RU2465004C1 - Use of human lactoferrin apo form as antihypoxant and hypoxia inducible factor-1 alpha stabiliser - Google Patents

Abstract

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

Description

The invention relates to medicine, in particular to the correction of hypoxic conditions. This correction is proposed to be carried out using a well-known drug lactoferrin for a new purpose.

The problem of pharmacological correction of hypoxic conditions is currently one of the priority ones. The relevance of the use of antihypoxants is explained by the extremely widespread occurrence of hypoxia, which occurs both in conditions of oxygen deficiency in the external environment and as a result of various pathological conditions associated with impaired functions of the respiratory, cardiovascular systems, as well as the transport function of blood. In all cases, ultimately, there is a decrease in oxygen delivery to tissues to a level insufficient to maintain cell function, metabolism, and structure. According to modern concepts, hypoxic conditions occur in almost any pathology [Bulletin of the Russian Academy of Medical Sciences, 1999, No. 3, pp. 18-25]. Since 1993, in the “Medicines” manual, antihypoxants have been singled out as an independent group.

It has now been shown that to prevent hypoxic conditions, compounds with the properties of chelators are promising, for example, the coumarin derivative under the laboratory code πQ226 [Vestn. Smolensk. Honey. Acad., 2000, No. 3, pp. 94-95].

Since the 70s of the last century, the drug desferal (DFO, N- {5- [acetyl (hydroxy) amino] pentyl} -N- [5 - ({4 - [(5-aminopentyl) (hydroxy) amino] -4-oxobutaneol } amino) pentyl] -N-hydroxysuccinamide, an iron chelator) is used for iron and aluminum poisoning, hemochromatosis in patients with certain types of chronic anemia (thalassemia and myeloblastic syndrome), which require multiple transfusions of red blood cells, which leads to an increase in toxic iron in tissues. In addition to desferal, commercially available synthetic pharmacological agents (iron chelators) —deferasirox (DFS) and deferiprone (DFP) are currently used in such pathologies [Hematologica, 2004, v. 89, No. 10, p.1187-1193].

The effectiveness of these three drugs is explained by a decrease in the level of ferritin in the blood in such patients with congenital chronic anemia, since an increased level of ferritin is a prognostic sign of the risk of developing hemochromatosis. However, synthetic drugs have a number of side effects, for example, DFO leads to growth retardation in children, clouding of the cornea, blindness and deafness, the drug is contraindicated in pregnancy and lactation [Reference Vidal, 1995, p. 418]. For DPP, agranulocytosis is a characteristic side effect, and DGF causes nausea, diarrhea and leads to renal dysfunction [Blood, 2006, v. 91, No. 3, p. 873-880].

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

In the case of hypoxia or iron deficiency, hydroxylases, enzymes hydroxylating GIF-1a in amino acid residues (proline, asparagine), are inactivated, which leads to stabilization of GIF-1a due to the impossibility of its further ubiquitinylation and proteosomal degradation.

Thus, during hypoxia or iron deficiency (including due to the action of the iron chelator DFO [Blood, 1993, v.82, No. 12, p.3610-3615]), GIF-1a is stabilized, which is transported to the nucleus, where forms a heterodimer with GIF-1 beta and activates the transcription of more than 200 genes, half of which are responsible for cell survival. The targets of GIF-1a, which is a key regulator of the pathophysiological response to stress and hypoxia, include genes involved in iron metabolism (transferrin and its receptor, ferritin, ceruloplasmin, hepsidin, hemoxygenase), in angiogenesis (endothelial growth factor - VEGF), in erythropoiesis (erythropoietin), in vasodilation (inducible nitric oxide synthase), in cell proliferation (insulin-like growth factor - IGF2), in glucose metabolism (glucose transporter - Glut1,3), in pH regulation (carboxylic anhydrase 9) and in cancer suppression (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. genes responsible for many processes in the body.

Drugs that stabilize GIF-1a are promising as preconditioning stimuli in the treatment of ischemia. So, the Far Eastern Federal District, which has been used for more than 30 years to correct iron intoxication and anemia, is currently undergoing clinical trials for strokes [Stroke, 2009, v.40, No. 3, p.90-91]. The neuroprotective properties of DFO in an animal model of Alzheimer's disease are also known [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 an effective preconditioning compound in neonatal and adult stroke models.

Given the protective effects of GIF-1a listed above, the use of drugs that cause its stabilization by reducing the iron pool is promising for maintaining tolerance to ischemia and neurodegeneration [Transl. Stroke Res., 2010, v. 3, No. 11, p. 19-30]. Prophylactic hydroxylase inhibitors stabilizing GIF-1a are effective nephroprotectors in cisplatin nephrotoxicity models, experimental glomerulonephritis, and diabetic nephropathy [J. Pharmacol Sci., 2009, v.109, p.24-31].

A protein of the transferrin family, lactoferrin (LF), synthesized in apo form and found in breast milk, in a number of exocrine secrets and secretory granules of neutrophilic leukocytes, is characterized by extreme multifunctionality. In particular, LF has the properties of antimicrobial, transcriptional, immunomodulating, growth and antitumor factors.

A distinctive property of LF among other transferrins is its high affinity for iron ions (K _a 10 ²⁰ l / mol) and the stability of its iron complex in an acidic environment up to pH 3.0.

However, until now, insufficient attention has been paid to the chelating properties of LF: the ability to sequestrate the 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 the reactions of Fenton chemistry - the formation of hydroxyl radicals that damage all types of biomolecules )

Preparations containing LF are used in medical practice, but for a different purpose. So, the injection drug "Laprot" (registration certificate dated 15.12.2006, N LS-002374) is used in the complex therapy of multiple organ failure of various origins, purulent-septic complications after surgical interventions: 50 mg of apoLF human iv for 5 days (resolution Federal Service for Supervision of Health and Social Development of December 29, 2007 N, FS-2007/249). However, currently there is no clear understanding of the mechanisms of pleiotropic action of LF.

Given that proteins with antioxidant properties can be effective antihypoxants, for example, the main plasma antioxidant ceruloplasmin [Expert. Wedge. Farm., 2003, vol. 66, No. 3, pp. 62-65], the authors first conducted a series of experiments to study the antihypoxic properties of LF and revealed that its apo form triggers the mechanism of iron-dependent stabilization of GIF-1a. Thus, the authors demonstrated the antihypoxic effect of the apo-form of LF (apoLF) in female C57Black mice (weight 20-25 g) in a model of acute normobaric hypoxia with hypercapnia [Psychopharm. and biol. narcology, 2005, v.5, No. 3, pp. 979-983], which was created by placing mice in an airtight pharmacy booth with a capacity of 200 ml.

Highly purified apoLF was isolated from breast milk and was administered ip in a dose of 5 mg per mouse. Control mice were injected with saline or albumin in the same doses. Resistance to hypoxia was judged by the time elapsed before the animals lost their posture and the first signs of agonal breathing appeared. ApoLF at a dose of 5 mg significantly increased the life time to 140% (p <0.05), transferring 75% of mice from the group of low-resistance to the group of medium- and highly resistant to hypoxia animals.

Two facts deserved special interest: iron-rich LF did not have such an effect (see Table 1), and the maximum protective effect of the apo-form of LF occurred a day after the administration of the drug. This makes us think that the main mechanism for preventing apoLF of hypoxia lies in its chelating properties that cause activation of unknown protective genes. The iron chelator and hypoxia mimetic, DFO, were found to be effective antihypoxants (Table 1). The selected doses of apo-LF and DFO (5 mg per mouse) correspond to the effective doses that prevent endotoxemia from the literature [Innate Immun. 2010, v.16, No. 2, p. 67-79].

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

Of particular interest is the fact that monthly mice fed breast milk instead of water (2 mg / ml apo-LF) or cow's milk supplemented with apo-LF showed stabilization of GIF-1a in the studied organs (liver, lungs, heart, brain, and kidney, according to Western blotting with monoclonal antibodies against GIF-1a, see Table 2) on days 3 and 6 of such a diet. Unlike synthetic iron chelators (according to published data, DFO does not penetrate 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 injections, and DFT, 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].

Toxic drugs have not yet been established toxic doses. With the intravenous administration of the drug lactoferrin, known on the pharmacological market as the drug "Laprot", which is usually administered in courses of 50 mg intravenously, there were no allergic reactions in doses up to 100 mg. Cow apoLF in the form of tablets is used abroad as an antianemic agent and an oncosuppressant in cancer patients and for prophylactic purposes at the risk of oncology without detecting side effects. It is known about the use of 200 mg of LF daily for 3 months [Oncologist. 2010, v.15, No. 8, p. 894-902] and up to 3 g of LF per day for a year [Cancer Prev. Res. (Phila), 2009. v.2, No. 11, 975-983] in cancer patients.

Given that the stabilization of GIF-1a leads to the activation of adaptive mechanisms in cells, it seems promising to use preparations containing human lactoferrin for a new purpose: for the prevention of neurodegenerative, ischemic and hypoxic conditions, as well as for chronic kidney diseases. The drug can be administered intranasally and in tablet form. Due to the fact that lactoferrin penetrates the blood-brain barrier, as well as into the bloodstream through the gastrointestinal tract, the lactoferrin contained in the preparations is easily bioavailable.

Table 1 Mouse life time (min) in acute normobaric hypoxia with hypercapnia Group of animals Number of animals Life time, min Standard deviation, min Significance of differences from the low-resistance group Highly resistant 19 26 2 P <0.01 Low resilience 45 eighteen 2 Low resistance, administration of apo-LF one day before hypoxia eleven 23 2 P <0.05 Low resistance, administration of apo-LF 1 hour before hypoxia eleven 22 four Low tolerance, administration of albumin the day before hypoxia 12 17 3 Low resistance eleven twenty 2 the introduction of iron LF the day before hypoxia Low resistance, the introduction of DFO the day before hypoxia 5 25 3 P <0.05

table 2 Identification of immunoreactive zones of GIF-1a in animal organs Animals Liver Lungs Brain Heart Kidney Mice (apo-LF 5 mg / mouse intraperitoneally) + + + + + Mice (DFO 5 mg / mouse intraperitoneally) + + + + + Mice (fiz. Rr intraperitoneally) - - - - - Muscle (2 mg / ml apo-LF in breast milk) + + + + + Mouse (cow's milk) - - - - - Mouse (cow's milk with the addition of apo-LF 2 mg / ml) + + + + +

Experiments to study resistance to hypoxia were carried out on old, low-resistance to hypoxia 18-month-old mice. With the introduction of apo-LF in these animals, stabilization of GIF-1a was revealed and, in parallel, the lifespan increased in conditions of acute normobaric hypoxia with hypercapnia. It is known that in old mice and rats, the activity of prolyl hydroxylases that cause destabilization of GIF-1a is increased (Am. J. Physiol. Regul. Integr. Coll. Physiol. 2009. V.297, 158-165). Such animals are low resistant to hypoxia, not responding to the stabilization of GIF-1a and the expression of its targets in response to a hypoxic stimulus. However, a hypoxia mimetic (cobalt chloride) is able to stabilize GIF-1a in such animals. Apo-LF can be proposed as a stabilizer of GIF-1a and an effective preconditioning stimulus in elderly people.

The administration of apo-LF does not only stabilize GIF-1a (the data in Fig. 1a for the identification of immunoreactive zones by Western blotting for GIF-1a in mouse tissue homogenates 24 hours after iv administration of 5 mg of apo-LF per mouse - lanes 1 -3, lanes 4-6 - positive control after iv administration of 5 mg of desferal per mouse, lanes 7-9 - negative control after iv administration of 200 μl of physiological saline, M - stained molecular weight marker (135 kDa): 1 , 4, 7 - the brain, 2, 5, 8 - the liver and 3, 6, 9 - the kidneys), but also an increase in the synthesis of target genes of this transcription factor, erythropoietin and ceruloplasmin (data on Fig. 1b for the identification of immunoreactive zones by Western blotting for ceruloplasmin in mouse tissue homogenates 24 hours after administration of apo-LF - lanes 1-3, lanes 4-6 - positive control after administration of desferal, lanes 7- 9 - negative control after administration of saline, M - stained molecular weight marker (135 kDa): 1, 4, 7 - brain, 2, 5, 8 - liver and 3, 6, 9 - kidneys). An increase in the concentration of erythropoietin in the blood serum of mice and rats was detected using enzyme-linked immunosorbent assay. An increase in ceruloplasmin in the brain, liver, heart, lungs and kidneys was detected using Western blotting with antibodies to mouse CP. Both proteins are responsible for adaptation to hypoxia and the survival of neurons in inflammation and neurodegeneration.

Claims (1)

The use of the apo-form of human lactoferrin as an antihypoxant and stabilizer of hypoxia-inducible factor-1 alpha.

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