RU2435574C1 - Method of treating anaemia, associated with failure of porphyrins synthesis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to hematology, and can be applied for treatment of hereditary anemias, associated with failure of porphyrine synthesis. For this purpose per orally introduced is 5-aminolevulinic acid or its hexyl ether in doses, which insure compensation of insufficient biosynthesis of 5-aminolevulinic acid by organism.

EFFECT: method insures fast and effective increase of hemoglobin level in treatment of said pathology in experiment.

3 ex, 4 dwg

Description

The invention relates to medicine, more specifically to hematology, and can be used in the treatment of hereditary anemia associated with impaired synthesis of porphyrins.

Porphyrin biosynthesis is a complex process, in the case of heme biosynthesis consisting of nine consecutive enzymatic reactions. The first specific intermediate in the pathway of heme biosynthesis is 5-aminolevulinic (5-amino-4-oxopentanoic) acid (ALA), which is formed from glycine and succinyl-CoA by the action of the enzyme ALA synthetase. To maintain a normal level of hemoglobin in the blood, a healthy person produces an average of about 0.2 g of ALA daily. A feature of the regulation of heme biosynthesis is that its deficiency leads to the activation of ALA synthetase, an increase in the activity of which leads to an increase in ALA biosynthesis and, ultimately, to an increase in heme biosynthesis.

A genetic defect in ALA synthetase causes a lack of ALA in the body, which leads to ineffective erythropoiesis, the development of anemia, leads to triggering mechanisms of increased absorption and metabolism of iron, and a pathological increase in iron content.

In the human body, there are two different forms of the enzyme ALA synthetase - hepatic and erythrocytic. The liver form is expressed mainly in the liver and produces ALA for heme synthesis, which is then used in heme-containing liver proteins - various cytochromes. The red blood cell, respectively, is expressed in the cells of the erythroid series and produces ALA for the synthesis of hemoglobin, which is more than 80% of ALA produced by the body. Enzymes differ in how they are regulated in the body and have different genes encoding them.

The erythrocyte ALK synthetase is encoded by a gene located on the X chromosome. More than 25 different mutations of the gene encoding ALA synthetase are known, which gives a great variety in the nature of the damage to the enzyme and, therefore, in the clinical picture of the disease. The disease affects mainly men. Patients either die at a young age from anemia or, if the anemia is not severe, suffer from diseases associated with an excess of iron, such as diabetes mellitus, cirrhosis of the liver, hormonal and heart failure.

In world practice, vitamins of the B6 group are used to treat such hereditary anemia, since pyridoxal-5'-phosphate is a coenzyme of ALA synthetase (Alcindor T, Bridges KR. Sideroblastic anaemias. Br J Haematol. 2002, 116 (4), 733-743 ) However, treatment is not always effective, because it depends on the nature of the damage to the function of the enzyme ALA synthetase. So, with some mutations of the ALA synthetase gene, anemia may be resistant to therapy with B6 vitamins. In this case, only maintenance therapy is possible, aimed at removing pathologically excess iron from the body. For this purpose, desferal (iron-binding drug) injections are used for long courses of 3-6 times a year. This is a very difficult and expensive method of treatment, moreover, aimed only at temporarily eliminating the consequences of the underlying pathology.

The objective of the invention is the treatment of hereditary anemia associated with impaired synthesis of porphyrins. The problem is solved by the use of ALA or its hexyl ether for treatment by oral administration into the body.

The use of ALA or its ester interventions to solve the problem is not an obvious solution. So, with a deficiency of one of the heme biosynthesis enzymes following ALA synthetase, a vicious pathological circle also forms: a lack of hemin causes an avalanche-like overproduction of ALA and, depending on the type of the missing enzyme, other porphyrin metabolism products that have a high degree of toxicity. As a result, there are diseases that are collectively called porphyria. Porphyria are predominantly hereditary diseases. They are based on the mutation of certain genes responsible for the activity of enzymes catalyzing heme synthesis. The genetics of porphyria is well studied; for each form of the disease, one or another biochemical or enzymatic defect causing it is established. Acute intermittent porphyria (an autosomal dominant type of inheritance) caused by a deficiency of porphobilinogen deaminase, the third enzyme in the biosynthesis chain, is more common than others. The high content of ALA in the blood and, as a result, in the urine is one of the main diagnostic criteria for the disease.

Moreover, the phototoxicity of ALA metabolites and ALA esters is based on their use in medicine as a sensitizer for fluorescence diagnostics and photodynamic therapy of a number of oncological diseases [J Eur Acad Dermatol Venereol., 2007, 21 (3), 293-302; RF patent No. 2191010]. This application is based on the fact that in most tumor cells, the activity of porphobilinogen deaminase increases and the activity of ferrochelatase decreases, which in turn leads to an increased content of fluorescent protoporphyrin IX (PP-IX), which is especially pronounced with the additional administration of ALA. A single oral administration of a large dose of ALA causes the accumulation of PP-IX in the tumor for several hours, its high level is maintained for 1-2 hours, while in normal cells, PP-IX is quickly utilized by converting it into heme. The result is a high fluorescence contrast of the tumor and the surrounding tissue, reaching 10-15 times the magnitude for various tumors, which is an important factor in the visualization of neoplasms during photodiagnostics and determines the local therapeutic effect without damaging the surrounding tissue when exposed to light during photodynamic therapy.

Thus, the high content of ALA in the body as a result of a pathological violation of heme biosynthesis or as a result of the introduction of ALA from the outside can lead to specific phototoxic damage to the body. However, we have shown that ALA and its hexyl ether can be used for the treatment of hematological diseases, namely for the treatment of hereditary anemia associated with impaired porphyrin synthesis.

Toxicity and ALA hexyl ALA esters with a single oral administration was investigated on mice hybrids F ₁ males and neinbrednyh male rats. It was shown that a single oral administration of drugs in doses from 10 to 1500 mg / kg did not lead to the death of mice during the entire observation period (14 days), at doses of 10 and 100 mg / kg there were no clinical signs of intoxication during the entire observation period . At doses of 500 and 1500 mg / kg for 3 days after administration of the drugs, external signs of toxicity were observed in animals.

Based on these data on the study of “acute” toxicity in mice, ALA and ALA hexyl esters are classified as low toxic pharmacological substances.

Morphological studies of blood cells and internal organs showed that, with a single oral dose of 1250, 625 mg / kg and lower, the drugs did not have toxic effects on peripheral blood, liver, kidneys, gastrointestinal tract and central nervous system (according to the analysis of behavioral reactions) in rats .

When administered orally seven times in total doses of 1,500 and 3,000 mg / kg, the preparations did not have a toxic effect on peripheral blood, liver and kidneys. Neurotoxicity (changes in behavioral reactions) and gastrointestinal toxicity were also absent.

To ensure regular administration, the drug can be administered with food several times a day, as in the examples below. With a single single administration, as can be seen from Figure 1, in rats after 10 hours the ALA content in the body is still quite high. After 24 hours, it is significantly reduced. Given the higher metabolic rate in rodents in relation to humans, for humans, regular administration will be once a day, or even less.

Knowing the average blood content in the human body (7% of body weight), the average hemoglobin in the blood is 160 g / l, the average red blood cell life is 120 days, and the molecular weights of hemoglobin and ALA, given that the synthesis of one molecule heme consumes 4 ALA molecules, and taking into account ineffective erythropoiesis of 10%, it can be calculated that 3.3 mg of ALA per kg of body weight is formed daily in the human body to replenish blood hemoglobin.

In mice, the average erythrocyte life time is 30 days (A.A. Kudryavtsev, L.A. Kudryavtseva. Clinical hematology of animals, M .: Kolos (1974), pp. 374-389), and accordingly, the daily formation of ALA will be 13, 4 mg / kg, i.e. four times higher.

Effective daily doses for humans are also expected to be four times less.

Doses causing a positive effect in mice in our examples are 5-50 times higher than the calculated amounts of their own biosynthesis. The use of daily doses less than the calculated amount of normally synthesized ALA will be obviously ineffective. The use of doses greater than that proven in the examples is unjustified and potentially dangerous.

In humans, we expect ALA to be effective in doses of 3 to 150 mg / kg ALA.

In our examples, the doses providing coverage for insufficient ALA biosynthesis by the body exceed the calculated amount of ALA necessary to maintain its normal level. The introduction of endogenous ALA for the treatment of the disease may be associated with a risk of poisoning with porphyrin metabolism products of the type of porphyria. Therefore, during the treatment, it is advisable to control the concentration of ALA in the body in order to select an individual dose.

The therapeutic efficacy of ALA and ALA hexyl ester is shown in a model for replacing ALA synthesis with chloramphenicol against the background of hyperplasia of erythroid cells caused by phenylhydrazine. This biological model reflects the acute development of anemia caused by a deficiency of the erythrocyte ALK synthetase enzyme.

Intraperitoneal administration of phenylhydrazine to animals causes intravascular hemolysis of red blood cells. Long-term administration of phenylhydrazine in small doses leads to the development of chronic hemolytic anemia, which, in turn, leads to the occurrence of compensatory erythroid hyperplasia (reticulocytosis in peripheral blood up to 30-40%) and provides a situation in which mainly erythroid cells are present in the bone marrow (Firkin FC. Mitochondrial lesions in reversible erythropoietic depression due to chloramphenicol. J Clin Invest. 1972, 51 (8), 2085-2092). Such a compensatory mechanism against a background of chronic hemolytic anemia provides a certain constant level of blood hemoglobin and thereby the life of animals.

The introduction of chloramphenicol against this background causes a sharp decrease in the activity of the enzyme ALA synthetase in reticulocytes (Rosenberg A, Marcus O. Effect of chloramphenicol on reticulocyte delta-aminolaevulinic acid synthetase in rabbits. Br J Haematol. 1974, 26 (1), 79-83) . Reticulocytosis does not develop. Due to suppression of erythropoiesis, there is no compensation for hemolytic anemia, the hemoglobin level decreases evenly, until the death of the animal.

At the same time, regular administration of ALA or ALA esters to animals with anemia caused their survival and increased hemoglobin levels in the blood.

The present invention is illustrated by the following drawings.

Figure 1 - change in the concentration of ALA in the urine of rats that received an intragastric single dose of ALA:

curve 1-210 mg / kg,

curve 2-370 mg / kg.

Figure 2 - change in the concentration of hemoglobin in the blood of mice:

curves 1 - control group: phenylhydrazine intraperitoneally;

curves 2 - group 2a: phenylhydrazine intraperitoneally, chloramphenicol with food;

curves 3 - group 2b: phenylhydrazine intraperitoneally, chloramphenicol with food, after 7 days - ALA orally.

Figure 3 - survival curves of mice:

curve 1 - control group,

curve 2 - the experimental group receiving ALA (4 g / kg of feed).

Figure 4 - survival curves of mice:

curve 1 - control group,

curve 2 - experimental group treated with ALA hexyl ester (0.72 g / kg feed).

The present invention is illustrated by the following examples.

Example 1

Two groups of mice - control (4 mice) and experimental (6 mice) - with an average weight of 33 g received a complete synthetic diet containing chloramphenicol (HF) in concentrations of 0 (control) and 5 g / kg of dry food. Starting from the third day, all animals began to receive intraperitoneal injections of phenylhydrazine at 48 mg / kg body weight with a frequency of three times a week.

On the eighth day, the second (experimental) group was divided into two subgroups. The second subgroup (26) began to administer an oral ALA solution at a dose of 700 mg / kg daily. All groups of animals reduced the load of phenylhydrazine - injected 3 times a week at 24 mg / kg of body weight.

The results of hemoglobin measurements are shown in figure 2; the resulting picture is similar to the results of the work (Firkin FC. Mitochondrial lesions in reversible erythropoietic depression due to chloramphenicol. J Clin Invest. 1972; 51 (8), 2085-2092), where rabbits were used and chloramphenisole was administered intraperitoneally. Regular administration of phenylhydrazine causes a drop in hemoglobin levels in all groups. However, if in the control group hemoglobin stabilizes at a certain level at which chronic hemolytic anemia is compensated by erythroid hyperplasia (group 1, curves 1), in mice receiving chloramphenicol with a diet (group 2a, curves 2), compensation does not occur. The hemoglobin level in their blood drops, and no later than the 13th day all animals die. The introduction of ALA reduces the mortality of mice receiving chloramphenicol with a diet (group 2b, curves 3): on the 21st day, two out of three animals remained alive, they showed an increase in hemoglobin level.

Example 2

Two groups of mice, experimental and control, 8 animals with an average weight of 23 g, received a standard food containing 5 g / kg of chloramphenicol. The food of the experimental group contained an additional 4 g / kg ALA; thus, the daily dose of animals from the experimental group was ~ 400 mg / kg ALA. Three times a week, all animals received intraperitoneal injections of a phenylhydrazine solution at a dose of 60 mg / kg body weight. The duration of the experiment was 30 days.

Survival results are shown in figure 3. From the 10th to the 30th day of observation, 5 out of 8 animals of the control group fell (curve 1). At the same time, all animals of the experimental group remained alive (curve 2). A comparative statistical analysis of survival curves was carried out according to the logrank test. The obtained differences in survival were significant (p = 0.025).

Example 3

In the presented experiment, hemolytic anemia was caused in small doses of phenylhydrazine in animals and in the control and experimental groups with the development of a compensatory mechanism against the background of chloramphenicol administration. Then the experiment was started, the experimental group with the diet was additionally injected with ALA hexyl ester, while significantly increasing the dose of phenylhydrazine.

Two groups of mice, experimental and control, 6 animals with an average weight of 49 g, received a standard food containing 5 g / kg of chloramphenicol. Three times a week, all animals received intraperitoneal injections of a phenylhydrazine solution at a dose of 40 mg / kg body weight. After such preparation, which lasted 20 days, an experiment was started: HE ALA was added to the food of the experimental group in the amount of 0.72 g / kg of dry food, and thus, the daily dose of animals from the experimental group was ~ 72 mg / kg; the phenylhydrazine load was increased to 33 mg / kg daily. Survival results are shown in figure 4. In the control group (curve 1), all animals died in the first four days of the experiment. In the experimental group (curve 2), by the sixth day, 3 out of 6 mice survived.

A comparative statistical analysis of survival curves was carried out according to the logrank test. The obtained differences in survival were significant (p = 0.025).

Thus, 5-ALA and its hexyl ether ensure the survival of animals with anemia associated with impaired porphyrin synthesis.

Claims (1)

The use of 5-aminolevulinic acid or its hexyl ester for the treatment of hereditary anemia associated with impaired synthesis of porphyrins by their oral administration.

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