Patent Publication Number: US-2009238895-A1

Title: Nutritional and/or pharmaceutical preparation for use in prophylaxis and treatment of disturbances in microelements absorption from the alimentary canal

Description:
An object of the invention is nutritional and/or pharmaceutical preparation for the use in the prophylaxis and treatment of disturbances in microelements absorption from the alimentary canal and the use of pharmaceutical preparation as a factor stimulating microelements absorption from the alimentary canal into the blood ensuring proper—physiological—curative level of these compounds in the blood in people and animals. 
     Alfa-keto-glutarate acid (AKG), glutamine derivative, is a key compound in Krebs cycle. 
     AKG, the mediate product in the main metabolic cycle, is transformed into glutaminic acid by the complex of glutamine dehydrogenase. The last is in turn transformed into glutamine in the presence of glutamine synthetase. 
     In the rapid process of izocitrate oxidative decarboxylation with the glutamate dehydrase stable and non-toxic AKG is generated: 
       Izocitrate+NAD +    α-ketoglutarate+CO 2 +NADH 
     Alpha-ketoglutarate acid (AKG) is thought to be the adequate exogenous precursor of endogenously synthetized glutamine and through glutamate acid which is necessary to the synthesis of other amino acids with 5-carbons chain, such glutamine, arginine, proline, histidine. These amino acids are transformed into a-glutamate which then is oxidatively deamninated with the glutamate dehydrase. AKG originates in the effect of these reactions. 
     Beside the function as energy donor alpha-ketoglutarate acid (AKG) plays also the protective role for proteins not allowing to their catabolizm. He works as “sweeper” of ammomum ions. Alpha-amino groups (residues) are transformed into ammonium ions in the process of oxidative deamination of glutamate with the glutamate dehydrase. AKG plays an important role in the process of organism detoxification and in the retention of ammonium ion protecting the break-up of proteins. Glutamine, the main precursor of AKG in the organism, is a main source of energy for the intestine epithelial cells. Its definite amount (concentration) is necessary to maintain the continuous rate of cells division necessary to keep main absorption functions. 
     The greatest part of necessary glutamine used by enterocytes originates directly in the intestine but food and blood are also its important source. In the case of glutamine deficiency in the alimentary canal its reserve is mobilized on the way of muscular tissue decomposition.
 
Many studies on animals and people with the intestine function disturbances and supporting treatment with glutamine. were performed. Glutamine supplied to primarily starved and then fed intravenously rats had had the positive effect on the improving of the intestine wall structure and decreased bacterial translocation to the circulatory system. There was also observed that glutamine has regenerative properties in the damaged wall of intestine in people after radiation and cytotoxic treatment.
 
It is known that bacteriemia caused by the translocation of the intestinal bacterial flora is often observed after operations when the dysfunction of alimentary canal occurs. That was proved that in such cases glutamine given per os or intravenously in animals and people has the protective activity minimalizing microbes dislocation. These observations leads to conclusion that in the metabolic stress the administration of glutamine as a therapeutic agent can have positive results.
 
However in practical practice glutamate is still difficult for using because is unstable in solution and poorly soluble.
 
To avoid these problems the interest of investigators has directed into glutamine precursors.
 
The nutritional or/and pharmaceutical preparation according to the invention distinguishes itself that contains alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide of glutamine and other amino acids or/and tripeptides of glutamine and other amino acids or/and glutamate with other amino acid or/and salts of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptides of glutamine or glutamate with other amino acid in a dose 0.01-0.5 g/kg/day and given orally stimulates the absorption of iron, zinc, coppers, manganese, strontium, calcium, phosphorus and other microelements from the alimentary canal to the blood ensuring their proper—physiological—curative level in the blood in people and animals.
 
     It is profitable if the nutritional or/and pharmaceutical preparation is curative and preventive in some diseases, especially in anaemia, postpartum enterostasis, heart dysfunction, atherosclerosis, tetany, osteoporosis, arthritis, intestine dysfunction, muscular dysfunction, dementia, the diminution of immunity, increased rates of bacterial, viral and mycotic infections increased morbidity of neoplastic and other diseases in people and animals and also increases the productiveness and the efficiency of farm animals as swine, poultry, sheep, cows, horses, goat and others. 
     Other nutritional or/and pharmaceutical preparation according to the invention distinguishes itself that contains iron (Fe) or other ions of bivalent metals being the alimentary microelements in a dose 0.0001-0.01 g/kg/day and increases the absorption into the blood and the metabolism in entereocytes of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptides of glutamine and other amino acids or/and tripeptides of glutamine and other amino acids or/and glutamate with other amino acid or/and salts of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptides of glutamine or glutamate with other amino acid ensuring their physiological—curative—preventive level in blood in people and animals.
 
It is profitable in other nutritional or/and pharmaceutical preparation if the nutritional or/and pharmaceutical preparation has therapeutic and preventive properties in some diseases, especially in anaemia, especially in anaemia, postpartum enterostasis, heart dysfunction, atherosclerosis, tetany, osteoporosis, arthritis, intestine dysfunction, muscular dysfunction, dementia, the diminution of immunity, increased rates of bacterial, viral and mycotic infections, increased morbidity for neoplastic and other diseases in people and animals and also increases the productiveness and the yield of farm animals as swine, poultry, sheeps, cows, horses, goats and others.
 
The use of the nutritional or/and pharmaceutical preparation according to the invention distinguishes itself that the preparation containing alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines and other amino acids or/and tripeptides of glutamine and other amino acids or/and glutamate with other amino acid or/and salts of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines or glutamate with other amino acid in a dose 0.01-0.5 g/kg/day administered orally is used as a stimulator of absorption of iron, zinc, coppers, manganes, strontium, calcium, phosphorus and other microelements from the alimentary canal to the blood.
 
The first other use of the nutritional or/and pharmaceutical preparation, according to the invention, distinguishes itself that the preparation containing alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines and other amino acids or/and tripeptides of glutamine and other amino acids or/and glutamate with other amino acid or/and salts of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines or glutamate with other amino acid in a dose 0.01-0.5 g/kg/day is used to assure proper—physiological—curative level of these substances in the blood in people and animals having therapeutic and preventive properties in the following diseases: anaemia, postpartum enterostasis, heart dysfunction, atherosclerosis, tetany, osteoporosis, arthritis, intestine dysfunction, muscular dysfunction, dementia, the diminution of immunity, increased rates of bacterial, viral and mycotic infections, increased morbidity for neoplastic and other diseases in people and animals.
 
The second other use of the nutritional or/and pharmaceutical preparation, according to the invention, distinguishes itself that the preparation containing alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines and other amino acids or/and tripeptides of glutamine and other amino acids or/and glutamate with other amino acid or/and salts of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines or glutamate with other amino acid in a dose 0.01-0.5 g/kg/day is used to increase the productiveness and yield of farm animals as the swine, poultry, sheeps, cows, horses, goats and others. The third other use of the nutritional or/and pharmaceutical preparation, according to the invention, distinguishes itself-that the preparation containing alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines and other amino acids or/and tripeptides of glutamine and other amino acids or/and glutamate with other amino acid or/and salts of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines or glutamate with other amino acid in a dose 0.01-0.5 g/kg/day is used to increase the absorption of manganese (Mn) to the bile and liver what makes possible the use of this preparation as a component of oral contrast used during examination and diagnosing of the neoplasms of the liver and other tissues of the alimentary canal in people and and animals.
 
The fourth other use of the nutritional or/and pharmaceutical preparation, according to the invention distinguishes itself the preparation containing iron (Fe) or other ions of bivalent metals being the alimentary microelements in a dose 0.0001-0.01 g/kg/day, increasing the absorption into blood and metabolism in entereocytes of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines and other amino acids or/and tripeptides of glutamine and other amino acids or/and glutamate with other amino acid or/and salts of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines or glutamate with other amino acid is used to assure their physiological—curative—preventive level in blood in the following diseases: anaemia, postpartum enterostasis, heart dysfunction, atherosclerosis, tetany, osteoporosis, arthritis, intestine dysfunction, muscular dysfunction, dementia, the diminution of immunity, increased rates of bacterial, viral and mycotic infections, increased morbidity for neoplastic and other diseases in people and animals.
 
The fifth other use of the nutritional or/and pharmaceutical preparation, according to the invention, distinguishes itself that contains iron (Fe) or other ions of bivalent metals being alimentary microelements in a dose 0.0001-0.01 g/kg/day and increases the absorption into blood and the metabolism in entereocytes of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines and other amino acids or/and tripeptides of glutamine and other amino acids or/and glutamate with other amino acid or/and salts of alpha-keto-glutarate or/and glutamine or/and glutamate or/and ornithine of alpha-keto-glutarate, dipeptide glutamines or glutamate with other amino acid is used for the protection and the increase of the productiveness and yield of such farm animals as swine, poultry, sheeps, cows, horses, goats and others. In compliance with the invention the influence of AKG on the absorption of iron and other microelements was examined. Iron and the other microelements, for example zinc, copper, manganese are necessary to the synthesis of hemoglobin, mioglobine, cytochromes, and other enzymes. The malabsorption of iron and other microelements is a main cause of many diseases, for example anaemia, postpartum enterostasis, heart dysfunction, atherosclerosis, tetany, osteoporosis, arthritis, intestine dysfunction, muscular dysfunction, dementia, the diminution of immunity, increased rates of bacterial, viral and mycotic infections, increased morbidity for neoplastic diseases.
 
The influence of AKG as a component of intravenous nutrition was intensively examined in different patients&#39; clinical condition. The intravenous nutrition formulas with the standard composition and with the addition of branch chain amino acids (BCAA), glutamine and AKG were investigated. That was proved that BCAA has no significance in the parenteral nutrition while glutamine and alfa-ketoglutarate administered parenterally increase the level of the constitutional glutamine in the muscles and decrease the negative nitrogen balance.
 
AKG reduces also the ammonia level what is essential in the medical care. High concentration of the ammonia in blood (ammonaemia) leads to hepatocirrhosis; however the most serious negative activity of this compound is its influence on the brain. Increased level of ammonia can lead to brain oedema and coma.
 
The invention is presented in examples on- the base of performed examinations: FIG.  1 —the diagram of the increased absorption of iron (Fe) from the ileum in pigs, FIG.  2 —the increased absorption of iron (Fe) into blood after oral administration of AKG in people, and FIG.  3 —increased absorption of zinc (Zn) into blood after oral administration of AKG in people, FIG.  4 —increased absorption of manganese (Mn) (gmol/kg of body weight) from the intestine after enteral administration of AKG in rats.
 
    
    
     EXAMPLE I. 
     Animals 
     Investigations were performed on 3 piglets of the Swedish race Landrace weighting 30-35 kg. Animals originated from the university herd (Swedish Agricultural University, Dept. of Agricultural Biosystems and Technology, Lund). Young pigs were fed 2 times a day at the same time (9:00-10:00 a.m. and 3:00-4:00 p.m.) with a standard fodder (Vaxfor, Lantmanen, Sweden) with a daily dose 50 g/kg body weight. They were watered ad libitum. Animals were held separately in boxes with automatically regulated 12 hour&#39;s periods of day and night. 
     The Surgical Operation 
     Sterile catheters and T-fistulas used in the experience were made of silicone tubes with maintained medical standards (Dow Cornning).
 
Piglets were starved for 9-12 hours before the operation.
 
Before the operation animals received a sedative drug—azoperone (Stresnil, 2 mg/kg body weight). Then the inhalation induction of general anaesthesia was performed (Fluothan 0.5-1.5 vol %). Duodenal and pelvic fistulas were made in all animals and AKG infusion was administered. With the catheters inserted to the portal vein and the common carotid-artery the blood samples for later analyses were collected.
 
     Two incisions were done: one 10-15 cm close to the right hunger fossa, second 2-3 cm between the brachial joint and the angle of the mandible. Duodenal fistula was placed behind the opening of the pancreatic duct to the duodenum. Pelvic T-fistula (int. diameter 3.18 mm and ext. diameter 4.64 mm—both fistulas) was introduced to the pelvic intestine at the end of the caeco-iliac ligament. 
     The catheter to the portal vein (int. diameter 0.64 mm and ext. diameter 1.19 mm) was introduced through the liver and fixed with several sutures (0-2 Catgut, Ethicon, England).
 
The catheter placed in portal vein and both intestinal fistulas were brought out the body in the line of incision. After dissection of zygomatic vein the catheter was introduced in the cardiac direction on the depth of 7 cm. The vein was closed with the double ligature (0-3 silicone-, Ethicon, England) and brought outside the body with the surgical needle in the dorsal part of the neck.
 
The abdomen and the neck wounds were closed with two layers of sutures: the peritoneum (only in the abdomen wound) and muscles were closed with twisted suture (0-0 Catgut , Ethicon), the skin was closed with the interrupted sutures (Suturamid 2-0, Ethicon).
 
After operation young pigs were placed in separate boxes. During 5 postoperative days animals were fed twice a day, they received water ad libitum. The operation was performed by the skilled surgeon in accordance with surgical techniques.
 
     Infusions/Doses: 
     solution 1-35.1 g AKG/I pH=5.0: 0.15 g/kg body weight
 
solution 2-5.64 g FeSO 4 /I pH=2.0: 10 mg/kg body weight
 
solution 3-35.1 g AKG/I , FeSO 4 /I pH=2.0: 0.15 g+10 mg/kg body weight
 
The above infusions, repeating twice, were administered to animals in two-days intervals according to the rule of “Latin square” (table 1)
 
                     TABLE 1                  The plan of experience                             Day of   Infusions                                     experience   AKG   FeSO 4     AKG/FeSO 4                                                   1   5d   6d   7d           3   5b   6b   7b           5   7d   5d   6d           7   7b   5b   6b           9   6d   7d   5d           11   6b   7b   5b                       d - infusion to the duodenum           b - infusion to the pelvic intestine           5, 6, 7 - the number of animals            
Before infusion animals were starved. The daily dose of food was given during evening feeding at 3:00 p.m.
 
All infusions were started at 9:00 a.m. Blood samples were taken 30 minutes before and 15, 30, 60 and 120 minutes after the infusion.
 
2. Infusions composition.
 
     
       
         
           
               
               
            
               
                   
                   
               
               
                   
                 studia 
               
            
           
           
               
               
               
               
            
               
                   
                 solution 1 
                 solution 2 
                 solution 3 
               
            
           
           
               
               
            
               
                   
                 pH 
               
            
           
           
               
               
               
               
            
               
                   
                 5 
                 2 
                 2 
               
               
                   
                 mmol/l 
                 mmol/l 
                 mmol/l 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 AKG −−   
                 240 
                 — 
                 240 
               
               
                   
                 Na +   
                 373 
                 159 
                 88 
               
               
                   
                 K +   
                 55 
                 — 
                 13 
               
               
                   
                 Ca ++   
                 — 
                 — 
                 — 
               
               
                   
                 Mg ++   
                 — 
                 — 
                 — 
               
               
                   
                 H +   
                 0.01 
                 10 
                 10 
               
               
                   
                 Cl −   
                 — 
                 182.5 
                 — 
               
               
                   
                 Fe ++   
                 — 
                 37 
                 37 
               
               
                   
                 SO4 2   −−   
                 — 
                 37 
                 37 
               
               
                   
                   
               
            
           
         
       
     
     Results 
     Results are presented on  FIG. 1  and Tables 3, 4.
 
AKG administered enterally significantly increases iron absorption from the intestine into the blood ( FIG. 1 , Table 3), FeSO 4  increases the absorption of AKG from the intestine into the blood (Table 4).
 
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 The iron concentration (μmol/l) in plasma of young pigs receiving FeSO4 
               
               
                 and FeSO4/AKG infusions to the duodenum and the pelvic intestine 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 FeSO4 infusion 
                   
               
            
           
           
               
               
               
               
               
            
               
                 Time 
                 To the duodenum 
                   
                 To pelvic intestine 
                   
               
            
           
           
               
               
               
               
               
            
               
                 (min) 
                 Zygomatic vein 
                 Portal vein 
                 Zygomatic vein 
                 Portal vein 
               
               
                   
               
               
                 −30 
                  18.2 ± 1.53 a   
                 16.33 ± 1.04 a   
                 19.67 ± 7.97 a   
                 19.17 ± 8.69 a   
               
               
                 15 
                 29.17 ± 5.51 b   
                  28.5 ± 4.09 b   
                 18.83 ± 6.81 a   
                 18.00 ± 5.41 a   
               
               
                 30 
                 39.50 ± 4.09 c   
                 37.67 ± 5.3 cb   
                 18.83 ± 6.81 a   
                 17.67 ± 6.53 a   
               
               
                 60 
                  43.0 ± 4.82 c   
                 40.17 ± 4.62 c   
                 18.00 ± 7.47 a   
                 16.83 ± 6.81 a   
               
               
                 120 
                 39.5 ± 3.5 c   
                 37.17 ± 4.37 c   
                 15.67 ± 6.43 a   
                 15.33 ± 7.18 a   
               
               
                   
               
            
           
           
               
               
            
               
                 Time 
                 FeSO4/AKG infusion 
               
            
           
           
               
               
               
               
               
            
               
                 (min) 
                 Zygomaticus vein 
                 Portal vein 
                 Zygomaticus vein 
                 Portal vein 
               
               
                   
               
               
                 −30 
                 20.33 ± 5.62 a   
                 20.50 ± 5.89 a   
                 20.47 ± 7.85 a    
                 20.00 ± 8.05 a    
               
               
                 15 
                 98.83 ± 29.72 d   
                 95.50 ± 29.51 d   
                 36.27 ± 25.54 bc   
                 32.00 ± 24.89 bc   
               
               
                 30 
                 96.00 ± 27.88 d   
                 88.67 ± 23.71 d   
                 36.93 ± 27.49 bc   
                 33.17 ± 27.76 bc   
               
               
                 60 
                 93.17 ± 17.96 d   
                 85.83 ± 15.50 d   
                 34.30 ± 26.01 bc   
                 29.83 ± 22.21 bc   
               
               
                 120 
                 80.50 ± 17.06 d   
                 75.67 ± 15.43 d   
                 27.67 ± 19.06 bc   
                 25.67 ± 18.45 bc   
               
               
                   
               
               
                 (average ± SD, n = 3, observations = 6) 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 AKG concentration (μg/l) in plasma of young pigs receiving FeSO4 
               
               
                 and FeSO4/AKG infusions to the duodenum and the pelvic intestine 
               
            
           
           
               
               
               
            
               
                 Time 
                 To the duodenum 
                 To pelvic intestine 
               
            
           
           
               
               
               
               
               
            
               
                 (min) 
                 Zygomatic vein 
                 Portal vein 
                 Zygomatic vein 
                 Portal vein 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 AKG infusion 
               
            
           
           
               
               
               
               
               
            
               
                 −30 
                 1.87 ± 0.64 a   
                  3.38 ± 0.54 b   
                 1.47 ± 0.65 a   
                 3.80 ± 2.57 b   
               
               
                 15 
                 11.32 ± 6.94 d   
                 28.62 ± 2.84 f   
                 2.96 ± 0.29 b   
                 5.33 ± 2.48 b   
               
               
                 30 
                 13.62 ± 3.91 d   
                 22.86 ± 4.08 f   
                 2.55 ± 1.26 b   
                 4.06 ± 2.55 b   
               
               
                 60 
                     7.72 ± 2.69 cb   
                 13.53 ± 3.04 e   
                 2.58 ± 0.26 b   
                 4.18 ± 0.63 b   
               
               
                 120 
                 4.82 ± 3.39 b   
                     11.11 ± 6.70 ce   
                 2.60 ± 0.73 b   
                 4.05 ± 0.16 b   
               
            
           
           
               
               
            
               
                   
                 FeSO4/AKG infusion 
               
            
           
           
               
               
               
               
               
            
               
                 −30 
                 3.06 ± 0.93 b   
                  4.78 ± 1.71 b   
                 3.44 ± 1.51 b   
                 4.53 ± 1.40 b   
               
               
                 15 
                 28.53 ± 21.89 f   
                  47.80 ± 23.62 g   
                     13.99 ± 16.35 def   
                     15.96 ± 20.72 def   
               
               
                 30 
                 17.80 ± 8.53 c   
                 27.41 ± 9.80 f   
                     9.67 ± 8.17 bce   
                      9.77 ± 10.60 bce   
               
               
                 60 
                 9.62 ± 4.09 c   
                 16.60 ± 4.86 e   
                 3.43 ± 1.23 b   
                     5.27 ± 4.12 bc   
               
               
                 120 
                 4.89 ± 5.03 b   
                     7.74 ± 1.77 cb   
                 4.89 ± 5.03 b   
                     6.51 ± 5.70 bc   
               
               
                   
               
               
                 (average ± SD, n = 3, observations = 6) 
               
            
           
         
       
     
     EXAMPLE II 
     Research was performed on 4 volunteers.
 
At the first day, after randomization and after taking the initial blood samples, volunteers drank on an empty stomach 200 mg zinc sulphate—ZnSO4—dissolved in 200 ml of H 2 O (Sovezink, Tika Läkemedel AB, Lund) and a tablet of iron formate—Fe(HCOO) 2  in a dose 200 mg (Erco-Fer, Orion). Immediately after the drugs consumption volunteers drank 1 litre of 10% glucose in 0.9% NaCl solution. Next blood samples were taken 30, 60 120 and 180 minutes after glucose drinking. Two days later these volunteers consumed identical doses of iron and zinc and drank thereafter 1 litre of 10% AKG-soda salt solution.
 
Results: Results are presented on  FIGS. 2 and 3 . AKG administered orally increases quantitative absorption of Fe2+ and Zn2+ ions from the alimentary canal into the blood in people.
 
     EXAMPLE III 
     Experiments were performed on 24 rats not eating for night before the experience. The induction of general anaethesia was done with ketamine 50 mg/kg (Ketalar).
 
The catheters to the duodenum, the back part of jejunum and to the biliary duct were inserted in animals. The experience was performed simultaneously in 6 animals.
 
After 30-minutes period of post-operative stabilization 6 animals received MnCl 2  to the duodenum, next 6 received MnCl 2  to the back part of jejunum and next 6 animals received MnCl 2  with the addition of AKG-soda salt in a dose 0.5 g/kg body weight to the duodenum. The last group of 6 animals received MnCl 2  with AKG-soda salt in a dose 0.5 g/kg body weight to the back part of intestine.
 
The content of manganese in the bile collected 2 hours after the infusion and in the liver dissected directly after the end of the bile collection and after the euthanasia of the animal was examined.
 
     Results 
     Results indicate that AKG increases the absorption of the manganese (Mn) from the back part of jejunum ( FIG. 4 )