Fruit Derived Juice With Low Levels Of Potassium For Patients With Chronic Kidney Disease

FRUIT DERIVED JUICE WITH LOW LEVELS OF POTASSIUM FOR PATIENTS WITH CHRONIC KIDNEY DISEASE

FIELD OF THE INVENTION

The invention relates to a method of producing a low potassium fruit juice which provides vasorelaxation, NADPH-oxidase mediation and repeated infection reduction characteristics and more particularly a method of producing a fruit juice which is suitable for consumption by patients suffering from progressive kidney failure including end stage renal failure who are placed on a restricted potassium diet. BACKGROUND TO THE INVENTION

Patients suffering from kidney failure, typically described as stages 1 to 5, where stage 5 represents chronic kidney disease (CKD) characterised as End Stage Renal Failure (ESRF) generally requiring dialysis pending kidney transplant, have decreased ability to excrete potassium and therefore maintaining blood ion balance and as a result they tend to suffer from hyper-phosphatemia. In extreme cases high serum potassium levels can give rise to heart failure and in the worst cases can result in death.

It has been estimated that the overall healthcare cost in the EU (2007) of end stage haemodialysis treatment for CKD patients ranges between €33 to €56bn per year, accounting for between 3 to 5% of total EU healthcare expenditure (all 27 states). The current (2007) annual cost is rising at 8 % per year and is spread over a relatively small End Stage Haemodialysis Treatment patient population of 250,000. Therefore a small cost improvement in treatment regime and/or increased patient outcome for stage 3, 4 and 5 CKD patients, can dramatically effect treatment costs.

About 20 % of haemodialysis patients die within the first year of their treatment. Inflammation is highly prevalent in this patient population and inflammation-related proteins, such as C- reactive protein (CRP) and certain cytokines and chemokines, are primary risk markers. Preliminary research has demonstrated that polyphenol-rich drinks administered to End Stage Haemodialysis (ESHD) patients was shown to reduce neutrophil NADPH oxidase activity, oxidized LDL (oxLDL) and MCP-1 levels. Nevertheless, the juice administered in the preliminary research contained higher than acceptable levels of potassium and cannot be stably administered to ESHD patients as food for special medical purposes to manage the characteristic inflammatory state of CKD patients undergoing haemodialysis treatment. As a result it is desirable to produce a juice with anti-inflammatory characteristics which also contains low levels of potassium. It is thought that that uraemia or the hemodialysis procedure itself induces an inflammatory state, corresponding to approximately 5-10-fold higher CRP concentrations than in the general population. In the presence of such a major chronic inflammatory insult, conventional statin therapy might not effectively lower CRP levels as it does in studies among patients who are not on dialysis. Thus, identifying treatments that decrease inflammation in HD patients is an important issue in the clinic, which can translate into improved patient outcomes. The risk of cardiovascular events in the general population is substantially greater among patients with evidence of inflammation than among those who show little or no inflammation3,4. In the 4D study, however, no significant effect of administering atorvastatin on the primary outcome (composite of death from cardiac causes, non-fatal myocardial infarction, or non-fatal stroke) was observed when patients were subdivided according to quartiles of baseline CRP concentration5.

Similarly, in the AURORA study, subgroup analyses in CRP tertiles did not find a significant effect of rosuvastatin on the incidence of the primary end point (cardiovascular death, Nonfatal myocardial infarction, or non-fatal stroke) as compared with placebo in any of these tertiles6.

A major cause of atherosclerosis (dyslipidaemia) has been extensively studied in Haemodialysis (HD) patients. Typically, HD patients have relatively normal total cholesterol and LDLcholesterol (LDL-c) plasma concentrations accompanied by high triglyceride (TG) and low HDLcholesterol (HDLc). While these abnormalities may contribute to atherosclerosis and cardiovascular mortality, the qualitative forms of dyslipidaemia of HD patients also plays a role. Approximately 20% - 40% of chronic HD patients have elevated TGs and reduced HDL-C12. As regards to the apoprotein profile in HD patients, ApoC-lll is significantly increased; ApoB and ApoE are moderately elevated, whereas ApoA-l and ApoA-ll are depressed13. The increase in ApoB is accounted for by TG-rich ApoB-containing lipoproteins in the VLDL and IDL range, as opposed to cholesterol-rich LDL14. Increased VLDL and IDL is most likely to be the result of diminished activity of lipoprotein lipase, also favoured by the increased ApoCIII content15. Conversely, CETP plasma levels were found to be increased in approximately one third of HD patients, which may contribute to the low level of HDLc found in this population16.

Haemodialysis patients can also have an atherogenic lipid profile in the absence of hyperlipidaemia, as shown by the increased prevalence of hypertriglyceridemia and higher VLDL-c and IDL-c and lower HDLc levels, despite LDL-c may be decreased17. Other studies have shown that HD patients tend to have elevated Lp(a) and particularly oxLDL18-22. In summary, the dyslipidaemia in HD is characterized by relatively normal total cholesterol (TC) and LDL-c, elevated TG, and low HDL-c, which in general, is similar to that in the non- dialysis dependent CKD population.

Data describing the association between dyslipidaemia and death in dialysis patients may seem contradictory. For instance, large cross-sectional studies have found no association between baselines TC or TG and CV disease in dialysis patients23,24, and two prospective studies in HD patients showed no association between baselines TG, TC, LDL-c, or HDLc and future atherosclerotic events25,26. By contrast, both cross-sectional studies27 and prospective28 studies of HD patients have shown dyslipidaemia is positively associated with coronary artery disease or death. The potential role of malnutrition/inflammation on mortality is important in interpreting conflicting data. In a large retrospective study of HD patients, the relation between TC and death took the form of a U-shaped curve, which appeared more linear after adjustment for serum albumin30. This apparently paradoxical relation may be wholly or partially due to increased mortality among patients with evidence of inflammation/malnutrition, which typically results in lower cholesterol levels32. In a prospective report of HD patients, low cholesterol was found to be independently associated with higher C-reactive protein (CRP) and mortality at 10 years in those with low albumin29. However, in the subgroup of patients with albumin >45 g/L, high cholesterol was associated with increased mortality. In a second prospective study, where patients were classified by the presence or absence of inflammation and/or malnutrition at baseline, based on serum levels of albumin, CRP or interleukin-6, an increment in baseline TC decreased all-cause mortality in the presence of inflammation/malnutrition, but in the absence of inflammation/malnutrition, there was a strong, positive, graded relation of TC with all-cause mortality and CV mortality. These studies demonstrate that hypercholesterolemia is a risk factor for all-cause and CV mortality in patients with ESRD, but the association can be masked by concomitant inflammation and/or malnutrition.

As in the general population, Lp(a) has been positively associated with atherosclerotic CV disease in HD patients33. There are few data describing the effect of other, less traditional, lipid risk factors (such as small dense LDL, oxidized LDL, and VLDL/IDL) on mortality in dialysis patients.

The treatment of dyslipidaemia in HD patients has been demonstrated to be as safe and efficacious in HD patients as in non-dialysis CKD patients and the general population5,34,35. The studies show that statins significantly reduce TC and LDL-c and increase HDL-c, whereas the effect in TG is variable. Treatment with statins has been also shown to decrease oxidized LDL levels and to shift LDL from the small dense to the larger, buoyant less atherogenic form35"38.

Statins also exert pleiotropic effects, including a reduction of CRP and an increase of serum albumin36"38. Observational studies demonstrated that statin use in the ESRD population is independently associated with reduced CV- and total mortality in both treated with HD or peritoneal dialysis (PD)39, 40. Three randomized controlled trials have been designed to establish if hypolipidemic treatment reduce mortality in dialysis patients. The "4D" Study was conducted with 1255 HD patients with type II diabetes, comparing atorvastatin 20 mg/day with placebo on the composite primary outcome of death from cardiac causes, non-fatal myocardial infarction, and stroke5. During the 4-year follow-up period, no apparent benefit with statin treatment was observed in HD patients. The authors suggested that the lack of beneficial effect could be due to non-traditional pathogenic pathways contributing to CV disease, making statin therapy unhelpful when postponed until dialysis5.

The AURORA study randomized 2776 patients undergoing maintenance hemodialysis to rosuvastatin 10 mg/day or placebo, and the end points were time to death from any cause and time to major CV event (non-fatal stroke, non-fatal myocardial infarction, or CV death)6. During a median follow-up period of 3.8 years, rosuvastatin lowered LDL-c by 39 mg/dl, but yielded a non-significant 4% reduction in the primary outcome of cardiovascular death, Nonfatal myocardial infarction or non-fatal stroke. There was also no significant effect on all- cause mortality6. The last, recently terminated trial is the SHARP study . This study enrolled 9,270 patients with CKD of whom 3,023 patients were receiving maintenance dialysis at randomization (2,527 heamodialysis and 496 peritoneal dialysis). Patients were randomly assigned to simvastatin 20 mg plus ezetimibe 10 mg daily versus matching placebo. The key pre- specified outcome was first major atherosclerotic event (non-fatal myocardial infarction or coronary death, non-haemorrhagic stroke, or any arterial revascularisation procedure). After a median follow-up of 4.9 years, in patients randomized to ezetimibe/simvastatin LDLc was reduced in 32 mg/dl, and experienced a significant 17% reduction in major atherosclerotic events compared with the placebo group41. In subgroup analyses, there was no evidence that the proportional effects on major atherosclerotic events differed between patients on dialysis and non-dialysis-CKD patients. However, SHARP did not have sufficient power to assess the effects on major atherosclerotic events separately in dialysis and non-dialysis patients41. Thus, no firm conclusion could be drawn from this study for HD patients. Taken all these data together, including the negative results found in the above 4D and AURORA studies, in contrast to non-dialysis-CKD patients, it is not clear the benefit of statin treatment in HD patients, at least in those not clearly hypolipidemic. It has been suggested that the advanced atherosclerotic state in chronic dialysis patients and the increased percentage of sudden death due to arrhythmia plays a significant role and that it is a condition not modifiable by statins42. Conversely, this lack of benefit might be attributed to the deleterious effects of hemodialysis as inducers of neutrophil activation and inflammation, and malnutrition.

The lack of benefits noted in studies that enrolled hemodialysis populations in contrast to the other populations with different stages of CKD might be attributed to different reasons43. In non-dialysis-dependent CKD, cardiac disease is predominantly due to coronary artery occlusive disease, which is amenable to statin treatment. In contrast, in hemodialysis, sudden death due to cardiac arrhythmias accounts for the majority of cardiovascular deaths44. This is attributed to myocardial fibrosis and left ventricular hypertrophy which lead to conduction abnormalities and subsequent ventricular arrhythmias and is blood plasma potassium concentration dependent45. The beneficial effect of statins on cardiovascular end points can be attributed to cholesterol-dependent and/or cholesterol-independent effects46"48. The cholesterol independent effects include decreasing inflammation, enhancing endothelial function, inhibiting smooth muscle proliferation, exerting direct anti-thrombotic properties and stabilizing pre-existing atherosclerotic plaque. However, the aforementioned cardiovascular risk factors increase as renal function declines, rendering statins less effective in dialysis patients. All these reasons contribute to the differences in the effects of statins seen in various stages of CKD.

The loss of normal kidney function is known to lead to anaemia in ESRD patients (van der Putten et al., 2008). The condition is exacerbated by haemodialysis-induced inflammation and can leave the patient being more prone to infection. To correct the anaemia erythropoietin is routinely used to stimulate red cell production and elevate plasma haemoglobin. In addition to the routine costs of dialysis, ESRD patients also need regular treatment injection with erythropoietin. The dose of erythropoietin is influenced by the general health of the patient, with HD-associated inflammation or infection being associated with the development of resistance and the requirement of a higher cost (De Francisco et al., 2009). An indication of the importance of 'inflammation' status on the costs of erythropoietin per patient comes from the finding that use of atorvastatin HD patients was associated with a reduction 'erythropoietin-resistance' (Chiang et al., 2009).

The generalised inflammatory state in ESRD patients, and the presence of raised level of oxidative species, has a direct impact on the function of the cardiovascular system and the vascular endothelium. These cells are known to release many key molecules, e.g., nitric oxide, that influences the function of platelets and immune cells, in addition to the underlying vascular smooth muscle (Hewitson et al., 2007). In ESRD patients impairment of endothelial cell function is known to contribute to the increased vascular stiffness, poor regulation of blood flow to key organs and increased cardiovascular morbidity (Laurant and Boutouyrie 2007). In general, interventions that either preserve or improve endothelial cell function by reducing oxidative stress are associated with better outcomes (Gross et al., 2001 ; Schiffren ef a/., 2007).

Inflammation underlies a wide variety of physiological and pathological processes. Infection and tissue injury are the classic inducers of inflammation, which results in the recruitment of leukocytes and certain cytokines and chemokines to the affected tissue site. Tissue stress also induces an adaptive response, which has been referred to as para-inflammation49. This response relies mainly on tissue-resident macrophages and presents as low grade inflammatory response.

Para-inflammation is responsible for the chronic inflammatory conditions that are associated with most prevalent human diseases and, particularly, CKD49. Mild-to-moderate inflammation is considered as a hallmark of CKD50,51. Although in the general population traditional risk factors, such as dyslipidaemia, hypertension and diabetes, explain most of the excess risk for CV mortality, in CKD patients inflammation has also a relevant impact on survival and cardiovascular complications52"54. Numerous studies have proven that elevated levels of CRP predict all-cause and cardiovascular mortality in patients on hemodialysis55"57. Only one early study did not confirm these findings, due to the short period of follow-up58. Moreover, in HD patients, CRP has been described to be predictive of sudden death and stroke59. Further support for the association between inflammation and poor outcomes is evident from two large studies involving HD patients, linking white-blood-cell count with all-cause mortality 60,

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Oxidative stress is an important cardiovascular risk factor in CKD patients62"64. Oxidative stress is brought about by an imbalance between production of oxidants and availability of antioxidant defence mechanisms, and plays a critical role in the pathogenesis of atherosclerosis and vascular disease65,66. This is particularly important in hemodialysis patients, in whom reactive oxygen species (ROS) are overproduced and antioxidant defence mechanisms are impaired64,67. Oxidized LDL (oxLDL) plasma concentration is a predictor of atherosclerosis in both the general population68"71 and HD patients72,73. Plasma concentration of oxLDL is significantly higher in HD patients than in healthy subjects22 and, importantly, oxLDL concentration is directly correlated with the severity of the underlying acute coronary syndrome69. Thus high concentration of oxLDL is present even though HD patients were receiving poly-vitamin supplements to maintain vitamin C and vitamin E plasma concentrations within normal values22. When administered at very high doses, however, of vitamin E is able to reduce oxLDL plasma concentration in HD patients74. These data indicate that circulating LDL is exposed to an intense attack by free radicals in HD patients. The causes of this may be various, however, the haemodialysis procedure itself causes oxidative stress by generating ROS through activation of circulating neutrophils75,76. In fact, coating dialysis membranes with vitamin E has been shown to effectively reduce superoxide anion production by neutrophils76, 77 and to lower the plasma concentration of oxLDL75,76 and

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NADPH-oxidase is a main source of superoxide radicals, which promote LDL oxidation. The enzyme is particularly active in neutrophils and monocytes, where it plays an important role in host defence. A direct association between increased phagocytic NADPH-oxidase activity and elevated circulating oxLDL was found in patients with metabolic syndrome79. In particular, NADPH-oxidase-dependent superoxide production appears to be abnormally high in mononuclear cells from patients with both non-dialysis dependent CKD80 and end-stage CKD . Consequently, both modulation of NADPH-oxidase to prevent overproduction of ROS81 and supplementation with antioxidants82,83 have been proposed as reasonable strategies to prevent the deleterious effects of oxidative stress in haemodialysis patients. Haemodialysis (HD) patients are subjected to dietary restrictions aimed to reduce potassium intake, with limitations in fruits and vegetables. These restrictions also result in an obvious reduction in the intake of vitamins and other micronutrients from plant origin84. Polyphenols comprise a wide variety of plant compounds that occur naturally in foods, which are absorbed to a significant extent by the intestine85. Bioavailability of dietary polyphenols has been demonstrated previously by many authors86"90. After ingestion, polyphenols have been detected in plasma both in their soluble form and physically associated with lipoproteins92"94. Oral administration of polyphenol-rich apple extracts (Evesse™ EPC, CORESSENCE Ltd, UK), freeze dried apple granules (Evesse™ WAG, CORESSENCE Ltd UK) and apple juice (Evesse™ Juice, CORESSENCE Ltd, UK) has demonstrated high bio-availability of (-)- epicatechin monomers in particular, which have been shown to mediate NADPH-oxidase and the give rise to vasodilation in healthy volunteers exhibiting signs of oxidative stress. Oral administration of polyphenol-rich red grape juice (high in potassium -185 mg/100ml) has been shown to increase both HDL-c and Apo A-l levels and to reduce LDL-c and Apo B-c22, this last effect being attributed to the stimulated LDL-receptor activity95. In addition, flavonoids confer antioxidant protection22,96,97, inhibit platelet function98,99, reduce both thrombus formation100 and the concentration of inflammatory biomarkers101 , and inhibit the activation of nuclear transcription factor NF-kB102. In both healthy volunteers and in HD patients, it has been demonstrated that acute ingestion of a polyphenol-rich high-potassium red grape juice reduced the plasma concentration of oxLDL22,74. This effect (>50% reduction) was much greater than that in LDL-c (<20% reduction). In contrast, treatment with atorvastatin in HD patients reduces LDL-c levels more intensely (50%) than oxLDL (25%)109. These results clearly indicate that the mechanisms by which polyphenols and statins reduce oxLDL plasma concentrations are different. A possible mechanism involved in the action of polyphenols on NADPH-oxidase as a main source of superoxide anion in blood. Polyphenols, more specifically flavan-3-ols, and more specifically circulating metabolites of (- )-epicatechin monomers have been shown to inhibit the activity of NADPH-oxidase by directly quenching free radicals but also to reduce the expression of different NADPH- oxidase subunits at the transcription level, as demonstrated both in different cell types in 'iro110,111 and also in animals112,113. It has been shown that the acute administration of a polyphenol-rich high potassium juice to HD patients significantly reduced the activity of NADPH-oxidase in circulating neutrophils74. Furthermore, changes in NADPH-oxidase activity were shown to directly correlate with those in oxLDL . These results suggest that the decrease of oxLDL is mediated by the reduction of NADPH-oxidase activity. MCP-1 has been recognized as an important factor in the progression of atherosclerosis114. It has been found that the concentration of MCP-1 significantly decreased after short-term regular ingestion of high potassium grape juice in HD patients, is correlated with the decrease of neutrophil NADPHoxidase activity74. The parallel changes with two inflammation-related parameters reflect a common sensing mechanism to ingested flavan-3-ols. Consequently it has been proposed the involvement of a redox-sensitive mechanism in the expression of vascular inflammatory gene products, in response to pro-inflammatory stimuli115. Furthermore, it has recently shown in HD patients that the expression of NADPH-oxidase p22phox subunit in leukocytes is positively correlated with the plasma concentration of CRP, another inflammatory marker116. Therefore we consider that both NADPH-oxidase subunit mRNA levels and classic inflammatory markers (acute-phase proteins, cytokines and chemokines) to be useful in assessing the para-inflammation state in HD patients and underlie the potential benefit of the ingestion of polyphenol-rich low potassium drinks and fruit juices which possess the ability to mediate NADPH-oxidase. Further results of previous work showed that oral supplementation with a concentrated polyphenol-rich potassium-rich juice decreased NADPH oxidase-dependent superoxide production in patients with end- stage renal disease (ESRD). Nevertheless, chronic ingestion of high-potassium juices cannot be recommended for CKD patients.

Therefore, patients with progressive kidney failure are subject to strict dietary restrictions designed to limit their intake of potassium and cannot freely eat vegetables or fruits and cannot consume fruit juices containing high levels of potassium. Furthermore, patients who have an imposed dietary restriction on potassium, tend to consume meals which are nutritionally unbalanced and forego healthy antioxidant and polyphenols which are known to improve peripheral vasculature and to impact on markers of oxidative stress and to reduce rates of infection. Whilst it is known that fruit juices can be treated with ion exchange resins to remove potassium ions, Japanese Patent 61-209573, US6387425 amongst others, the balance of micro and macro nutritional components can be seriously disturbed such that the resultant product formulations do not satisfy their use as healthcare products, prescribed by doctors for consumption by CKD patients or administered prophylactically to early stage kidney disease patients. In general, reduced potassium juice is subject to a limit on potassium reduction of 90% of the original potassium content to maintain palatability. In the case of some juices which possess high antioxidant capacity such as pomegranate juice, the potassium content can exceed 600mg per 100ml. While this is well below the recommended maximum intake of 1500 mg/day for patients undergoing haemodialysis three times a week, it represents a high intake level of potassium in relation to other dietary intake which may be consumed under the dietary restrictions and continued consumption would seriously compromise HD patient dietary regimes. In general, fruit juices may be subjected to up to 90% potassium reduction through processing, however, the resultant juice is often highly acidic and unpalatable. As a result, the technology developed to manage potassium content has not used beyond 30% reduction of that originally contained in the juice, which technically produces juices in the range of 60 mg/100ml to 180 mg/100ml of potassium content, which is considered dangerous for sustained HD patient consumption. In addition, juices subject to ion-exchange potassium modification do not generally contain high levels of (-)-epicatechin monomers and therefore do not demonstrate a clear NADPH-oxidase mediation and vasodilation effect.

Therefore, it is desirable to produce a palatable fruit juice composition that provides low levels of potassium, preferably less than 60 mg/100ml more favourably less than 22 mg/100ml and more favourably 10 mg/100ml or zero mg/100ml, which does not compromise taste whilst providing additional benefits of vasodilation (or conversely reduced vascular stiffness) resulting from the interaction of circulating metabolites of flavan-3-ols, in particular, (-)-epicatechin monomers with cellular-bound endothelium NADPH-oxidase and reduced infection rates when consumed by CKD patients.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a fruit derived juice which comprises low levels of potassium for use in administration to patients suffering from chronic kidney disease.

According to a further aspect of the invention, there is provided a juice as defined herein, for use in dietary supplementation and/or prevention of inflammation in patients undergoing haemodialysis.

DETAILED DESCRIPTION OF THE INVENTION Treatment Of Ckd Patients In one embodiment, the low-potassium juice is or comprises a naturally pressed juice or an extract of a plant (for example the fruit, leaves or stem). In one embodiment, the fruit is an apple. In one embodiment, the low-potassium juice is a characteristic of the fruit selected, such as an apple described in WO 2008/017845, the contents of which is incorporated herein by reference. Further, the juice may be produced or comprise an extract obtainable and described as in WO 2008/017842, the contents of which are incorporated herein by reference.

In one aspect, the present invention relates to the use of a juice comprising naturally low levels of potassium which provides an acceptable dietary level of potassium for patients undergoing haemodialysis.

It has been surprisingly found that apples (Malus prumila Mill) that contain high levels of flavan-3-ols can be used to produce juices containing low levels of potassium. As such the use of such juices can aid the dietary management of CKD patients subject to haemodialysis. Thus, in one embodiment, the apple is Malus prumila Mill.

In a further aspect, the present invention relates to a juice which specifically comprises (-)- epicatechin can be used in the treatment and/or prevention of peripheral arterial disease developed by patients undergoing haemodialysis or progressing through the progressive management stages of diagnosed kidney failure.

In a further aspect, the present invention relates to juices which provide high levels of antioxidants which reduce levels of oxidative stress by mediating NADPH-oxidase.

Other aspects of the invention are apparent from the below description.

It is also noted that the teachings of each heading are not necessarily limited to that particular section and in fact may be combined.

In one aspect of the present invention there is provided the use of a composition comprising a flavan-3-ol compound which induces peripheral vascular smooth muscle relaxation.

Flavan-3-ols

In one embodiment, the juice comprises a flavan-3-ol compound. Flavan-3-ols are a group of compounds characterised by the following general formula (I):

(I)

wherein either of the benzyl rings may be optionally further substituted by one or more hydroxyl groups. Where this is the case, the compound is also considered to be a polyphenol. Flavan-3-ols are to be distinguished from flavonoid compounds, which comprise a ketone moiety on the pyrano ring. It will be understood that flavan-3-ols according to formula (I) contain two chiral centres at the 2 and 3 positions of the pyrano ring. Furthermore, it will be understood that as a result of the presence of two substitutions on the pyrano ring, flavan-3-ols according to formula (I) can be present in different epimeric forms. Epimers are diasteroisomers that differ in configuration of only one stereogenic centre. For example, the flavan-3-ol compound commonly referred to a catechin actually exists as four different stereoisomers.

Furthermore, flavan-3-ols according to formula (I) can be linked together as polymers and these compounds are generally referred to as oligomers. In one embodiment, the flavan-3-ol compound is (-)-epicatechin.

It will be appreciated that the juice according to the invention may either be administered under the supervision of a medical practitioner or without the direction of a medical practitioner. It will also be appreciated that the juice according to the invention may be consumed as part of dietary management of both haemodialysis and peritoneal dialysis patients.

Administration of the juice according to the invention provides the advantage of reducing the number of first and second recurrent admissions to hospital of haemodialysis patients due to infections. Vascular Relaxation

In one embodiment, the juice may be used in vascular relaxation in patients undergoing haemodialysis. According to the present invention, vascular relaxation means that the compound gives rise to smooth muscle relaxation in a dose dependent manner. In particular, vascular relaxation means that the composition exhibits smooth muscle relaxation when ingested which is not exhibited by epicatechin-rich compositions in which the major component is the (+)- epicatechin stereoisomer as may be exhibited by certain grape juices, extracts and compounds. Thus although vascular relaxation resulting from the ingestion of (-)-epicatechin stereoisomers is known to be the result of metabolites, effectively defining (-)-epicatechin as a pro-drug.

In one embodiment, the present invention relates to the use of a juice comprising flavan-3-ol oligomers with degree of polymerisation less than or equal to 2 which give rise to vascular smooth muscle relaxation when ingested. In one embodiment, the juice comprises a flavan- 3-ol oligomer with a molecular weight less than 600 Da.

In a further embodiment, the present invention relates to the use of a juice produced from apple which has naturally low levels of potassium which do not require further processing using ion exchange to limit the potassium content. Thus, in one embodiment the juice contains potassium between zero and 60 mg per 100 ml.

In one embodiment, the juice comprises (-)-epicatechin in an amount of at least 3.5 wt% per 100ml of juice. In one embodiment, the composition comprises (-)-epicatechin in an amount of at least 7.0 wt% per 100 ml of juice. In one embodiment, the composition comprises (-)- epicatechin in an amount of at least 10.0 wt% per 100 ml of juice. In one embodiment, the composition comprises (-)-epicatechin in an amount 3.5 wt% per 100 ml to 10 wt% per 100ml of juice.

In one embodiment, the juice comprises (-)-epicatechin in an amount 3.5 wt% per 100 ml to wt% of juice together with less than 60 mg per 100 ml of juice of potassium. In one embodiment, the juice comprises (-)-epicatechin in an amount 3.5 wt% per 100 ml to 10 wt% per 100ml of juice together with less than 22 mg per 100 ml of potassium, less than 10 mg per 100 ml of potassium and zero mg per 100 ml of juice. In one embodiment, the juice is produced without the need for additional processing using ion exchange stripping of potassium. In one embodiment, the juice is produced to potassium content between 10mg and zero mg per 100 ml of juice using ion exchange processing. In one embodiment, the juice contains a bioactive flavan-3-ol consists essentially of (-)- epicatechin. In this embodiment, "consists essentially of" means that although other flavan-3- ols and low molecular weight polyphenols may be present, they are present in such low concentrations that they do not materially affect the efficacy of the juice for ingestion by CKD patients. In one embodiment, the composition of the juice consists of (-)-epicatechin. In one embodiment, the composition of the juice does not comprise flavanol compounds.

In one embodiment, the composition of the juice comprises (-)-epicatechin and at least one other flavan-3-ol compound selected from the group consisting of (+)-catechin, (-)_catechin, (+)-epicatechin, (+)-gallocatechin, (-)-gallocatechin, (-)+epigallocatechin and (+)- epigallochatechin.

In one embodiment, (-)-epicatechin is present in the composition as a monomer. In a further embodiment, all of the flavan-3-ol compounds are present in the juice as monomers. In one embodiment, the juice is used to mediate the inflammation state of a CKD patient undergoing haemodialysis. In a further embodiment, the juice is used to mediate the vascular smooth muscle interactions between NADPH-oxidase and Nitric Oxide production across the endothelium of the vascular lumen. In a further embodiment, the juice mediates infection rates in HD patients. In one embodiment, the juice is ingested in an amount of at least 100 ml containing 3.5 wt% of (-)-epicatechin and less than 60 mg of potassium. In one embodiment, the juice is ingested in an amount of less than 150 ml containing 10.5 wt% of (-)-epicatechin and less than 33 mg of potassium. In one embodiment, the juice is ingested in an amount of less than 150 ml containing 10.5 wt% of (-)-epicatechin and less than 15 mg of potassium. In one embodiment, the juice is ingested in an amount of less than 150 ml containing 10.5 wt% of (- )-epicatechin and less than 15 mg of potassium. In one embodiment, the juice is ingested in an amount of less than 150 ml containing 10.5% wt% of (-)-epicatechin and zero wt% of potassium.

In one embodiment, the (-)-epicatechin containing juice is or comprises the pressing using conventional or enzyme assisted methods well known in the art, of the whole or part of a plant (for example the fruit). In one embodiment, the composition is or comprises an apple. For example, the present invention comprises the juice process from an apple described in WO 2008/017845, the contents of which are incorporated herein by reference. Further, the composition of the juice of the present invention may be or comprise an extract obtainable and described in WO 2008/017842, the contents of which are incorporated herein by reference.

In one embodiment, the juice comprises up to 10 mg per 100 ml of calcium. Further Aspects

In a further aspect, the present invention provides a juice which may be produced as an aseptic juice with a shelf life of 28 days at + 5 Deg C. In one embodiment, the juice of the invention may be subjected to ultra-filtration to at least 10,000 Da. which may be stored at ambient temperature for longer than 28 days. In a further aspect, the present invention provides a juice which may be produced as an aseptic juice without ultra-filtration. In a further aspect of the invention, the juice may be produced as an aseptic juice by ultrafiltration and micro-filtration to 0.2 μηι. In a further embodiment, the present invention provides a juice which may be dried into a powder for rehydration at a future date. Thus, in a further embodiment the juice is cloudy, clarified, concentrated or dried to powder form.

In one embodiment, the juice is administered either before or after a haemodialysis session for up to 3 times per week.

In one embodiment, the juice is administered to a patient undergoing haemodialysis for administration on non-dialysis days.

EXAMPLE

The present invention will now be described with reference to the following non-limiting example.

Example 1 - Evaluation of Potassium and calcium content of an aseptic juice produced from an apple (Malus pumila Mill) comprising a flavan-3-ol compound, (-)- epicatechin, sold under the trade name Evesse™ (available from Coressence Ltd., One Glass Wharf, Bristol, BS2 0ZX, UK). A selection of fruits as described in WO 2008/017845, the contents of which is incorporated herein by reference, were subjected to pressing in a large scale Bucker apple press model Filter Press HPX 5005L Enzyme processing was accomplished using a non-GMO pectinmethyllesterase, derived from a strain of Aspergillus niger, specifically designed to remove the methyl side groups from pectin molecules, while leaving the long-chain pectin molecules intact. The purpose of the use of this enzyme was to increase the yield of juice with a stable cloud due to the preservation of the long-chain stable pectin. The resultant juice was then pasteurised in a flash pasteurisation system heating the juice to at least 73 Deg C for 15 seconds. The resultant juice was aseptically packed into 1000 litre sealed containers. Three 5 litre samples were recovered from the batch and sent for systematic triplicate analysis, the results are shown in Table 1 below:

Table 1

Iron 0.7 mg/kg

Phosphorus 9.0 mg/100g

Glucose 1.98 g/100g

Galactose <0.1 g/100g

Fructose 7.53 g/100g

Sucrose 0.68 g/100g

Lactose <0.1 g/100g

Maltose <0.1 g/100g

Aspartic acid 0.599 mg/kg

Threonine 0.279 mg/kg

Serine 0.293 mg/kg

Glutamic acid 0.624 mg/kg

Proline 0.255 mg/kg

Glycine 0.255 mg/kg

Valine 0.259 mg/kg

Methionine 0.077 mg/kg

Isoleucine 0.216 mg/kg

Leucine 0.400 mg/kg

Tyrosine 0.095 mg/kg

Phenylananine 0.250 mg/kg

Histidine 0.214 mg/kg

Lysine 0.326 mg/kg

Arginine 0.201 mg/kg

It will be seen from Table 1 that the potassium content of the juice is 22 mg per 100g which is equivalent to 20.9 mg per 100 ml. Furthermore it will be seen that the juice contained 82.8 mg/kg of calcium (8 mg per 100 ml).

It will be recognised that patients suffering from kidney failure have decreased functions of excreting potassium and of maintaining blood ion balance so they tend to suffer from hyperphosphatemia or hypocalcemia and hence the administration of calcium carbonate to such patients is necessary. For example a low potassium juice described in US 6,387,425 describes the addition of 50 mg per 100 ml of calcium carbonate. It may be observed that the low potassium juice produced in this example contains up to 20% of the calcium levels without fortification. REFERENCES

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154. Chaudhuri AA, So AY, Sinha N, et al. MicroRNA-125b Potentiates Macrophage Activation. J Immunol 2011 ;187:5062-8.

155. Kim SY, Kim AY, Lee HW, et al. miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARgamma expression. Biochem Biophys Res Commun 2010;392:323-8. All publications mentioned herein are incorporated by reference. Various modification and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology, food science or related fields are intended to be within the scope of the following claims:

CLAIMS

1. A fruit derived juice which comprises low levels of potassium for use in administration to patients suffering from chronic kidney disease.

2. The juice according to claim 1 , wherein the fruit is an apple.

3. The juice according to claim 2, wherein the apple is Malus pumila Mill. 4. The juice according to any one of claims 1 to 3, which contains potassium between zero and 60 mg per 100 ml.

5. The juice according to any one of claims 1 to 4, which comprises up to 10 mg calcium per 100 ml.

6. The juice according to any one of claims 1 to 5, which comprises a flavan-3-ol compound.

7. The juice according to claim 6, wherein the flavan-3-ol compound is present in an amount of at least 3.5 wt% per 100 ml.

8. The juice according to claim 6 or claim 7, wherein the flavan-3-ol compound is present in an amount from 3.5 wt% to 10 wt% per 100 ml. 9. The juice according to any one of claims 6 to 8, wherein the flavan-3-ol compound is (-)-epicatechin.

10. The juice according to any one of claims 1 to 9, which is cloudy, clarified, concentrated or dried to powder form.

1 1. A juice according to any one of claims 1 to 10, for use in dietary supplementation and/or prevention of inflammation in patients undergoing haemodialysis.

12. A juice according to claim 1 1 , for use in vascular relaxation in patients undergoing haemodialysis.

13. A juice according to any one of claims 1 to 12, which is administered either before or after a haemodialysis session for up to 3 times per week. 14. A juice according to any one of claims 1 to 13, wherein the juice is administered to a patient undergoing haemodialysis for administration on non-dialysis days.

AMENDED CLAIMS

received by the International Bureau on

1 October 2013 (01.10.2013)

CLAIMS

1 , A fruit derived juice which comprises low levels of potassium for use In the treatment of chronic kidney dleease, characterised In that said fruit Is an apple which Is Malus pumlla s Mill,

2. The juice for use according to claim 1 , which contains potassium between zero and 80 mg per 100 ml. 0 3, The juice for use according to claim 1 or claim 2, which comprises up to 10 mg calcium per 100 ml,

4. The juice for use according to any one of claims 1 to 3, which comprises a flavan-3-ol compound.

5

5. The juice for use according to claim 4, wherein the flavan-3-ol compound Is present In an amount of at least 3 5 wt% per 100 ml.

Θ. The juice for UBB according to claim 4 or claim 5, wherein the flavan-3-ol compound0 Is present In an amount from 3.6 wt% to 10 wt% per 100 ml.

7. The juice for use according to any one of claims 4 to Θ, wherein the flavan-3-ol compound Is (-)-eplcatechln. 5 8. The juice for use according to any, one of claims 1 to 7, which Is cloudy, clarified, concentrated or dried to powder form.

Θ. The juice for use according to any one of claims 1 to 8, wherein the juice Is administered to a patient undergoing haemodlalysis as a dietary supplement and/or for the0 prevention of Inflammation.

10. The juice for use according to claim 9, wherein the Juice Is administered to a patient undergoing haemodlalysis for vascular relaxation,

AMENDED SHEET (ARTICLE 19)

31

11. The juice (or use according to any one of claims 1 to 10, wherein the juice Is administered to a patient either before or after a haemodlalysis session for up to 3 times per week, s 12, The juice for use according to any one of claims 1 to 11 , wherein the Juice Is administered to a patient undergoing haemodlalysis on non-dlalysle days,

AMENDED SHEET (ARTICLE 19)

32

Statement under Article 19(1) PCT

1. Novelty

We submit that amended claims 1 to 12 are novel over the prior art cited by the

Examiner by virtue of the fact that the claims require the juice to be derived from an apple which Is Malus pumlle Mill. None of the prior art documents cited by the Examiner disclose a juice derived from Malus pumlle Mill for use In the treatment of chronic Kidney disease, This fact is acknowledged by the Examiner who has not cited any documents as novelty destroying for previous claim 3 (the claim previously directed to Malus pumlle Mill.

Therefore, we submit that the presently amended claims are novel over the prior art cited by the Examiner,

2. Inventive Step

The problem Intended to be solved by the Invention Is as set out on page 1 , lines 6 to 8 of the application as filed, namely the treatment of patients with end stage kidney failure who have been placed on a restricted potassium diet.

The solution provided by the present Invention Is a fruit juice derived from an apple which Is Malus pumlle Mill.

As described In section 1 above, the technical difference between the solution provided by the present Invention and the compositions of the prior art Is that none of the compositions of the prior art disclose the use of a juice derived from Malus pumlle Mill for the treatment of chronic kidney disease.

The technical effect of this difference Is that the juice derived from Malus pumlle Mill has been surprisingly Identified to contain low potassium levels and therefore finds great utility In the treatment of kidney failure in patients who have been placed on a restricted potaeBlum diet. This surprising finding Is clearly stated on page 11 , lines 13 to 16 of the application as filed;

"It hes been surprisingly found that apples (Malus prumlla Mill) that contain high levels of fla Bn'3'Ol can be used to produce Juices containing low levels of potassium, As such the use of such Juices cen eld the dietary management of CKD patients subject to heemodlelysls. "

The Examiner has alleged that the skilled person "would try the apple of D10 to D12 to get the advantage of Its composition". However, we respectfully disagree. Although the skilled person could have tried the apple In light of the high flavan-3-ol content, the skilled person would not have been motivated to try the apple of D10 to D12 In the expectation of success that a fruit Juice having low levels of potassium would result,

Therefore, we submit that the presently amended claims are Inventive over the prior art cited by the Examiner.

Heme VII and VIII

We submit that the objections raised by the Examiner under Items VII and VIII are now moot In view of the reformulation of the claims as "Compound X for use In the treatment of disease V" claims,

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