Use of human lactoferrin apo form as antihypoxant and hypoxia inducible factor-1 alpha stabiliser

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly, the use of human lactoferrin apo-form as an antihypoxant and a hypoxia inducible factor-1 alpha stabiliser.

EFFECT: invention provides the use of the natural iron chelator lactoferrin, no toxicity, hypoallergenicity, an ability to penetrate through the bowel into the blood flow and through the blood-brain barrier.

2 tbl, 1 dwg

 

The invention relates to medicine, in particular to the correction of hypoxic conditions. This correction can be carried out with the help of application of the known drug lactoferrin for a new purpose.

The problem pharmacological correction of hypoxic States currently refers to the priority. The relevance of the application of antihypoxants due to the extremely wide distribution of the hypoxia that occurs in conditions of oxygen deficiency in the external environment, and as a result of various pathological conditions associated with dysfunction of the respiratory, cardiovascular systems, as well as transport function of blood. In all cases, ultimately, there is a reduction of oxygen delivery to the tissues to a level insufficient to maintain function, metabolism and cell structure. According to modern concepts hypoxic conditions arise in almost any pathology [Bulletin of the Russian Academy of medical Sciences, 1999, No. 3, p.18-25]. Since 1993, in "Drugs" antihypoxants in a separate group.

Currently, it is shown that to prevent hypoxic conditions are promising compounds with properties of chelators, for example, a derivative of coumarin under laboratory cipher πQ226 [Vestn. Smolensk. The honey. Acad., 2000, No. 3, s-95.

With the 70-ies of the last century medicinal product Desferal (DFO, N-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-oxobutyl}amino)pentyl]-N-hydroxysuccinimide, the iron chelator) is used in case of poisoning by iron and aluminum, hemochromatosis in patients with certain types of chronic anemia (thalassemia and myeloblastic syndrome), which require multiple transfusions of red blood cells, leading to increased toxicity of iron in the tissues. In addition Desferal, currently with similar pathologies used commercially available synthetic pharmacological agents (chelators of iron) - deferasirox (FID) and deferiprone (Suite) [Hematologica, 2004, v.89, No. 10, p.1187-1193].

The effectiveness of these three drugs is explained by lower levels of ferritin in the blood of such patients with congenital chronic anemia, because elevated levels of ferritin is a prognostic sign the risk of developing hemochromatosis. However, synthetic drugs have side effects, such as DFO leads to stunted growth in children, clouding of the cornea, blindness and deafness, the drug is contraindicated in pregnancy and lactation [Reference Vidal, 1995, s]. For sequential function typical side effect is agranulocytosis, and FID causes nausea, diarrhea and leads to renal dysfunction [Blood, 2006, .91, No. 3, p.873-880].

Since 1993 it is known that DFO is pharmacological mimetic of hypoxia, causing stabilization of hypoxia-inducible factor-1 alpha (HYPHAE-1A). HYPHAE-1A is a pleiotropic transcription factor that is constantly synthesized in the cell and undergo degradation when normoxia and in the absence of iron deficiency.

In the case of hypoxia or iron deficiency inactivated hydroxylase enzyme, gidrauxiliruetsa HYPHAE-1A amino acid residues (Proline, asparagine), which leads to stabilization SWFS-1A due to the impossibility of its further ubiquitination and proteosomal degradation.

Thus, during hypoxia or iron deficiency (including, by the action of the iron chelator DFO [Blood, 1993, v.82, no. 12, p.3610-3615]) there is a stabilization SWFS-1A, which is transported into the nucleus where it forms heterodimer with HYPHAE-beta and activates the transcription of more than 200 genes, half of which are responsible for the survival of the cells. Targets HYPHAE-1A, which is a key regulator of pathophysiological response to stress and hypoxia, are genes involved in the metabolism of iron (transferrin and its receptor, ferritin, ceruloplasmin, hepsidin, heme oxygenase), angiogenesis (growth factor endothelium - VEGF), erythropoiesis (EPO), vasodilation (inducible synthase nitric oxide), cell proliferation (insuli podobny growth factor - IGF2), glucose metabolism (glucose Transporter - Glut1,3), in the regulation of pH (carboxylic anhydrase 9) and oncosuppressor (p53, P21, P27) [Exp. Neorol. 2009, v.216, No. 1, p.7-15; Antioxidants and Redox Signaling, 2010, v.12, No. 4, p.445-458], i.e. the genes responsible for many processes in the body.

Drugs, stabilizing HYPHAE-1A, are promising as preconditioners incentives in the treatment of ischemia. So, the far Eastern Federal district, which for more than 30 years used for the correction of intoxication iron and anemia, is currently undergoing clinical trials for stroke [Stroke, 2009, v.40, No. 3, p.90-91]. It is also known about the neuroprotective properties of DFO on animal models of Alzheimer's disease [Eur. J. Neurosci., 2009, v.29, No. 7, p.1323-1334] and Parkinson's disease [Ann. NY Acad. Sci., 2004, v.1012, p.306-325; Neourochem. Int., 2010, v.57, no. 3, p.198-205]. DFO is effective preconditioners connection in models of neonatal and adult stroke.

Given the above protective effects HYPHAE-1a, use of drugs that cause its stabilization by reducing the pool of iron is promising to maintain tolerance to ischemia and neurodegeneration [Transl. Stroke Res., 2010, v.3, No. 11, p.19-30]. Inhibitors of prolyl-hydroxylase stabilizing HYPHAE-1a, are effective nephroprotection models cisplatino nephrotoxicity, experimental glomerulonephritis, diabetic nephropathy [J. Pharmacol. Sci., 2009, v.109, p.4-31].

Protein family transferrin, lactoferrin (LF), synthesized in the APO-form and is detectable in breast milk, in some exocrine secretion and secretory granules of polymorphonuclear leukocytes, is extremely multifunctional. In particular, LF has antimicrobial properties, transcription, immune, growth and anti-tumor factors.

A distinctive property of LF among other transferring is its high affinity for iron ions (Kand1020l/mol) and the stability of its iron complex in acidic medium until the pH to 3.0.

But still gelatine properties of LF not paid enough attention: the ability to sekvestrirovatj iron ions, necessary for the growth of microorganisms was considered only in the light of its bactericidal-bacteriostatic and antioxidant properties (chelating iron, LF prevents reactions Antonovsky chemistry is the formation of hydroxyl radicals, damaging all kinds of biomolecules).

Drugs, containing LF, used in medical practice, but for another purpose. So, injecting drug Laprot" (registration certificate dated 15.12.2006, N LS-002374) used in the treatment of multiple organ failure of various origins, purulent-septic complications after surgical interventions: 50 the g apaf person/in 5 days (resolution of the Federal service on surveillance in healthcare and social development from 29.12.2007 N, FS-2007/249). However, there is currently no clear idea of the pleiotropic mechanisms of action of LF.

Given that proteins with antioxidante properties can be an effective antihypoxants, for example, the primary antioxidant plasma ceruloplasmin [Expert. The wedge. Pharm., 2003, t, No.3, pp.62-65], the authors conducted a series of experiments to study the antihypoxic properties of LF and found that its APO-form triggers zelenodoliskiy stabilization SWFS-1A. Thus, the authors demonstrated antihypoxic effect APO-form LF (apaf) females C57Black mice (weighing 20-25 g) in a model of acute normobaric hypoxia with hypercapnia [Psychopharm. and Biol. narcology, 2005, volume 5, No. 3, s-983], which was created by placing mice in a sealed pharmacy stangles with a capacity of 200 ml

Highly Adolf was isolated from breast milk and put in/b in a dose of 5 mg per mouse. Control mice were injected W/saline or albumin in the same doses. About resistance to hypoxia was assessed by time elapsed before losing animals pose and appearance of the first signs of agonal breathing. Apaf at a dose of 5 mg was significantly increased life expectancy of up to 140% (p<0.05)by transferring 75% of mice from group discostick in the group's medium - and highly resistant to hypoxia animals.

Of particular interest was earned two facts: rich in iron LF not had the such actions (see Table 1), and the maximum protective effect APO-form LF came a day after the injection. This suggests that the main mechanism for preventing Adolf hypoxia is its chelating properties, causing activation of unknown protective genes. Effective antihypoxic drug was the iron chelator and the hypoxia mimetic - DFO (table 1). The selected dose of APO-LF and DFO (5 mg per mouse) correspond to the effective doses prevents endotoxemia adopted in the literature [Innate Immun. 2010, v.16, No. 2, page 67-79].

Assuming that gelatine properties of LF and its delayed (after 24 hours) antihypoxic effect provide its activity as a transcription factor, the authors found that APO-LF (5 mg/mouse intraperitoneally), as well as pharmacological mimetic of hypoxia, a synthetic iron chelator DFO (positive control), causes stabilization of HYPHAE-1a in mice and rats in the investigated organs (liver, lungs, heart, brain and kidneys, according to Western blotting with monoclonal antibodies against HYPHAE-1a, see table 2).

Of particular interest is the fact that monthly mice treated with water instead of breast milk (2 mg/ml APO-LF) or cow's milk with the addition of APO-LF showed stabilization SWFS-1a in the investigated organs (liver, lungs, heart, brain and kidneys, according to Western-blotty the ha monoclonal antibodies against HYPHAE-1a, see table 2) on the 3rd and 6th day of this diet. Unlike synthetic iron chelators (DFO according to literature data does not penetrate through the cell membrane), LF in adult rats penetrates through the gastrointestinal tract into the bloodstream through the lymphatic system [Exp. Physiol., 2004, v.89, p.263-270] and the blood-brain barrier [J. Vet. Med. Sci., 2008, v.70, no. 3, p.313-315] when administered orally. While the effect of DFO is achieved only in the case of intravenous or subcutaneous injection, and sequential function used in the form of tablets since 1987, is ineffective in the treatment of thalassemia compared with DFO [Ann. Hematol., 2006, v.85, No. 5, p.315-319].

For drugs LF is still not established toxic doses. When intravenous drug lactoferrin, known on the pharmaceutical market as a drug "Lapot", which usually introduce courses on 50 mg intravenously, there was no evidence of allergic reactions in doses up to 100 mg of Adolf cows in the form of tablets used abroad as Antianemic agent and oncosuppressor in cancer patients for preventive purposes, at the risk of the cancer without detection of side effects. Is known about the use of 200 mg LF daily for 3 months [Oncologist. 2010, v.15, No. 8, p.894-902] and up to 3 g LF of the day in the year [Cancer Prev. Res. (Phila), 2009. v.2, No. 11, 975-983] in cancer patients.

Given that stabilization SWFS-1a leads to activation of the adaptive mechanism is smow in cells it seems promising to use drugs, containing lactoferrin person, for a new purpose: to prevent neuro-degenerative, ischemic and hypoxic conditions, and chronic kidney disease. The drug can be administered intranasally in the form of tablets. Due to the fact that lactoferrin crosses the blood-brain barrier and into the bloodstream through the gastrointestinal tract, lactoferrin contained in preparations, easily bioavailable.

Table 1
The lifetime of the mice (min) in acute normobaric hypoxia with hypercapnia
The group of animalsThe number of animalsLifetime minStandard deviation, minThe reliability of differences between groups discostick
Highly stable19262P<0.01
Nizkosoleva45182
Nizkosoleva, the introduction of APO-LF per day to hypoxia11232P<0.05
Nizkosoleva, the introduction of APO-LF 1 hour before hypoxia11224
Nizkosoleva, the introduction of albumin per day to hypoxia12173
Nizkosoleva,11202
introduction iron LF per day to hypoxia
Nizkosoleva, the introduction of the far Eastern Federal district the day before hypoxia5253P<0.05

Table 2
Identification of immunoreactive areas HYPHAE-1A vorgang animals
AnimalsLiverLightThe brainHeartKidney
Mouse (APO-LF 5 mg/mouse intraperitoneally)+++++
Mouse (DFO 5 mg/mouse intraperitoneally)+++++
Mouse (nat. R-R intraperitoneally)-----
Mice (2 mg/ml APO-LF in the composition of breast milk)+++++
Mice (cow's milk)-----
Mice (cow's milk with the addition of APO-LF 2 mg/ml +++++

Studies of resistance to hypoxia was held at the old discostick to hypoxia 18-month-old mice. With the introduction of APO-LF in these animals was found to stabilize the HYPHAE-1a and parallel to the increased lifetime in acute normobaric hypoxia with hypercapnia. It is known that in aged mice and rats increased the activity of prolyl-hydroxylase, causing destabilization of the HYPHAL-1a (Am. J. Physiol. Regul. Integr. TCS. Physiol. 2009. V.297, 158-165 (in Russian)). Such animals are nizkosolevaya to hypoxia that does not meet the stabilization SWFS-1A and the expression of its targets in response to hypoxic stimulus. However, the hypoxia mimetic (cobalt chloride) is capable of stabilizing the HYPHAE-1A in these animals. You can offer APO-LF as a stabilizer HYPHAE-1A and effective preconditioners stimulus in the elderly.

The introduction of APO-LF causes not only the stabilization of the HYPHAL-1a (data 1A to identify immunoreactive areas of Western blot testing to HYPHAL-1a in tissue homogenates of mice 24 hours after the/b introduction 5 mg APO-LF on mouse - tracks 1-3, tracks 4-6 - positive control after the/b introduction 5 mg Desferal mouse, tracks 7-9 - negative control after the/b introduction 200 ál physio is oricheskogo solution, M - stained molecular weight marker (135 kDa): 1, 4, 7 - brain, 2, 5, 8 and liver 3, 6, 9 - buds), but also increase the synthesis of target genes of this transcription factor, erythropoietin and ceruloplasmin (data .1b to identify immunoreactive areas of Western blot testing for ceruloplasmin in tissue homogenates of mice 24 hours after injection of APO-LF - tracks 1-3, tracks 4-6 - positive control after the introduction of Desferal, tracks 7-9 - negative control after administration of saline, M - stained molecular weight marker (135 kDa): 1, 4, 7 - brain, 2, 5, 8 and liver 3, 6, 9 - buds). Increasing the concentration of erythropoietin in the serum of mice and rats detected by using enzyme-linked immunosorbent assay. The increase in the content of ceruloplasmin in the brain, liver, heart, lungs and kidneys detected using Western blotting with antibodies to the CPU mouse. Both proteins are responsible for adaptation to hypoxia and survival of neurons during inflammation and neurodegeneration.

The use of APO-form of human lactoferrin as antihypoxic drug and stabilizer hypoxia-inducible factor-1 alpha.



 

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