DE4426165C2 - Use of lactoferrin for the prophylaxis and therapy of liver dysfunction - Google Patents

Description

The invention relates to the use of lactoferrin for prophylaxis and therapy Pie of liver dysfunction and / or liver parenchymal damage and Therapy of their subsequent states.

DE 40 08 033 A1 already describes the use of the applicant Lactoferrin as a therapeutic agent to combat and Reduction of the toxic effects of endotoxin on Endo toxemia known. The proposed there as additional therapy Lactoferrin is also suitable for patients with Disruption of endotoxin elimination via the liver. The funk tion disorders of the liver itself are neither thera prophylactically they are still prevented.  

Lactoferrin is a glycoprotein with an average molecular weight of 77,000 D. It can reversibly bind two molecules of iron. However it is also able to add other divalent or trivalent metal ions instead of iron to store.

Lactoferrin was first isolated from milk in 1960. The lactoferrin content of Breast milk is approx. 1 mg / dl. Lactoferrin was also found in the blood, as well as in the granules of the neutrophil granulocytes, in the tear fluid, in the stomach juice and detected in the secretions of the intestinal tract. Was in the blood Lactoferrin found in a concentration of 40-200 µg / dl.

Lactoferrin is found in the blood, mainly from the granules of neutrophils Granulocytes is released, especially as part of infections or Ent ignitions have a certain importance for the transport of iron to the cells des RES (Van Snick JL, Masson L, Heremans JF. The involvement of lactoferrin in the hyposideremia of acute inflammation. J Exp Med 1974; 140: 1068). As The rapid release of lactoferrin from the granulocytes was found in animal experiments with the administration of bacteria or endotoxin a tight time Relationship to endotoxin administration and sepsis, which is why the increase in lacto ferric concentration in the blood as an early indicator of endotoxemia or Septicemia is considered (Gutteberg TJ., Rokke O., Joergensen T .; Andersen O .: Lactoferrin as an indicator of septicemia and endotoxemia in pigs. Scand J Infect Dis (1988), 20: 659).

Another important meaning of lactoferrin, especially in the neonatal period is seen in the regulation of iron absorption from the intestinal tract hen (Brock JH, lactoferrin in human milk: its role in iron absorption and pro tection against enteric infections in the newborn infant. Arch Dis Child (1980) 55: 417-422), since the lactoferrin of breast milk transfers the iron to the Cells of the intestinal mucosa can give off.  

However, the main biological importance of lactoferrin is in its bacteriostatic effect seen (Arnold RR, Brewer M, Gauthier JJ. Bacte ricidal activity of human lactoferrin: sensitivity of a variety of microorganisms. Infection and Immunity (1980) 28: 893; Arnold RR, Russel JE, Champion WJ, Gauthier JJ. Bactericidal activity of human lactoferrin: Influence of physical conditions and metabolic state of the target microorganisms. Infection and Immunity (1981) 32: 655; Gutteberg T.J., Rokke O., Andersen O., Joergensen T .; Early fall of circulating iron and rapid rise of lactoferrin in septicemia and en dotoxemia: an early defense mechanism. Scand J Infect Dis, (1989) 21: 709). Due to its bacteriostatic effect, this is due to breast milk guided lactoferrin in the newborn plays an important role as a protective mecha nism against infections of the intestinal tract. The bacteriostatic effect be rests on the high iron binding capacity of lactoferrin. Iron is a we significant growth factor for the bacteria. Due to the complex formation of the Iron with lactoferrin is the concentration of free iron in the area the bacteria are kept below the level needed for bacterial growth is such. This leads to an inhibition of bacterial growth. With The bacteriostatic effect of lactoferrin decreases with increasing iron content. Because in animal experiments iron-free lactoferrin in bacteremia and endotoxemia Gutteberg et al. assumed that the iron-free lactoferrin, due to its bak teriostatic effect, the task of an early defense mechanism of the Organism against bacteria (Gutteberg T.J., Rokke O., Andersen O., Joergensen T .; Early fall of circulating iron and rapid rise of lacto ferrin in septicemia and endotoxemia: an early defense mechanism. Scand. J. Infect. Dis. (1989), 21: 709).

Evidence that lactoferrin is also used as a liver protection therapeutic can not be found in the literature.

The liver has a variety of functions in the organism. This includes so probably the synthesis of plasma proteins as well as detoxification and elimi nation of toxic metabolites, such as B. ammonia and bilirubin. Either toxic and inflammatory damage to the liver cells, but also a ver reduction of functionally intact liver tissue due to parenchymal defects, the for example by fatty liver, cirrhosis of the liver or also by Liver cell necrosis can result, depending on the extent of the damage to a more or less severe restriction of liver function. Either by reducing the functionally intact liver tissue in the context of  Liver parenchyma damage, as well as with impaired liver cell function toxic or inflammatory damage to the liver cells will cause damage to the Detoxification of ammonia also the conjugation function and thus the Eli Mination of bilirubin and the synthesis of proteins more or less strongly impaired.

Usually the ammonia that gets into the blood gets in the liver Degraded urea or detoxified with glutamic acid. A restriction or failure of liver function is therefore associated with an increase in concentration of ammonia in the blood. During the concentration of ammonia in the blood of healthy people is approx. 30 to 100 µg / dl, the ammonia level can increase or decrease liver function up to 500 µg / dl. The ammonia intoxication then becomes particularly difficult for the patient major problem if with portal hypertension that is in the frame cirrhosis can develop through surgery to prevent shunts Bypassing the liver. Because the ammonia intoxication is too high neurological disorders up to encephalophathy can, in particular, take measures to prevent increased Increase in the concentration of ammonia in the blood is one of the essential therapeu tasks in all liver dysfunctions.

In addition to the therapeutic measures to reduce the increase in However, it also includes blood ammonia in toxic concentration ranges the requirements for successful therapy of liver dysfunction, that the metabolic situation of the intact residual tissue of the liver be especially with regard to protein synthesis and elimination of bilirubin is improved, so good conditions for regeneration of the liver webes can be created. Even in patients who have liver trans plantation is expected: Measures for ver improvement in liver function can have a favorable effect on the further course nen.

The invention has for its object an organic Liver damage resulting in liver dysfunction fixed, with a therapeutic and prophylactic agent to treat that restores the function of the liver. This is solved by the features of the main claim. Lactofer rin can be administered parenterally or administered enterally the. When given enterally, lactoferrin can be combined with other substances be niert and also be administered together with nutrient solutions.  

In animal experiments it was first shown that the intraperito neal administration of lactoferrin the extent of development of toxic liver cell damage can be reduced. Surprisingly, ge could continue are shown that in the enteral administration of lactoferrin via a Not only probe unclear temperatures in patients with no discernible septi hearth but also liver dysfunction in a positive way can be influenced. In some patients with hepatic insufficiency the bottom of an alcohol-toxic liver damage that a lactoferrin sol was administered per os, a rapid regression of the increased activity of the liver enzymes was observed in the blood, whereas the activity of these Enzymes in patients with a comparable clinical picture who do not have Lak toferrin had remained unchanged.

Other patients with hepatic impairment have been administered of lactoferrin also evidence of an improvement in protein synthesis found the liver. These patients were found shortly after Be After the enteral administration of lactoferrin, an increase in the concentration of transferrin, fibrinogen and albumin in the blood, which are synthesized in the liver become.

Plasma bilirubin levels also showed dysfunction in patients liver decreased significantly with lactoferrin. As further indication for an improved detoxification function was given by Lactoferrin also has a decrease in blood ammonia concentration in patients measured with a severe functional impairment of the liver.

Treatment of patients with lactoferrin is preferably carried out several times a day Days. A pure lactoferrin solution can be administered here the. However, it is also possible to combine the lactoferrin with other sub punch and proteins, e.g. B. in connection with enteral solutions To administer nutrition. The lactoferrin solution can either be drunk or applied via a gastric or duodenal tube, one continuous application is possible. Depending on the severity of the liver function the therapeutic dose of lactoferrin should be approximately 2 g to 9 g per Day, but can also be increased if necessary. With lighter ones Forms of liver dysfunction or also with prophylactic measures If necessary, smaller amounts of lactoferrin can be given.  

In the case of dysfunction, which is caused by therapy with lactoferrin It can be expected to have a beneficial effect on liver function inflammatory diseases of the liver as well as toxic liver cell damage act. Lactofer ring administration is also more beneficial in the case of parenchymal damage Expected effect. Such parenchymal damage can be caused by stasis over, with a fatty degeneration of the liver cells, with a liver fibrosis, with liver cirrhosis or liver cell necrosis. This he Diseases can both interfere with the synthesis function of the liver as well as a restriction of the conjugation and detoxification function ren, which results in complications of hyperbilirubinaemia and / or Can develop hypeammonemia. The increase in ammonia in the blood is in even more so in cases where portal hypertension Circulations between the portal vein system and the venous system neither train spontaneously nor be created by shunt operations.

The use of lactoferrin according to the invention is not only therapeutic but also prophylactically possible. Both with inflammatory liver cranes as well as in the beginning of liver insufficiency, which is within the scope liver parenchymal damage, such as B. liver fibrosis, liver cirrhosis or can also develop a stasis liver, the metabolic function the liver is positively influenced by the prophylactic administration of lactoferrin. This improves the detoxification function of the liver cells threatening complications can be prevented early and thus the further development of liver cell necrosis can be delayed.

In laboratory chemistry, the positive effect of lactofererring u. a. in a Reduk tion of the ammonia concentration and the bilirubin level, as well as in an Ab increase in the activity of liver enzymes in the blood. The green The effect of lactoferrin on liver cell function is also in a verb recognition of protein synthesis, for which purpose the concentration cations of transferrin, albumin and fibrinogen are used as parameters can be. Through the prophylactic administration of lactoferrin in patients with portal hypertension, both before and after portal shunt Operations to avoid the neurological problems caused by the Increase in blood ammonia can be caused.

Also for the aftercare of patients who have a liver transplant  was carried out, the use of lactoferrin as a liver protection preparation possible because this gives you the opportunity to detoxify and substance to improve the alternating function of the transplanted liver.

The following examples illustrate the invention:

example 1

Wistar rats underwent short-term general anesthesia gene probe placed. A lactoferrin solution (lactoferrin (human), dissolved in 0.9% NaCl) in a dose of 160 mg / kg.

The application of this lactoferrin dose was in the therapy group (A) (n = 10) and in a second therapy group (B) (n = 12) at 4-hour intervals carried out.

The animals in control group (C) (n = 12) each received the instead of lactoferrin same amount of casein (160 mg / kg) in 0.9% NaCl at 4 hourly intervals over the probe.

Another therapy group (D) (n = 10) was given the lactoferrin in the same Dosage (160 mg / kg) administered intraperitoneally.

The animals were treated with Thera 8 hours after the start of the lactoferrin therapy piegruppe (B) and the control group (C) galactosamine (D-galactosamine Hydrochloride in 0.9% NaCl) at a dose of 300 mg / kg intraperitoneally applied. The animals in therapy group (A) received instead of galactosa min only 0.9% NaCl i.p. In the animals of the therapy group (D) (lactoferrin i.p.) the galactosamine application took place 4 hours after the first lac gift of toferrin.

After the i.p. Lactoferrin therapy was used in the application of galactosamine Therapy group (B) and casein administration in the control group (C) as a rule moderate time intervals of 4 hours and for the therapy group (D) in Inter continued for 6 hours each. The animals of not using galactosamine treated group (A) also received the same intervals as Group (B) applied the lactoferrin via the probe. The last application was carried out in all groups 24 hours after administration of galactosamine or NaCl (Group A).  

12 hours and 24 hours after the galactosamine or NaCl application venipuncture was performed to collect blood. By doing the activity of the following enzymes was determined in venous blood: GOT, GPT, LDH, alkaline phosphatase (AP).

After 30 hours, ie 6 hours after the last blood draw, the animals were put to sleep with an ether overdose. The liver was immediately removed and examined histologically.

• I.) The following mean values were found for the activity of the enzymes determined in the serum:
  • a) Lactoferrin group (A) (lactoferrin po, no galactosamine):
    12 hour control
    GOT: 46 ± 11 [U / l]; GPT: 21 ± 9 [U / l]; LDH: 172 ± 81 [U / l]
    AP: 129 ± 21 [U / l]
    24 hour control
    GOT: 49 ± 8 [U / l]; GPT: 19 ± 11 [U / l]; LDH: 203 ± 73 [U / l]
    AP: 121 ± 28 [U / l].
  • b) Lactoferrin group (B) (lactoferrin po galactosamine ip):
    12 hours after ip administration of galactosamine
    GOT: 398 ± 67 [U / l]; GPT: 439 ± 71 [U / l]; LDH: 1,168 ± 288 [U / l];
    AP: 318 ± 54 [U / l]
    24 hours after ip administration of galactosamine
    GOT: 443 ± 72 [U / l]; GPT: 497 ± 94 [U / l]; LDH: 1,432 ± 345 [U / l];
    AP: 297 ± 62 [U / l].
  • c) Casein control group (C) (casein per os, galactosamine ip):
    12 hours after ip administration of galactosamine
    GOT: 779 ± 107 [U / l]; GPT: 847 ± 121 [U / l]; LDH: 1,968 ± 428 [U / l];
    AP: 576 ± 63 [U / l]
    24 hours after ip administration of galactosamine
    GOT: 863 ± 94 [U / l]; GPT: 1,136 ± 183 [U / l]; LDH: 2,406 ± 409 [U / l];
    AP: 634 ± 87 [U / l].
  • d) ip lactoferrin group (D) (lactoferrin ip galactosamine ip):
    12 hours after ip administration of galactosamine
    GOT: 432 ± 52 [U / l]; GPT: 489 ± 61 [U / l]; LDH: 1,364 ± 288 [U / l];
    AP: 286 ± 43 [U / l]
    24 hours after ip administration of galactosamine
    GOT: 476 ± 67 [U / l]; GPT: 526 ± 95 [U / l]; LDH: 1,197 ± 309 [U / l];
    AP: 326 ± 51 [U / l].

The activity of the enzymes released by the liver was significantly higher after ip administration of galactosamine in the animals (groups B and D) to which lactoferrin was administered than in the lactoferrin group (A), which received lactoferrin but no galactosamine would have. However, compared to the untreated casein control group (C), the activity of the liver-released enzymes in the blood of the animals treated with lactoferrin per os or ip (groups β and D) was significantly lower than in the case of those not treated with lactoferrin Casein control group (C). Between the group that received lactoferrin enterally (group B) and the group that received lactoferrin intraperitoneally (group D), the activity of the enzymes released by the liver in the blood showed no statistically significant difference.

• II.) Microscopic examination of the liver revealed
  • a.) Lactoferrin group (A) (lactoferrin po, no galactosamine) inconspicuous histological picture, no evidence of fatty liver formation.
  • b.) Lactoferrin group (B) (lactoferrin po galactosamine ip) minor signs of fatty liver formation, but marked cellular infiltrates. Areas circumscribed in three of 12 animals with marked fatty liver cells and beginning necrotic changes.
  • c.) Casein control group (C) (casein per os, galactosamine ip):
    clear signs of fatty liver formation; Signs of diffuse hepatocellular necrosis: inflammatory infiltrates in the entire liver
  • d.) ip lactoferrin group (D) (lactoferrin ip galactosamine ip)
    Signs of moderate degree of fatty liver formation; cellular infiltrates in the area of the entire liver, which are, however, less pronounced than in the casein control group; only isolated necrotic changes.
    The necrotic changes in the circumscribed areas are only slightly more pronounced in two animals.

Both through enteral and intraperitoneal application of Lactoferrin has a protective effect on the liver. This enables the Significantly reduce the effects of toxic damage to the liver, which is caused by galactosamine.

Example 2

A lactoferrin solution (lactoferrin bovin in 0.9% NaCl) was found in 7 patients a dose of 200 mg (daily dose: 800 mg) partly per os, partly via one Probe administered at 6 hourly intervals for a total of 4 days. At these patients, who had cirrhosis of the liver, had had one hemorrhagic shock is a severe restriction of liver function developed the u. a. a sharp rise in blood ammonia levels would have. Before the lactoferral ring started, these patients had blood ammonia levels every 12 hours over a period of in total measured 24 hours. Only if the ammonia level is initially above the Was within the normal range and there was no tendency to improve during this period, d. H. spontaneous decrease in blood ammonia concentration was evident, 24 hours after the start of monitoring with the application of lacto ferrin started.

All patients who had received lactoferrin had been on for 24 hours a clear decrease in concentration after the start of lactoferrin application of the ammonia measured in the blood. The ammonia concentration remained at these Patients in the normbe during the entire lactoferrin application period rich (Tab. I).

In a comparison collective of a total of 12 patients who had an identical Had basic problems and a similar degree of hepatic impairment the same and the usual therapeutic measures men (paromomycin sulfate, lactulose) as in the lactoferrin group  Lactoferrin was not administered to these patients.

In the patients from this comparison group, the blood ammonia remained values without additional lactoferrin therapy during the first three days of Observation period increased significantly above the norm and only showed there after a gradual tendency to decrease. At the end of the monitoring however, the ammonia levels in the blood of this patient group are still significant higher than for the lactoferror group (Tab. I).

Table I

Blood ammonia values (µg / dl) in patients with liver failure. Lactoferrin therapy group (n = 7) and untreated control group (n = 12). The mean values and the standard deviation are given

Example 3

When lactoferrin was given enterally, there was also a favorable influence on bilirubin levels in the blood in acute liver failure, which had increased as part of liver failure. A further increase in bilirubin could be prevented by the lactoferene administration.

• a) In a 59-year-old woman had liver failure developed hyperbilirubinaemia. The bilirubin concentration in the blood was increased to 8.2 mg / dl. At this point, the application  started by lactoferrin. The lactoferrin was in a dosage of Administer 200 mg each at 8 hourly intervals using a gastric tube enough. After the start of therapy, a further increase in the Bilirubins observed, its concentration 48 hours after the start of the Lactoferrin therapy was 8.4 mg / dl. At this point, however, continued a continuous decrease in the level of bilirubin in the blood. The Lactofering was administered over a total of 12 days. On the 13th day after the start of therapy, the bilirubin concentration in the patients' blood was tin only 3.2 mg / dl. Overall, the patient's condition had that was somnolent at the start of lactoferrin therapy, under the dose significantly improved by lactoferrin.
• b) In a 54-year-old man with cirrhosis of the liver, there was a continuous increase in the level of bilirubin in the blood in the context of acute liver failure. Lactoferrin was administered to this patient via a gastric tube at a dose of 200 mg each at a time interval of 6 hours. At the start of lactoferrin administration, the bilirubin concentration in the blood was 10.6 mg / dl. Approx. 48 hours after the start of lactoferrin administration, the bilirubin concentration was 9 mg / dl. The lactoferrin application was stopped when the bilirubin concentration in the blood was only 7.1 mg / dl 4 days after the start of lactoferrin administration. After the lactoferrin dose was stopped, however, a continuous increase in the bilirubin concentration in the blood was measured.
  6 days after the end of lactoferrin administration, the bilirubin concentration in the blood was already 17.9 mg / dl.

Example 4

In a 46-year-old woman who had acute viral hepatitis was administered approximately 7 days after the first manifestation of the disease Lactoferrin per os. began. It became lactoferrin (bovine) in one Dosage given 4 times 1 g per day.

At the beginning of lactoferrin administration, an activity of 835 [U / l] and for GPT 1.316. [U / l] measured. A value was set for the Gamma GT found from 146 [U / l]. Lactoferrin was administered for a total of 7 days carried out. There was no further increase in AK when lactoferrin was given to monitor the activity of these enzymes in the blood. There was a gradual decrease  the activity of these enzymes in the blood.

At the end of lactoferrin administration, i.e. H. about 2 weeks after clinical First manifestation of hepatitis was an activity of 612 [U / l] for GOT GPT: 708 [U / l] and measured for gamma GT: 94 [U / l].

After stopping the lactoferrin dose was initially in the further course no significant change in the still increased enzyme activities in the blood take care. Only three weeks after the lactoferrin dose was stopped further, significant decrease in enzyme activity was observed.

Example 5

With the enteral administration of lactoferrin, an improvement in the restricted protein synthesis in the liver could also be observed in severe liver dysfunction. Albumin, fibrinogen and transferrin were measured in the blood as parameters for protein synthesis

• a) In a 53-year-old man who had acute liver failure as a result of severe cirrhosis of the liver, lactoferrin was administered at a dose of 1500 mg / day. At the start of lactoferrin administration, the following concentrations were measured for albumin, fibrinogen and transferrin: albumin 2.1 g / dl, fibrin gene: 41 mg / dl; Transferrin 83 mg / dl.
  The following concentrations were measured in the blood 48 hours after the start of lactoferrin administration: albumin 2.9 g / dl, fibrinogen: 108 mg / dl; Transferrin 132 mg / dl.
• b) In a 59-year-old man with liver failure with high-grade cirrhosis, lactoferrin was administered via a probe at a dose of 1200 mg / day. At the start of lactoferrin administration, the following concentrations were measured for albumin, fibrinogen and transferrin: albumin 2.5 g / dl; Fibrinogen: 72 mg / dl; Transferrin 61 mg / dl.
  The following concentrations were measured after a total of 6 days of lactoferrin administration: albumin 3.2 g / dl; Fibrinogen: 152 mg / dl; Transferrin 158 mg / dl.

Claims (6)

1. Use of lactoferrin for prophylaxis and The Therapy of liver dysfunction and / or liver paralysis chemical damage and its consequences, unless an endo toxinemia is the indication for administration. 2. Use of lactoferrin according to claim 1, characterized characterized that the lactoferrin per os or over a probe is administered. 3. Use lactoferrin according to any of the previous ones existing claims, characterized in that it is with liver parenchyma damage around a fatty degeera tion of the liver cells. 4. Use of lactoferrin according to claim 1 or 2, characterized in that it is in the liver radio disorder is the consequences of a congestion liver. 5. Use of lactoferrin according to claim 1 or 2, characterized in that the liver parenchyma damage has led to portal hypertension. 6. Use of lactoferrin according to claim 1 or 2 for the prophylaxis and therapy of intoxication by Am moniac in liver dysfunction.