DE102007003524A1 - Medicines for the treatment and / or prevention of arteriosclerosis - Google Patents

Abstract

The present invention relates to a medicament for the treatment and / or prevention of arteriosclerosis, to a process for the preparation of such a medicament and to a process for the therapeutic and / or prophylactic treatment of a subject afflicted with atherosclerosis and / or at risk of arteriosclerosis ,

Description

The The present invention relates to a medicament for the treatment and / or Prevention of arteriosclerosis, a method of preparation of such a medicament as well as a method of therapeutic and / or prophylactic treatment of a living thing, that of atherosclerosis affected and / or at risk of arteriosclerosis.

drug for the treatment and / or prevention of arteriosclerosis, Process for the preparation of such medicaments and process for therapeutic and / or prophylactic treatment of atherosclerosis The subject is well known in the art.

The Arteriosclerosis is a highly complex, active pathological process in its center an inflammatory reaction in the walls the blood-carrying vessels of an affected Individual stands. The emergence of atherosclerosis can occur in several Phases are divided.

The early phase of arteriosclerosis is characterized by a so-called endothelial dysfunction. A series of different ones Risk factors such. Smoking, obesity, physical Inactivity, hyperlipoproteinemia and type II diabetes and other factors not yet identified to damage the endothelium. The permeability endothelium for lipoproteins and other circulating Plasma substances increase as a result. As a result, endothelial cells become Activates and expresses more and more so-called adhesion molecules on the cell surface. Above all, so-called Selective first the temporary contact of certain Blood cells such as monocytes and T lymphocytes with the endothelium. By another group of adhesion molecules, the so-called Cellular Adhesion Molecules (CAMs), it comes to solid Attachment of these cells to the vessel wall. Especially at vascular branches - the places, at which more often atherosclerotic lesions emerge - mechanical forces also play a role. Increased shear forces can increase the formation of endothelial nitrogen (NO). NO acts as a vasodilator and has anti-inflammatory properties. About that In addition, increased shear forces increase Increased formation of adhesion molecules with the consequences described above.

in the Monocytes and, to a lesser extent, T-lymphocytes are more likely to progress into the subintimal space. This intrusion is by a another group of molecules, to which z. The chemokine Monocyte chemoattractant protein-1 (MCP-1) is mediated. It comes to the differentiation of monocytes in macrophages in the subintimal Room.

in the damaged endothelium also causes the oxidation of Low-density lipoprotein (LDL), thereby forming oxLDL. The oxLDL is released into the subintimal space where it is the macrophages that have emerged there from monocytes are loaded. These macrophages become so-called by the loading with oxLDL Foam cells transformed, the characteristic cells of arteriosclerotic plaques, as additional components u. a. T lymphocytes and from the media Immigrated smooth muscle cells included.

One Such arteriosclerotic plaque is deposited on the arterial walls and is doing by a stabilizing fibrous cap consisting of smooth muscle cells and extracellular matrix coated. The arteriosclerotic plaque is now in the center of an inflammatory Reaction in which it is used to produce a variety of inflammatory mediators such as cytokines, chemokines, proteases, etc. It can Tissue necrosis come in the vicinity of which lime salts are deposited become. This allows the vessel lumen to complete Closure constricted with the result of circulatory disorders become.

in the further course of arteriosclerosis it comes to the distribution of matrix metalloproteinases (MMPs) by macrophages and foam cells. The proteolytic activity of MMPs occurs an increased degradation of extracellular matrix in the arteriosclerotic environment. The increased degradation or the reduced new formation of the extracellular matrix lead to thinning of the fibrous cap arteriosclerotic plaque with consecutive plaque instability and threatening rupture danger.

Of the Plaque can be caused by the thinning of the fibrous Tearing the cap, causing the thrombogenic lipid nucleus and Collagen are exposed in the vessel wall. The This causes conditional activation of the hemostasis system occlusive and non-occlusive thrombus formation, d. H. to activate the coagulation cascade, in the center of which so-called tissue factor (TF) stands. Clinically manifest the plaque rupture with thrombus formation as unstable angina pectoris or acute myocardial infarction.

It is known that in the case of arteriosclerosis, the calcium phosphate budget also has an important significance. Thus, it has been described that calcium-phosphate balance disorders contribute to vascular calcification, which is observed in atherosclerosis, cf. Ketteler et al .: Novel insights into vascular calcification. Kidney. Int. Suppl. 2006 (105): 5-9 ; Ketteler et al .: Inhibitors of calcification: general aspects and implications in renal failure, Pediatr. Nephrol. 2005 20 (3): 383-8 ,

Currently Arteriosclerosis is usually about the administration treated by lipid-lowering or statins. This is it to a group of agents that ultimately endogenous cholesterol synthesis inhibit. The statins include u. a. Atorvastatin, cerivastatin, Fluvastatin, lovastatin (mevinolin), mevastatin (compactin), pravastatin and simvastatin. These substances influence in different ways the lipid metabolism, z. By competitive inhibition of the key enzyme cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, by reducing cholesterol biosynthesis in the liver, by Propagation of LDL receptors on the liver cell and by modification the lipoprotein composition. Statins have a big one Influence on the composition of serum lipids and lead u. a. to a slight increase in the concentration of so-called "High density lipoproteins" (HDL) and a strong reduction the LDL concentration. Overall circulate through the effect of Statins less fats in the blood, so the arteriosclerotic Plaques less fat and store the risk of thrombosis and the consequent dangers thereby sink.

Although a number of other positive attributes are attributed to the statins, they have come under criticism for their conspicuous accumulation of rare but serious side effects on muscles and kidneys. Therefore, in particular the active ingredient cerivastatin (eg ^Lipobay® , Zenas®) was ^withdrawn from the market in Germany in August 2001 and alternatives are being sought.

Criscuoli et al .: Glunicate (Lg 13979) protects the arterial wall from cholesterol-induced arterosclerotic changes in the rabbit without affecting plasma lipids. Arteriosclerosis 1984, 53: 59-68 describe the use of nicotinic acid to protect the arterial wall against cholesterol-induced changes in atherosclerosis. However, the protective effect of nicotinic acid was not significant in any of the cases shown.

Feinblatt et al .: Treatment of arteriosclerosis and vague abdominal distress with niacinamide hydroiodide (without side-effects). Amer. jour. Dig. Dis. 1955; January: 5-6 , suggest the treatment of arteriosclerosis by a combination of iodides and nicotinamide hydroiodides. However, such treatment has not proven in practice.

In front It is an object of the present invention to provide a Provide substance with which to treat arteriosclerosis or this can be prevented, and with the above-mentioned disadvantages the known lipid-lowering or other substances reduced or can be largely avoided.

These Task is accomplished by the provision of nicotinic acid amide solved.

The Inventors have recognized that nicotinic acid amide is the cellular Absorption of phosphate and the accumulation of calcium phosphate in inhibits the cell. This reduces the risk that the solubility product calcium phosphate is exceeded, less calcium phosphate fails and the risk of arteriosclerosis and the occurrence Calcified arteriosclerotic plaques is significantly reduced.

These Knowledge of the inventors was surprising since the nicotinic acid amide, also referred to as nicotinamide, pyridine-3-carbamide or niacinamide is attributed so far completely different properties become.

To describe eg. Eto et al .: Nicotinamide prevents the development of hyperphosphataemia by suppressing intestinal sodium-dependent phosphate transporters in rats with adenine-induced renal failure. Nephrol. Dial. Transplant. 2005; 20: 1378-1384 in that nicotinamide in rats inhibits the sodium-dependent phosphate transporter NaPiIIb and thus the uptake of phosphate into the organism.

Tenenhouse HS and Chu YL: Hydrolysis of nicotinamide-adenine dinucleotide by purified renal brush-border membranes. Mechanism of NAD + inhibition of brushborder membrane phosphate-transport activity. Biochem. J. 1982; 204: 635-638 , further claim that nicotinic acid amide inhibits the renal absorption of Phosphate inhibitor and thus increase the renal excretion of phosphate.

The from Eto et al. (supra) and Tenenhouse and Chu (supra) Effects of nicotinic acid amide should therefore be the plasma concentration of phosphate, which is also claimed by Eto et al. However, the inventors have exactly the opposite effect of nicotinic acid amide can watch. This was surprisingly by the inventors found that nicotinamide the concentration of Phosphate in plasma increased.

at The findings of the inventors are therefore a departure from the view expressed in the prior art the properties of nicotinic acid amide.

For Nicotinic acid amide has also been described that a Lack thereof lead to the disease of the so-called. Pellagra This can be referred to as dermatitis with hyperpigmentation in the area sun-exposed skin. The administration of nicotinic acid amide is therefore proposed for the treatment of pellagra.

Various authors suggest the administration of nicotinic acid amide to extend the life span or to delay the onset of aging; see. Crane and Low: Plasma membrane redox and control of sirtuin. Age 2005; 27: 147-152 . Bitterman et al .: Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J. Biol. Chem. 2002; 277: 45099-45107 (in yeast), and Kang et al .: Nicotinamide extends replicative lifespan of human cells. Aging Cell 2006; 5: 423-436 (in human cells).

Although Feinblatt et al. (supra) and Criscuoli et al. (a.O.) a Relationship between arteriosclerosis and niacinamide hydroiodide or Arteriosclerosis and nicotinic acid was due to the one by Eto et al. (a.O.) and Tenenhouse and Chu (supra) can not be expected that also Nicotinsäureamid antiarteriosclerotic effect shows. In addition, nicotinic acid and nicotinamide hydroiodide low molecular weight compounds where minor modifications are already making a complete change their biological effects. So For example, the inventors also have isonicotinic acid amide tested anti-arteriosclerotic effects and found, that such versus nicotinamide only slightly modified compound as a substance against arteriosclerosis is unsuitable.

The The object underlying the invention is achieved by the provision completely dissolved by nicotinic acid amide.

The The object is further achieved by a method for the production a drug for treatment and / or prevention of atherosclerosis comprising the following steps: (1) providing nicotinic acid amide, and (2) formulation of the nicotinic acid amide in a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are fully described in the art. For example, reference is made to Bauer et al.: Textbook of Pharmaceutical Technology, 6th ed., Wissenschaftliche Verlagsgesellschaft mbH Stuttgart 1999 ; Rowe et al .: Handbook of pharmaceutical excipients 5th ed., Pharmaceutical Press and American Pharmacist Association 2006 , The content of the aforementioned publications is incorporated by reference into the application.

at the use according to the invention or the inventive It is preferred if the drug is for a Application is formed, which is selected from the Group consisting of: oral, rectal, parenteral, local, transdermal.

These Measure has the advantage that the drug depending on desired application form already in a suitable manner is designed. The attending physician thereby has a choice various forms of the drug are available, whereby the concrete form depends on the condition of the patient, the respective one Treatment program and other factors.

In front In this background, it is also preferable if the drug is formed in a form which is selected from the Group consisting of: powder, tablet, juice, drops, capsule, suppositories, Solution, solution for injection, aerosol, ointment, conditioner, Plaster, pellet, dragee.

With this measure, a formulation variant is already provided in an advantageous manner, the immediate use of nicotinamide for the treatment or prevention of arteriosclerosis se allows.

at the use according to the invention or the inventive It is also preferred if the drug is used Nicotinamide in a concentration selected is selected from the group consisting of (each given in weight of Active substance nicotinic acid amide based on the total weight of the drug): 1 μg / g, 10 μg / g, 100 μg / g, 1 mg / g, 10 mg / g, 100 mg / g, 1 g / g.

These Measure has the advantage that the drug is the active ingredient Nicotinic acid amide already in a concentration, which is used to treat or prevent atherosclerosis particularly suitable.

Further it is preferred if the absolute amount of the active ingredient nicotinic acid amide in a dosage unit of the drug between approx. 1 and approx. 3000 mg, preferably between about 10 and about 1500 mg, more preferably between about 100 and about 750 mg, and most preferably at about 500 mg.

With Nicotinamide is already being used for this measure provided in such an amount that in the patient without further the desired nicotinamide levels can be achieved. Under a dosage unit is According to the invention, an above-mentioned formulation variant denotes, for example, a tablet, a dragee, a capsule, etc.

Further it is in the use according to the invention or preferred method of the invention, if the medicine additionally another against atherosclerosis having effective substance.

These Measure has the advantage that by adding the antiarteriosclerotic Effects of the medicament according to the invention can still be improved. So it is possible by the combination with another substance an intervention in the atherosclerosis in another place to cause the clinical picture by synergistic effects even more effective to combat.

at the further substance is preferably according to the invention a lipid-lowering agent (statin), which is more preferably selected is from the group consisting of atorvastatin, cerivastatin, fluvastatin, Lovastatin (mevinolin), mevastatin (compactin), pravastatin and Simvastatin.

These Measure has the advantage that the drug is such contains more substance that has been proven to be antiarteriosclerotic Effect shows and routinely used. According to the invention by the interaction of nicotinic acid amide and the further substance possibly synergistic Effects are achieved.

In front This background concerns a further subject matter of the present invention Invention a method for therapeutic and / or prophylactic Treatment of a living being affected by arteriosclerosis or / and at risk of arteriosclerosis, the following Comprising: (1) providing nicotinic acid amide, (2) introduction of nicotinic acid amide into the animal, and (3) if necessary, repeating steps (1) and (2) several times.

The Nicotinic acid amide can be used in this process in the form of provided drug according to the invention and be brought into the living being. The ones described above Features, characteristics and advantages apply in this respect this method accordingly.

It it is understood that the above and the following yet to be explained features not only in each case specified combination, but also in other combinations or can be used in isolation, without the scope of the present To leave invention.

The The invention will now be described in more detail with reference to exemplary embodiments explains what makes up more features, features and benefits. Reference is made to the attached Figures showing the following:

1 shows the fluid intake of the test animals before and after administration of nicotinic acid amide,

2 shows the food intake of the experimental animals before and after administration of nicotinic acid amide,

3 shows the faeces dry weight of the test animals before and after administration of nicotinic acid amide,

4 shows the body weight of the test animals before and after administration of nicotinic acid amide,

5 shows the urinary flow rate of the experimental animals before and after administration of nicotinic acid amide,

6 shows the phosphate concentrations in the urine of the test animals before and after the administration of nicotinic acid amide,

7 shows the phosphate excretion via the urine of the test animals before and after the administration of nicotinic acid amide,

8th shows the phosphate excretion via the faeces of the test animals before and after the administration of nicotinic acid amide,

9 shows the abundance of the transport protein NaPiIIb in the first and second half of the small intestine of the experimental animals before and after administration of nicotinic acid amide,

10 shows the phosphate concentration in the plasma of the experimental animals before and after administration of nicotinic acid amide,

11 shows a calibration curve for the measurement of free phosphate on cultures of intestinal epithelial cells,

12 shows the phosphate uptake by intestinal epithelial cells in the control (culture medium with phosphate and without test substance) compared to cells in culture medium (culture medium without phosphate and without test substance),

13 shows the influence of three different concentrations of nicotinic acid amide on the uptake of phosphate by intestinal epithelial cells,

14 shows the influence of three different concentrations of isonicotinic acid amide on phosphate uptake by intestinal epithelial cells, and

15 shows the influence of three different concentrations of thiamine hydrochloride on phosphate uptake by intestinal epithelial cells.

embodiments

 1. Material and methods

1.1 Animal experiments

1.1.1 Experiments on animals

All Animal experiments were conducted in accordance with the guidelines of the American Physiological Society and in accordance carried out with the German Animal Welfare Act and by approved by the local authorities.

1.1.2 Metabolic Cages

The experiments were performed on 6-10 month old mice maintained on a control diet (C1314, 0.2% Na ⁺ , 1% K ⁺ , 0.9% Ca, Altromin, Heidenau, Germany). Before and during the experiments, the mice had free access to tap water with and without nicotinamide (0.1-2 mg / g) as indicated. To assess renal and fecal excretion, the mice were individually placed for 24 hours for urine collection with free access to drinking water in metabolic cages (Tecniplast Hohenpeissenberg, Germany). The inner wall of the metabolic cages was siliconized and the urine was collected under water-saturated oil.

Around To obtain blood samples, the animals were treated with diethyl ether (Roth, Karlsruhe, Germany) and about 150 μl Blood was heparinized by puncturing the retro-orbital plexus Taken from capillaries.

Faeces were dried at about 80 ° C for about 3 hours, and then the faeces dry weight was determined. 5 ml of 0.75 M HNO ₃ was added to the faeces and the sample was shaken for 48 hours on an electric shaker. The samples were then centrifuged at 3500 rpm for 10 minutes and 1 ml of the supernatant was collected. The supernatant was again centrifuged at 14,000 rpm for 5 minutes. The supernatant was stored at -20 ° C until analysis. The supernatant was analyzed as described below.

1.1.3 Determination of phosphate concentrations in the plasma, urine and faeces

The concentration of phosphate (P _i ) in plasma, urine and faeces was determined using a photometric kit (Roche, Mannheim, Germany) according to the manufacturer's instructions with adjustments to the different origin of the samples.

All Substances came from Sigma (Taufkirchen, Germany) or Roth (Karlsruhe, Germany).

1.1.4 Western blot analysis of abundance of the NaPiIIb protein

Intestinal tissue was taken from the first and second half of the small intestine. Subsequently, the epithelial tissue was scraped off and the total protein was isolated from untreated and nicotinamide-treated mice (n = 3 animals each, pooled and doubly determined). The small intestinal segments were rinsed with ice-cold saline solution and opened lengthwise. The mucosa was scraped with a glass slide in buffer containing (in mM): 250 saccharose, 20 tris (pH 7.5), 5 EGTA and protease inhibitor cocktail (Roche, Mannheim, Germany). The suspension was homogenized with an omnimixer for 7 seconds. MgCl ₂ was added to the homogenate at a final concentration of 10 mM. The suspension was shaken on ice and then centrifuged at 1600 g for 15 minutes. The plasma membranes remaining in the supernatant were collected by centrifugation at 20,000 g for 30 minutes. The resulting pellet was suspended in pH 7.4 buffer containing (in mM): 125% sucrose, 10% tris (pH 7.5), 2.5% EGTA, 2.5% MgSO ₄ . This suspension was homogenized with 50 "up-down" strokes with a glass homogenizer and centrifuged at 20,000 g for 30 minutes. The final pellet containing the cleaned brush border membrane vesicles (BBMV) was homogenized by forcing the suspension through 25 and 28 gauge needles and subsequent solubilization All steps were performed at 4 ° C the samples were frozen at -80 ° C for later use The membrane protein was determined as described by Bradford The total protein (20-30 μg / μl) was separated by SDS-PAGE (8% Tris-Glycine) on nitrocellulose membranes blocked overnight in blocking buffer (5% non-fat milk in PBS containing 0.15% Tween) and incubated overnight at 4 ° C with monoclonal anti-NaPiIIb antibody (diluted 1: 1000 in B10 citration buffer) (Alpha Diagnostics , Germany) After incubation with horseradish peroxidase (HRP) -conjugated anti-mouse secondary antibody (Amersham, Freiburg, Germany), the bands with enhanced chemolu Mescence (ECL) visualized according to the manufacturer's instructions.

1.1.5 Statistical analysis

The Data is presented as means ± SEM, n represents the number of independent experiments. All Data were analyzed for their significance using the unpaired Student's t-test with Welch correction tested if required.

1.2 cell culture experiments

1.2.1 Test substances

nicotinamide (NA): DSM Nutritional Products, product no. 0587848, CAS no. 98-92-0, Lot 404023; Stock solution: 10 mg / ml in culture medium (MEM) without additives; Substance is readily soluble, pH-neutral

acid amide (INA): Acros Organics, product no. 12258,0000, CAS no. 1453-82-3, Lot A0214246; Stock solution: 10 mg / ml in culture medium (MEM) without additives; Substance is readily soluble, pH-neutral

thiamine hydrochloride (THC): DSM Nutritional Products, product no. 0413038, CAS no. 67-03-8, Lot UQ50308195; Stock solution: 10 mg / ml in culture medium (MEM) without additives; Substance is easily soluble, however acidic pH of 4.82. Therefore, by adding a few drops 1 N NaOH neutralized.

Out the stock solutions were made by further dilution with culture medium incubation solutions with one concentration of 5 mg / ml and 1 mg / ml. All solutions were 10 times concentrated, d. H. the concentrations in the cell biological Tests were thus 100 μg / ml, 500 μg / ml and 1000 μg / ml.

1.2.2 Cell line used and routine culture

humane Intestinal epithelial cells, cell line Caco-2 (Human Caucasian colon adenocarcinoma; ECACC 86010202, DSMZ ACC 169); Passage 67 internally. The Caco-2 cells were mass cultures in minimum essential medium with L-glutamine (MEM) with the addition of 5% fetal bovine serum and 100 U / ml penicillin and 100 μg / ml streptomycin and 1% NEAA (nonessential amino acid solution, 100 times).

1.2.3 Phosphate stock solution for Exposure of the cells

• • KH₂PO₄ wasserfrei 10,0 g/l = 100 μg/ml im Kulturmedium zur Inkubation
• • Na₂HPO₄ × 2H₂O 57,5 g/l = 575 μg/ml im Kulturmedium zur Inkubation

The 100-fold concentrated phosphate stock solution for incubation of the cells contained, based on the phosphate concentration in Dulbecco's phosphate-buffered saline for cell culture:

• • KH ₂ PO ₄ anhydrous 10.0 g / l = 100 μg / ml in the culture medium for incubation
• • Na ₂ HPO ₄ × 2H ₂ O 57.5 g / l = 575 μg / ml in the culture medium for incubation

1.2.4 Determination of inorganic phosphate

Reagents and solutions

• • 1 mM Na ₂ HPO ₄ to prepare the calibration curve (solution 1): 178 mg Na ₂ HPO ₄ × 2H ₂ O in 1 liter distilled water. dissolved storage at 4 ° C in the refrigerator.
• • 0.5 MH ₂ SO ₄ aus konz. H ₂ SO ₄ (18 M): 28 ml conc. H ₂ SO ₄ in 900 ml distilled water. poured under stirring and cooling, if necessary, and distilled to 1000 ml with distilled water. filled
• • 0.42% (NH ₄ ) ₆ Mo ₇ O ₂₄ × 4H ₂ O: 4.2 g (NH ₄ ) ₆ Mo ₇ O ₂₄ × 4H ₂ O dissolved in 0.5 MH ₂ SO ₄ and diluted to 1000 ml with 0.5 MH ₂ SO ₄ filled. Store in a brown glass bottle at 4 ° C in the refrigerator.
• • 10% ascorbic acid solution (each freshly made on the day of the experiment): 1.11 g of ascorbic acid in 10 ml distilled water solved
• • Phosphate reagent (freshly prepared each day): 10% ascorbic acid solution and 0.42% molybdate solution mixed in the ratio 1 + 6 (6 ml of 10% ascorbic acid solution + 36 ml molybdate solution)

Calibration curves and execution the provision

• • Sample A: 10 μl solution 1 + 990 μl aqua dest. = 1 μM in the test
• Sample C: 50 μl solution 1 + 950 μl Distilled water = 5 μM in the test
• • Sample D: 100 μl solution 1 + 900 μl Distilled water = 10 μM in the test
• • Sample F: 200 μl solution 1 + 800 μl Distilled water = 20 μM in the test
• • Sample G: 300 μl Solution 1 + 700 μl Distilled water = 30 μM in the test
• • Sample H: 500 μl Solution 1 + 500 μl Distilled water = 50 μM in the test

To 100 .mu.l of the sample or distilled water. for the blank were pipetted 900 .mu.l phosphate reagent and for 60 min at 37 ° C in a shaking water bath in the dark incubated. This was followed by measurement of the absorbance at 820 nm with an ATI Unicam UV / Vis UV4 spectrometer.

1.2.5 Carrying out cell culture experiments

• Caco-2 cells were seeded at a density of 750,000 cells / culture dish (growth area per culture dish 55 cm) and for 3 days until reaching approximately 90% confluency in Minimum Essential Medium (MEM) with the addition of 5% fetal calf serum and 100 U / ml penicillin and 100 μg / ml Strep tomycin and 1% NEAA (non-essential amino acid solution, 100-fold).
• The Caco-2 cells were then incubated with minimum essential medium (MEM) without further additives (contains 140 mg / l sodium dihydrogen phosphate × H ₂ O) ± 100, 500 resp. 1000 μg / ml test substance preincubated for 15 min at 37 ° C. in the incubator.
• • The culture medium was aspirated and fresh MEM with 100 μg / ml potassium dihydrogen phosphate and 575 μg / ml Disodium hydrophosphate dihydrate from the 100X concentrated phosphate stock solution ± 100, 500 respectively. 1000 μg / ml test substance added and for Incubated for 60 min at 37 ° C in the incubator.
• • The incubation medium was aspirated, once with washed 10 ml per dish of 0.9% saline, the cells are detached by short-term trypsin / EDTA treatment (5 ml trypsin / EDTA for 5 min at 37 ° C) and suspended (+ 5 ml saline solution)
• The cell suspension was centrifuged (7 min at 240 × g), the supernatant with a Pasteur pipette aspirated and the cell sediment taken up in 1 ml saline
• • Samples were stored in Eppendorf cups at -20 ° C frozen and stored until phosphate determination
• • After thawing on the day of phosphate determination were the Eppendorf cups with the cell samples to destroy the Cell membranes are flash frozen briefly in liquid nitrogen (about 30 sec) and then the samples again at room temperature melted
• • The corresponding volume (dilution 1:10) for phosphate determination was added
• • Measurement at the ATI Unicam UV / Vis Spectrometer UV4 at a wavelength of 820 nm
• • The investigations were in triple parallel approach carried out

1.2.6 Internal sample names

• • Untreated cells in culture medium (KM)
• • saline (NaCl)
• • 15 min MEM without TS + 60 min IM without TS (K)
• • 15 min MEM with 100 μg / ml TS + 60 min IM with 100 μg / ml TS (TS100)
• • 15 min MEM with 500 μg / ml TS + 60 min IM with 500 μg / ml TS (TS500)
• • 15 min MEM with 1000 μg / ml TS + 60 min IM with 1000 μg / ml TS (TS1000)

• Abbreviations: IM = incubation medium = culture medium with phosphate 1: 100; TS = test substance, d. H. NA = nicotinamide, INA = Isonicotinamide and THC = thiamine hydrochloride.

2 results

 2.1 Animal experiments

In the 1 to 8th are the arithmetic mean ± SEM of fluid intake (ml / 24 hours; 1 ), food intake (mg / 24 h; 2 ), faeces dry weight (g / 24 hours, 3 ), body weight (g; 4 ), the urinary flow rate (ml / 24 hours; 5 ), urinary phosphate concentration (mg / dl; 6 ), urinary phosphate excretion (mg / 24 h; 7 ) and feces phosphate excretion (mg / 24 h; 8th ) of the experimental animals before (control) or one or two weeks after the administration of 0.1, 0.3, 0.5, 1 or 2 [mg / g body weight] nicotinic acid amide. * indicates a statistically significant (p <0.05) difference from the value before administration of nicotinic acid amide.

2.1.1 Fluid intake

As in 1 also illustrates the administration of low doses of nicotinamide a slight decrease in fluid intake of the experimental animals, the effect at 0.3 mg / g body weight for one week reaches the significance. Conversely, administration of 0.5 mg / g body weight of nicotinic acid amide tends to result in a slight but significant increase in fluid intake.

2.1.2 Food intake

Likewise, dietary intake of the test animals after administration of 0.1 to 0.3 mg / g body weight of nicotinamide decreases slightly and nicotine amide readily after administration of 0.5 mg / g body weight; see. 2 ,

2.1.3 Faeces dry weight

In addition to the effect on food intake, administration is 0.1 to 0.3 mg / g Body weight Nicotinsäureamid rather to a decrease in the faeces dry weight of the experimental animals, but this effect does not reach statistical significance; see. 3 ,

2.1.4 Body weight

The administration of nicotinic acid amide did not result in significant changes in body weight of the experimental animals; see. 4 ,

2.1.5 urine flow

The urinary flow rate of the test animals significantly decreased nicotinamide after two weeks of administration of 0.1 to 0.3 mg / g body weight; see. 5 ,

2.1.6 urinary phosphate concentration

The administration of 0.1 and 0.3 mg / g body weight nicotinamide increased the urinary phosphate concentration of the experimental animals, the effects being significant one and two weeks after the initiation of treatment; see. 6 ,

2.1.7 urinary phosphate excretion

Similarly, 0.1 mg / g body weight of nicotinamide increased urinary phosphate excretion of the experimental animals, this effect being significant one week after initiation of treatment; 7 ,

2.1.8 Faecal phosphate excretion

After administration of nicotinamide 0.3 mg / g body weight, fecal phosphate excretion of the experimental animals increases significantly; 8th ,

2.1.9 Expression of the phosphate transporter NaPiIIb

Subsequently, Western blot experiments were performed to investigate whether nicotinamide treatment affects the expression of the phosphate transporter NaPiIIb in the gut. The result of such an experiment is in 9 shown. The upper bars show the original blots, the lower bars the arithmetic mean ± SEM of NaPiIIb abundance (arbitrary units) before administration of nicotinamide (0 days) and 7 days after treatment with 2 mg / g body weight of nicotinamide. # indicates statistical significance (d <0.05) between the first and second halves of the small intestine.

As illustrated in this figure, protein abundance was evident higher in the first (jejunal) than in the second (ileal) Half of the small intestine before administration of Nicotinamide. The nicotinamide treatment leads to completely disappear the NaPiIIb protein from the boundaries of the small intestinal border.

2.1.10 Phosphate concentration in plasma

Next, it was examined whether the administration of nicotinic acid amide resulted in a decrease in the plasma phosphate concentration of the test animals. The result of a corresponding experiment is in 10 shown. Shown are the arithmetic mean ± SEM of the plasma phosphate concentration (mg / dl) before (control) or one or two weeks after the administration of 0.1, 0.3, 0.5, 1 or 2 [mg / g body weight] nicotinic acid mid , * indicates a statistically significant (p <0.05) difference from the value before administration of nicotinic acid amide.

there shows that at higher doses (≥ 0.5 mg / g) Nicotinic acid amide to a decrease in plasma phosphate concentration comes. This effect reaches statistical significance two weeks after the start of administration of 0.5 and 1 mg / g body weight Nicotinic acid amide and one week after administration of 2 mg / g body weight nicotinic acid amide. in the Contrary to the degrading effect of nicotinic acid amide at ≥ 0.5 mg / g, increased administration of Nicotinamide at low dosage (0.1 mg / g and 0.3 mg / g) the plasma phosphate concentration. This effect reached Statistical significance two weeks after 0.1 and 0.3 mg / g body weight Nicotinamide administration.

2.2 cell culture experiments

Around to check if the increase in plasma phosphate concentration at low doses of nicotinamide with a reduced Inclusion of phosphate in the cells is associated with the inventors a cellular test system with intestinal epithelial cells (cell line Caco-2), at which the phosphate uptake of the cells in Dependence of nicotinic acid amide quantitatively can be determined. Furthermore, two more test substances in their ability to study the phosphate uptake of Cell, namely isonicotinic acid amide and thiamine hydrochloride.

2.2.1 Phosphate calibration curve

First, a phosphate calibration curve was created to document the linear measurement range. Table 1 below shows the phosphate measured values at a dilution of 1:10. Calibration curve phosphate determination (1:10) sample [Pi] in μM readings OD 820 nm medium value SD A 1 0.112 0.103 0,111 0.112 0.005 C 5 0,179 0.171 0.172 0,179 0,004 D 10 0.245 0.257 0,251 0.245 0,006 F 20 0.362 0.365 0.359 0.362 0,003 G 30 0.482 0.473 0.459 0.482 0,012 H 50 0.804 0.786 0.816 0.804 0,015 Tab. 1: Tabular representation of the phosphate measured values at a dilution of 1:10 for documentation of the linear measuring range

11 shows a graphical representation of the phosphate measured values from Table 1 for documentation of the linear measuring range.

at the subsequent phosphate measurements were the maximum optical densities (OD) at 820 nm at 0.8 and were therefore still in the linear range of the calibration curve.

2.2.2 Phosphate uptake by intestinal epithelial cells (Controls)

In 12 is the phosphate uptake by intestinal epithelial cells (Caco-2) in the control (K = culture medium with phosphate 1: 100 without test substance) compared to cells in culture medium (KM = culture medium without phosphate 1: 100 and without test substance) shown. The phosphate uptake of the cells is 27.63 ± 6.6% (mean ± standard deviation) and is significant (p <0.05, Student's t-test). Thus, it is shown that all the effects measured with the test substances are actually due to a 1: 100 increase in the phosphate accumulated by the one-hour incubation with phosphate by the cells.

2.2.3 Phosphate uptake by intestinal epithelial cells in the presence of nicotinic acid amide

In 13 shows the influence of three different concentrations of nicotinic acid amide (NA) on phosphate uptake by intestinal epithelial cells (Caco-2). Indicated is the mean ± standard deviation (n = 3). It can be clearly seen that nicotinic acid amide causes a dose-dependent reduction of phosphate uptake with increasing nicotinic acid amide concentration by about 22 to 26%. Even at the lowest test concentration of 100 μg / ml, the reduction is already significant (p <0.05, Student's t-test).

This Experiment confirms that an increase the plasma phosphate concentration with a reduction in phosphate uptake accompanied by nicotinic acid amide.

The inventors have now recognized that the decreased cellular uptake of phosphate reduces the cellular accumulation of calcium phosphate (CaHPO ₄ ) and thus the risk of arteriosclerosis.

2.2.4 Phosphate uptake by intestinal epithelial cells in the presence of isonicotinamide

In 14 the influence of three different concentrations of isonicotinic acid amide (INA) on the uptake of phosphate by the intestinal epithelial cells is shown. The mean ± standard deviation (n = 3) is given again.

there shows that isonicotinamide the phosphate uptake the cell is largely unaffected. So is with the Concentrations 500 μg / ml or 1000 μg / ml none Deviation from control detectable (p <0.05; Student's t-test).

2.2.5 Phosphate uptake by intestinal epithelial cells in the presence of thiamine hydrochloride

In 15 the effect of three different concentrations of thiamine hydrochloride (THC) on phosphate uptake by intestinal epithelial cells is shown. Indicated is the mean ± standard deviations (n = 3).

there shows that thiamine hydrochloride is a dose-dependent Promote phosphate uptake by about 29 to 9% with reverse Proportionality causes, d. H. the lower the test concentration, the greater the promotion of phosphate uptake. Only at the lowest test concentration of 100 μg / ml However, the funding is significant (p <0.05, Student's t-test).

2.2.6 All test substances in comparison / summary

The results obtained with the three substances tested for phosphate uptake into the cells are summarized in Table 2 below. Phosphate uptake under nicotinamide sample Measured values OD 820 nm corrected, ie minus OD of 0.9% saline solution Average SD Mean Pi recording in% (K = 100%) SD Pi recording in% NaCl 0.08 0.112 0.094 0,095 0.016 KM 0.257 0.226 0,241 0,241 0.016 72.37 6.43 K 0.352 0.309 0,339 0.333 0,022 100.00 6.62 NA100 0,278 0.234 0,269 0.260 0.023 78.08 8.94 NA500 0.266 0.236 0,262 0,254 0.016 76.38 6.40 NA1000 0,270 0.247 0.224 0.247 0.023 74.07 9.32 Phosphate uptake under isonicotinic acid amide sample Measured values OD 820 nm corrected, ie minus the OD 0.9% saline solution Average SD Mean Pi recording in% (K = 100%) SD Pi recording in% NaCl 0.08 0.112 0.094 0,095 0.016 KM 0.257 0.226 0,241 0,241 0.016 72.37 6.43 K 0.352 0.309 0,339 0.333 0,022 100.00 6.62 INA100 0,238 0.287 0.263 0,262 0,025 78.78 9.34 INA500 0.307 0.331 0.348 0.328 0,021 98,60 6.27 INA1000 0.321 0.369 0.335 0.342 0,025 102.56 7.20 Phosphate uptake under thiamine hydrochloride sample Measured values OD 820 nm corrected, ie minus OD of 0.9% saline solution Average SD Mean Pi recording in% (K 100%) SD Pi recording in% NaCl 0.08 0.112 0.094 0,095 0.016 KM 0.257 0.226 0,241 0,241 0.016 72.37 6.43 K 0.352 0.309 0,339 0.333 0,022 100.00 6.62 THC100 0.396 0.459 0,430 0.428 0.032 128.53 7.37 THC500 0.361 0.428 0.394 0.394 0.034 118.32 8.50 THC1000 0,330 0.394 0.362 0.362 0.032 108.61 8.85 Tab. 2: Tabular representation of all measured values for phosphate uptake under the influence of the three different concentrations of the three different test substances.

The Inventors could by means of animal experiments and by means of a cellular Test system prove that nicotinamide the inclusion significantly reduce phosphate from the plasma into the cell can and therefore an effective substance for treatment and prophylaxis of atherosclerosis represents. Impressively, nicotinic acid amide the only test substance that allows the phosphate uptake into the cells reduced at all concentrations tested.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

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Claims (17)

Use of nicotinamide for Preparation of a medicament for treatment and / or prevention of arteriosclerosis. Use according to claim 1, characterized that the drug is designed for an application which is selected from the group consisting of: oral, rectal, parenteral, local, transdermal. Use according to claim 1 or 2, characterized that the drug is formed in a form selected is from the group consisting of: powder, tablet, juice, drops, Capsule, suppositories, solution, solution for injection, Aerosol, ointment, conditioner, patch, pellet, dragee. Use according to one of claims 1 to 3, characterized in that the drug Nicotinsäureamid in a concentration selected from the group consisting of (in each case given in weight of the active substance Nicotinic acid amide based on the total weight of the drug): 1 μg / g, 10 μg / g, 100 μg / g, 1 mg / g, 10 mg / g, 100 mg / g, 1 g / g. Use according to one of claims 1 to 4, characterized in that the absolute amount of the active ingredient Nicotinic acid amide in a unit dose of the drug between about 1 and about 3000 mg, preferably between about 10 and about 1500 mg, more preferably between about 100 and about 750 mg, and most preferably about 500 mg. Use according to one of claims 1 to 5, characterized in that the drug additionally has another active against atherosclerosis substance. Use according to claim 6, characterized that the further substance is a lipid-lowering agent (statin). Use according to claim 7, characterized that the lipid-lowering agent is selected from the group consisting from: atorvastatin, cerivastatin, fluvastatin, lovastatin (mevinolin), Mevastatin (compactin), pravastatin and simvastatin. Process for the preparation of a medicament for Treatment and / or prevention of arteriosclerosis, the the following steps: (1) provision of nicotinic acid amide, and (2) Formulation of nicotinic acid amide in one pharmaceutically acceptable carrier. Method according to claim 9, characterized in that that the drug is designed for an application which is selected from the group consisting of: oral, rectal, parenteral, local, transdermal. Method according to claim 9 or 10, characterized that the drug is formed in a form selected is from the group consisting of: powder, tablet, juice, drops, Capsule, suppositories, solution, solution for injection, Aerosol, ointment, conditioner, patch, pellet, dragee. Method according to one of claims 9 to 11, characterized in that the drug Nicotinsäureamid in a concentration selected from the group consisting of (in each case given in weight of the active substance Nicotinic acid amide based on the total weight of the drug): 1 μg / g, 10 μg / g, 100 μg / g, 1 mg / g, 10 mg / g, 100 mg / g, 1 g / g. Method according to one of claims 9 to 12, characterized in that the absolute amount of the active ingredient Nicotinic acid amide in a unit dose of the drug between about 1 and about 3000 mg, preferably between about 10 and about 1500 mg, more preferably between about 100 and about 750 mg, and most preferably about 500 mg. Method according to one of claims 9 to 13, characterized in that the drug additionally has another active against atherosclerosis substance. Method according to claim 14, characterized in that that the further substance is a lipid-lowering (Stalin). Method according to claim 15, characterized in that that the lipid-lowering agent is selected from the group consisting from: atorvastatin, cerivastatin, fluvastatin, lovastatin (mevinolin), Mevastatin (compactin), pravastatin and simvastatin. Method for therapeutic and / or prophylactic Treatment of a living being affected by arteriosclerosis or / and at risk of arteriosclerosis, the following Steps: (1) provision of nicotinic acid amide, (2) Introduction of nicotinic acid amide into the animal, and (3) if necessary, repeated repetition of steps (1) and (2).