.

[0122] Naturally, and as set out elsewhere herein, all aspects as disclosed herein in relation to a composition for use also applies to a method for preventing and / or treating a disorder or disease, particularly renal failure in a subject, wherein said method comprises administering a pharmaceutically effective amount of a composition to a subject in need thereof.

[0123] The skilled person may decide on different dosages and treatment schemes suitable for the subject depending on the health condition of the subjects and other relevant parameters. Herein examples of suitable doses are given but it is not limited thereto.

[0124] Accordingly, a composition according to the present disclosure may be provided to said subject a few times a day, such as about 3 to 4 times a day, or whatever is convenient and wherein in total, said subject may consume between 2 to 8 doses or tablets / pills a day, such as 3 to 4 or 3 to 6 doses or tablets / pills per day. Such a one dose of said composition comprises a total weight of about 400 to 800 mg, such as about 500 to 700 mg, such as about 647 mg, as illustrated in Table 1 , as calculated for a subject weighing about 70 kg. This is only an example of a one-unit dose form, and the disclosure is not limited thereto.

[0125] It is envisaged that the composition of the disclosure can be given to a subject on a regular basis as part of a normal diet, for longer periods of time, such as months or years, or as part of a diet where the subject intends to lose weight.

[0126] There is also provided herein the use of a composition comprising: i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root for preventing or reducing gain of body weight in a subject.

[0127] Examples of wt% of the ingredients of said composition are provided elsewhere herein.

[0128] Using a composition as disclosed herein, may have the effect of reducing the appetite in said subject. Said composition may also preserve a normal glucose metabolism. In addition, when said composition is used for preventing and / or reducing gain of body weight in a subject, said composition may be administered to said subject as described elsewhere herein.

[0129] In the described LINX-WD model, further described below in the experimental section, it could be demonstrated that chronic consumption of a special high-salt Western Diet (WD) also rich in fat and fast carbs in young mice with 50% less nephrons (UNX) induced significantly increased body weight and impaired cardiovascular phenotype (increased blood pressure, endothelial dysfunction, leakage of heart specific peptides, metabolic phenotype (increased fat deposition and reduced glucose-clearance) and renal deterioration phenotype (pathologically reduced glomerular filtration rate, mesangial and tubular damage and albuminuria).

[0130] Surprisingly, the supplementation with FLX (group UNX-WD-FLX), i.e. the composition of the present invention, caused less accelerated gain of body weight and fat re-deposition, partially preserved blood pressure elevation, less compromised endothelium-dependent relaxation, much less leakage of Troponins from the heart, less reduction of renal dysfunction and glomerular damages in the kidneys and significant effects on glucose homeostasis.

[0131] Further aspects of the disclosure:

[0132] Embodiment 1: Use of a composition comprising: i) an extract of Rhodiola rosea root, ii) L- arginine, ii) L-citrulline; and iv) an extract of beet root, for preventing or reducing gain of body weight, or for reducing body weight, in a subject.

[0133] Embodiment 2: The use of embodiment 1, wherein said composition further comprises vitamin C and / or magnesium citrate.

[0134] Embodiment 3: The use of embodiment 1 or 2, wherein said composition comprises: i) from about 10 wt% to about 20 wt%, such as about 15.5 wt%, of an extract of Rhodiola rosea root, based on the total weight of the composition; ii) from about 22 wt% to about 32 wt%, such as about 27 wt%, of L-arginine, based on the total weight of the composition; iii)bfrom about 12 wt% to about 25 wt%, such as about 19 wt% or 19.3 wt% of L-citrulline, based on the total weight of the composition; and iv) from about 10 wt% to about 20 wt%, such as about 15.5 wt%, of an extract of beet root, based on the total weight of the composition.

[0135] Embodiment 4: The use of embodiment 3, wherein said composition further comprises: v) from about 2 wt% to about 6 wt%, such as about 4 wt%, of vitamin C, based on the total weight of the composition; and / or vi) from about 2 wt% to about 6 wt%, such as about 4 wt%, of magnesium citrate, based on the total weight of the composition, and vii) optionally from about 10 wt% to 20 wt%, such as about 15 wt%, of one or more additional ingredients, such as one or more of a stabilizing agent, an anti-caking agent, a preservative and an emulsifier, based on the total weight of the composition.

[0136] Embodiment 5: The use of any one of embodiments 1 to 4, wherein said composition reduces appetite in said subject.

[0137] Embodiment 6: The use of any one of embodiments 1 to 5, wherein said composition preserves a normal glucose metabolism.

[0138] The examples below further illustrate the present invention but should not be considered as limiting the invention, which is defined by the scope of the appended claims.

[0139] EXPERIMENTAL SECTION

[0140] Materials and methods

[0141] Mice

[0142] Commercially available conventional young male C57BL / 6J mice (Janvier Labs) were housed in an animal facility. They were maintained in conditions of controlled temperature, humidity, and light-and-dark cycle (12 / 12 h), with ad libitum access to food and water. The animals were received at 21 days of age, and after 7 days of acclimatization, sham surgery or unilateral nephrectomy (UNX) was performed to remove 50% of the available nephrons (details described below) at 28 days of age. This was done to mimic accelerated deterioration of renal function in the animals.

[0143] After 7 days and complete surgical recovery, the animals (BW=21g) were randomly divided into 3 different groups: (1) Sham surgery with standard rodent chow (n= 18, SHAM); (2) Unilateral nephrectomy with consumption of Western diet rich not only in sugar and fat but also in salt (n= 18, UNX-WD) and (3) Unilateral nephrectomy with consumption of the same Western diet rich in salt and in which a supplement, FLX, was given to the mice throughout the study (n=8, UNX-WD-FLX).

[0144] Unilateral Nephrectomy

[0145] After the acclimatization period, at 28 days of age, sham surgery or unilateral nephrectomy (UNX) was performed. For induction and maintenance of anesthesia, isoflurane concentrations of ~5% and 1.5% (vol / vol), respectively, were used (Pharmacia & Upjohn, Stockholm, Sweden). The mice were placed on a heating table and the abdominal cavity was accessed through the linea alba. The left kidney was located with a binocular stereomicroscope (Carl Zeiss Microscopy, Stockholm, Sweden), and the renal artery and vein were ligated with 6-0 suture thread, then the ureter was ligated with the same suture thread and the kidney was dissected and removed from the abdominal cavity. After checking for bleeding, the abdominal muscles and skin were sutured separately. The mice were monitored until complete recovery from anesthesia and the analgesia protocol was performed with buprenorphine twice a day until surgical recovery.

[0146] Methods

[0147] After starting the experimental protocol with dietary interventions (~35 days of age), the animals were monitored twice a week, body weight gain, water and feed consumption, and other noteworthy considerations were recorded. The animals were followed for 12 weeks.

[0148] The group UNX-WD-FLX was given a dietary supplement (referred to herein as FLX or FLEXO, see Table 1), which contains rhodiola rosea, amino acids, beetroot (beta vulgaris) extract and NO enhancing compounds (L-arginine and L-citrullinee) in the drinking water (250 mg / L) whereas the control group was given only the same diet and regular tap water (LINX-WD) ad libitum. Functionally, body weight and water consumption were monitored weekly, and cardiovascular, metabolic, renal function parameters and mitochondrial function in heart and kidneys were evaluated at the end of the 3 month intervention period.

[0149] The FLX supplement (FLEXOVITAL™, BIOCONCEPT AB) was a composite compound comprising as active ingredients Rhodiola rosea extract (100 mg), L-arginine (175 mg), L- citrulline (125 mg) and beet root extract (100 mg), per 500 mg of gross product. A recommended daily intake to a human weighing about 70 kg is 3-4 times daily of 1 tablet of said 500 mg gross product. The dose to the experimental animals was based upon such a 3 x 500 mg dose daily and adjusted accordingly in relation to body weight.

[0150] Measurements of body weight gain, DEXA (Dual-energy X-ray absorptiometry) for differentiation of body mass composition (Baseline and 5 weeks), fasting and non-fasting glucose and glucose tolerance tests, blood pressure, serum and urinary biomarkers such as creatinine, CA, P, Na, K, GFR (Glomerular Filtration Rate) and proteinuria, Troponin I, etc. were made intermittently during the course of the follow-up. These procedures are further described below under the section “analysis”.

[0151] At the end of the experiments, animals were sacrificed and a piece of the aorta (aortic ring) was captured for measurement of endothelial dependent vasodilation ex vivo, using a myograph after stimulation with acetylcholine (Ach). Heart, kidney, liver and brain tissues were secured for histology including light microscopy (hematoxylin, eosin and collagen) and immune staining followed by immunofluorescence (IFL) for detection of fibrosis, infiltration of inflammatory cells, deposition of metabolites etc. These procedures are also further described below under the section “analysis”.

[0152] Analysis

[0153] Metabolic function

[0154] Body composition

[0155] Body composition was measured by dual-energy x-ray absorptiometry (DEXA), using a Medikors InAnlyzer densitometer (MEDIKORS Inc., Republic of Korea). Fat and lean mass were calculated in relation to body weight. Additionally, only the liver was dissected and submitted to DEXA evaluation to quantify the percentage and distribution of fat.

[0156] Metabolic parameters

[0157] The metabolic parameters evaluated were glucose concentrations in fasting (5-hours) and non-fasting mice, intraperitoneal glucose (ipGTT) tolerance tests and the skeletal muscle fibers glucose uptake ex vivo.

[0158] Mice were fasted for 5 h (morning fasting) for ipGTT. Blood glucose levels were measured by Freestyle Lite Blood Glucose Meter (Abbott Diabetes Care Inc) at 0, 15, 30, 60, and 120 min after 50% D-glucose solution (2 g / kg body weight) intraperitoneal injection for ipGTT.

[0159] For the evaluation of glucose uptake in muscle fibers, skeletal muscles soleus and extensor digitorum longus (EDL) are accurately isolated from mouse legs and kept in a relaxant solution: Krebs-Ringer-Phosphate-Hepes (KRPH) buffer (20mM HEPES, 5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCI2, 136 mM NaCI, 4,7 mM KCI, pH 7,4) containing 2% BSA (KRPHB), for 60 minutes at room temperature. Then insulin 20 mllnits / ml was added and incubate at 30°C for 20 minutes. After, 2-deoxyglucose (2-DG) 1mM was added and incubate at 30°C for 20 minutes, the muscles were washed with PBS solution and the glucose uptake assay was performed following the manufacture instructions (Glucose Uptake Fluorometric Assay Kit, Catalog Number MAK084, Sigma-Aldrich).

[0160] Fat analysis

[0161] Triglycerides and cholesterol levels in plasma samples was analyzed by the Triglyceride Colorimetric Assay Kit (No. 10010303 - Cayman Chemical, Michigan, USA) and Cholesterol Fluorometric Assay Kit (No. 10007640 - Cayman Chemical, Michigan, USA), respectively. Plasma was diluted and protocols performed according to the manufacturer's instructions. Gonadal white adipose tissue (gWAT) and subcutaneous white adipose tissue (scWAT) were collected immediately after termination. After rinsing with saline to remove furs, gWAT and scWAT were fixed with 4% paraformaldehyde for 48 hours and then transferred to PBS. All samples were cut into 5 pm thick sections, de-paraffinized in xylene, and rehydrated in serial dilutions of ethanol and stained with hematoxylin-eosin. Adipocyte area size and diameter of gWAT and scWAT was analyzed respectively by Adiposoft plugin using Fiji Imaged Software. The inclusion range of adipocyte size is 30 pm to 120 pm for gWAT, and 20 pm to 120 pm for scWAT. Six random areas were chosen from each section for Image J analysis.

[0162] Cardiovascular function

[0163] Blood pressure

[0164] Coda High Throughput Noninvasive Tail Monitoring System (Kent Scientific, Torrington, CT, USA) was used for conscious blood pressure monitoring, according to the manufacturer's protocol. The system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. Systolic (SAP), diastolic (DAP) and mean blood pressure (MAP) was quantified by 45 repetitions. Averaged data from each animal was used for analysis.

[0165] Mitochondrial function

[0166] Kidney and heart mitochondria were isolated by differential centrifugation, as described elsewhere (Schiffer et al., 2018). Mitochondrial function was evaluated using high-resolution respirometry (Oroboros 2K, Innsbruck, Austria). Preserved mitochondrial integrity was confirmed by measuring respiratory control ratio (RCR) defined as maximal complex I mediated maximal respiratory capacity (pyruvate (5mM), malate (2mM) and ADP (2.5 mM)) divided by leak state without adenylates (pyruvate, malate). Maximal complex I (Cl) dependent respiration was evaluated in presence of pyruvate (5 mM) malate (2 mM) ADP (2.5 mM), complex II (CH) dependent respiration in presence of succinate (10 mM) ADP (2.5 mM) + rotenone (0.5pM). Maximal CI+CII dependent respiration was measured in presence of pyruvate, malate, succinate and ADP. Maximal complex IV (CIV) activity was evaluated by adding ascorbate (2 mM), TMPD (0.5 mM) in presence of antimycin (2.5 pM) and ADP (2.5 mM) and cytochrome c (10 pM). Leak respiration was measured in the presence of pyruvate (5 mM), malate (2 mM) and oligomycin (2.5 pM). Mitochondrial H2O2-production was measured spectrofluorometrically using the amplex red system (5 pM, Amplex ultraredTM) together with horseradish peroxidase (1 U / ml) (Sigma-Aldrich P 8250). Calibration of the H2O2 signal prior to each experiment was performed by adding standard solution of hydrogen peroxide (180 nM). Mitochondrial oxygen efficiency (P / O-ratio) was determined in presence of Cl substrates and ATP (2mM) by steady-state infusion of nonsaturating levels of ADP using the TIP2K titration injection micropump. The amount of mitochondria in the chamber was adjusted so that the respiration during the ADP infusion corresponded to approximately 50% of maximal state 3 respiration. P / O-ratio was calculated as the rate of infused ADP divided by the oxygen consumed during steady state at the last minutes of infusion.

[0167] Background correction was made for the infused oxygen present in the ADP solution.

[0168] Inflammatory and cardiac injury biomarkers

[0169] As a marker of direct myocardial injury, the levels of cardiac troponin I (CTnl) were quantified in plasma samples using the colorimetric sandwich ELISA assay (No. NBP3-00456, Novous Biologicals, CO, USA). Interleukin 6 (IL-6) was quantified as an inflammatory marker by the IL-6 Mouse ProQuantum Immunoassay Kit (Catalog # A43656 - ThermoFisher, Stockholm Sweden). Procedures and dilutions were performed according to the manufacturer's instructions.

[0170] Kidney function

[0171] Glomerular filtration rate

[0172] The glomerular filtration rate (GFR) was determined using clearance kinetics of plasma FITC-inulin. FITC-inulin (Sigma F3272) 1% was dissolved in PBS solution in dark and then sterilized by filtration through a 0.22-pm filter. FITC-inulin solution was injected ~100pL of FITC-solution in the tail vein and blood samples were taken in heparinized glass capillaries at time-points 1 , 3, 5, 10, 15, 35, 55 and 75 minutes after injection. Blood samples were centrifugated to obtain plasma and were buffered to PBS pH 7.4 with 500 mM 4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES). Fluorescent intensity was determined using a Multi-Mode Microplate Readers (SpectraMax iD3, USA) with excitation at 480 nm and emission at 530 nm. The linear correlation between fluorescent intensity and inulin concentration was established by measuring fluorescent intensity of an FITC-inulin solution of known concentration and a standard curve for blood samples was generated.

[0173] Kidney function biomarkers

[0174] Creatinine levels in plasma and urine were measured by the Creatinine colorimetric assay kit (No. 700460, Cayman Chemical, Michigan, USA) or Creatinine colorimetric assay kit (No. 500701, Cayman Chemical, Michigan, USA) relied on the Jaffe's reaction, respectively. Procedures and dilutions were performed according to the manufacturer's instructions. Albumin levels, a useful marker for early detection of chronic kidney diseases, was analyzed in urine, using a fluorometric assay kit (No. ab241017, Abeam, Cambridge, UK). The samples were handled according to the manufacture. Urea in plasma was detected by a colorimetric detection kit, BUN (No. EIABUN, Invitrogen, ThermoFisher, Stockholm Sweden). Plasma was diluted as recommended in the instruction manual from the manufacturer. Urine specific gravity was measured by refractometry, employing analogue refractometer (KERN ORA-P, Zollernalbkreis, Germany). Plasma and urinary levels of nitrate and nitrite were analyzed by high-performance liquid chromatography (HPLC; ENO-20; EiCOM) as described previously [PMID: 32032718], In brief, this method separated nitrite and nitrate by reverse phase / ion exchange chromatography followed by nitrate reduction to nitrite by cadmium and reduced copper. Griess reagent was then used for derivatization of reduced nitrite to form diazo compounds and analyzed by a visible detector at 540 nm.

[0175] Histopathological evaluation

[0176] Heart, kidney, liver, and duodenum were collected and fixed with 4% paraformaldehyde for histopathological analysis. After fixation, the samples were embedded in paraffin and cut using a microtome (5pm). Heart slides were staining with hematoxylin-eosin, Masson’s trichrome and Piero Sirius Red. Kidney slides were staining with hematoxylin-eosin, Masson’s trichrome and periodic acid-Schiff (PAS). Liver and duodenum slides were staining with hematoxylin-eosin. All slides were blindly evaluated under light microscopy by a specialized histopathologist. Initially, tissue morphology was evaluated and parameters such as the presence of fibrosis, necrosis and inflammatory infiltrate were investigated, and then, quantitative methods were used. For the liver, the fat deposition was calculated by the percentage of the area with fat in the hepatic tissue, 5 random fields were evaluated per animal (20X objective). For the evaluation of the duodenum, the length of the villi and the depth of the crypts were measured in 5 random fields per animal (10X objective), the villus:crypt ratio was used for analysis of the area of intestinal absorption. The histopathological evaluation of the kidney was divided into tubular and glomerular evaluation. For the tubules, histologically from two divisions, first the tubular evaluation, where a score from 0 to 10 was assigned, based on the distribution of previously described alterations such as: tubular vacuolization, dilatation and atrophy, loss of brusher border and thickening of tubular basement membrane. The histopathological evaluation of the kidney was divided into tubular and glomerular evaluation. For the tubules, a score from 0 to 10 was assigned, based on the distribution of previously described alterations, such as: tubular vacuolization, dilatation and atrophy, loss of brush border and thickening of tubular basement membrane. For the renal glomeruli, the area and perimeter were quantified to assess glomerular dilation, and the glomerular injury was characterized by the presence of one or more of the following changes: 1- glomerulosclerosis, 2 - mesangial hypercellularity and matrix expansion and 3 - irregular thickening of the glomerular basement membrane. Eight fields were quantified per sample and the results were expressed as the percentage of injured compared to healthy glomeruli. Histopathological evaluations were performed using Axioscope Microscope and Camera Axiocam 208 color (Carl Zeiss Microscopy, Stockholm, Sweden), and quantifications using Fiji / lmageJ Software and the Adiposoft plugin.

[0177] Statistics

[0178] Data are presented as the mean ± SD unless otherwise indicated. Statistical analysis was performed by using one-way or two-way ANOVA, followed by Tukey's multiple comparisons test for comparisons among the groups. Comparison between only two groups was performed using the unpaired t test (highlighted when used). A P value of less than 0.05 was considered statistically significant. All statistical analysis was performed using GraphPad Prism, version 9.2.0 (GraphPad Software).

[0179] Results

[0180] The following differences were seen between the groups LINX-WD and UNX-WD-FLX:

[0181] Body weight

[0182] The weight gain seen in group (UNX-WD-FLX) was significantly less than in the experimental group (UNX-WD). This means that providing the animals with a composition of the present disclosure prevented weight gain in the animals as compared to when the animals were not given the composition. These results are shown in Figure 2.

[0183] Fat mass and lean body mass

[0184] Figure 3 shows, by DEXA measurements of fat / lean mass, that these were increased in the group (UNX-WD) but that they were reversed in the group (UNX-WD-FLX) group. Again this shows that the composition of the present disclosure had beneficial effects on the fat content of the animals.

[0185] Adipocyte area

[0186] Figure 4 shows that there was an increase of the adipocyte area in the group (UNX-WD), but that the adipocyte area was significantly reduced in the group UNX-WD-FLX (Fig 4).

[0187] Glucose levels

[0188] Figure 5 illustrates the observation of an increase in fasting and non-fasting glucose in group (UNX-WD), and that both fasting and non-fasting glucose were significantly lowered in group (UNX-WD-FLX) Blood pressure

[0189] Figure 6 shows that there was an increase in blood pressure in group (UNX-WD), but a significantly lower blood pressure, SBP, DBP and MAP in group (UNX-WD-FLX). Vasodilation

[0190] As can be seen in Figure 7, Endothelial-dependent vasodilation became significantly compromised in group (UNX-WD) but was surprisingly significantly reversed in subjects of group (UNX-WD-FLX).

[0191] Renal function

[0192] Figure 8 shows the pronounced reduction in GFR in the UNX-WD group and significant protection of GFR in the FLX group. Tubular and glomerular injury

[0193] As illustrated in Figure 9, tubular and glomerular injury caused a reduction in Glomular Filtration Rate (GFR) in the group (LINX-WD) but was partially protected in subjects of group (UNX-WD-FLX). 58% reduction of GFR in the group (UNX-WD), vs. 23% in group (UNX-WD- FLX). 4 / 8 (UNX-WD-FLX) subjects had non-affected GFR, whereas all animals in the group (UNX-WD) showed a reduced GFR.

[0194] Renal glomerular damage

[0195] Similarly, subjects in group (UNX-WD) developed renal glomerular damage consisting of mesangial proliferation and increased amount of ECM (strikingly similar to renal damages seen in obesity and in diabetic injury), whereas there was a clear protection in FLX treated subjects, group (UNX-WD-FLX).

[0196] Urinary excretion of albumin

[0197] Albuminuria increased in group (UNX-WD) and was partially protected by FLX in group (UNX-WD-FLX). These results are shown in Figure 12.

[0198] Mitochondrial function

[0199] Mitochondrial function in the heart and kidney tissue was tested with five different markers. The major finding was in the P / O ratio in renal tissue, which was compromised in group (UNX-WD) and clearly reversed in group (UNX-WD-FLX). These results are shown in Figure 13.

[0200] Troponin

[0201] Figure 14 shows the effects on plasma Troponin I values, with virtually normal range in the sham group and a 2-3 fold increase in the experimental UNX-WD group. In subjects treated with FLX (UNX-WD-FLX) we observed an almost total normalization of Troponins levels.

[0202] Inflammation, IL-6

[0203] Figure 15 shows IL-6 levels in all groups. In the SHAM and the UNX-WD groups 10 / 20 subjects had an IL6 levels > 0.1 pg / ml, whereas in the UNX-WD-FLX group there was 1 / 5 with IL6 > 0.1 pg / ml in plasma. This is a strong indication that FLX has a lowering influence on inflammatory activity in this setting.

[0204] Discussion

[0205] In the described UNX-WD model, it could be demonstrated that chronic consumption of a special high-salt Western Diet (WD) also rich in fat and fast carbs in young mice with 50% less nephrons (UNX) induced significantly increased body weight and impaired cardiovascular phenotype (increased blood pressure, endothelial dysfunction, cardiac troponin leakage), metabolic phenotype (increased fat deposition and reduced glucose- clearance) and renal deterioration phenotype (pathologically reduced glomerular filtration rate, mesangial and tubular damage and albuminuria), mitochondrial compromise and degree of systemic inflammation.

[0206] Surprisingly, the supplementation with FLX (group UNX-WD-FLX), i.e. the composition of the present disclosure, caused less accelerated gain of body weight, reduced and caused redeposition of tissue fat, partially preserved blood pressure elevation, resulted in less compromised endothelium-dependent relaxation, resulted in almost complete normalization of cardiac troponin leakage, caused less reduction of renal dysfunction and glomerular damages in the kidneys, reduced systemic inflammation and had significant effects on glucose homeostasis.

[0207] Hence, the above results show that a composition of the present invention has a reducing or preventing effect on the weight gain normally associated with the intake of a western diet. The composition also has beneficial effects on subjects with a reduced kidney function I renal failure / uremia and associated biological injuries, when such subjects are exposed to a diet high in fat and sugar.

[0208] All in all, the results indicate a potential use of the present composition as an inducer of weight loss, or as a means for preserving a steady body weight even if a subject is being exposed to a diet that it high in fat and salt. The positive effects on the body weight provides additional positive effects as overweight is often associated with other conditions such as high blood pressure, disturbed glucose mechanism, renal injury and cardiac stress and injury. Furthermore, subjects were partially compromised with regard to renal function, which progressed in the experimental group along with associated organ and tissue damages, which were largely protected by the composition. Endothelial dysfunction, renal damage and reduced renal function, reduced mitochondrial function were also largely protected by the use of the composition. Of note, the triggering of cardiac troponin leakage was virtually normalized by the use of the composition, indicating that cardiac stress leading to cardio myocyte damage could be prevented by the composition.

[0209] Herein, it was shown that positive effects were achieved by the composition on virtually all the negatively affected health indicators, which emerged in the experimental group.

Claims

CLAIMS1. A composition comprising i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root, for use in preventing and / or treating renal failure in a subject.

2. The composition for use of claim 1, wherein said renal failure comprises uremia.

3. The composition for use of claim 1 or 2, wherein said composition comprises: i) from about 10 wt% to about 20 wt%, such as about 15.5 wt%, of an extract of Rhodiola rosea root, based on the total weight of the composition; ii) from about 22 wt% to about 32 wt%, such as about 27 wt%, of L-arginine, based on the total weight of the composition; iii) from about 12 wt% to about 25 wt%, such as about 19 wt% or 19.3 wt% of L- citrulline, based on the total weight of the composition; and iv) from about 10 wt% to about 20 wt%, such as about 15.5 wt%, of an extract of beet root, based on the total weight of the composition.

4. The composition for use of any one of claims 1 to 3, wherein said composition further comprises vitamin C and / or magnesium citrate.

5. The composition for use of any one of claims 1 to 4, wherein said composition further comprises: v) from about 2 wt% to about 6 wt%, such as about 4 wt%, of vitamin C, based on the total weight of the composition; and / or vi) from about 2 wt% to about 6 wt%, such as about 4 wt%, of magnesium citrate, based on the total weight of the composition, and vii) optionally from about 10 wt% to 20 wt%, such as about 15 wt%, of one or more additional ingredients, such as one or more of a stabilizing agent, an anti-caking agent, a preservative and an emulsifier, based on the total weight of the composition.

6. The composition for use of any one of the preceding claims, wherein said subject suffers from organ and / or tissue damage and wherein said composition further prevents and / or treats said organ and / or tissue damage.

7. The composition for use of any one of the preceding claims, wherein said subject is suffering from overweight, particularly obesity, and further wherein said composition prevents weight gain and / or reduces body weight in said subject.

8. The composition for use of any one of the preceding claims, wherein said composition preserves a normal glucose metabolism and / or treats and / or prevents hyperglycemia in said subject.

9. The composition for use of any one of the preceding claims, wherein said subject is or has been exposed to a diet high in fat and / or carbohydrates and / or a diet high in salt.

10. The composition for use of any one of the preceding claims, wherein said composition preserves endothelium-dependent vascular relaxation and / or further prevents and / or treats hypertension in said subject.

11. The composition for use of any one of the preceding claims, wherein said composition preserves a normal blood pressure in said subject.

12. The composition for use of any one of the preceding claims, wherein said composition further prevents and / or treats heart disease related to renal failure or uremia in said subject.

13. The composition for use of any one of the preceding claims, wherein said composition further prevents and / or treats systemic inflammation related to renal failure or uremia in said subject.

14. A method for preventing and / or treating renal failure in a subject, said method comprising administering a pharmaceutically effective amount of a composition comprising i) an extract of Rhodiola rosea root, ii) L-arginine, iii) L-citrulline; and iv) an extract of beet root, to a subject in need thereof.

15. The method of claim 14, wherein said renal failure comprises uremia.

16. The method of claim 14 or 15, wherein said composition comprises: i) from about 10 wt% to about 20 wt%, such as about 15.5 wt%, of an extract of Rhodiola rosea root, based on the total weight of the composition; ii) from about 22 wt% to about 32 wt%, such as about 27 wt%, of L-arginine, based on the total weight of the composition; iii) from about 12 wt% to about 25 wt%, such as about 19 wt% or 19.3 wt% of L- citrulline, based on the total weight of the composition; and iv) from about 10 wt% to about 20 wt%, such as about 15.5 wt%, of an extract of beet root, based on the total weight of the composition.

17. The method of any one of claims 14 to 16, wherein said composition further comprises vitamin C and / or magnesium citrate.

18. The composition of any one of claims 14 to 17, wherein said composition further comprises: v) from about 2 wt% to about 6 wt%, such as about 4 wt%, of vitamin C, based on the total weight of the composition; and / or vi) from about 2 wt% to about 6 wt%, such as about 4 wt%, of magnesium citrate, based on the total weight of the composition, and vii) optionally from about 10 wt% to 20 wt%, such as about 15 wt%, of one or more additional ingredients, such as one or more of a stabilizing agent, an anti-caking agent, a preservative and an emulsifier, based on the total weight of the composition.

19. The method of any one of claims 14 to 18, wherein said subject suffers from organ and / or tissue damage and wherein said method further comprises preventing and / or treating said organ and / or tissue damage in said subject.

20. The method of any one of claims 14 to 19, wherein said subject suffers from overweight, particularly obesity, and further wherein said method comprises preventing weight gain and / or reducing body weight in said subject.

21. The method of any one of claims 14 to 20, wherein said method further comprises preserving a normal glucose metabolism and / or treating and / or preventing hyperglycemia in said subject.

22. The method of any one of claims 14 to 21, wherein said subject is or has been exposed to a diet high in fat and / or carbohydrates and / or a diet high in salt.

23. The method of any one of claims 14 to 22, wherein said method further comprises preserving endothelium-dependent vascular relaxation and / or preventing and / or treating hypertension in said subject.

24. The method of any one of claims 14 to 23, wherein said method further comprises preserving a normal blood pressure in said subject.

25. The method of any one of claims 14 to 24, wherein said method further comprises preventing and / or treating heart disease related to renal failure or uremia in said subject.

26. The method of any one of claims 14 to 25, wherein said method further comprises preventing and / or treating systemic inflammation related to renal failure or uremia in said subject.

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