HK1140423B - Use of known pharmacologically active chemical compounds - Google Patents

Description

Use of known pharmacologically active compounds

Technical Field

The present invention relates to a new use of known pharmacologically active compounds. More specifically, the present invention relates to the novel use of alpha-ketoglutaric acid, amides, salts thereof and mixtures thereof for the preparation of a pharmaceutical preparation or a food or feed supplement for the therapeutic in vivo improvement of vascular elasticity, in particular arterial elasticity, in a subject in need thereof.

Background

Gastric surgery

With the escalation of obesity in the western world, the prospect of an invasive form of weight loss as a means to reduce mortality and improve co-morbidities is increasingly promising. Indeed, some advocate that invasive weight loss is the only accepted mode of weight loss, as the non-surgical approach reported in the NIH declaration in 1992 has been successful at 5% or less in obese patients. Gastric bypass surgery has been shown to result in excessive weight loss of up to 70%, with long-term weight loss lasting 10-14 years.

More recently, Coates and her colleagues reported that invasive surgery, such as gastric bypass surgery, may affect bone health. In a study of 25 patients with gastric bypass surgery, a marked increase in bone turnover markers and a decrease in bone mineral density were found in the hip trochanter.

Gastric bypass surgery is also known to be beneficial in reducing hypertension, a common problem in morbid obesity. In a follow-up visit with 67 morbidly obese patients enrolled as having hypertension (bp > 160/90mm Hg), it was shown that gastric bypass surgery reduced preoperative hypertension in 44 individuals (66%).

Vascular elasticity in disease

Vascular elasticity, particularly arterial elasticity, has long been known to be associated with hypertension and related conditions. Arteriosclerosis has been shown to be an independent predictor of the progression of hypertension in non-hypertensive subjects, i.e., arteriosclerosis is a risk factor for developing hypertension independent of other known risk factors (Dernellis and Panaretou, Hypertension.2005; 45: 426-. Retinal vascular disease may be due to hypertensive retinopathy, i.e., to systemic hypertension.

Pulmonary Hypertension (PH) refers to an increase in blood pressure in the pulmonary artery, pulmonary vein, or pulmonary capillary (collectively referred to as pulmonary blood vessels) that results in shortness of breath, dizziness, fainting, and other symptoms, all of which can be exacerbated by exercise. Depending on the cause, pulmonary hypertension can be a serious disease with significantly reduced exercise tolerance and can lead to right heart failure. Pulmonary arterial hypertension (WHO class I) involves vasoconstriction or sclerosis of blood vessels connecting to the lungs and of blood vessels within the lungs. This makes it more difficult for the heart to pump blood through the lungs. Over time, the affected blood vessels become stiffer and thicker in a process known as fibrosis. This further raises the blood pressure in the lungs and impairs their blood flow. In addition, increased cardiac load can lead to right ventricular hypertrophy, which can progress to right ventricular failure.

Aneurysms are localized hyperemic expansions (balloon-like expansions) of a blood vessel caused by disease or weakened vessel walls. Aneurysms are most commonly found in the basilar arteries of the brain (Willis's cycle) and in the aorta (the major arteries flowing from the heart), known as aortic aneurysms. The swelling in the blood vessel can rupture at any time and lead to disease or death. The larger the aneurysm, the more likely it is to rupture.

Although treatments (both pharmaceutical and surgical) for hypertension, pulmonary hypertension, ventricular hypertrophy, and aneurysms exist, there is still a need for improved treatments with better efficacy and/or fewer side effects and/or better cost effectiveness.

Disclosure of Invention

Now, the inventors show that obese individuals have altered elasticity of their arteries after gastric surgery. Bypass surgery alters nutrient uptake affecting arterial structures (in a similar manner to that seen in the skeletal system), with adverse consequences for the elasticity and strength of the artery. However, no effective treatment for these side effects has been reported. Accordingly, there is an unmet need to prevent these side effects of gastric surgery. Furthermore, it has also been found that the same treatment can be used in other subjects (e.g., elderly subjects) in need of increased vascular elasticity.

Accordingly, the present invention provides the use of at least one substance selected from the group consisting of:

a) alpha-ketoglutaric Acid (AKG);

b) a pharmaceutically acceptable salt of alpha-ketoglutaric acid;

c) amides of amino acids, dipeptides or tripeptides with alpha-ketoglutarate and pharmaceutically acceptable salts thereof; and

d) a pharmaceutically acceptable physical mixture of α -ketoglutaric acid or a pharmaceutically acceptable salt thereof and at least one amino acid or a pharmaceutically acceptable salt thereof.

The blood vessel is preferably an artery.

In certain embodiments, the subject is in need of treatment and/or prevention of hypertension, pulmonary hypertension, cardiovascular disease, retinal vascular disease, heart failure, atherosclerosis, ventricular hypertrophy, stroke, aneurysm, renal failure, nephrosclerosis, or other disease associated with hypertension.

In one embodiment, the invention relates to the use of at least one substance selected from the group consisting of: alpha-ketoglutaric Acid (AKG); a pharmaceutically acceptable salt of alpha-ketoglutaric acid; amides of amino acids, dipeptides or tripeptides with alpha-ketoglutarate and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable physical mixture of alpha-ketoglutaric acid or a pharmaceutically acceptable salt thereof and at least one amino acid. According to still further embodiments, the gastric surgery is gastric bypass surgery, gastrectomy, partial gastrectomy, or gastric ligation.

According to still further embodiments, alpha-ketoglutaric acid or an alkali metal salt or alkaline earth metal salt thereof, or a combination thereof, is used.

According to one embodiment of the invention, sodium α -ketoglutarate is used. According to another embodiment, calcium α -ketoglutarate is used.

According to still further embodiments, the dose of the substance administered to the patient is 1-1000mg/kg body weight/day, 10-400mg/kg body weight/day, or 10-100mg/kg body weight/day.

In another aspect of the present invention, there is provided a method of treatment for increasing vascular elasticity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of at least one substance selected from the group consisting of:

a) alpha-ketoglutaric Acid (AKG);

b) a pharmaceutically acceptable salt of alpha-ketoglutaric acid;

c) amides of amino acids, dipeptides or tripeptides with alpha-ketoglutarate and pharmaceutically acceptable salts thereof; and

d) a pharmaceutically acceptable physical mixture of α -ketoglutaric acid or a pharmaceutically acceptable salt thereof and at least one amino acid or a pharmaceutically acceptable salt thereof.

The blood vessel in the method is preferably an artery.

The subject to whom the treatment is administered may be a subject in need of treatment and/or prevention of hypertension, pulmonary hypertension, cardiovascular disease, retinal vascular disease, heart failure, atherosclerosis, ventricular hypertrophy, stroke, aneurysm, renal failure, nephrosclerosis, or other diseases associated with hypertension.

According to the present invention, it was unexpectedly found that alpha-ketoglutaric Acid (AKG); a pharmaceutically acceptable salt of alpha-ketoglutaric acid; amides of amino acids, dipeptides or tripeptides with alpha-ketoglutarate and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable physical mixture of alpha-ketoglutaric acid or a pharmaceutically acceptable salt thereof and at least one amino acid or a pharmaceutically acceptable salt thereof, are useful for the therapeutic treatment of the side effects of vascular elasticity and strength in vivo in individuals having undergone gastric surgery. According to still further embodiments, the gastric surgery is gastric bypass surgery, gastrectomy, partial gastrectomy, or gastric ligation.

In yet another embodiment, the present invention relates to a method for treating side effects on vascular elasticity and strength in an individual who has undergone gastric surgery, said method comprising administering to a subject in need thereof an effective amount of at least one substance selected from the group consisting of: alpha-ketoglutaric Acid (AKG); a pharmaceutically acceptable salt of alpha-ketoglutaric acid; amides of amino acids, dipeptides or tripeptides with alpha-ketoglutarate and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable physical mixture of alpha-ketoglutaric acid or a pharmaceutically acceptable salt thereof and at least one amino acid.

According to still further embodiments, the gastric surgery is gastric bypass surgery, gastrectomy, partial gastrectomy, or gastric ligation.

In yet another embodiment, the present invention relates to a method of treating side effects on vascular elasticity and strength in an individual suffering from a condition associated with malnutrition or an elderly individual, the method comprising administering to a subject in need of such treatment or prevention an effective amount of at least one substance selected from the group consisting of: alpha-ketoglutaric Acid (AKG); a pharmaceutically acceptable salt of alpha-ketoglutaric acid; amides of amino acids, dipeptides or tripeptides with alpha-ketoglutarate and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable physical mixture of alpha-ketoglutaric acid or a pharmaceutically acceptable salt thereof and at least one amino acid.

According to still further embodiments, alpha-ketoglutaric acid or an alkali metal salt or alkaline earth metal salt thereof, or a combination thereof, is used.

According to one embodiment of the invention, sodium α -ketoglutarate is used. According to another embodiment, calcium α -ketoglutarate is used.

According to still further embodiments, the dose of the substance administered to the patient in the methods of the invention is 1-1000mg/kg body weight/day, 10-400mg/kg body weight/day, or 10-100mg/kg body weight/day.

Drawings

The invention is further explained in the following description with the aid of preferred embodiments, examples and figures:

FIG. 1 shows the recorded values of the elastic recoil of the aortic segment in bypass-operated rats (B) and sham-operated rats (S) administered AKG (+ AKG) or vehicle (-AKG). The recording was performed with a force transducer connected to an a/D converter with a sampling rate of 1000 samples/second. Each point represents the mean ± SE. The significant differences between the mean values are as follows: a and d ═ p < 0.05, b ═ p < 0.01, and c ═ p ═ 0.01. Animals assigned to the four groups were as follows: B-AKGn ═ 6, B + AKG n ═ 11, S-AKG n ═ 12, and S + AKG n ═ 12.

Fig. 2 shows a typical experimental diagram of an aortic segment subjected to a series of stretch/relaxation cycles. The maximum applied stretching force was about 0.14% as measured in rat aorta (13-14 kPa). Note that the slope of the line for the first stretch/relax cycle is compared to the slope of the line for the second and third cycles. This slope represents the elastic recoil force inherent in the aortic segment (about 16% of the manually applied tension). Clearly, repeating the stretch/relaxation cycle in this range results in a decrease in elasticity.

Figure 4 shows a first stretch series: values were recorded as elastic recoil of the aortic segment of control and (A) Na-AKG and (B) Ca-AKG treated mice. The recording was performed with a force transducer connected to an a/D converter with a sampling rate of 1000 samples/second. Each point represents the mean ± SE. The significant differences between the mean values are as follows: p < 0.05, P < 0.01, c < 0.001. Animals assigned to each of the three groups were n-6.

Figure 5 shows a second stretch series: values were recorded as elastic recoil of the aortic segment of control and (A) Na-AKG and (B) Ca-AKG treated mice. The recording was performed with a force transducer connected to an a/D converter with a sampling rate of 1000 samples/second. Each point represents the mean ± SE. The significant differences between the mean values are as follows: p < 0.05, P < 0.01, c < 0.001. Animals assigned to each of the three groups were n-6.

Detailed Description

It is an object of the present invention to provide an effective and safe treatment useful for increasing vascular elasticity in a subject in need thereof. In a preferred embodiment, the vessels with increased elasticity are arteries, but the elasticity of veins, capillaries, venules and arterioles can also be increased by the present invention.

Since there is a defined correlation between vascular elasticity, in particular arterial elasticity, and hypertension and pulmonary hypertension, the provided treatment for increasing vascular elasticity can be used for the production of a medicament for the treatment and/or prophylaxis of hypertension and pulmonary hypertension. Hypertension is also known to be a contributing factor to cardiovascular disease, retinal vascular disease, heart failure, stroke, atherosclerosis, renal failure, nephrosclerosis, and other diseases. Also, pulmonary hypertension is known to be a causative factor in right ventricular hypertrophy. Thus, the provided treatments for improving vascular elasticity can be used for the preparation of a medicament for the treatment and/or prevention of said diseases and conditions where hypertension and pulmonary hypertension are a causative or risk factor, as well as other diseases and conditions where hypertension and pulmonary hypertension are a causative or risk factor. The provided treatments may also be used in the preparation of medicaments for the treatment and/or prevention of other conditions in which vascular elasticity is known to be impaired.

It is another object of the present invention to provide an effective and safe treatment of the side effects on blood vessel elasticity and strength caused by gastric surgery.

The term "treat" in its various grammatical forms in connection with the present invention refers to preventing, curing, reversing, attenuating, alleviating, ameliorating, inhibiting, minimizing, stopping or stopping the side effects of the condition being treated.

The term "side effects" in connection with gastric surgery in the context of the present invention refers to adverse effects on vascular properties (e.g. elasticity and/or strength of arteries) that occur after gastric surgery. For example, decreased elasticity of arteries is seen after gastric bypass surgery.

The term "malnutrition" means a medical condition caused by inadequate or inadequate diet, usually caused by insufficient, malabsorption or excessive loss of nutrients.

Even with a full diet, certain conditions associated with malnutrition can occur. For example, gastrointestinal tract function may be impaired due to aging or other diseases. In such cases, the impaired digestion may be due to, or lack of production of, host digestive enzymes, e.g., in the stomach, intestine, pancreas, etc.; insufficient bile formation; inadequate gastric pH (impaired HCl production) or other causes. Villous atrophy due to old age, diet (e.g., intolerance to gluten), or disease disruption of the villus may be a direct cause of malnutrition due to impaired absorption. Conditions involving bacterial overgrowth or lack of gut bacteria may also be a cause of malnutrition. Malnutrition is also caused by several other causes such as bowel cancer, surgery, toxins, genetics, circulatory (blood and lymph) problems, anorexia, etc. In any case, malnutrition and malnutrition-related disorders can lead to cachexia (kachexia) and reduce vital functions.

The term "elderly" in the context of the present invention means an age group in an organism (e.g. a human) in which age-related decline begins to become apparent. In the case of humans, the aged age may be defined as being over 40 years old, preferably over 50 years old, more preferably over 60 years old, or most preferably over 65 years old.

"improving the elasticity of the blood vessel" means that the elasticity of the blood vessel becomes large, that is, the hardness of the blood vessel becomes small. The term also includes the increase in tensile strength of the blood vessel.

Examples of gastric procedures relevant to the present invention include, but are not limited to, gastric bypass surgery, gastrectomy, partial gastrectomy, and gastric ligation.

Thus, according to one aspect of the present invention, there is provided the use of at least one substance selected from the group consisting of: alpha-ketoglutaric Acid (AKG); a pharmaceutically acceptable salt of alpha-ketoglutaric acid; amides of amino acids or di-or tripeptides with alpha-ketoglutaric acid and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable physical mixture of alpha-ketoglutaric acid or a pharmaceutically acceptable salt thereof and at least one amino acid or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the pharmaceutical formulation is directed to increasing the elasticity of an artery.

In one embodiment, the pharmaceutical formulation relates to a subject who has undergone gastric surgery.

In one embodiment, the pharmaceutical formulation relates to a subject having a disorder associated with malnutrition.

In one embodiment, the pharmaceutical formulation is directed to an aging subject.

According to one embodiment of the invention, alpha-ketoglutaric acid or an alkali metal salt or alkaline earth metal salt thereof or a combination thereof is used.

Preferably, sodium α -ketoglutarate is used. Even more preferably, calcium alpha-ketoglutarate is used. Sodium alpha-ketoglutarate is absorbed more rapidly and has a higher peak blood level after enteral administration, whereas calcium alpha-ketoglutarate is absorbed more slowly and has a longer lasting effect after enteral administration. Example 2 shows that calcium alpha-ketoglutarate is more effective than sodium alpha-ketoglutarate in certain conditions.

In yet another aspect, the present invention relates to a method for increasing vascular elasticity (e.g. for treating and/or preventing hypertension, pulmonary hypertension, cardiovascular disease, retinal vascular disease, heart failure, atherosclerosis, ventricular hypertrophy, stroke, aneurysm, renal failure, nephrosclerosis and diseases associated with hypertension), the method comprising administering to a subject in need of such treatment or prevention an effective amount of at least one substance selected from the group consisting of: alpha-ketoglutaric Acid (AKG); a pharmaceutically acceptable salt of alpha-ketoglutaric acid; amides of amino acids or di-or tripeptides with alpha-ketoglutaric acid and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable physical mixture of alpha-ketoglutaric acid or a pharmaceutically acceptable salt thereof and at least one amino acid or a pharmaceutically acceptable salt thereof.

According to certain embodiments, the subject has undergone gastric surgery, has a condition associated with malnutrition, or is an elderly subject.

According to some embodiments of these aspects, α -ketoglutaric acid or an alkali metal salt or alkaline earth metal salt thereof, or a combination thereof, is administered. Preferably, sodium alpha-ketoglutarate is administered. More preferably, calcium alpha-ketoglutarate is administered.

The pharmaceutical formulations of the active ingredients used in the present invention may be administered to vertebrates, including mammals and birds, for example rodents such as mice, rats, guinea pigs or rabbits; birds, such as turkeys, hens or chicks, and other broilers and free farming animals; cattle, horses, pigs or piglets and other farm animals; dogs, cats and other pets, particularly humans.

Administration can be carried out in different ways depending on the type of vertebrate being treated, the condition of the vertebrate being treated in need of such treatment, and the specific indication to be treated.

In one embodiment, administration is in the form of a food or feed supplement (e.g., a dietary supplement and/or an ingredient in the form of a solid food and/or beverage). Other embodiments may be a suspension or solution, such as a beverage as described further below. Furthermore, the forms may be capsules or tablets, such as chewable or soluble tablets (e.g. effervescent tablets) as well as powders and other dry forms well known to those skilled in the art, such as pills (e.g. pellets) and granules.

Administration can be carried out parenterally, rectally, or orally as a food or feed supplement, as described above. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or fixed oils.

The food and feed supplements may also be emulsified. The active therapeutic ingredient can then be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Furthermore, the compositions may contain minor amounts of auxiliary substances which enhance the effectiveness of the active ingredient, such as wetting or emulsifying agents, pH buffering agents, if desired.

Different forms of parenteral food or feed supplements may be provided, for example, in solid food, liquid or lyophilized or other dry formulations. It may comprise diluents with different buffers (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives (e.g. albumin or gelatin) to prevent surface adsorption; detergents (e.g., tween 20, tween 80, pluronic F68, cholate); solubilizers (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite); preservatives (e.g., Thimerosal, benzyl alcohol, paraben); bulking substances (bulking substances) or tonicity modifiers (e.g. lactose, mannitol); a polymer (e.g., polyethylene glycol) covalently attached to the composition; a complex with a metal ion; or incorporating the material into or onto a particulate formulation of a polymeric compound (e.g., polylactic acid, polyglycolic acid, a hydrogel, etc.); or incorporated onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroids.

In one embodiment, the food or feed supplement is administered in any of the methods of the invention in the form of a beverage or a dry composition.

The beverage comprises an effective amount of the active ingredient together with a nutritionally acceptable water-soluble carrier such as minerals, vitamins, carbohydrates, fat and protein. If the beverage is provided in dry form, all of these ingredients are provided in dry form. The provided beverage ready for consumption further comprises water. The final beverage solution may also have a controlled tonicity and acidity, such as a buffer solution in accordance with the general recommendations of the preceding paragraph.

The pH range is preferably from about 2 to about 5, especially from about 2 to about 4, to prevent bacterial and fungal growth. Sterile beverages with a pH of about 6-8 may also be used.

The beverages can be provided alone or in combination with one or more therapeutically effective compositions.

According to yet another embodiment, the pharmaceutical preparations as a medicament for oral and rectal use may be in the form of tablets, troches, capsules, powders, aqueous or oily suspensions, syrups, elixirs, aqueous solutions and the like, containing the active ingredient in admixture with pharmaceutically acceptable carriers and/or additives such as diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers useful in the methods and uses disclosed herein.

In addition, "pharmaceutically acceptable carriers" as used herein are well known to those skilled in the art and may include, but are not limited to, 0.01-0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's solution glucose, glucose and sodium chloride, lactated ringer's solution or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

In the amide of an amino acid or a dipeptide or tripeptide with alpha-ketoglutaric acid, the amino acid or the amino acid forming the dipeptide or tripeptide may be any amino acid present as a component in the natural peptide. The same applies to the pharmaceutically acceptable physical mixture of alpha-ketoglutaric acid or a salt thereof and at least one amino acid. Preferably, the amino acid is selected from arginine, ornithine, leucine, isoleucine and lysine.

The amino acids are preferably used in their L-configuration.

Examples of amides of alpha-ketoglutarate with amino acids, dipeptides or tripeptides include, but are not limited to, amides of alpha-ketoglutarate with an amino acid selected from glutamine, glutamic acid, arginine, ornithine, lysine, proline, isoleucine and leucine; amides of alpha-ketoglutaric acid with glutamine and dipeptides of any of glutamic acid, arginine, ornithine, lysine, proline, isoleucine and leucine; and amides of alpha-ketoglutaric acid with glutamic acid and dipeptides of any of arginine, ornithine, lysine, proline, isoleucine and leucine.

Examples of physical mixtures of alpha-ketoglutaric acid or a salt thereof with at least one amino acid include, but are not limited to, mixtures of at least one selected from alpha-ketoglutaric acid and sodium, potassium, calcium and magnesium salts thereof with any of glutamine, glutamic acid, arginine, ornithine, leucine, isoleucine, lysine and proline, and any combination of said amino acids.

Generally, the molar ratio of alpha-ketoglutaric acid or salt thereof to amino acid in the physical mixture is from 1: 0.01 to 1: 2, preferably from 1: 0.1 to 1: 1.5, most preferably from 1: 0.2 to 1: 1.0.

Depending on the active ingredient to be used; the condition to be treated; the age, sex, body weight, etc. of the patient to be treated may vary, but the dose range is usually 1 to 1000mg/kg body weight/day, or 10 to 400mg/kg body weight/day, preferably 10 to 100mg/kg body weight/day.

The above-described embodiments can be freely combined with each other. Therefore, the details and the particular cases described above and in the claims apply equally to any other embodiment of the invention, mutatis mutandis. While the invention has been described in terms of certain disclosed embodiments, those of skill will envision other embodiments, modifications, or combinations not specifically described, which are also within the scope of the claims appended hereto.

All references cited herein are hereby incorporated by reference in their entirety.

The expressions "comprising", "including", "containing" and "containing" as used herein are to be understood as including but not limited to the items described.

The invention will now be further illustrated by examples which should not be construed as limiting the scope of the invention.

Examples

Example 1

The aim of this study was to address 1) the effect of bypass surgery connecting the esophagus to the duodenum on arterial tone in rats; 2) the long-term effects of the procedure on blood pressure; and 3) any beneficial effect of AKG intake in reversing any arterial tone changes resulting from bypass surgery.

Preparation of animals and aorta

Adult male Sprague Dawley rats housed in the animal facility of the department of comparative physiology at the university of longde are used. Animals were kept under the same conditions, 12/12 photoperiod, and the animals weighed 479 + -5 g. Rats were fed rodent pellets ad libitum (Altromin No.1314 spezialfurterwerke, Lage, germany) and had free access to water. Rats were grouped as follows: bypass surgery without AKG (B-AKG) (n ═ 6); bypass surgery, administration of AKG (B + AKG) (n ═ 11); sham surgery, no AKG (S-AKG) (n-12); in sham surgery, AKG (S + AKG) (n-12) was administered.

Rats were exposed to 95% CO₂Rats were sacrificed by cervical dislocation. Rats were sacrificed according to local and national regulations.

The dissected abdominal aorta portion (located anterior to the right and left common iliac arteries) was carefully cleaned to remove adherent tissue. The aorta is cut into approximately 6-9mm segments, 3-4mm in diameter at rest, and then each segment is firmly attached at one end to the force sensor and at the other end to a metal staple on a fixture, as in Harrison et al, Reprod Fertil Dev.1997; 9(7): 731-40, Harrison and Flatman Am J physiol.1999Dec; 277(6Pt 2): r1646-53. The aortic segment had a weight range of 8-25mg (average 14.32mg) and an average diameter of 3.75 + -0.08 mm.

The aortic segment was immersed in an oxygenated thermostatically controlled chamber (37 ℃) of 5.5 and 3.2cm internal depth and diameter, respectively, containing 44ml of phosphate buffer (0.15M PBS, pH 7.4) containing 136.91mM NaCl, 2.68mM KCl, 8.08mM Na₂HPO₄And 1.66mM NaH₂PO₄. The force was measured using an FTO3 force displacement transducer (Grass Instrument, West Warwick, RI) connected to a self-made bridge amplifier connected to an 8S PowerLab A/D converter (AD Instruments, Chalgrove, Oxfordshire, UK). The functional range (functional range) of the converter is 0-0.05kg, with a confidence force (reliable force) of 2mg, equal to 0.004% of the functional range. The 8S PowerLab A/D converter is connected to an iBook G4 running Chart5v.5.4 software (AD Instruments, Australia). Data recording was performed at a sampling rate of 40000 data samples per second (40KHz), with the input impedance of the amplifier being 200M omega differential.

Determination of force

The aortic segment was suspended vertically in triplicate. The recording signal of the unstretched aortic segment is adjusted to zero by means of a compensation scale fixed to the preamplifier unit. Approximately 5 stepwise increases in tension (each time approximately 0.09N) were then applied to each aortic segment until a final maximum tension of 0.49N (as measured using the FT03Grass force sensor) was reached. This final tension level is far from near the physiological maximum force recorded in the aortic segment. The aortic segment was then allowed to relax completely and then the application of the stepwise increasing tension was repeated 2 more times. The aortic segment was then removed and weighed. A recording speed of 1000 data samples per second was used.

After a stepwise increase in tension, it can be seen that the recorded image immediately shows a very small drop in the aorta tissue, since the aorta tissue shows a certain degree of elastic recoil. The drop in this recorded map over time is measured.

Tension calculation

The laplace equation assumes that the tension (T) on the wall of an empty cylinder is proportional to the cylinder radius (r) and the pressure (p) on the wall caused by the flow inside the cylinder, so that T ═ p × r. If it is assumed that the tension on the wall of the aortic segment is equal to the tension recorded by the force sensor as a result of manual stretching, the pressure causing such tension increase can be calculated using the laplace equation and the measured radius of each aortic segment.

Statistical analysis

Data are expressed as mean ± SE. Statistical significance of differences between mean values was tested using student's paired t-test and an additional gaussian normal distribution test. Data were found to be normally distributed and uniform in variance. Differences with P values > 0.05 were not considered significant.

Results

Tension and pressure measurements

Typically, at full tension, the tension applied to the aortic segment is about 0.034N/mg wet weight (0.49N maximum tension/14.32 mg average tissue weight). Thus, using laplace's equation and an average aortic radius of 1.87mm, the resulting pressure is on the order of 0.018 kPa. Such pressure increases are 0.18% of those typically measured in the human artery (10kPa), and about 0.14% of those measured in the rat (13-14kPa) (Carroll et al 2006; Duka et al 2006).

The average tension increase of the manual step was 0.09N (4.95X 10)^-3N/mg wet weight), typically, the aortic segment has a recoil force of 0.015N, which is about 16% of the manually applied tension.

Sham operated control rats

The elasticity of the control aorta was significantly higher than that of bypass-operated rats (P ═ 0.007; 1.9X 10 for the B-AKG group and S-AKG group, respectively^-7±0.2×10^-7N/ms/mg wet weight and 4.9X 10^-7±0.8×10^-7N/ms/mg wet weight). In the control group, AKG intake had no effect on this phenomenon (P ═ 0.44).

Bypass operation rat

The aorta elasticity of both the AKG-ingested and non-ingested bypass rats was shown to be lower than that of the control rats (P ═ 0.037; 3.1 × 10 for the B + AKG group and S-AKG group, respectively^-7±0.4×10^-7N/ms/mg wet weight and 4.9X 10^-7±0.8×10^-7N/ms/mg wet weight). AKG intake had a significant effect in bypass operated rats, increasing arterial elasticity to levels between control rats and bypass operated rats (B-AKG); p ═ 0.047; the B-AKG group and the B + AKG group were 1.9X 10, respectively^-7±0.2×10^-7N/ms/mg wet weight and 3.1X 10^-7±0.4×10^-7N/ms/mg wet weight (see FIG. 1).

Drawing series

In all arteries studied, the initial stretch sequence (e.g., applying tension, then relaxing) results in a decrease in elasticity with subsequent application of tension. This effect can be compared to the kind of injury for which a sudden increase in blood pressure is expected (see figure 2).

Discussion of the related Art

The results of this study clearly show that bypass surgery has an adverse effect on arterial elasticity. Furthermore, to our knowledge, it is the first time that such surgery can result in significant changes in arterial tone. Similar changes can also occur with stretch commensurate with a sudden rise in blood pressure.

Bypass surgery

In this experiment, none of the bypass-operated rats was obese, or there was any difference from the sham-operated control group. Both groups were operated on, so any stress associated with the operation was common to them. However, with regard to surgical bypass, which connects the esophagus with the duodenum, there is a difference from group to group. This type of bypass surgery is comparable to the Roux-en-Y gastric bypass surgery, in which most of the stomach and duodenum is bypassed in a manner that allows secretion of a portion of the stomach (proximal portion), pancreas, gall bladder, and duodenum. This is currently the most common method of selecting bariatric surgery to treat morbid obesity (Adrian et al 2003).

Gastric bypass surgery is expected to limit food intake and thereby prevent obesity. This effect is very pronounced (coats et al 2004, Cowan and1998, Fernstrom et al 2006), but physiological consequences must also be taken into account.

In addition to acting as a food reservoir and mechanically breaking down food, the stomach is the site of digestion and secretion. When food enters the stomach, there is a stage where digestive enzymes from saliva are still active. The absence of this stage in digestion can affect, among other things, the breakdown of starch. Although this results in less energy being produced, it should not be necessary or present a major problem in digestion. In general, this is also true for the mechanical breakdown of food particles.

A more serious aspect is the loss of gastric secretion. The major components in the stomach are pepsin and lipase, intrinsic factor and hydrochloric acid. Pepsin is essential for protein breakdown, the enzyme requiring hydrochloric acid to activate secreted pepsinogen to pepsin. Thus, the lack of the stomach can severely affect amino acid absorption, resulting in amino acid deficiency. Another aspect is the release of minerals and vitamins bound to enzymes. If the micronutrients are not released, they cannot be further absorbed along the digestive tract. Lipase from the stomach breaks down triglycerides, but even if this does not happen, triglycerides will still encounter lipase from the pancreas and the fatty acid demand should be met.

However, in the absence of the stomach, regulation of pancreatic secretion or bile may be reduced, as the acidity of the solution entering the duodenum regulates such secretion. The stomach also regulates the amount of food that enters the duodenum at any given time. Transport is dependent on the carbohydrate, protein or fat content, with the minimum amount of fat and the maximum amount of carbohydrate passing at any given time. This mechanism ensures efficient digestion and regulates the rate of movement through the intestine. With bypass surgery, this mechanism is eliminated and overall digestion is impaired. As the acidity and flow rate change, certain components may be poorly or less absorbed.

Vitamin B12 is released from proteins and internal factors are secreted in the stomach. Intrinsic factors are essential for the absorption of B12 in the ileum. Vitamin B12 is normally abundant, but suddenly after surgery this may not be the case. It has also been reported that in humans, 70% of bypass surgery patients develop vitamin B12 deficiency at the time of postoperative follow-up (Lynch et al 2006, Shah et al 2006). These authors also reported anemia, which may be partly due to iron deficiency (less released from proteins especially when the environment is not sufficiently acidic) and partly due to vitamin deficiency. Calcium and folate deficiency following bypass surgery has also been reported (Lynch 2006, Parkes 2006, Shah 2006). It is unclear whether this is due to malabsorption caused by the absence of the duodenum or due to the lower availability of both substances. In the rat study of the present invention, the duodenum should be functional, but changes in fluidity and pH can affect the breakdown of nutrients in the remaining intestine and exhibit different absorption patterns. The flow of hypertonic substances into the intestine can significantly affect the fluidity. This is supported by the following assumptions: the influx of hypertonic food into the small intestine may be responsible for "dumping syndrome" (which may lead to vomiting) in some gastric bypass patients (Lynch et al 2006).

In our study, the stress effect of surgery was considered using sham operated rats as a control group. Furthermore, prior to surgery, any rat was not obese or had high blood pressure, and we can assume that the arteries had normal elasticity prior to bypass surgery.

In humans, a decrease in blood pressure occurs after bypass surgery (Coates, Buffington 1998, Fernstrom et al 2006, Foley et al 1992). This is likely to be due primarily to weight loss, but may also be due in part to alterations in metabolism and hormonal balance. Thus, the changes in arterial tone that occur in our bypass-operated rats are unlikely to be due to hypertension, since blood pressure is expected to decrease slightly or remain normal.

After surgery, our measurements clearly show a decrease in arterial elasticity.

The problem now is what happens after surgery? What can affect the arterial wall? Gokce et al (2005) reported a long-term improvement in endothelium-dependent flow-mediated relaxation through weight loss, which is significantly better than the weight loss of drug treatment in gastric bypass surgery patients. In our rats that were not overweight prior to surgery, we only examined changes in body absorption of nutrients and the effects of metabolism and hormonal balance.

Cations that can affect blood pressure (e.g., calcium and potassium) bind to a large extent to charged proteins and are likely to be present at lower than normal concentrations.

Possibly explained in other respects. If the protein is not or only partially decomposed, there will be insufficient amino acids transported to the intestine. This necessarily affects the turnover of proteins. In the arterial wall, elasticity is due in part to the connective tissue in the wall. The tissue is constantly remodeling and if damaged, it will be repaired. If the amount of amino acids used for remodeling or repair is insufficient, the wall will lose its elasticity over time. Another factor contributing to this result may be the different hormonal balances that arise due to the different signals emitted by the digestive tract. This may be due to the same reasons that lead to the following: in persons with gastric bypass, increased bone turnover and increased bone resorption is seen, followed by decreased bone mass. Part of the reason may be that the proteins required for a healthy transition cannot be digested by gastric bypass rats. Higher bone turnover also demonstrates that the hormone-regulated cycle is reprogrammed. If the elasticity is reduced, it will be the structure or percentage of the elastic fibers that causes this overall change.

Stretching action

The fluid pressure P in the vessel wall is equal to the wall tension T divided by the radius of curvature r plus the external pressure pn, i.e. P ═ pn + T (1/r), as defined by laplace's law. If one chooses to ignore external pressure and any support from surrounding tissue, only a reasonably sized cylindrical vein or artery is treated, then the equation can be simplified to P ═ T/r. Furthermore, it is well known that the tension developed depends on the thickness of the vessel wall, that is, on the amount of membrane and muscle tissue that constitute the vessel wall. Thus, if a constant pressure is maintained, it is expected that the thickness of the vessel wall should vary with the vessel radius according to a simplified equation. However, in reality, the pressure within the circulation system is not constant, and in fact it drops due to frictional losses. However, nevertheless, the larger and smaller vessels follow the rules set by the simplified equation over a period of time, where the wall thickness is proportional to the vessel size.

AKG action

It is known that certain amino acids are metabolized in the intestinal wall-suggesting that AKG is preferentially used for AA, and therefore supplementation of AKG in bypass rats increases AA absorption compared to bypass rats not administered AKG.

Conclusion

The results of this study indicate that this type of bypass surgery significantly affects arterial elasticity.

AKG intake had a positive effect on arterial tone in bypass-operated rats, but not in controls.

In both control and bypass operated rats, sudden high passive tension has a lasting effect on arterial elasticity, making blood vessels less prone to sudden changes in blood pressure.

Example 2

Method of producing a composite material

The purpose of this study was to elucidate whether the effects observed in the study of example 1 were limited to subjects who had undergone gastric surgery. Experimental subjects requiring increased arterial tone for more general reasons (i.e. age) are now being investigated.

Local ethical approval

The study was approved by the ethical review committee of animal experiments at university of longde (ethical permit M14-05) and was conducted in accordance with the rules of the european community relating to the protection of experimental animals.

Preparation of animals and aorta

Female NMRI mice (50 weeks old at the beginning of the experiment) were housed in the animal facilities of the department of cell and organism biology at the university of london, sweden. Animals were kept under the same conditions for 12/12 hours photoperiod. Mice were fed rodent pellets ad libitum (Altromin No.1314 spezialfurterwerke, Lage, germany) and water was freely available. Mice were randomized into one of three groups and housed for 182 days until they reached 76 weeks of age, at which time they weighed 28 ± 7 g. Mice in the first group eat rodent pellets and (2% w/v) Na₂-AKG 2H₂O (n ═ 6), while mice in the second group eat rodent pellets and (2% w/v) Ca-AKG H₂O (n ═ 6). Mice assigned to the third group eat only rodent pellets, considered a control group (n-6). AKG levels consumed as a food supplement were 2% of voluntary food intake in mice, with daily food intake of about 10-15% of body weight.

Mice were exposed to 95% CO₂The mice were sacrificed by cervical dislocation. The dissected abdominal aorta portion (located anterior to the right and left common iliac arteries) was carefully cleaned to remove adherent tissue. The aorta is cut into segments of approximately 4.5mm, with a diameter of 1mm at rest, and then each segment is firmly attached at one end to the force sensor and at the other end to a metal staple on a fixture, as in Harrison et al, reprod Fertil dev.1997; 9(7): 731-40, Harrison and Flatman Am J physiol.1999Dec; 277(6Pt 2): r1646-53. The aortic segment was weighed to an average of 2.75mg using a weight-recording scale (accurate to 0.01 mg).

The aortic segment was immersed in an oxygen-filled thermostatically controlled chamber (37 ℃) having an internal depth and diameter of 5.5 and 3.2cm, respectively, containing 44ml of phosphate buffer (0.15M PBS, pH 7.4) containing 136.91mM NaCl, 2.68mM KCl, 8.08mM Na₂HPO₄And 1.66mM NaH₂PO₄. The force was measured using an FTO3 force displacement sensor (GrassInstrument, West Warwick, RI) connected to a self-made bridge amplifier interfaced to an 8S PowerLab a/D converter (adestruments, Chalgrove, Oxfordshire, U K). The functional range of the converter is 0-0.05kg with a confidence force of 2mg, equal to 0.004% of the functional range. The 8S PowerLab A/D converter was connected to the iBook G4 running Chart5v.5.4 software (ADInstructions, Australia). Data recording (40KHz) was performed at a sampling rate of 40.000 data samples per second, with the input impedance of the amplifier being 200M omega differential.

Determination of force

The aortic segment was suspended vertically in triplicate. The recording signal of the unstretched aortic segment is adjusted to zero by means of a compensation scale fixed to the preamplifier unit. Approximately 5 stepwise increases in tension (each time approximately 0.09N) were then applied to each aortic segment until a final maximum tension of 0.49N (as measured using the FT03Grass force sensor) was reached. This final tension level is far from near the physiological maximum force recorded in the aortic segment. The aortic segment was then allowed to relax completely and then the application of the stepwise increasing tension was repeated 2 more times tightly. The aortic segment was then removed and weighed.

After a gradual increase in tension, it can be seen that the recorded images show a very small drop immediately, since the aortic tissue shows a certain degree of elastic recoil. The drop in this log was measured over time using an average slope calculation as part of Chart v.5.4 software (AD Instruments, australia). The mean slope (μ g/ms) is the time derivative of the data points in the map selection, which can be calculated from the best-fit least-squares line.

Tension calculation

The laplace equation assumes that the tension (T) on the wall of an empty cylinder is proportional to the cylinder radius (r) and the pressure (p) on the wall caused by the flow inside the cylinder, so that T ═ p × r. If it is assumed that the tension on the wall of the aortic segment is equal to the tension recorded by the force sensor as a result of manual stretching, the pressure causing such tension increase can be calculated using the laplace equation and the measured radius of each aortic segment.

Thus, the measurement of the average slope (. mu.g/ms) obtained from each aortic sample was converted to newtons (N/ms) and then adjusted according to the sample weight to give the final elastic recoil force value in N/ms/mg wet weight.

Statistical analysis

Data are expressed as mean ± SE. Statistical significance of differences between means was tested using one-way ANOVA and an additional gaussian normal distribution test. Data were found to be normally distributed and uniform in variance. Differences with P values > 0.05 were not considered significant.

Results

Tension determination

Typically, when fully stretched, the tension applied to the aortic segment is 0.178N/mg wet weight (0.49N max tension/2.75 mg average tissue weight). Thus, using Laplace's equation and an average aortic radius of 1.0mm, the resulting pressure is on the order of 0.178 kPa.

The average tension increase of the manual step was 0.09N (4.95X 10)^-3N/mg wet weight), typically, the aortic segment has a recoil force of 0.015N, which is about 16% of the manually applied tension.

Control mice

For the first and second series of stretches, the control aorta elasticity was 3.3X 10 respectively^-5±7.8×10^-7N/ms/mg wet weight and 3.4X 10^-6±9.4×10^-7N/ms/mg wet weight. The repeated stretching protocol resulted in about a 90% reduction in elastic retractive force for the second series compared to the first series (figure 5).

Na-AKG mouse (A)

For the first and second series of stretches, the elasticity of the aortic segment of Na-AKG-ingested mice was 4.3X 10, respectively^-5±1.6×10^-6N/ms/mg wet weight and 3.7X 10^-6±1.1×10^-6

N/ms/mg wet weight. Na-AKG intake had a significant effect on arterial elasticity compared to control mice (see figure 4). The repeated stretching protocol resulted in a 91% reduction in elastic retractive force for the second series compared to the first series (figure 5).

Ca-AKG mouse (B)

For the first and second series of stretches, the elasticity of the aortic segment of the Ca-AKG ingested mice was 6.4X 10, respectively^-5±2.7×10^-6N/ms/mg wet weight and 3.8X 10^-6±1.2×10^-6N/ms/mg wet weight. Ca-AKG intake had a significant effect on arterial elasticity compared to control mice (see figure 4). The repeated stretching protocol resulted in a 94% reduction in elastic retractive force for the second series compared to the first series (figure 5).

Stretch series and arterial stiffness (robustness)

In all arteries studied, the initial stretch sequence (e.g., applying tension, followed by relaxation) results in a decrease in elasticity when tension is subsequently applied. This effect can be compared to the type of injury that is expected to cause a sudden rise in blood pressure (see fig. 5).

Table 1: stiffness of the arterial segment to stretching. Serial number of stretches (in triplicate) of the cut aortic segment subjected to progressively reached maximum tension of 0.49N without rupture.

	First repetition	Second repetition	Average (%)
				Control	Four in six	Four in six	66.7
Na-AKG(A)	Four in six	Five in six	75.0
				Ca-AKG(B)	Four in six	Five in six	75.0

[0165] Discussion of the related Art

The results of this study clearly show that alpha-ketoglutarate treatment has a beneficial effect on the arterial tone of older mice. Furthermore, to our knowledge, this is the first reported treatment that can target the stiffness of the aorta.

Animal(s) production

In this study, adult animals were selected whose week age was comparable to that of the aged human subjects. In this study, when the aorta was dissected from the mice, it was evident that arterial deposition occurred such that the aorta appeared almost white to translucent and even after dissection they retained the tubular morphology.

Blood pressure and tension

In rats at 6 months of age and above, blood pressure obtained by intubation of the abdominal aorta or the iliac or carotid arteries was recorded with an average value of 119mmHg (upper limit of 150mmHg, lower limit of 92mmHg) (Durant, 1927). The authors also described a correlation between age and blood pressure up to 6 months of age, and the recorded blood pressure did not rise any further after 6 months, although body weight increased further. This blood pressure value in small rodents is very close to the resting blood pressure of a human subject, with a systolic blood pressure of typically 120mmHg (16 kPa). Furthermore, the pressure rise value of the arterial segment in this study was 1.8% as commonly exhibited in the human artery, and about 1.4% as measured in rats (13-14kPa) (Carroll et al 2006; Duka et al 2006).

The aortic layer (aortic media) contains stratified smooth muscle cells, which are connected tangentially to the elastic layer; the change in smooth muscle tone provides a dynamic or functional modulation of stiffness by changing the distribution of forces between elastic and collagen fibers (McEniery et al 2007). Indeed, at lower levels of arterial pressure, the stresses generated within the aortic wall are predominantly borne by the elastic fibres, whereas at higher levels of arterial pressure, the stresses are generally borne by the more rigid collagen fibres. Thus, one effect of aging is to engage collagen fibers at lower levels of arterial pressure, resulting in a concomitant increase in pulse pressure.

The tension generated in the artery depends on the thickness of the vessel wall, that is, on the amount of connective and muscular tissue contained in the wall. Thus, if a constant pressure is maintained, the laplace equation predicts that the thickness of the vessel wall should vary with the vessel radius. However, in reality, the pressure within the circulation system is not constant, and in fact it is reduced by frictional losses. Nevertheless, over time, the larger and smaller vessels follow Laplace's law according to the simplified equation.

Laplace's equation indicates that the fluid pressure P in the vessel wall is equal to the wall tension T divided by the radius of curvature r plus the external pressure P_nI.e. P ═ P_n+ T (1/r). If one chooses to treat only reasonably sized cylindrical arteries, ignoring external pressure and any support from surrounding tissue, the equation can be simplified to P ═ T/r. In this study, the aortic segments were dissected out of the abdominal aorta (anterior to the right and left common iliac arteries) and of a diameter such that they meet the Laplace's Law requirements. Aged artery

Aging affects organs, tissues and cell types in an organism in different ways, which can be thought of in various ways as the differential rate of functional decline (Calabresi et al 2007). Within the vessel wall of the aorta, age-related structural changes occur, including hardening and thickening of the media and increased lumen diameter (Marin & Rodriguez-Martinez, 1999; Dao et al 2005), and these changes are often different along the arterial tree (Hajdu et al 1990; Moreau et al 1998; Laurant et al 2004). Changes in smooth muscle cell number, increased collagen deposition, and altered elastin structure are all characteristic manifestations in the aorta of aged rats (Jacob, 2003; Dao et al 2005). Indeed, many studies have reported a decrease in the number of smooth muscle cells with age (Cliff, 1970; Orlandi et al 1993), an increase in type I and type III collagen in arteries and a relative decrease in elastin density (Jacob, 2003; Dao et al 2005; Marin, 1995).

Notably, the stretching methods used in this study showed that the second series of repeated stretches had much weaker elastic retractions than the first series of repeated stretches. This point indicates and emphasizes that the old aortic segment has or lacks the ability to cope with a relatively mild stretching period corresponding to an increase in blood pressure of 0.178 kPa. Thus, the second stretch can be considered an indicator of toughness, which in this case does not appear to be present in the aortic segment of aged mice. In humans, by the age of 60, each individual undergoes an average of two billion aortic stress cycles (average heart rate × age) (McEniery et al 2007), where the damage caused by these stress cycles requires immediate regulation and repair, involving elastin, collagen and smooth muscle components of the vessel wall. In this study, it was not possible to modulate smooth muscle tone, nor had any opportunity to repair elastin and collagen fibers, and therefore the fact that after the first series of stretches the control aorta lost almost 90% of the elastic recoil (N/ms/mg wet weight), for AKG treated mice a similar level of elastic recoil was found in the second series, indicating how fragile the macrovascular recoil is in older mice.

Arterial elastic recoil and AKG

Although traditional antihypertensive agents have been reported to reduce arteriosclerosis (primarily by virtue of the indirect effect of reducing mean blood pressure), the relative insensitivity of peripheral arteries to hardening with age is generally attributed to the much lower ratio of elastin to smooth muscle and collagen, although it may also reflect other biological processes, such as the ability of the arteries to remodel themselves (McEniery et al 2007).

Alpha-ketoglutaric acid is the rate-limiting intermediate in the Krebs cycle, which plays a key role in cellular energy metabolism. It also functions as a source of glutamate and glutamine, and stimulates protein synthesis and inhibits protein degradation (Hammarqvist et al, 1991). In terms of collagen metabolism, AKG not only acts as a cofactor for prolyl-4-hydrolase, which catalyzes the formation of 4-hydroxyproline, which is essential for the formation of the collagen triple helix, but also promotes collagen synthesis by increasing the proline pool from glutamate (Son et al 2007).

The Ca-AKG group worked better compared to the Na-AKG group, which can be explained by the longer availability of AKG provided in the Ca-AKG salt. The Ca salt acts to slowly release AKG ions, controlling the presence of AKG ions in the intestinal lumen, since it has a solubility of 2g/100ml, while Na-AKG has a solubility 50 times that of Ca-AKG. Thus, AKG anions in the form of Na-AKG can be utilized more quickly. In this case, most of AKG is simply converted to energy when blood levels exceed about 10 μ g/ml. Blood levels of AKG can easily exceed 10 μ g/ml after enteral administration of Na-AKG. This is not or hardly observed after enteral administration of Ca-AKG. When AKG is provided in the form of Ca-AKG, it is released slowly and over a longer period of time, and therefore has more time to convert to proline and other amino acids, rather than to energy.

Recently, AKG has been considered as a natural ligand for a G-protein coupled receptor (CPR99) which is currently known to be expressed in kidney, testis, and smooth muscle (He et al, 2004). AKG, as a ligand for G-protein coupled receptors, can link TCA cycle intermediates to both metabolic state and protein/collagen synthesis, which may in fact be demonstrated to be an intrinsic cause of the beneficial effects on aortic wall elasticity observed in the present invention.

Conclusion

The results of this study indicate that AKG is effective not only in increasing arterial tone in subjects who have undergone gastric surgery (example 1), but also in increasing arterial tone in other subjects with reduced arterial tone. In this case, the subject is an aging rodent, which is considered to be a relevant model for a human subject with reduced arterial tone, typically an aging subject.

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Claims (11)

1. Use of calcium alpha-ketoglutarate for the preparation of a pharmaceutical preparation or a food or feed supplement for increasing vascular elasticity in a subject.

2. The use of claim 1, wherein the blood vessel is an artery.

3. The use of claim 1 or 2, wherein the subject is in need of treatment and/or prevention of hypertension, pulmonary hypertension, retinal vascular disease, ventricular hypertrophy, or aneurysm.

4. The use of claim 1 or 2, wherein the subject has undergone gastric surgery.

5. The use of claim 4, wherein the gastric surgery is gastric bypass surgery, gastrectomy, or gastric ligation.

6. The use of claim 4, wherein the gastric surgery is a partial gastrectomy.

7. The use of claim 1 or 2, wherein the subject has a condition associated with malnutrition.

8. The use of claim 1 or 2, wherein the subject is an elderly human.

9. Use according to claim 1 or 2, wherein the dose administered to the patient is in the range of 1-1000mg/kg body weight/day of calcium α -ketoglutarate.

10. Use according to claim 1 or 2, wherein the dose administered to the patient is in the range of 10-400mg/kg body weight/day of calcium α -ketoglutarate.

11. Use according to claim 1 or 2, wherein the dose administered to the patient is in the range of 10-100mg/kg body weight/day of calcium α -ketoglutarate.