RU2448712C2 - Prostaglandin/cyclooxygenase pathway modulation - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmaceutics and medicine and concerns using 7α-hydroxy-epiandrosterone or 7β-hydroxy-epiandrosterone for preparing a drug for an inflammatory state specified in inflammation, inflammation disease of peripheral organs specified in liver or kidneys, inflammation respiratory disease, diabetes mellitus, inflammation-associated pain, inflammatory skin disease, multiple sclerosis, rheumatoid arthritis, osteoarthritis and complications thereof.

EFFECT: development of the drug preparation for the inflammatory state.

4 cl, 18 tbl, 7 ex

Description

The present invention relates to a number of compounds that have been found to modulate metabolic pathways that determine the activity of cyclooxygenase and prostaglandinsynthases, as well as the synthesis of prostaglandins, and, thus, can be used for the treatment and prevention of various diseases and disorders that were immune to the previous ones treatment regimens.

Prostaglandins (PG), also defined as prostanoids, are a widespread group of oxygenated lipids that modulate cell functions physiologically and pathologically. PG biosynthesis results from the release of arachidonic acid (AA), the main fatty acid catalyzed by phospholipase A2 (PLA2), from cell membranes. Released arachidonic acid (AA) under the influence of cyclooxygenase (COX) turns into an unstable oxygenated intermediate prostaglandin (PGH2). The resulting intermediate prostaglandin PGH2 can be converted into various prostaglandins, such as prostaglandin E2 (PGE2), prostacyclin (PGI2), prostacyclin F2α (PGF2α) or prostaglandin D2 (PGD2), under the influence of specific enzymes called prostaglandin synthases (for example, PGE synthase PGE-S, PGD synthase or PGD-S, etc.).

Cyclooxygenase (COX) is present in three isoforms: COX-1, COX-2 and COX-3, which is a spliced version of COX-1. COX-1 is mainly expressed in most tissues, while COX-2 induction usually occurs under the influence of pro-inflammatory cytokines and stress. The results obtained have conclusively proved that SOX-2 is responsible for the harmful pro-inflammatory effect of prostanoids. Therefore, inhibition of COX-2 is considered the main target when creating drugs for the treatment of inflammatory diseases. However, studies have shown that COX-2 also plays an important role in homeostasis of major organs and tissues.

Recent clinical studies have shown that prolonged treatment with COX-2 inhibitors increases the likelihood of stroke and myocardial infarction, which are complications arising from the blocking of the vasoprotective effect of PGI2 formed by COX-2. However, blocking COX enzymes also reduces the production of PGD2. In peripheral tissues, PGD2 stimulates vasodilation and inhibits platelet aggregation. PGD2 is the most common prostaglandin in the brain, and a recent study showed that PGD2 mediates a neuroprotective effect in hippocampal neurons. Therefore, blocking of COX-2-formed PGD2 can increase the likelihood of stroke and myocardial infarction observed during clinical trials using COX-2 inhibitors.

PGD2 exists for a very short period of time and undergoes dehydration in vivo and in vitro to form biologically active prostaglandins of the J2 series, including 15-deoxyprostaglandin J2 (15d-PGJ2). This prostaglandin is a natural chemically stable anti-inflammatory derivative of PGD2. 15d-PGJ2 is a high affinity peroxisome proliferator activated receptor ligand (PPAR) of the PPARγ subtype, which is a ligand-dependent nuclear transcription factor involved in a variety of cell functions, including anti-inflammatory action. 15d-PGJ2 suppresses several inflammatory genes, such as genes for nitric oxide synthase, prostaglandin E synthase (PGES) and tumor necrosis factor α (TNFα), using PPARγ-dependent and PPARγ-independent mechanisms. In rat chondrocytes, 15d-PGJ2 almost completely ceases cytokine-stimulated PGE2 synthesis and PGES expression, which suggests that 15d-PGJ2 is an anti-inflammatory messenger that disables the production of pro-inflammatory prostaglandin PGE2 in these cells. Regardless of the current mechanism, 15d-PGJ2 is present in vivo in the resolution phase of the inflammatory process, which also suggests its functioning as a feedback regulator of the inflammatory reaction. In addition, it was found that the introduction of 15d-PGJ2 reduces the development of experimentally induced colitis in rats, which is a model of inflammatory bowel disease. Therefore, it should be expected that the compounds and conditions that shift the balance towards the production of prostaglandins PGD2 and 15d-PGJ2 should have an anti-inflammatory effect.

In the central nervous system, the anti-inflammatory effects of 15d-PGJ2 can be beneficial in the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis, as well as stroke, spinal cord injury and traumatic brain injury, when inflammation causes brain damage and cell death . In all these cases, brain damage is due to excessive activation of microglia. Microglia, natural resident immunocompetent cells, play an important role in inflammatory processes that occur in the central nervous system. Uncontrolled activation of microglia can have toxic effects on brain cells due to the release of various substances such as inflammatory cytokines (IL-1β, TNF-α, IL-6), NO, PGE2 and superoxide. As a result of several studies, it was found that 15d-PGJ2 can inhibit the production of inflammatory cytokines and NO by activated microgliocytes and astrocytes, which suggests that prostaglandins can play an important role in preventing brain damage due to excessive activation of gliocytes. Evidence that the administration of 15d-PGJ2 before and during the experimentally induced autoimmune encephalitis (EAE), an animal model of multiple sclerosis, significantly reduces the severity of EAE, further suggesting that 15d-PGJ2 can effectively prevent brain damage in case of inflammatory neurodegenerative diseases.

Recent studies have shown that 15d-PGJ2 reduces inflammation of brain tissue, impaired behavior and death of neurons after intracerebral hemorrhage in rats and protects the brain from reperfusion due to ischemia in an experimental model of stroke. Intraventricular administration of 15d-PGJ2 reduces the volume of cerebral infarction, inhibits apoptosis of the brain and neurons, inhibits the activation of NF-κB and increases the level of heme oxygenase-1 (HO-1), a strong endogenous antioxidant, depending on PPARγ. The above results, demonstrating the neuroprotective effect of 15d-PGJ2 in the case of ischemic stroke, are confirmed by a recent study showing that subjects suffering from acute ischemic stroke have higher plasma levels of 15d-PGJ2 than normal subjects and that the content is elevated Plasma 15d-PGJ2 is associated with a better neurological condition in the early and late stages of the disease. Increased plasma 15d-PGJ2 was also associated with a smaller volume of heart attack, and this effect was not dependent on other important prognostic indicators. Therefore, it can be assumed that the compounds and conditions that shift the balance of prostaglandin production towards 15d-PGJ2 should have a beneficial effect in the treatment of both chronic inflammatory neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and multiple sclerosis, and acute illnesses such as stroke, spinal cord injury and traumatic brain injury.

Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disease characterized by the deposition of extracellular Aβ plaques (amyloid β) and the formation of intracellular plexuses in the brain. Aβ plaques are composed mainly of amyloid β and Aβ peptides. The accumulation of Aβ causes an inflammatory reaction, which is supposed to contribute to the pathogenesis of AD and an increase in neuronal damage. In addition, it is believed that elevated levels of soluble Aβ peptides in the brain cause neuronal dysfunction and cognitive impairment prior to plaque deposition. Therefore, the formation of Aβ peptides in the brain is the main target in the pathogenesis of AD.

The formation of Aβ induces a protease that cleaves the larger β-amyloid precursor protein (βAPP) at the N-terminus of the Aβ peptide. Said protease, also referred to as β-secretase or β-site APP-cleaving enzyme (BACE1), is a transmembrane aspartyl protease. BACE1 protein and β-secretase (C-terminal β-fragment) levels are elevated in the brain of subjects suffering from sporadic Alzheimer's disease. Therefore, a decrease in Aβ production in the brain of subjects suffering from Alzheimer's disease is the main therapeutic goal.

The scientific literature has reported that BACE1 mRNA and protein levels increase under the influence of pro-inflammatory mediators and decrease with drugs that are agonists of the γ receptor activated by the peroxisome proliferator (PPARγ). Recently, it was found that a decrease in PPARγ causes an increase in β-secretase mRNA levels as a result of an increase in the activity of the BACE1 gene promoter, while overexpression of PPARγ, as well as PPARγ activators, specifically modulates the transcription of BACE1 by suppressing the activity of the BACE1 gene promoter, which implies that PPARγ may be a BACE1 inhibitor. In addition, treatment of transgenic hAPP mice with PPARγ agonists reduced both BACE1 mRNA and intracellular β-amyloid levels in neurons of the cerebral cortex.

In addition, it was found that overexpression of PPARγ reduces Aβ secretion in cultured cells as a result of activation of ubiquitous and proteasome-mediated destruction of βAPP precursor protein and activation of PPARγ directly affects the stability of Aβ added to cell culture. Such a decrease in resistance suggests that activation of PPARγ may trigger the inclusion of a mechanism for rapid cell clearance for the amyloid peptide.

The data obtained indicate a significant role of PPARγ in modulating the formation of β-amyloid and the presence of a protective mechanism due to which the activation of PPARγ reduces the production of β-amyloid peptides in the brain.

15d-PGJ2, a high affinity peroxisome proliferator activator (PPAR) ligand for the PPARγ subtype, inhibits several inflammatory genes by PPARγ-dependent and PPARγ-independent mechanisms. Therefore, it can be assumed that the conditions under which the endogenous production of 15d-PGJ2 is increased should suppress inflammatory processes in the brain and reduce the formation of amyloid peptide in the brain of subjects suffering from Alzheimer's disease. Therefore, compounds and conditions that enhance the endogenous production of 15d-PGJ2 should have a beneficial effect in the treatment of Alzheimer's disease.

Prostaglandins of the J2 series are potent inducers of nerve growth factor (NGF) and the formation of nerve growth factor isolated from the brain (BDNF), and stimulate the growth of axons under the influence of NGF in cell culture. The ability of 15d-PGJ2 to stimulate axons is not associated with PPARγ, since the synthetic agonist and antagonist of PPAR-γ does not alter the axon-stimulating effect of 15-deoxy-PGJ2. In animal experiments, it was shown that the introduction of NGF into the ventricles of the brain protects cholinergic neurons, stimulates axon growth and improves cholinergic function. Similarly, the introduction of NGF into the ventricles of the brain reduces the death of neurons in the hippocampus of gerbils suffering from cerebral ischemia. It was found that BDNF stimulates the survival and growth of developing neurons in vitro and improves the function of motor neurons in animal models. In animals in which forebrain ischemia was caused, BDNF reduced ischemic damage to neurons. Unfortunately, due to the poor penetration of these neurotrophins through the blood-brain barrier, clinical trials did not allow a significant effect. However, it should be expected that conditions or treatment to increase endogenous levels of 15d-PGJ2 should improve the local production of these growth factors and stimulate the growth of neurons and, therefore, facilitate the recovery of neurons in the spinal cord and brain.

Elevated levels of inflammatory markers are associated with vascular ischemia, and inflammation mediates the pathogenesis of acute coronary artery syndromes. Inflammation plays a significant role in the onset and development of atherosclerosis, and can also play a major role in the development of thrombosis as a result of the activation of the coagulation process. Therefore, conditions and compounds that reduce vascular inflammation can have a beneficial effect on cardiovascular diseases in which inflammation mediates cell death or damage, such as coronary artery disease.

prostaglandin D synthase mRNA (PGDS) is expressed in the heart. Thus, locally produced PGD2 can cause the formation of 15d-PGJ2 in myocytes or surrounding cells. PPARγ is also present and functions in myocytes of the heart. A recent study showed that 15d-PGJ2, a natural metabolite of PGD2, has an anti-inflammatory effect in heart myocytes, modulating IL-1β-stimulated COX-2, PGE-S and iNOS depending on PPAR. 15d-PGJ2 blocked IL-1β stimulated PGE2 production, but did not modify the stimulation of IL-1β prostaglandins PGI2 or PGF2α, which implies that the effect of the PPARγ ligand is specific for PGE-S. Blocking IL-1β-induced PGE-S under the influence of 15d-PGJ2 supposedly reduces the production of pro-inflammatory prostaglandin PGE2 in heart tissue. In addition, it was found that 15d-PGJ2 increases the expression of heme oxygenase-1 (HO-1) in heart myocytes and reduces the magnitude of myocardial infarction in the animal model of rat myocardial infarction caused by reperfusion during regional ischemia. Among the various ligands studied, PPARγ 15d-PGJ2 caused the most pronounced decrease in infarction. Although this action is mediated by PPARγ, the increased expression of the antioxidant and the cytotoxic protein HO-1 is independent of PPARγ. In general, the results suggest that conditions and compounds that increase endogenous levels of 15d-PGJ2 can reduce vascular inflammation and thus have a beneficial effect in the treatment of cardiovascular diseases.

More and more evidence suggests that adipose tissue is the main source of circulating inflammatory factors, especially in the case of obesity. Fat produces pro-inflammatory adipocytokines, including TNF-α, leptin, PAI-1, IL-6 and angiotensinogen. TNF-α is the main activator of NFκB. TNF-α also inhibits insulin signaling, thus causing insulin resistance. PAI-1 levels predict CAD and diabetes and are a major factor in thrombosis in case of obesity. IL-6 stimulates the production of C-reactive protein (CRP) in the liver and increases the levels of highly sensitive (hs) CRP in the serum of obese subjects. HsCRP predicts myocardial infarction, stroke, peripheral artery disease, and sudden death. Angiotensinogen is a precursor to Ang II, which, as is well known, activates numerous mechanisms of vascular damage. As a rule, the levels of all these adipokines are increased in insulin-resistant subjects suffering from visceral obesity, creating a pro-inflammatory environment. Therefore, type 2 diabetes mellitus (T2D) and obesity are inflammatory conditions.

Adipose tissue expresses the highest levels of PPARγ compared to other tissues. PPARγ ligands stimulate the differentiation of fat cells and the absorption of free fatty acids by adipose tissue. These ligands also contribute to the weakening of the pro-inflammatory environment by reducing the expression of TNF-α, PAI-1 and IL-6 and increasing the expression of adiponectin in fat. Thus, the activation of PPARγ directly suppresses inflammation in vascular cells and has an indirect effect by regulating gene expression in adipose tissue.

T2D and metabolic syndrome are characterized by resistance to insulin in the peripheral tissues, including skeletal muscle, liver and adipose tissue. Activation of PPARγ by certain synthetic ligands, such as thiazolidinediones, improves insulin sensitivity and lowers blood glucose, triglycerides, and free fatty acids without stimulating insulin secretion in rodent animal models of T2D. PPARγ agonists also reduce insulin resistance in peripheral tissues in humans and have been used effectively to treat subjects suffering from type 2 diabetes mellitus (T2D).

15-Deoxyprostaglandin J2 (15d-PGJ2) is a naturally occurring chemically resistant anti-inflammatory prostaglandin, which appears to be the putative endogenous high affinity ligand for the peroxisome proliferator proliferator PPARγ subtype. Compounds and conditions that increase the endogenous production of 15d-PGJ2 are expected to suppress vascular inflammation and improve insulin susceptibility in type 2 diabetes.

Pro-inflammatory cytokines, such as the tumor necrosis factor alpha (TNF-α), are overexpressed in the case of psoriasis and atopic dermatitis. TNF-α plays an important role in the onset and development of inflammation, and recently obtained experimental data show that the occurrence of lesions in the experimental model of psoriasis is mediated by TNF-α. The data suggesting the role of TNF-α in the pathogenesis of psoriasis are confirmed by recent clinical trials showing that administration of a monoclonal antibody (mAb) against TNF-α (infliximab) or a soluble TNF receptor fusion protein (etanercept) improves the condition of patients psoriasis.

15d-PGJ2, a chemically stable metabolite of prostaglandin PGD2, is a high affinity peroxisome proliferator activated receptor (PPARγ) ligand.

15d-PGJ2 suppresses several pro-inflammatory genes in activated macrophages, microgliocytes and human astrocytes, including genes for inducible NO synthase (iNOS) and tumor necrosis factor α (TNF-α), and this suppression is at least partially dependent on PPARγ expression. In addition, it was found that synthetic PPARγ ligands, such as insulin-sensitizing thiazolidinediones, improve the condition of patients with psoriasis. In general, the results suggest that compounds and conditions that enhance the endogenous production of 15d-PGJ2 may have an effective effect in the treatment of psoriasis.

Recently obtained data show that certain synthetic PPARγ agonists have moderate antiproliferative activity against many cancer cell lines isolated from human epithelium. In addition, recently obtained data indicate that normal prostate epithelial cells and T lymphocytes are more resistant to apoptosis under the influence of these PPARγ ligands. Given the above specific cancer action, the possible use of PPARγ agonists as chemotherapeutic agents deserves serious attention.

In addition, it was found that 15d-PGJ2, a natural PPARγ ligand, has an antitumor effect. For example, 15d-PGJ2 significantly inhibits cell growth and causes apoptosis of several types of cancer cells, including cancer cells of the colon and rectum, stomach, breast and liver. Mechanistic studies suggest that this growth inhibitory effect is mediated by mechanisms independent of PPARγ. However, regardless of the acting mechanism of the compound, and conditions that increase the endogenous levels of 15d-PGJ2, are expected to inhibit tumor growth and development.

Elevated levels of prostaglandin E2 (PGE2) have been detected in various malignancies. Various data, in addition to detecting elevated levels of PGE2 in tumors, suggest that PGE2 plays a role in the onset and development of cancer. For example, PGE2 can stimulate cell proliferation and motility by inhibiting apoptosis and immune surveillance. It is important to note that PGE2 can also, at least partially, induce the formation of blood vessels, increasing the production of pro-angiogenic factors, including endothelial vascular growth factor. The above results are consistent with the fact that higher levels of PGE2 in cancer tissue samples are largely consistent with the occurrence of metastases and increased tumor vascularization.

Recent studies using experimental animals also suggest that PGE2 may stimulate carcinogenesis. Genetic destruction of the prostaglandin PGE2 receptor EP2 has been found to reduce the number and size of experimental tumors, while other studies have shown that treatment with a monoclonal antibody against PGE2 inhibits the growth of transplanted tumors. Based on the foregoing, it can be expected that the compounds and conditions that inhibit the enzymatic pathways that cause an increase in the content of PGE2 in the tumor should also inhibit tumor growth.

The synthesis of PGE2 from arachidonic acid requires two sequential enzymes, cyclooxygenase (COX) and prostaglandin E synthase (PGES). Increased PGES expression has been detected in several human cancers, which suggests that aberrant PGES expression causes increased production of PGE2, which promotes cell proliferation and tumor growth. As reported in the scientific literature, 15d-PGJ2 almost completely inhibits cytokine-induced PGE2 synthesis and expression of membrane PGE synthase. Therefore, compounds and conditions that increase endogenous 15d-PGJ2 levels are expected to reduce PGE2 synthesis as well as cell proliferation and tumor growth and have a beneficial effect in the treatment of cancer.

The inventors of the present invention previously showed that 7β-hydroxyepiandrosterone (7β-OH-EPIA), an endogenous 7-hydroxysteroid, both have neuroprotective and cardioprotective effects (WO 02/00224, WO 02/00225 and WO 03/015791). This steroid protects nerve cells from in vitro death (organotypic hippocampal slice cultures (OTHSC) and PC12 cells) and the brain from damage in numerous in vivo experimental models and provides effective protection against regional ischemia-induced myocardial infarction in perfused rat hearts. In general, the results suggest that 7β-OH-EPIA can have a beneficial effect in the prevention and treatment of neurodegenerative diseases such as stroke, spinal cord injury, traumatic brain injury and Alzheimer's disease, and cardiovascular diseases such as heart attack myocardium (MI).

The authors of the present invention recently showed that incubation of PC12 cells with indomethacin, a cyclooxygenase inhibitor (COX), completely reversed the protection provided by 7β-OH-EPIA against ischemia. The results suggest that COX activity is necessary for the neuroprotective effect of 7β-OH-EPIA.

The authors of the present invention showed that incubation of human blood cell monocytes (hMBC) with nanomolar concentrations of 7β-OH-EPIA causes an almost 10-fold increase in prostaglandin production of 15-deoxy-Δ12,14-J2 (15d-PGJ2). A similar effect of 7-hydroxysteroid, apparently, is specific for 15d-PGJ2, since this steroid does not significantly change the production of prostaglandin E2 (PGE2) in these cells. In contrast, incubation of hMBC with the tumor necrosis factor-α (TNFα) proinflammatory cytokine increases the production of both prostaglandins by about 3 times. In addition, co-incubation with TNF-α and 7β-OH-EPIA causes an increase in 15d-PGJ2 similar to the increase observed using only 7β-OH-EPIA, while the addition of nanomolar concentrations of 7β-OH-EPIA completely eliminated the increase in PGE2 under exposure to TNFα.

It has been reported in the scientific literature that 15d-PGJ2 almost completely inhibits cytokine-induced PGE2 synthesis and expression of membrane PGE synthase (mPGES) in rat chondrocytes, which suggests that 15d-PGJ2 is an anti-inflammatory messenger that disables the production of pro-inflammatory prostaglandin PGE2 cells . Therefore, the results obtained may indicate that the inhibition of TNFα-induced production of pro-inflammatory prostaglandin PGE2 under the influence of 7β-OH-EPIA is mediated by an increased release of 15d-PGJ2.

15d-PGJ2 is a high affinity peroxisome proliferator activated receptor ligand (PPAR) of the PPARγ subtype, a ligand-dependent nuclear transcription factor involved in a variety of cell functions, including anti-inflammatory, neuroprotective, cardioprotective, metabolic and antitumor effects. The inventors have found that compounds such as 7β-OH-EPIA are capable of selectively facilitating the production of 15d-PGJ2 and thus can have a beneficial effect in case of inflammation and skin diseases, such as inflammatory bowel disease and psoriasis, in case of neurological cardiovascular and metabolic diseases such as stroke, spinal cord injury, traumatic brain injury, Alzheimer's disease, Parkinson's disease, coronary artery disease of the heart and type 2 diabetes in which s inflammation causes dysfunction and cell death, and in the case of various types of cancer in which increased production RGE2 contributes to cell proliferation and tumor growth.

Thus, one object of the present invention is the use of a substance that enhances the production of 15-deoxyprostaglandin J2 for the manufacture of a medicament for the treatment or prevention of conditions mediated by elevated levels of prostaglandin E2 or other metabolites of cyclooxygenase and prostaglandin synthase or for the treatment or prevention of conditions whose deterioration due to reduced or decreased availability of 15-deoxyprostaglandin J2.

Another object of the present invention is the use of a substance that facilitates the production of 15-deoxyprostaglandin J2 and selectively inhibits the production of prostaglandin E2 in the presence of an inflammation-causing agent for the manufacture of a medicament for the treatment or prophylaxis of conditions mediated by elevated levels of prostaglandin E2 or other cyclooxygenase-2 metabolites, either for the treatment or prevention of conditions whose worsening is due to a decreased or decreased low availability of 15-deoxyprostaglandin J2.

Another object of the present invention is the use of a substance that enhances the production of 15-deoxyprostaglandin J2 for the manufacture of a medicament intended to stimulate axonal proliferation or to treat peripheral neuropathy.

Peripheral neuropathy may result from treatment with chemotherapeutic agents, such as cisplatin, or from other causes, such as diabetic neuropathy.

Another object of the present invention is the use of a substance that enhances the production of 15-deoxyprostaglandin J2 and activates PPAR-gamma, for the preparation of a medicinal product intended for the treatment of a condition requiring activation of PPAR-gamma.

Another object of the present invention is the use of a compound that enhances the production of 15-deoxyprostaglandin J2 for the preparation of a medicament for the treatment or prevention of cancer.

Another object of the present invention is the use of a compound that enhances the production of 15-deoxyprostaglandin J2 for the manufacture of a medicament for inhibiting the proliferation of cancer cells, inducing apoptosis of cancer cells or inhibiting the growth and development of a tumor.

The cancer cells to which this object of the present invention is most relevant include cancer cells of the colon and rectum, stomach, breast, liver, prostate, bladder, thyroid and esophagus.

In addition, the compounds of the present invention can be used for the treatment and prevention of:

pain associated with inflammation;

diseases of the peripheral arteries (including disorders that prevent blood flow to the limbs or caused by peripheral atherosclerosis) and their complications, such as critical limb ischemia;

coronary artery disease and its complications, such as coronary heart disease (for example, stable and unstable angina) and myocardial infarction (MI);

cardiovascular diseases and their complications, such as stroke and transient ischemic cerebrovascular accident (TIA);

ischemia of the liver and kidney, for example, atherosclerotic renal artery stenosis;

metabolic diseases such as type 2 diabetes and its deposition, such as peripheral artery disease, coronary artery disease, vascular kidney disease and diabetic neuropathy;

obesity and its complications, such as type 2 diabetes, peripheral artery disease and coronary artery disease;

inflammatory respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD), for example, chronic bronchitis;

chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and peripheral neuropathies;

acute neurological degenerative conditions, such as traumatic brain injury and spinal cord injury;

inflammatory bowel disease;

inflammatory diseases characterized by degeneration of articular cartilage, such as rheumatoid arthritis, primary and secondary osteoarthritis, and their complications;

wounds; and

toxicity or peripheral neuropathies caused by chemotherapeutic agents.

These substances can preferably be used to treat diabetes and its complications; coronary artery disease; pain associated with inflammation; inflammatory skin diseases; spinal cord injuries; peripheral neuropathy; multiple sclerosis; inflammatory bowel disease; rheumatoid arthritis; metabolic syndrome X, obesity, acromegaly and wound healing.

Such substances may also be suitable for the treatment or prophylaxis of conditions such as inflammatory diseases of peripheral organs, for example, liver and kidney, caused, for example, by ischemia of the liver and kidney.

Other inflammatory conditions that can be treated with these substances include inflammatory diseases of the respiratory tract, such as asthma, rhinitis, bronchitis, and chronic obstructive pulmonary disease (COPD).

The present invention is illustrated by the accompanying drawings, in which 7β-hydroxyepiandrosterone is defined as 7β-OH-EPIA.

Figure 1 presents the combined data of the average percentage of cell death obtained as a result of 4 separate experiments in example 1, which are expressed as mean ± standard error of the mean.

Figure 2 shows the average percentage of cell death in individual experiments described in example 1.

Figure 3 shows the average percentage of cell death in individual experiments described in example 1.

Figure 4 shows the average percentage of cell death in individual experiments described in example 1.

5 (a) and 5 (b) show the effect of increasing concentrations of 7β-OH-EPIA on prostaglandin PGD2 levels found in supernatants of peripheral blood mononuclear cells incubated with 7β-OH-EPIA in the presence and absence of TNF-α as described in example 2.

6 (a) and 6 (b) show the effect of increasing concentrations of 7β-OH-EPIA on prostaglandin PGE2 levels found in supernatants of peripheral blood mononuclear cells incubated with 7β-OH-EPIA in the presence and absence of TNF-α as described in example 2.

FIGS. 7 (a) and 7 (b) show the effect of increasing concentrations of 7β-OH-EPIA on prostaglandin 15d-PGJ2 levels found in supernatants of peripheral blood mononuclear cells incubated with 7β-OH-EPIA in the presence and absence of TNF -α, as described in example 2.

Fig. 8 shows the effect of 7β-hydroxy-EPIA administration on (A) myeloperoxidase (MPO) and oxidative stress markers, namely (B) Prot CO, (C) T-folds and (D) GSH antioxidant defense marker in tissue colon, as described in more detail in example 4.

Figure 9 shows the level of 15d-PGJ2 in the colon (A) and the quantitative determination of the relative expression of COX-2, mPGES-1 and H-PGDS mRNA (B) at different time periods of administration of 7β-hydroxy-EPIA in Example 4.

Figure 10 shows the effect of 7β-hydroxy-EPIA on the synthesis of prostaglandin E2, D2 and 15d-PGJ2 in the colon during the administration of DSS in Example 4.

11 shows the expression in the colon of mRNA COX-2, mPGES-1 and H-PGDS during induction of colitis in example 4.

Compounds that can be used in the present invention include compounds of formula (I):

Where

the dotted circle indicates that the ring containing it can be fully saturated or may have one, two or three carbon-carbon double bonds;

the dashed line indicates that said bond may be a carbon-carbon single or double bond;

R ¹ means a hydrogen atom or a methyl group;

R ² , R ³ and R ⁴ have the same or different meanings and mean an oxo group, a hydroxyl group, a mercapto group, a hydrogen atom, a halogen atom, an alkoxyl group, an aryloxy group or an acyl group;

their pharmaceutically acceptable salts and esters.

The numbers (3) and (7) in the above formula are for reference only and are used to determine the numbering system used. Of course, when R ² is an oxo group, a dashed circle can only mean a fully saturated ring (in the sense of no double bonds in the ring) or one or two double bonds.

Of these compounds, preferred compounds are compounds of formula (II):

(where R ¹ , R ² , R ³ and R ⁴ have the above meanings) and their esters.

Another preferred class of these compounds are compounds of formula (III):

(where R ^2a means an oxo group, a hydroxyl group, a mercapto group or a halogen atom; and R ¹ , R ³ and R ⁴ have the above meanings) and their esters.

Another preferred class of these compounds are compounds of formula (IV):

(where R ¹ , R ² , R ³ and R ⁴ have the above meanings) and their esters.

Another preferred class of these compounds are compounds of formula (V):

where R ² , R ³ and R ⁴ have the above meanings.

Examples of compounds of formula (II) include 7-hydroxytestosterone having the formula (IIa):

and its esters. The 7-hydroxyl group in this compound may be in the alpha or beta configuration, or a mixture of two isomers may be used.

Examples of compounds of formula (III) include 7α-hydroxydehydroepiandrosterone (7α-hydroxy-DHEA) having the formula (IIIa):

and its esters, 7β-analogue having the formula (IIIb):

and its esters, 7β-hydroxypregnenolone having the formula (IIIc):

and its esters, 7α-hydroxyl analogue and its esters.

Examples of compounds of formula (IV) include 7-hydroxyepiandrosterone, which may be in the form of 7α or 7β isomers, and esters thereof. 7β-hydroxyepiandrosterone, hereinafter referred to as 7β-OH-EPIA, has the formula (IVa):

and 7α-hydroxyepiandrosterone, hereinafter referred to as 7α-OH-EPIA, has the formula (IVb):

Examples of compounds of formula (V) are 7β-hydroxy-17β-estradiol having the formula (Va):

and its esters, 7α-analogues and their esters, and 7β-hydroxyestron having the formula (Vb):

and its esters, 7α-analogues and their esters.

When R ² , R ^2a , R ³ or R ⁴ in the compounds of the present invention is a halogen atom, such an atom may be a fluorine, chlorine, bromine or iodine atom, preferably a chlorine atom.

When R ² , R ^2a , R ³ or R ^{4 is an} alkoxy group, such a group may be a straight or branched chain group, preferably containing 1-6 carbon atoms. Examples of such groups include methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, sec-butoxyl, isobutoxyl, tert-butoxyl, pentyloxy and hexyloxy groups.

When R ² , R ^2a , R ³ or R ^{4 is an} aryloxy group, such a group is preferably a phenoxy or naphthyloxy group.

When R ² , R ^2a , R ³ or R ^{4 is} an acyl group, such a group may be, for example, an aliphatic acyl group or an aromatic acyl group. Examples of aliphatic acyl groups include groups containing 1-6 carbon atoms, such as formyl, acetyl, propionyl, butyryl, valeryl, isovaleryl, pivaloyl and hexanoyl groups. Examples of aromatic acyl groups include benzoyl, naphthoyl and toluoyl groups.

It should be noted that when the compound contains a group of the formula —OR, where R is any of the above groups and atoms shown for R ² , etc., the active type is apparently a compound containing a free hydroxyl group. Thus, instead of a hydroxyl group, any group that can be converted in vivo to a hydroxyl group can be used.

These compounds can be prepared by various well-known methods using starting steroids. For example, such compounds can be obtained by the methods described in European patent EP 1294382.

The compounds of formula (I) described in European patent EP 1294382, publications WO 2002/000224 and WO 2002/000225, are intended for the treatment or prevention of chronic and acute neurodegenerative diseases or conditions, and the compounds described in publication WO 2002/015791 are intended for treatment or prevention of chronic and acute cardiodegenerative diseases or conditions, and such treatment or prevention is, of course, excluded from the present claims only in relation to compounds of formula (I).

Another class of compounds that can be used in the present invention include compounds of formula (VI):

Where

X means a group of the formula> CR ⁵ R ⁶ or, when R ^{10 is} not a hydrogen atom, a group of the formula> SO ₂ ;

Y represents a group of the formula> NH or> CR ⁵ R ⁶ ;

Z is a group of the formula> C = O, a group of the formula> CH _2, or a direct bond;

R ⁵ means a hydrogen atom and R ⁶ means a hydrogen atom, a carboxyl group or a hydroxyl group; or

R ⁵ and R ⁶ together mean an oxo group, methylenedioxy group or hydroxyimino group;

R ⁷ means a hydrogen atom or a lower alkyl group;

R ⁸ means two hydrogen atoms, or an oxo group, or a hydroxyimino group;

R ⁹ means a hydrogen atom, a lower alkyl group or a halogen atom;

R ¹⁰ means a hydrogen atom, a lower alkoxyl group or a carboxyl group;

R ¹¹ and R ¹² have the same or different meanings and mean a hydrogen atom, a lower alkyl group or a halogen atom;

their salts and esters, when these compounds contain a carboxyl group.

In the compounds of formula (VI), Z may mean a direct bond, and in this case, Z may be part of a 5-membered ring fused to a 5-membered nitrogen-containing heterocyclic ring, or may be a group of the formula> CH ₂ or> C = O, and in this case, Z is part of a 6-membered ring.

When R ⁷ , R ⁹ , R ¹¹ or R ¹² means a lower alkyl group, such a group may be a straight or branched chain alkyl group containing 1-10, preferably 1-6 carbon atoms. Examples of such groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 2-methylbutyl, 1-ethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1- methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, hexyl, isohexyl, heptyl, octyl, nonyl and decyl groups, of which methyl is preferred Ordering, propyl, butyl and hexyl groups, more preferred are methyl and ethyl groups, and most preferred is a methyl group.

When R ⁹ , R ¹¹ or R ¹² is a halogen atom, such an atom may be a fluorine, chlorine, bromine or iodine atom, of which fluorine and chlorine atoms are preferred.

When R ¹⁰ means a lower alkoxyl group, such a group may be a straight or branched chain alkoxyl group containing 1-10, preferably 1-6 carbon atoms. Examples of such groups include methoxy, ethoxy, propoxy, isopropoxy, butoxyl, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy, 2-methylbutoxy, 1-ethylpropoxy, 4-methylpentyloxy, 3-methylpentyloxy, 2-methylpentyloxy, 2 methylpentyloxy, 3,3-dimethylbutoxyl, 2,2-dimethylbutoxyl, 1,1-dimethylbutoxyl, 1,2-dimethylbutoxyl, 1,3-dimethylbutoxyl, 2,3-dimethylbutoxyl, 2-ethylbutoxyl, hexyloxy, isohexyloxy, heptyl nonyloxy and decyloxy groups, of which we prefer the methoxy, ethoxy, propoxy, butoxy and hexyloxy groups, more preferred are methoxy and ethoxy groups, and most preferred is a methoxy group.

Of the compounds of the present invention, those in which

X is a group of the formula> CR ⁵ R ⁶ where R ⁵ is a hydrogen atom and R ⁶ is a hydrogen atom, a hydroxyl group or a carboxyl group, or R ⁵ and R ⁶ together are an oxo group or methylenedioxy group;

Y is a group of the formula> CR ⁵ R ⁶ where R ⁵ is a hydrogen atom and R ⁶ is a hydrogen atom or a carboxyl group;

R ⁷ means a hydrogen atom;

R ⁸ means two hydrogen atoms or an oxo group;

R ⁹ means a hydrogen atom;

R ¹⁰ means a hydrogen atom, a C ₁ -C ₄ alkoxyl group or a carboxyl group; and R ¹¹ and R ¹² have the same or different meanings and mean a hydrogen atom or a C ₁ -C ₄ alkyl group;

their salts and esters.

Table 1 below provides specific examples of the compounds of the present invention.

The most preferred compounds of formula (VI) are the compounds indicated in the above table under the numbers 4, 6, 7, 8, 10, 14, 15, 16, 17, 20, 21, 22 and 23.

Compounds of the present invention containing a carboxyl group, for example when R ⁵ or R ¹⁰ means a carboxyl group, can form esters that can be prepared by standard esterification methods. There are no particular restrictions on the nature of the ester, provided that in the case of medical use of the resulting compound, such a compound must be pharmaceutically acceptable, that is, it should not be less active or unacceptably less active, more toxic or unacceptably more toxic than the parent compound . However, if this compound is intended for non-medical use, for example, as an intermediate in the preparation of other compounds, the above limitation is not valid, while there are no restrictions on the nature of the esters that can be obtained.

Examples of ester groups include

alkyl groups containing 1-20 carbon atoms, more preferably 1-10 carbon atoms shown for R ⁷ , R ⁹ , R ¹¹ or R ¹² , and higher alkyl groups well known in the art, such as dodecyl, tridecyl, pentadecyl , octadecyl, nonadecyl and icosyl groups;

cycloalkyl groups containing 3-7 carbon atoms, such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl groups;

aralkyl groups in which the alkyl part contains 1-3 carbon atoms and the aryl part is a carbocyclic aromatic group containing 6-14 carbon atoms, which may be substituted or unsubstituted; examples of such aralkyl groups include benzyl, phenethyl, 1-phenylethyl, 3-phenylpropyl, 2-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl, 2- (1-naphthyl) ethyl, 2- (2-naphthyl) ethyl, benzhydryl ( there are diphenylmethyl), triphenylmethyl, bis (o-nitrophenyl) methyl, 9-antrylmethyl, 2,4,6-trimethylbenzyl, 4-bromobenzyl, 2-nitrobenzyl, 4-nitrobenzyl, 3-nitrobenzyl, 4-methoxybenzyl and piperonyl groups;

alkenyl groups containing 2-6 carbon atoms, such as vinyl, allyl, 2-methylallyl, 1-propenyl and isopropenyl groups;

halogen-substituted alkyl groups containing 1-6, preferably 1-4 carbon atoms, such as 2,2,2-trichloroethyl, 2-haloethyl (e.g. 2-chloroethyl, 2-fluoroethyl, 2-bromoethyl or 2-iodoethyl), 2 , 2-dibromethyl and 2,2,2-tribromethyl groups;

substituted silylalkyl groups, for example, 2-tri (C ₁ -C ₄ ) -alkylsilylethyl groups, in particular a 2-trimethylsilylethyl group;

substituted and unsubstituted phenyl groups, such as, for example, phenyl, tolyl and benzamidophenyl groups;

substituted and unsubstituted phenacyl groups, such as, for example, a phenacyl group or a p-bromophenacyl group;

cyclic and acyclic terpenyl groups, such as, for example, geranyl, non-aryl, linalyl, wick, mentyl (in particular, m- and p-methyl), thuyl, caryl, pinanyl, boryl, norkaryl, norkinanyl, norbornyl, menthenyl, camphenyl and norbornenyl groups;

alkoxymethyl groups in which the alkoxy part contains 1-6, preferably 1-4 carbon atoms and may be substituted by one unsubstituted alkoxy group, such as methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl and methoxyethoxymethyl groups;

aliphatic acyloxyalkyl groups in which the acyl group is preferably an alkanoyl group and more preferably an alkanoyl group containing 2-6 carbon atoms and the alkyl part contains 1-6, preferably 1-4 carbon atoms, such as acetoxymethyl, propionyloxymethyl, butyryloxymethyl, isobutyryloxymethyl, pivaloyloxymethyl, 1-pivaloyloxyethyl, 1-acetoxyethyl, 1-isobutyryloxyethyl, 1-pivaloyloxypropyl, 2-methyl-1-pivaloyloxypropyl, 2-pivaloyloxypropyl, 1-iso butyryloxyethyl, 1-isobutyryloxypropyl, 1-acetoxypropyl, 1-acetoxy-2-methylpropyl, 1-propionyloxyethyl, 1-propionyloxypropyl, 2-acetoxypropyl and 1-butyryloxyethyl groups;

cycloalkyl-substituted aliphatic acyloxyalkyl groups in which the acyl group is preferably an alkanoyl group and more preferably an alkanoyl group containing 2-6 carbon atoms, the cycloalkyl substituent contains 3-7 carbon atoms and the alkyl part contains 1-6, preferably 1-4 carbon atoms such as cyclohexylacetoxymethyl, 1- (cyclohexylacetoxy) ethyl, 1- (cyclohexylacetoxy) propyl, 2-methyl-1- (cyclohexylacetoxy) propyl, cyclopentylacetoxymethyl, 1- (cyclopentylacetoxy) et Ordering, 1- (cyclopentylacetoxy) propyl and 2-methyl-1- (cyclopentylacetoxy) propyl groups;

alkoxycarbonyloxyalkyl groups, in particular 1- (alkoxycarbonyloxy) ethyl groups such as 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl, 1-propoxycarbonyloxyethyl, 1-isopropoxycarbonyloxyethyl, 1-butoxycarbonyloxyethyl, 1-isobutoxycarbonyl-1-isobutoxycarbonyloxy-1-isobutoxycarbonyloxy-1-isobutoxycarbonyl-1-isobutoxycarbonyloxy-1-butoxycarbonyloxy-1-butoxycarbonyloxy-1-butoxycarbonyloxy-1-butoxycarbonyloxy-1-butoxycarbonyloxy-1-butoxycarbonyloxy-1-butoxycarbonyl-1-butoxycarbonyloxy-ethyl butoxycarbonyloxyethyl, 1- (1-ethylpropoxycarbonyloxy) ethyl and 1- (1,1-dipropylbutoxycarbonyloxy) ethyl groups, and other alkoxycarbonylalkyl groups in which both alkoxy and alkyl these groups contain 1-6, preferably 1-4 carbon atoms, such as 2-methyl-1- (isopropoxycarbonyloxy) propyl, 2- (isopropoxycarbonyloxy) propyl, isopropoxycarbonyloxymethyl, tert-butoxycarbonyloxymethyl, methoxycarbonyloxymethyl and ethoxycarbonyloxymethyl groups;

cycloalkylcarbonyloxyalkyl and cycloalkyloxycarbonyloxyalkyl groups such as, for example, 1-metiltsiklogeksilkarboniloksimetilnaya, 1-metiltsiklogeksiloksikarboniloksimetilnaya, tsiklopentiloksikarboniloksimetilnaya, tsiklopentilkarboniloksimetilnaya, 1- (cyclohexyloxycarbonyloxy) ethyl, 1- (cyclohexylcarbonyloxy) ethyl, 1- (cyclopentyloxycarbonyloxy) ethyl, 1- (cyclopentylcarbonyloxy) ethyl, 1- (cycloheptyloxycarbonyloxy) ethyl, 1- (cycloheptylcarbonyloxy) ethyl, 1-methylcyclopentylcarbonyloxymethyl , 1-methylcyclopentyloxycarbonyloxymethyl, 2-methyl-1- (1-methylcyclohexylcarbonyloxy) propyl, 1- (1-methylcyclohexylcarbonyloxy) propyl, 2- (1-methylcyclohexylcarbonyloxy) propyl, 1- (cyclohexylcarbonyloxy) propyl, 2- (cyclohexyl) 2-methyl-1- (1-methylcyclopentylcarbonyloxy) propyl, 1- (1-methylcyclopentylcarbonyloxy) propyl, 2- (1-methylcyclopentylcarbonyloxy) propyl, 1- (cyclopentylcarbonyloxy) propyl, 2- (cyclopentylcarbonyloxy) propyl, 1- (1- methylcyclopentylcarbonyloxy) ethyl, 1- (1-methi cyclopentylcarbonyloxy) propyl, adamantiloksikarboniloksimetilnaya, adamantilkarboniloksimetilnaya, 1-adamantiloksikarboniloksietilnaya and 1-adamantylcarbonyloxyethyl groups;

cycloalkylalkoxycarbonyloxyalkyl groups, such as, for example, cyclopropylmethoxycarbonyloxymethyl, cyclobutylmethoxycarbonyloxymethyl, cyclohexylmethoxycarbonyloxymethyl, 1- (cyclopropylmethyloxybenzylmethoxycarbonyloxy) methyloxybenzylmethylbenzene

terphenylcarbonyloxyalkyl and terphenyloxycarbonyloxyalkyl groups, such as, for example, 1- (methyloxycarbonyloxy) ethyl, 1- (methyloxycarbonyloxy) ethyl, methyloxycarbonyloxymethyl, methylphenylcarbonyloxymethyl, 1- (3-pinanyloxycarbonyloxy) ethyl-3-phenyl-3-yl-1- (1) 3-pinanylcarbonyloxymethyl groups;

5-alkyl- or 5-phenyl- (2-oxo-1,3-dioxolen-4-yl) alkyl groups, such as, for example, (5-methyl-2-oxo-1,3-dioxolen-4-yl ) methyl, (5-phenyl-2-oxo-1,3-dioxolen-4-yl) methyl, (5-isopropyl-2-oxo-1,3-dioxolen-4-yl) methyl, (5-tert- butyl-2-oxo-1,3-dioxolen-4-yl) methyl and 1- (5-methyl-2-oxo-1,3-dioxolen-4-yl) ethyl groups; and

other groups, in particular groups that are readily removable in vivo, such as phthalidyl, indanyl and 2-oxo-4,5,6,7-tetrahydro-1,3-benzodioxolen-4-yl groups.

In addition, compounds of the present invention containing a carboxyl group can be converted by standard methods into salts with a base. There are no particular restrictions on the nature of such salts, provided that the compounds intended for medical use must be pharmaceutically acceptable. However, if this compound is to be used for non-medical purposes, for example, as an intermediate in the preparation of other compounds, the above limitation is invalid, while there are no restrictions on the nature of the salts that can be obtained. Examples of such salts include salts with an alkali metal such as sodium, potassium or lithium; salts with an alkaline earth metal such as barium or calcium; salts with another metal, such as magnesium or aluminum; ammonium salts; salts with organic bases, such as a salt with methylamine, dimethylamine, triethylamine, diisopropylamine, cyclohexylamine or dicyclohexylamine; and salts with a basic amino acid such as lysine or arginine. Pharmaceutically acceptable salts are preferred.

The compounds of the present invention can also be converted by standard methods into salts with acids. There are no particular restrictions on the nature of such salts, provided that the compounds intended for medical use must be pharmaceutically acceptable. However, if this compound is to be used for non-medical purposes, for example, as an intermediate in the preparation of other compounds, the above limitation is invalid, while there are no restrictions on the nature of the salts that can be obtained. Examples of such salts include salts with mineral acids, in particular with hydrohalic acids (such as hydrofluoric acid, hydrobromic acid, hydroiodic acid or hydrochloric acid), nitric acid, perchloric acid, carbonic acid, sulfuric acid or phosphoric acid; salts with lower alkyl sulfonic acids such as methanesulfonic acid, trifluoromethanesulfonic acid or ethanesulfonic acid; salts with arylsulfonic acids such as benzenesulfonic acid or p-toluenesulfonic acid; salts with organic carboxylic acids such as acetic acid, fumaric acid, tartaric acid, oxalic acid, maleic acid, malic acid, succinic acid, benzoic acid, mandelic acid, ascorbic acid, lactic acid, gluconic acid or citric acid; and salts with amino acids such as glutamic acid or aspartic acid. Pharmaceutically acceptable salts are preferred.

The compounds of formula (VI) described in WO 2006/082409 are intended for the treatment or prophylaxis of chronic and acute neurodegenerative diseases or conditions, and such treatment or prophylaxis is, of course, excluded from the present claims only with respect to compounds of formula (VI).

The compounds of the present invention can be used to treat or prevent various chronic and acute diseases or conditions, and to achieve these goals can be obtained in the form of standard pharmaceutical preparations well known in this field. Thus, these compounds can be administered orally, for example, in the form of tablets, capsules, granules, powders, syrups, spray solutions and other well-known forms, or parenterally, for example, in the form of injections, spray solutions, eye drops, adhesive plasters or suppositories, and etc.

These pharmaceutical preparations can be obtained by standard methods and may contain known adjuvants commonly used in the art, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, etc., depending on the intended use and form of the preparation. The magnitude of the dose depends on the condition, age and body weight of the subject, as well as on the nature and severity of the disease to be treated, but in the case of oral administration to an adult, the total daily dose is usually from 0.01 to 50 mg / kg body weight (more preferably from 0 , 05 to 20 mg / kg body weight) and can be administered as a single dose or in divided doses, administered, for example, one to three times a day.

Typically, the compounds of the present invention can be used to treat or prevent various inflammatory conditions or conditions caused by metabolic pathways that cause inflammation. Examples of the use of these compounds include stimulation of axon growth; treatment or prevention of diabetes mellitus (in particular type 2 diabetes mellitus) and its complications; treatment and prevention of ischemic vascular diseases; treatment of pain associated with inflammation; treatment and prevention of inflammatory skin diseases, including psoriasis and wound healing. Other inflammatory conditions that can be treated or prevented, or whose severity can be alleviated by using the compounds of the present invention, include spinal cord injury, peripheral neuropathy, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, peripheral neuropathy, or chemotherapeutic toxicity such as cisplatin, or other causes such as diabetic neuropathy. Finally, conditions that can be treated or prevented, or whose severity can be alleviated by using the compounds of the present invention, also include various types of cancer.

The present invention is further illustrated by the following non-limiting examples.

Example 1

Protective effect of 7β-hydroxyepiandrosterone against ischemia in PC-12 cells: inhibition of indomethacin, a cyclooxygenase inhibitor (MOR)

The aim of this experiment was to investigate the ability of 7β-OH-EPIA to maintain neuroprotective efficacy in a hypoxia model while weakening indomethacin prostaglandin synthesis. The main experimental system used was ischemia-induced cytotoxicity in PC12 cells. The end point of the experiment was cell death.

Ischemia-induced PC-12 cell death was significantly reduced by 7β-OH-EPIA. Indomethacin (10 μM), which blocks the synthesis of prostaglandins, did not directly affect cell death caused by ischemia, but completely blocked the neuroprotective effect of 1 μM and 10 μM 7β-OH-EPIA, confirming the hypothesis that prostaglandin synthesis is necessary for the neuroprotective effect of 7β-hydroxy EPIA.

The experimental technique

Cell Culture PC-12

PC-12 cells were placed in type 1 collagen-sensitized flasks in PC-12 media of the following composition: RPMI 1640 without L-glutamine, with 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, additional glucose until final concentration of 4.5 g / l (RPMI usually contains 2 g / l), 10% heat-inactivated horse serum, 5% fetal calf serum and 50 units of penicillin / streptomycin. Wednesdays were changed every 2 days.

PC-12 Cell Assays

Confluent PC-12 cell cultures were differentiated by culturing for 7 days in serum-free PC-12 medium, but with 50 ng / ml NGF (NGF medium for PC-12). Cells were harvested, washed and counted, 1 × 10 ⁵ PC-12 cells / well were cultured overnight on a microtiter plate in NGF PC-12 medium without glucose. Then this medium was replaced with NGF medium for PC-12 without glucose containing the test compounds, and the tablets were kept under normal conditions for 30 minutes.

At this stage, the media and test substances intended to be in anoxic conditions were placed in a chamber and deoxygenated with 95% N ₂ /5% CO ₂ . The media on the plates was replaced with oxygen-free media and the plates were placed in an anaerobic chamber, into which 95% N ₂ /5% CO ₂ was supplied for 10 minutes, then sealed and incubated at 37 ° C overnight (18 hours). In control experiments, the cells were treated in the same way as in ischemic conditions, except that all incubations were performed in an atmosphere of 5% CO ₂ /95% air.

Cell viability was determined by exclusion using trypan blue.

results

PC-12 cells were relatively resistant to hypoxia. In this regard, more severe conditions were created for the combined reduction of oxygen-glucose (ischemia) to initiate toxicity (figure 1). 7β-OH-EPIA in this analysis exerted a cytoprotective effect depending on the dose, with significant neuroprotection observed at a dose of 1 μM (26% decrease in cell death) and 10 μM (53% decrease in cell death). Figure 1 presents the combined data of 4 experiments in which the above effect was detected. Figure 1 shows the average percentage of cell death obtained on the basis of the combined data of 4 separate experiments. Data are expressed as mean ± standard error of the mean. In the case of ischemia, cell death was equal to 76% ± 2.2%. A statistically significant decrease in cell death was observed using 1 μM 7β-OH-EPIA (26% decrease in cell death, p <0.001), 10 μM 7β-OH-EPIA (53% decrease in cell death, p <0.001), *** = p <0.001 compared with fully ischemic conditions.

The NMDA MK-801 receptor antagonist also reduced ischemia-induced toxicity of PC-12 cells (FIG. 2). Indomethacin cyclooxygenase inhibitor had an average protective effect at a dose of 100 μM (29% reduction in cell death), which was absent at lower concentrations (Fig.2). Figure 2 shows the average percentage of cell death. Data are expressed as mean ± standard error of the mean. Under completely ischemic conditions, cell death was equal to 79% ± 5.3%. A statistically significant decrease in cell death was observed using 100 μM indomethacin (IM) (29% decrease in cell death, p <0.05), 10 μM MK801 (62% decrease in cell death, p <0.001), ** = p <0, 01, *** = p <0.001 compared with fully ischemic conditions.

The neuroprotective effect of 7β-OH-EPIA was completely blocked by 10-100 μM indomethacin (IM) (figure 3), while the toxicity in these cultures did not differ from cultures under completely ischemic conditions. Figure 3 shows the average percentage of cell death. Data are expressed as mean ± standard error of the mean. Under completely ischemic conditions, cell death was 76% ± 4.2%. A statistically significant decrease in cell death was observed using 10 μM 7β-OH-EPIA (50% decrease in cell death, p <0.001), 10 μM MK801 (42% decrease in cell death, p <0.001), *** = p <0.001 compared with completely ischemic conditions.

As shown in FIG. 4, 10 μM indomethacin completely blocked the protective effect of 1 μM and 10 μM 7β-OH-EPIA. Figure 4 shows the average percentage of cell death. Data are expressed as mean ± standard error of the mean. Under completely ischemic conditions, cell death was 76% ± 4.8%. A statistically significant decrease in cell death was observed using 1 μM 7β-OH-EPIA (25% decrease in cell death, p <0.05), 10 μM 7β-OH-EPIA (50% decrease in cell death, p <0.001), * = p <0.05, *** = p <0.001 compared with fully ischemic conditions.

Conclusion

Ischemia-induced PC-12 cell death was significantly reduced by 7β-OH-EPIA. Indomethacin, which blocks the synthesis of prostaglindins, did not directly affect the death of PC-12 cells at concentrations up to 10 μM. However, with a further increase in concentration to 100 μM, an average neuroprotective effect was observed.

Indomethacin (10 μM) did not directly affect ischemia-induced cell death, but significantly attenuated the neuroprotective effect of 7β-OH-EPIA, confirming the hypothesis that prostaglandin synthesis is necessary for the neuroprotective effect of 7β-OH-EPIA.

Example 2

The effect of 7β-hydroxyepiandrosterone on the production of prostaglandins D2, E2 and 15-deoxy-Δ ^12.14 -J2 human mononuclear cells

The purpose of this experiment was to evaluate the ability of 7β-OH-EPIA to induce the biosynthesis of specific metabolites of arachidonic acid in human monocytes, namely prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and 15-deoxy-Δ ^12,14- prostaglandin J2 (15d-PGJ2 )

Blood monocytes were exposed to different concentrations of 7β-OH-EPIA in the absence or presence of tumor necrosis factor (TNF-α), a pro-inflammatory factor, and the amount of PGD2, PGE2, and 15d-PGJ2 produced was measured using an enzyme-linked immunosorbent assay.

7β-OH-EPIA (0.1 nM - 100 nM) caused, depending on the concentration, an increase in the production of PGD2 from normal mononuclear cells in human peripheral blood. TNF-α increased PGD ₂ production compared to control cells, and a similar increase occurred at the highest concentration of 7β-OH-EPIA. 7β-OH-EPIA (1 nM - 1000 nM), apparently, does not significantly affect the biosynthesis of PGE2 in normal mononuclear cells in human peripheral blood, but completely suppresses TNF-α-induced increase in PGE2 production. It was found that 7β-OH-EPIA (0.1 nM - 1000 nM) increases the production of 15d-PGJ2 in normal mononuclear cells in human peripheral blood by about 9-12 times in the absence of TNF-α and 2-2.5 times the presence of TNF-α compared with the corresponding control cells.

The experimental technique

Peripheral blood mononuclear cells

Mononuclear cells (monocytes and lymphocytes) were obtained by centrifugation in a density gradient of 48 ml of human whole blood with ficoll-hypak. Blood was placed on top of ficoll (density = 1.077 g / ml) and centrifuged at 400 g for 1 hour (22 ° C), after which the cells in the interphase were pipetted and transferred to clean tubes. The tubes were filled with RPMI 1640 culture medium (2 volumes), thoroughly mixed and centrifuged at 400 g for 5 minutes (22 ° C). The supernatant was removed, cell debris was resuspended in RPMI 1640 culture medium, and the volume was adjusted to obtain the appropriate number of cells in one incubation, as indicated in the Results section below. Cells were incubated in sterile plastic 1.5 ml tubes in a final volume of 1 ml of RPMI 1640 medium for 18 hours at 37 ° C in an air atmosphere with 5% CO ₂ and 100% humidity. Then 7β-OH-EPIA was added and the cells were incubated for another hour at 37 ° C, after which the recombinant human tumor necrosis factor α (TNF-α) was added and the cells continued to incubate for another 3 hours. 7β-OH-EPIA was obtained in dimethyl sulfoxide (DMSO), all other agents were obtained in RPMI 1640 medium. The required control samples contained the medium or DMSO in the same concentration, which was always <0.1% vol./about. The incubation was stopped by centrifuging the tubes with an acceleration of 11000 g for 30 seconds at 22 ° C, and supernatants were transferred to clean 1.5 ml tubes. Samples were immediately processed to determine PGD2 as described below or stored at −20 ° C. until PGE2 or 15d-PGJ2 was measured.

Enzyme immunoassays (EIA) of prostaglandins

The production of prostaglandins by human monocytes under the influence of 7β-OH-EPIA without stimulation or upon stimulation of TNF-α was determined by measuring extracellular eicosanoid levels using commercially available EIA kits.

Enzyme-linked immunosorbent assay PGD2

PGD2 analysis cannot be performed directly from cell supernatants, since this prostaglandin is chemically unstable and quickly decomposes into a number of J-series prostaglandins, including PGJ2, Δ ¹² -PGJ2 and 15-deoxy-Δ ^12,14 -PGJ2. To eliminate this problem, the unstable prostaglandin PGD2 was chemically treated to form a stable derivative, in this case, prostaglandin D2-methoxamine (PGD2-MOX), which was stored for subsequent analysis. Immediately after incubation, 1.5 ml tubes containing 100 μl of methyl oxime reagent reagent (methoxylamine hydrochloride (MOX-HCl) and sodium acetate dissolved in an ethanol / water solution with a ratio of 10:90 v / v were added 100 μl of sample supernatant The tubes were placed in a water bath and reacted for 30 minutes at 60 ° C. At the end of this period, the samples were stored at -80 ° C. PGD2 levels were determined using a Cayman Chemical prostaglandin D2-MOX enzyme immunoassay kit (Catalog number 512011).

Enzyme immunoassay PGE2

Extracellular PGE2 levels were determined using an R&D Systems PGE2 Parameter immunoassay kit according to the manufacturer's instructions for a highly sensitive experimental procedure.

Enzyme immunoassay 15d-PG2

Extracellular levels of 15d-PGJ2 were determined using an Assay Designs 15-deoxy-Δ ^12,14- prostaglandin J2 Correlate-EIA Prostaglandin enzyme-linked immunosorbent assay kit (catalog number 900-023) in accordance with the manufacturer's instructions.

Statistical analysis

Results are expressed as mean ± standard deviation for n = 3 incubations. The probability (P) that two sets of data differ from each other was determined using the unpaired Student t-test. The difference was considered significant at P <0.05. Statistical calculations were performed on an Apple Macintosh computer using Statview software from Abacus Concepts Inc.

results

The effect of 7β-OH-EPIA on the production of PGD2 by human mononuclear cells

Figure 5 (a) shows the effect of increasing concentrations of 7β-OH-EPIA (0.1 nM - 1000 nM) on PGD2 levels found in supernatants of 1 × 10 ⁷ peripheral blood mononuclear cells / ml, incubated for 4 hours with 7β -ON-EPIA. 7β-OH-EPIA, apparently, causes an increase in PGD2 production depending on the concentration, which reaches a maximum value at 100 nM of 7β-OH-EPIA (102 ± 24 pg / ml PGD2), which is significantly higher than that of the control sample ( 40 ± 13 pg / ml PGD2; P = 0.01).

5 (b) shows the effect of increasing concentrations of 7β-OH-EPIA (0.1 nM - 1000 nM) on PGD2 levels found in supernatants of mononuclear cells incubated in the presence of TNF-α (0.5 μg / ml). TNG-α caused a 2.3-fold increase in PGD2 compared to the control DMSO vehicle (P <0.05). At concentrations of 0.1 nM - 100 nM, 7β-OH-EPIA does not seem to affect TNF-α-induced PGD2 biosynthesis. At the highest concentration of 7β-OH-EPIA used in this experiment (1000 nM), PGD2 levels increased to 164 ± 31 pg / ml (P = 0.03 compared to using only TNF-α).

The effect of 7β-OH-EPIA on the production of PGE2 by human mononuclear cells

6 (a) shows the effect of increasing concentrations of 7β-OH-EPIA (1 nM - 1000 nM) on PGE ₂ levels found in supernatants of 6 × 10 ⁵ peripheral blood mononuclear cells / ml, incubated for 4 hours with 7β- OH-EPIA. 7β-OH-EPIA appears to increase PGE ₂ levels compared to control DMSO, however, such increases were not statistically significant.

6 (b) shows the effect of increasing concentrations of 7β-OH-EPIA (1 nM - 1000 nM) on PGE ₂ levels found in supernatants of mononuclear cells incubated in the presence of TNF-α (10 ng / ml). TNF-α stimulated a significant 1.97-fold increase in PGE2 compared to control DMSO (P = 0.001). At concentrations of 1 nM - 100 nM, 7β-OH-EPIA significantly inhibited TNF-α-induced biosynthesis of PGE2 from 167 ± 6 pg / ml under the influence of TNF-α alone to 75 ± 25 pg / ml, 82 ± 23 pg / ml and 74 ± 12 pg / ml for 1 nM, 10 nM and 100 nM of 7β-OH-EPIA, respectively (all P values are <0.02 compared to using only TNF-α). At the highest concentration of 7β-OH-EPIA used in this experiment (1000 nM), no effect on TNF-α-induced PGE2 production was detected.

The effect of 7β-OH-EPIA on the production of 15d-PGJ2 by human mononuclear cells

7 (a) shows the effect of increasing concentrations of 7β-OH-EPIA (0.1 nM - 1000 nM) on the 15d-PGJ2 levels found in supernatants of mononuclear cells incubated for 4 hours with 7β-OH-EPIA. 7β-OH-EPIA significantly increased 15d-PGJ2 levels by about 9-12 times at all concentrations used. The levels increased from 51 ± 6 pg / ml for the control DMSO to 506 ± 101 pg / ml, 539 ± 51 pg / ml, 520 ± 45 pg / ml, 450 ± 133 pg / ml and 590 ± 84 pg / ml, respectively, for 0 , 1 nM, 1 nM, 10 nM, 100 nM and 1000 nM 7β-OH-EPIA (all P values <0.05 compared with the control DMSO).

Fig. 7 (b) shows the effect of increasing concentrations of 7β-OH-EPIA (0.1 nM - 1000 nM) on 15d-PGJ2 levels found in supernatants of mononuclear cells incubated with TNF-α (10 ng / ml). At the concentration of the cytokine used in this experiment, TNF-α seems to stimulate a slight increase in 15d-PGJ2, however, this increase did not differ significantly from the control DMSO. At all concentrations used, 7β-OH-EPIA increased levels of 15d-PGJ2 in the presence of TNF-α. Levels of 15d-PGJ2 were increased from 157 ± 39 pg / ml in the presence of TNF-α alone to 348 ± 48 pg / ml, 334 ± 24 pg / ml, 356 ± 85 pg / ml, 406 ± 30 pg / ml and 318 ± 100 pg / ml in the presence of TNF-α, together with 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM 7β-OH-EPIA, respectively (all P values <0.05 compared to using only TNF-α) .

Example 3

7α-Hydroxy-DHEA, 7β-hydroxy-DHEA and 7β-hydroxy-EPIA are natural metabolites of dehydroepiandrosterone (DHEA) and epiandrosterone (EPIA). Since it is noted in the scientific literature that many steroids interfere with inflammatory and immune processes, the aim of the present invention was to study the effect of these hydroxysteroids on PG production and gene expression of related enzymes. Human peripheral blood monocytes (PBMCs) were cultured for 4 hours and 24 hours in the presence of each of these steroids (1-100 nM) with and without the addition of the pro-inflammatory cytokine TNF-α (10 ng / ml). The levels of PGE2, PGD2, and 15-deoxy-Δ ^12.14 -PGJ2 (15d-PGJ2) were measured in an incubation medium, and the content of cyclooxygenase (COX-2) and PGE synthase (m-PGES1) mRNAs was determined by quantitative PCR method with reverse transcriptase (RT-PCR). The addition of TNF-α caused increased PG production and increased levels of COX-2 and m-PGES1 mRNA. Of the three steroids studied, only 7β-hydroxy-EPIA decreased the expression of COX-2 and m-PGES1 with a significant decrease in PGE2 production and an increase in 15d-PGJ2 production. The results show that 7β-hydroxy-EPIA has an anti-inflammatory effect.

1.1. Obtaining and culturing monocytes of human peripheral blood (PBMC)

Whole blood taken from donors was collected in bags containing EDTA at Etablissement Francais du Sang (Brest, France). PBMCs were isolated from whole blood under sterile conditions for 36 hours after blood collection. Density gradient centrifugation was performed on ficoll (Eurobio). Cells were washed in RPMI 1640 medium (Eurobio) and suspended in RPMI 1640 medium containing 10% heat inactivated fetal calf serum (Eurobio), 2 mM glutamine (D. Dutscher), 100 units of penicillin / ml (D. Dutscher) and 100 μg streptomycin / ml (D. Dutscher). Monocytes were selected by adhesion to plastic for 1 hour, and approximately 10 ⁷ cells were cultured on 6-well tissue culture plates (3 ml medium / well). All incubations were performed in a humid chamber at 37 ° C and 5% CO ₂ . Cells were isolated and dispersed in 2 ml of fresh incubation medium containing either 7β-hydroxy-EPIA, or 7β-hydroxy-DHEA, or 7α-hydroxy-DHEA (in 20 μl of ethanol) in the presence or absence of 0.01 μg / ml TNF -α (Sigma-Aldrich). In control incubations, 20 μl of ethanol was used, but without a steroid. Supernatants were collected after incubation for 4 hours and 24 hours to measure the content of PG and cells were used for subsequent isolation of RNA. As a result of the one-stage extraction method using trizole reagent (Invitrogen, Cergy-Pontoise, France), total RNA was obtained.

1.2. Real-time reverse transcriptase polymerase chain reaction

cDNA was synthesized from RNA treated with TURBO DNase I (Ambion, Huntingdon, UK) using a Superscript first strand synthesis system kit (Invitrogen). RT-PCR amplification mixtures (50 μl) contained a 2.5-fold solution of RealMaster / 20-fold SYBR mixture (11.25 μl) (Eppendorf, Le Pecq, France) and 200 nM of the upper and lower seeds. Reactions were carried out in a fuser for reactions RealPlex ep gradient S (Eppendorf). The cyclization was carried out under the following conditions: 10 minutes at 95 ° C and 45 cycles at 95 ° C, 55 ° C and 68 ° C for 15 seconds, 30 seconds and 30 seconds, respectively. As a result of each analysis, a standard curve was constructed for four consecutive dilution points of the control cDNA. For quantitative analysis, the required HPRT1 gene was used. All oligonucleotide seeds (table 2) were synthesized using Genecust / Distribio (Evry, France). The specificity of the amplified product was monitored by the melting curve of the product and confirmed by performing agarose gel electrophoresis analysis.

1.3. Prostaglandin Measurements (PG)

To determine the levels of PGE2 (Oxford Biomedical Research, UK) and 15d-PGJ2 (Assay designs, Euromedex, France) in a culture medium, commercially available EIA kits were used. PGD2 levels were measured using an enzyme-linked immunosorbent assay for prostaglandin D2-MOX (Cayman Chemical, Euromedex). In this case, prior to analysis, the samples just obtained were immediately treated with MOX-HCl reagent, which converted PGD2 to PGD2-MOX, thus preventing any further chemical decomposition.

1.4. Statistical Data Analysis

All analyzes were performed three times and the results were presented as a mean curve ± standard error of the mean. Analysis of variance on one basis was performed based on multiple Duncan tests to compare differences between groups. Differences were considered statistically significant at p <0.05.

2.1. Exposure to 7α-hydroxy-DHEA

Human PBMCs were cultured with and without TNF-α for 4 or 24 hours. The levels of PGE2, PGD2 and 15d-PGJ2 were measured in the culture medium, and mRNA production of related genes (COX-2, m-PGES1) was measured in the cell. The data obtained are shown in Table 2. In the absence of TNF-α and the addition of 7α-hydroxy-DHEA in three different concentrations, there was no significant change in the levels of PGE2, PGD2 and 15d-PGJ2 in the cultures for 4 hours. Only 15d-PGJ2 level rose moderately after culturing for 24 hours. Incubation with 7α-hydroxy-DHEA significantly increased m-PGES1 mRNA levels in cultures after 24 hours.

The presence of TNF-α caused an expected increase in all prostaglandin levels after 24 hours. Incubation with 7α-hydroxy-DHEA for 4 hours did not change PG levels at all tested concentrations. In contrast, the levels of PGE2 and two PGD2-15d-PGJ2 were respectively significantly increased and decreased after incubation with 7α-hydroxy-DHEA and TNF-α for 24 hours compared to using only TNF-α. In addition, incubation with TNF-α (i.e., inflammatory activation) caused a marked increase in COX-2 and m-PGES1 mRNA production. Incubation with 7α-hydroxy-DHEA did not cause significant changes in mRNA levels compared to using only TNF-α.

2.2. Exposure to 7β-hydroxy-DHEA

Human PBMCs were cultured with and without TNF-α for 4 or 24 hours. The levels of PGE2, PGD2 and 15d-PGJ2 were measured in the culture medium, and mRNA production of related genes (COX-2, m-PGES1) was measured in the cell. The data obtained are shown in table 3. Incubation with 7β-hydroxy-DHEA in three concentrations for 4 hours in the absence of TNF-α did not cause significant changes in the levels of PGE2, PGD2 and 15d-PGJ2 in the culture medium. However, the levels of PGD2 and 15d-PGJ2, as well as m-PGES1 mRNA, increased after 24 hours of incubation with 7β-hydroxy-DHEA in culture.

The presence of TNF-α caused an expected increase in all prostaglandins (PG) after 24 hours. Incubation with 7β-hydroxy-DHEA for 4 hours did not cause further change in PG levels at any concentrations tested. In contrast, PGD2 and 15d-PGJ2 levels decreased significantly after incubation with 7β-hydroxy-DHEA and TNF-α for 24 hours compared to using only TNF-α. In addition, incubation with TNF-α (i.e., inflammatory activation) caused a significant increase in COX-2 and m-PGES1 mRNA production. Incubation with 7β-hydroxy-DHEA did not cause comparable changes in mRNA levels compared to using only TNF-α.

2.3. Exposure to 7β-hydroxy-EPIA

Human PBMCs were cultured with and without TNF-α for 4 or 24 hours. The levels of PGE2, PGD2 and 15d-PGJ2 were measured in the culture medium, and mRNA production of related genes (COX-2, m-PGES1) was measured in the cell. The data obtained are shown in table 4. Incubation with 7β-hydroxy-EPIA in three concentrations for 4 hours in the absence of TNF-α did not cause significant changes in the levels of PGE2, PGD2 and 15d-PGJ2 in the culture medium. However, the contents of PGD2 and 15d-PGJ2 markedly increased upon incubation of cell cultures with 7β-hydroxy-EPIA for 24 hours. The expression of COX-2 was not significantly altered by the indicated steroid after 4 or 24 hours, but the m-PGES1 mRNA levels decreased significantly and then increased after 4 hours and 24 hours, respectively.

The presence of TNF-α caused an expected increase in all prostaglandins (PG) after 24 hours. Incubation with 7β-hydroxy-EPIA for 4 hours did not cause a further change in PG levels at any concentrations tested. In contrast, after 24 hours, two lower doses of 7β-hydroxy-EPIA reduced PGD2 and increased 15d-PGJ2 compared to using TNF-α alone. Incubation with TNF-α (i.e., inflammatory activation) also caused a marked increase in COX-2 mRNA and m-PGES1. The increase in production of COX-2 mRNA and m-PGES1 under the influence of TNF-α was slowed by incubation of cell cultures with 10 and 100 nM 7β-hydroxy-EPIA for 24 hours.

In general, the results clearly show that 7β-hydroxy-EPIA has a significant anti-inflammatory effect.

Example 4

The impact of 7β-OH-EPIA on an experimental model of colitis

Administration to rats of sodium dextran sulfate (DDS) in drinking water for 6 consecutive days caused inflammation of the colon (colitis), characterized by a decrease in the length of the colon, increased activity of MPO, the absence of mucus in goblet cells and increased expression of COX-2 and mPGES-1- synthase and production of prostaglandin E2 (PGE2). The introduction of DSS also increased the content of oxidative stress markers, such as protein carbonyl (Prot CO) and T-folds in the intestine. The authors of the present invention provide evidence that the introduction of 7β-hydroxy-EPIA once a day for 7 days before the introduction of DSS can prevent the development of DSS-induced colitis. The administration of 0.01 mg / kg of 7β-hydroxy-EPIA completely prevented colitis and tissue inflammation, and the indicated effect of 7β-hydroxy-EPIA was associated with a marked decrease in oxidative stress markers and PGE2 production, which was due to an initial but temporary increase in COX- expression 2 and a stable increase in the production of anti-inflammatory prostaglandin 15d-PGJ2. The results show that 7β-hydroxy-EPIA has significant anti-inflammatory effects at very low doses in this generally accepted experimental model of inflammatory bowel disease (IBD).

The experimental technique

Animals

All methods and methods for performing the experiment were in accordance with the Directive 86/609 / CEE of the European Union on the use of laboratory animals. Male Wistar rats (180-200 g) purchased from Charles River (L'Arbresle, France) received laboratory rodent food and water as needed.

Drug Administration and Colitis Induction

After a 7-day adaptation period, animals were divided into two control groups (a control group of animals treated with pseudotherapy and a control group of animals that were injected with 7β-hydroxy-EPIA) and two groups of animals suffering from colitis (untreated colitis and colitis treated with 7β-hydroxy-EPIA). 7β-hydroxy-EPIA (0.01, 0.1 and 1 mg / kg body weight, dissolved in DMSO) or only DMSO (vehicle) was administered intraperitoneally once a day for 7 days, from 0 to 7 day. Colitis was induced from the 7th to the 14th day by adding DSS (molecular weight 36-50 kDa; MP Biomedicals, France) in drinking water. The two control groups received only tap water.

Macroscopic assessment of colon damage

On the 9th, 11th, 13th, and 14th day, body weight, colon length, stool consistency were recorded, and fresh rectal bleeding was assessed by visual inspection. The severity of lesions of the colon was evaluated blindly in accordance with the description given in the publication Mabley et al., 2001, Inflamm. Res .; 50: 561-569) (0: well-formed granules and no damage to the colon; 1: colon with a small amount of blood mixed with feces; 2: colon with a large amount of blood in feces; 3: colon filled with blood when lack of feces).

Histological examination

Part of the proximal colon (1 cm) was fixed in 4% formaldehyde (Labonord, Templemars, France) and embedded in paraffin. Tissue sections (5 μm) were prepared, which were then cleaned, hydrated and stained with hematoxylin / eosin or Alcian blue in accordance with standard histological methods for colon lesions and goblet cells, respectively.

Obtaining tissue homogenates

The colon was opened along the border of the mesenteric part and the epithelial cells were scraped off with the blunt edge of a glass slide, weighed, washed and centrifuged. Cell debris was homogenized in 9 volumes of TKE buffer (10 nM Tris buffer with HCl; 150 mM KCl; 1 mM EDTA; 0.25 mM PMSF, pH 7.4) and frozen at -80 ° C until subsequent use. The protein content of the homogenates was measured as described in Lowry et al. (J. Biol. Chem. 1951; 193: 265-74).

MPO activity

MPO activity was determined in homogenates by the o-dianisidine-based method described in Krawisz et al. (Gastroenterology 1984; 87 (6): 1344-50), which has been modified as described in Pelissier et al. (Steroids 2006; 71 (3): 240-8). MPO activity was expressed as the amount of enzyme required to change the absorbance per minute at 460 nm using the extinction coefficient for oxidized o-dianisidine (1.13 × 10 ⁴ mol ^-1 cm ^-1 ).

Biochemical determination of oxidative stress

Lipid peroxidation (T folds) was measured by the method described by Ohkawa et al. (Anal. Biochem. 1979; 95: 315-58), which has been modified as described in Albrecht et al., (1992, Toxicol. Lett; 63: 91-96). The optical density of the adduct of malondialdehyde and thiobarbituric acid with a ratio of 1: 2 (Sigma-Aldrich, St. Quentin-Fallavier, France) was measured at 532 nm (used extinction coefficient: 0.156 μmol ^-1 cm ^-1 ). The carbonyl content of oxidized proteins (Prot CO) was determined by the method described in Levine et al. (Methods Enzymol. 1990; 186: 464-78). The content of non-protein sulfhydryl groups (mainly GSH), taken as a marker of antioxidant protection in homogenates, was determined by the method described in Sedlak and Lindsay (Anal. Biochem. 1968; 25 (1): 192-205).

Prostaglandin immunoassays

To determine PGE2 levels (Oxford Biomedical Research, UK) and 15d-PGJ2 levels (Assay designs, Euromedex, France) in colon supernatants obtained from freshly prepared homogenates, commercially available EIA kits were used. PGD2 levels were measured using a prostaglandin EIA kit D2-MOX (Cayman Chemicdal, Euromedex). In this case, prior to analysis, the samples that were just prepared were immediately treated with the MOX-HCl reagent, which converted PGD2 to PGD2-MOX, thus preventing any further chemical decomposition.

Real-time reverse transcriptase PCR

Total RNA from freshly prepared colon samples (300 mg) was extracted using Trisol reagent (Invitrogen, Cergy-Pontoise, France). Poly (A) RNA was purified from total RNA using the MicroPoly (A) Purist kit (Amvion, Huntingdon, UK). cDNA was synthesized using a Superscript first strand synthesis kit (Invitrogen). Real-time PCR was performed using mixtures (25 μl) containing a 2.5-fold solution of a RealMaster / 20-fold SYBR mixture (11.25 μl) (Eppendorf, Le Pecq, France) and 300 nM upper and lower seeds . Reactions were carried out in a fuser for reactions RealPlex ep gradient S (Eppendorf). The cyclization was carried out under the following conditions: 10 minutes at 95 ° C and 40 cycles at 95 ° C, 55 ° C and 68 ° C for 15 seconds, 30 seconds and 30 seconds, respectively. As a result of each analysis, a standard curve was constructed for four consecutive dilution points of the control cDNA. Oligonucleotide seeds were synthesized using Genecust / Distribio (Evry, France). The specificity of the amplified product was monitored by the melting curve of the product and confirmed by performing agarose gel electrophoresis analysis. MRNA levels were expressed relative to the control DMSO after normalization to HPRT1.

Statistical analysis

All analyzes were performed three times for each animal and the results were presented in the form of a graph of the mean ± standard error of the mean. Analysis of variance on one basis was performed on the basis of multiple Duncan tests to compare differences between groups. Differences were considered statistically significant at p <0.05.

results

The study of induction of colitis in time

Administration to rats of 5% DSS in drinking water for 7 days (D7-D14) caused clinical and histological signs of colitis without the death of animals. All rats usually developed severe diarrhea 5 days after the induction of colitis (day 12) and rectal bleeding was observed the next day. In all rats that received DSS, on day 11, a decrease in body weight, epididymal adipose tissue mass and liver mass was recorded compared with control animals subjected to pseudo-treatment (respectively -5%, -17%, -8%; p < 0.05). A decrease in the above indicators was also noted on the 14th day (table 5). No change in spleen mass was noted. Compared with the control group of animals subjected to pseudo-treatment, rats treated with DSS also showed a significant shortening of the colon from the 11th day (-14%, p <0.05) to the 14th day (-26%; p <0 , 05) (see table 5) due to thickening of the colon tissue and lack of stool. MPO activity in the colon mucosa was measured as an indicator of neutrophil infiltration. On the 13th and 14th day in the group of animals suffering from colitis, a 9-fold and 7-fold increase in the activity of MPO in the mucous membrane was detected compared to control animals subjected to pseudo-treatment (p <0.05) (Fig. 8 ,BUT). Until the 13th day, there was no change in the activity of MPO. The results are comparable with the results of histological analysis. Indeed, the main signs of inflammation of the colon, namely latent deformation, neutrophil infiltration into the mucous membrane tissue and the loss of goblet cells containing less mucin, appeared in the group of animals suffering from colitis on the 13th day and became more pronounced on the 14th day after compared with the control group of animals subjected to pseudo-treatment (data not shown).

As shown in Fig. 8, two markers of oxidative stress, namely Prot CO, T-folds, and GSH antioxidant defense levels, were significantly increased compared to control levels on the 13th and 14th day in the colon mucosa animals suffering from colitis.

Protective effect of 7β-hydroxy-EPIA in case of colitis

Two low doses of 7β-hydroxy-EPIA (0.01, 0.1 mg / kg) prevented DSS-induced damage to the colon, as indicated by the cessation of diarrhea and rectal bleeding on day 14. Intraperitoneal injection of 0.01 mg / kg of 7β-hydroxy-EPIA once a day for 7 days before DSS administration restored body weight to control levels without changing the weight of the epididymis (table 5). Between the 9th and 14th days, the decrease in the length of the colon was less pronounced in the groups that were given two lower doses of the steroid (0.01, 0.1 mg / kg) compared with the group of animals suffering from colitis (data not shown for the 9th day, table 5). 7β-hydroxy-EPIA prevented goblet reduction in goblet cells on day 14 and histological changes, such as pathological crypts and neutrophil infiltration (data not shown), and significantly reduced MPO activity with all doses, 0.01, 0.1 and 1 mg / kg (Fig. 8, A). Oxidative stress parameters (Prot CO, T-folds) and GSH levels were the same on the 9th day (not shown) and the 11th day in all groups, namely in the control group of animals subjected to pseudotherapy, in the group of animals suffering from colitis, which was not treated, and in the group of animals suffering from colitis, which were injected with 7β-hydroxy-EPIA (0.01, 0.1 and 1 mg / kg) (Fig. 8, B, C and D). All oxidative stress markers and the antioxidant defense marker did not change in animals that were injected with 7β-hydroxy-EPIA (days D0-D7), while these parameters were significantly increased in the group of animals suffering from colitis (p <0.05). The results show that 7β-hydroxy-EPIA has a significant anti-inflammatory effect at very low doses in this experimental model of inflammatory bowel disease (IBD).

Table 5 Final body and organ mass, colon length, macroscopic assessment of colon damage in rats suffering from DSS-induced colitis (DSS (5%) was injected into tap water for 7 days (days 7-14). Data obtained at the end of the experiment (Day 14), expressed as mean ± standard error of the mean. Values not marked with a superscript (ac) in the line differ significantly (p <0.05). Colon lesion was assessed as described in Mabley et al. (2001, Inflamm. Res., 50, 561-569) (0: well-formed groups anula and lack of lesion of the colon; 1: colon with a small amount of blood in the feces; 2: colon with a large amount of blood in the feces; 3: colon filled with blood in the absence of feces). * mg / kg body weight Group Control group Colitis group Colitis group previously administered with 7β-hydroxy-EPIA 0 0 0.01 * 0.1 * one* The number of rats (n) fifteen 23 3 12 12 Body weight (g) 304.6 ± 6 ^a 279.6 ± 6.5 ^b 297.5 ± 9.5 ^a 272.1 ± 6 ^b 267.3 ± 7.7 ^b Epididymis mass (g) 2.5 ± 0.08 ^a 2.1 ± 0.1 ^b 2.5 ± 0.03 ^a 1.9 ± 0.14 ^b 1.9 ± 0.1 ^b Liver mass (g) 12.7 ± 0.4 ^a 11.1 ± 0.3 ^b 12.1 ± 0.3 ^a 12.3 ± 0.6 ^a 11.9 ± 0.4 ^a The mass of the spleen (g) 0.9 ± 0.05 ^a 0.9 ± 0.08 ^a 0.8 ± 0.02 ^a 1.03 ± 0.2 ^a 0.82 ± 0.06 ^a Colon Length (cm) 18.4 ± 0.6 ^a 13.7 ± 0.4 ^b 16 ± 0.01 ^s 15.6 ± 0.3 ^s 14.2 ± 0.6 ^b

Production of prostaglandins (PG) in colon tissue: exposure to β-hydroxy-EPIA

Administration of 7β-hydroxy-EPIA did not alter the levels of PGE2 and PGD2 in colon tissue in control rats without colitis (data not shown). In contrast, administration of 7β-hydroxy-EPIA from day 0 to day 7 caused a significant increase in 15d-PGJ2 levels from day 2 to day 14 compared to baseline. The administration of 0.1 mg / kg of 7β-hydroxy-EPIA caused a 51-fold increase in the level on the 2nd day, which gradually decreased from the 4th day to the 14th day (decrease from a 44-fold to a 5-fold value) ( Fig.9, A, Fig.10, C).

The administration of the inflammatory agent DSS from day 7 to day 14 caused a marked increase in the production of pro-inflammatory prostaglandin PGE ₂ on day 14, while the level of anti-inflammatory prostaglandin 15d-PGJ ₂ significantly decreased (figure 10).

Administration of 0.01 mg / kg of 7β-hydroxy-EPIA from day 0 to day 7 completely prevented DSS-induced PGE2 production in the colon and significantly increased the level of 15d-PGJ2 (Fig. 10).

Expression of COX-2 and PG synthase in groups of pseudo-sick animals and animals suffering from colitis: effects of previously introduced 7β-hydroxy-EPIA

Due to significant changes in prostaglandin levels with the introduction of DSS and 7β-hydroxy-EPIA, the authors of the present invention investigated the change in the expression of COX-2, mPGES-1 and H-PGDS as a result of preliminary steroid administration by quantifying specific mRNA by reverse transcriptase PCR RT-PCR) in real time. Investigated the transcription of these genes and compared with transcription of the binding gene HPRT1. In control rats not suffering from colitis, the administration of 0.1 mg / kg of 7β-hydroxy-EPIA caused a significant 1.5-fold increase in COX-2 mRNA levels after 15 hours with a significant decrease on the 2nd day and 4th day with subsequent return to the original values (Fig.9, B). The expression of mPGES-1 mRNA temporarily increased between the 6th and 15th hours and returned to its original level on the 2nd day. The synthesis of H-PGDS mRNA was not changed during the experiment.

After the induction of colitis under the influence of DSS on the 13th and 14th day, a 2.5-fold increase in the expression of COX-2 mRNA was observed (Fig. 11, A), while the amount of mPGES-1 mRNA significantly increased only by 13- th day (Fig. 11, B). With the introduction of DSS, the amount of H-PGDS did not change.

Preliminary administration of 7β-hydroxy-EPIA (from day 0 to day 7) inhibited the increase in mRNA synthesis of both COX-2 and mPGES-1 under the influence of DSS (Fig. 11).

The results show that the anti-inflammatory effect of 7β-hydroxy-EPIA is due to a simultaneous decrease in the production of PGE2 and an increase in the production of 15d-PGJ2. The long-term effects of 7β-hydroxy-EPIA on 15d-PGJ2, which took place in this study, suggest that 7β-hydroxy-EPIA causes a prolonged change in the expression of genes involved in inflammation and its resolution.

FIG. 8 shows the effects of 7β-hydroxy-EPIA on (A) myeloperoxidase (MPO) and oxidative stress markers, namely (B) Prot CO, (C) T-folds and (D) GSH antioxidant defense marker in colon tissue guts. Biomarker levels indicate that rats treated with DSS differed significantly from control animals on days 13 and 14 (p <0.05). 7β-Hydroxy-EPIA significantly reduced MPO activity on the 13th and 14th day compared with the group of rats suffering from colitis (p <0.05), and restored the parameters of oxidative stress (B, C and D) to control levels on 13th and 14th day with all doses administered. Values are expressed as mean ± standard error of the mean (A) and percentage for the group of pseudo-sick rats (B, C and D) in the presence of 3-23 rats in the group. In Fig. 8, A-8, D, black bars = control group of pseudo-sick rats; white columns = group of rats suffering from DSS-induced colitis; dark gray columns = DSS + 0.01 mg / kg 7β-hydroxy-EPIA; moderately gray columns = DSS + 0.1 mg / kg 7β-hydroxy-EPIA and light gray columns = DSS + 1.0 mg / kg 7β-hydroxy-EPIA.

The group of rats suffering from DSS-induced colitis, compared with the control group of all-rats (p <0.05) of the group of rats that were injected with 7β-hydroxy-EPIA, compared with the group of rats suffering from DSS-induced colitis (p <0, 05).

Figure 9 shows the level of 15d-PGJ2 in the colon (A) and the quantitative determination of the relative expression of COX-2, mPGES-1 and H-PGDS (B) mRNA at different periods of administration of 7β-hydroxy-EPIA. Values are expressed as mean ± standard error of the mean (n = 3-15) (A) and mRNA values are presented in comparison with mRNA values in the control group of pseudo-sick animals after normalization to HPRT1 (B). In Fig. 9, A black column = control group of pseudo-sick rats; dark gray column = 0.01 mg / kg 7β-hydroxy-EPIA; a moderately gray column = 0.1 mg / kg of 7β-hydroxy-EPIA and a light gray column = 1.0 mg / kg of 7β-hydroxy-EPIA. In Fig.9, In a dark gray column with white dots = SOX-2; light gray column with black dots = mPGES-1 and white column with black dots = H-PGDS.

Figure 10 shows the effect of 7β-hydroxy-EPIA on the synthesis of prostaglandins E2, D2 and 15d-PGJ2 in the colon during the administration of DSS. The introduction of DSS significantly increased the synthesis of PGE2 (A), D2 (B) and 15d-PGJ2 (C) (p <0.05) on the 13th and 14th day (p <0.05). Administration of 7β-hydroxy-EPIA for 7 days prior to administration of DSS decreased PGE2 synthesis on days 13 and 14 (A) with a significant increase in 15d-PGJ2 production at all doses administered (C). Data are expressed as mean ± standard error of the mean for n = 3-23. 10, A-10, C black column = control group of pseudo-sick animals; white column = group of animals suffering from DSS-induced colitis; dark gray column with white dots = DSS + 0.01 mg / kg 7β-hydroxy-EPIA; light gray column with black dots = DSS + 0.1 mg / kg 7β-hydroxy-EPIA; and a white column with black dots = DSS + 1.0 mg / kg 7β-hydroxy-EPIA.

Figure 5 shows the expression in the colon of mRNA COX-2, mPGES-1 and H-PGDS during induction of colitis. Colitis caused a significant increase in the synthesis of COX-2 mRNA on the 13th and 14th day (A), while the synthesis of mPGES-1 increased only on the 13th day (B). A return to baseline gene expression levels was observed at all doses of 7β-hydroxy-EPIA. 10, A-10, C white column = group of animals suffering from DSS-induced colitis; dark gray column = DSS + 0.01 mg / kg 7β-hydroxy-EPIA; moderately gray column = DSS + 0.1 mg / kg 7β-hydroxy-EPIA; and light gray column = DSS + 1.0 mg / kg 7β-hydroxy-EPIA.

Example 5

1. The effect of 7β-OH-EPIA on collagen-induced arthritis

In this experiment, the efficacy of controlling 7β-OH-EPIA inflammatory and pathological changes that occurred in a collagen-induced arthritis model was investigated. An animal model using mice demonstrates the clinical reminiscence of human arthritis.

Male DBA / 1 mice (10-12 weeks old) were injected with 100 μg of type II chicken collagen emulsified in Freund's complete adjuvant (CII / CFA) on day 0 by injection at the base of the tail. The introduction was carried out daily from the 20th to the 50th day by subcutaneous injection (groups 2, 3 and 4) and from the 0th to the 30th day by subcutaneous injection (group 5) as follows:

Experimental groups (n = 10 / group)

Group 1: Day 0 CII / CFA, no treatment - pseudo-treatment from day 20 to day 50 Group 2: Day 0 CII / CFA, administration of 1 μg / kg 7β-hydroxy-EPIA from day 20 to day 50 Group 3: Day 0 CII / CFA, administration of 10 μg / kg of 7β-hydroxy-EPIA from day 20 to day 50 Group 4: Day 0 CII / CFA, administration of 100 μg / kg of 7β-hydroxy-EPIA from 10th to 50th day Group 5: Day 0 CII / CFA, administration of 10 μg / kg 7β-hydroxy-EPIA from day 0 to day 30 Group 6: Day 0 CII / CFA, no treatment - pseudo-treatment from day 0 to day 30

At the end of the experiment, the hind limbs were removed, the knee homogenates were collected to measure prostaglandins (only groups 1 and 4), and the paws were fixed for examination of the pathology by standard methods. Each paw was cut halfway in the longitudinal direction, dissected and decalcified to make slices. Decalcified samples were subjected to standard processing, sections were made and one stained section was prepared for examination. Slices were obtained from both halves of each sample. Each paw was evaluated according to a standard rating system. Samples were evaluated blindly, having no idea of the experimental technique or group membership.

Clinical swelling of the joints was evaluated twice a week from the 21st to the 50th day. Each of the four limbs was assigned a number of points in accordance with the following rating system (joint clinical assessment of the disease):

0 normal condition one slight swelling of the entire joint or inflammation of a single finger 2 secondary swelling of the entire joint with redness and / or inflammation of several fingers 3 severe joint inflammation and redness covering many fingers four severe joint inflammation and redness, covering many fingers, obvious signs of bone deformation

The results are shown in table 6. From the table it can be seen that the introduction of 7β-OH-EPIA caused a noticeable decrease in joint swelling, as measured by the above method of clinical evaluation of the disease, compared with the control groups (control groups of pseudo-sick animals 1 and 6 were combined). This result was confirmed by pathological findings, indicating less swelling of the paws (table 7). Table 7 shows that the lowest pathology score, according to which 9 out of 10 animals had no histopathological change in the joints of the paws, occurred when 7β-hydroxy-EPIA was started to be administered simultaneously with the introduction of arthritis-inducing collagen ( Day 0), and continued treatment until the 30th day. Thus, treatment of 7β-hydroxy-EPIA diseases such as rheumatoid arthritis can have a beneficial effect.

2. Measurement of prostaglandins in knee homogenates

The levels of 15d-PGJ2 and PGE2 in the knee homogenates obtained above were measured by standard methods. As can be seen from table 8, the levels of PGE2 were lower in the group that was administered 0.1 mg / kg of 7β-OH-EPIA, compared with the control group not being treated. In the 0.1 mg / kg 7β-OH-EPIA group, 15d-PGJ2 levels were higher compared to the untreated control group.

Conclusion

7β-Hydroxy-EPIA had a clear anti-inflammatory effect. Treatment during the induction of the disease had the greatest effect. The same result was obtained for other test compounds, including steroids tested in this model, and reflects the relative ease of preventing joint damage compared to restoring existing changes. The condition of the animals that were given higher doses of 7β-hydroxy-EPIA was the same when the treatment was started on the 20th day. This degree of protection against the disease is high enough for the treatment regimen. The lowest dose of 7β-hydroxy-EPIA appears to have less impact on the disease, although statistical analysis is needed to determine if the result is a consequence of the dose-response relationship.

Analysis of the levels of prostaglandins E2 and 15d-J2 showed the presence of a pronounced effect of treatment. PGE2 levels were lower in treated mice compared to untreated control animals. In contrast, 15d-PGJ2 levels increased after treatment.

Clinical observation of the course of the disease and histopathology data are convincing evidence of the anti-inflammatory effect of 7β-hydroxy-EPIA in the considered model of arthritis. Treatment before the onset of the disease was the most effective, which finds almost unchanged confirmation in this model. However, the fact that 7β-hydroxy-EPIA can inhibit the development of the disease when administered during its onset distinguishes this compound from many competitive drugs and is very encouraging.

Table 6 The effect of 7β-hydroxy-EPIA on the histopathology of rats suffering from collagen-induced arthritis Treatment from the 20th to the 50th day Group n Histopathology scores (both paws on day 50) Pseudo-treatment - not being treated 10 1.35 7β-hydroxy-EPIA 1.0 μg / kg 10 0.60 7β-hydroxy-EPIA 10.0 mcg / kg 9 0.44 7β-hydroxy-EPIA 100.0 mcg / kg 10 0.85

Based on the results, it can be concluded that 7β-hydroxy-EPIA, administered at low doses, such as 1.0 μg / kg, can reduce joint damage.

Table 7 The effect of 7β-hydroxy-EPIA on the histopathology of rats suffering from collagen-induced arthritis Treatment from day 0 to day 30 Group n Histopathology scores (both paws on day 50) Pseudo-treatment - not being treated 9 1.22 7β-hydroxy-EPIA 10.0 mcg / kg 10 0.2 * * In 9 out of 10 animals, histopathological changes were completely absent

Based on the above results, it can be concluded that 7β-hydroxy-EPIA, administered at a dose of 10.0 μg / kg, almost completely prevents the development of the disease, that is, damage to the joints.

Table 8 The effect of 7β-hydroxy-EPIA (0.1 μg / kg per day, from the 20th to the 50th day) on joint swelling and prostaglandin content in rats suffering from collagen-induced arthritis, on the 50th day Untreated animals Animals treated Clinical assessment 7.50 ± 2.51 3.10 ± 2.96 PGE2 (pg / ml) 177.09 ± 70.08 95.61 ± 34.65 15d-PGJ2 (pg / ml) 19.12 ± 6.43 78.96 ± 22.18 PGE2 / 15d-PGJ2 9.26 1.21

Based on the above results, it can be concluded that 7β-hydroxy-EPIA alleviates the severity of clinical symptoms of collagen-induced arthritis and alters the production of prostaglandins in tissues in favor of resolving inflammation, protecting cells and tissue repair.

Example 6

The effect of 7β-OH-EPIA in a model of cisplatin-induced peripheral neuropathy

Cisplatin is an antimitotic compound used to treat various types of cancer. However, its use is limited due to several harmful effects, among which peripheral neuropathies are particularly unpleasant. Cisplatin-induced neuropathies are mainly sensory neuropathies. In subjects, the sensitivity in the distal extremities disappears with the subsequent occurrence of sensory ataxia. Histological studies revealed axon degeneration. The effect of cisplatin on sensory neurons in cell cultures caused a decrease in the density of the axon network and subsequent degeneration of the cell body. Nerve Growth Factor (NGF), a specific growth factor for sensory neurons, protects neurons from this intoxication. Thus, intoxication of sensory neurons in culture with cisplatin is an acceptable model for studying the neuroprotective effect of compounds in the case of peripheral neuropathies.

The neuroprotective effect of 7β-OH-EPIA was evaluated against rat sensory neurons in a peripheral neuropathy model.

Primary cultures of sensory neurons of dissociated ganglion of the posterior roots of the spinal cord were incubated with 3 μg / ml cisplatin in the presence and absence of 7β-OH-EPIA for 48 and 72 hours and the following parameters were evaluated:

- cell bodies of neurons stained with anti-MAP2 antibody (protein 2 associated with microchannels);

- network density of axons stained with anti-β-tubulin antibody.

Neuron cultures

Rat sensory neurons were obtained by the method described in the publication of Hall et al., 1997 [Hall et al. (J. Neurosci. 1997 Apr. 15; 17 (8); 2775-84]. Female rats (15 days pregnant) were euthanized by displacement of the cervical spine (Rats Wistar; Janvier, Le Genest-St-Isle, France) and the fetus was removed from the uterus The spinal cord with the posterior root ganglion (DRG) was removed and placed in ice-cooled Leibovitz medium (L15, Fisher 11415-049) containing penicillin 50 units / ml - streptomycin 50 μg / ml (PS, 1%) and bovine serum albumin (BSA 1%, Sigma A6003). DRG was isolated and dissociated by trypsin treatment for 20 minutes at 37 ° C (trypsin EDTA, 10-fold, 10%, Fisher 15400054) diluted in PBS without calcium and magnesium (Fi sher 2007-03). The reaction was terminated by adding Dulbecco's Modified Eagle Medium (DMEM, Fisher 21969-035) containing grade II DNase I (0.1 mg / ml, Roche diagnostic 104159) and fetal bovine serum (FBS 10% , Fisher 10270-098) A suspension of cells was triturated with a 10 ml pipette and centrifuged at 350 x g for 10 minutes at room temperature. The debris of dissociated cells was resuspended in a culture medium of a specific composition.

Viable cells were counted on a Neubauer cytometer using a trypan blue exclusion test (Sigma) and plated at a density of 30,000 cells / well in 96-well plates (Nunc). Wells were pre-sensitized with poly-L-lysine (10 μg / ml, Sigma P2636) in ultrapure sterile water (Merck Eurolab 60759.01).

Cells were left for adhesion for 2 hours and kept in a humid chamber at 37 ° C in an atmosphere of 5% CO ₂ /95% air.

Incubation of neuronal cultures with 7β-OH-EPIA

After cultivation for 5 days, the culture medium was replaced with a culture medium of a certain composition in accordance with the following different conditions:

- filler (DMSO 0.1%)

- filler (DMSO 0.1%) + cisplatin (3 μg / ml, Sigma ref: p4394)

test compound 7β-OH-EPIA (1 nM, 10 nM and 100 nM) + cisplatin (3 μg / ml)

- reference compound NGF (10 ng / ml) + cisplatin (3 μg / ml)

Survival of neurons was evaluated using six wells for each condition. After incubation for 48 hours and 72 hours, the neurons were fixed for 5 minutes in a solution of ethanol / acetic acid (95% / 5%) at -20 ° C and washed three times in PBS.

The neurotrophic effect of the compounds was monitored by incubating NGF (10 ng / ml) and 7β-OH-EPIA (1 nM, 10 nM and 100 nM) for 48 hours and 72 hours. At the end of the incubation, the cells were fixed for 5 minutes in a solution of ethanol / acetic acid (95% / 5%) at -20 ° C and washed three times with PBS.

Analysis of the number of cell bodies and axon networks in the field of view

The cell bodies of sensory neurons were labeled with anti-MAP-2 monoclonal antibody (Sigma M4403) and the axons of sensory neurons were labeled with anti-β-tubulin monoclonal antibody (Sigma T8660). These antibodies were diluted 1: 400 in an incubation solution (PBS with 5% FCS and 0.1% saponin, Sigma S-7900). These antibodies specifically label cell bodies of neurons and axons.

Cells were incubated for 2 hours, washed in PBS and incubated with goat IgG against goat mouse Alexa Fluor 488 (Molecular Probes A11001) diluted 1: 300 in the incubation solution to detect antibodies against MAP-2 and β-tubulin. Cell nuclei were stained with a fluorescent marker (Hoechst staining solution, SIGMA H6024, 1 μg / ml in an incubation solution for 1 hour).

For each condition, 2 pictures of each well were taken (12 pictures for each condition) in an In Cell Analyzer 1000 analyzer (Amersham Biosciences) using computer software for In Cell Analyzer 1000 3.2. A 10-fold increase was used for labeling MAP-2, a 20-fold increase was used for tagging β-tubulin. All images were taken for each labeling under the same conditions.

The analysis of the number of cell bodies labeled with anti-MAP-2 antibodies and the total length of axons labeled with anti-β-tubulin antibodies was performed using the In Cell Analyzer 1000 3.2 workstation software. The results were expressed as a percentage compared to the vehicle. Each group was compared using an unpaired t-test.

results

Protection provided by 7β-OH-EPIA against cisplatin-induced loss of axon density

Incubation with 3 μg / ml cisplatin for 48 hours

The axon density was on average 5459 μm of the total axon length in the field of view after incubation of sensory neurons in the medium (“filler”), that is, without cisplatin, for 48 hours. Incubation with cisplatin for 48 hours reduced the total length of the axons in the field of view to approximately 4585 μm. This decrease in axon network density by cisplatin was statistically significant (-16%, p <0.001) compared to vehicle.

Incubation with 10 ng / ml NGF for 48 hours prevented cisplatin-induced axon loss and caused a significant increase in axon length compared to cultures incubated only in medium.

Incubation with 1 nM and 10 nM 7β-OH-EPIA protected the sensory neurons from cisplatin-induced axon loss after 48 hours. The indicated effect was statistically significant. The total axon length was 5378 μm and 5549 μm after incubation for 48 hours with 1 nM and 10 nM 7β-OH-EPIA, respectively, which corresponds to a decrease in cisplatin-induced toxicity of 90.7% and 101%, respectively.

Incubation with 3 μg / ml cisplatin for 72 hours

Sensory neurons in the filler medium without cisplatin were on average equal to 5320 μm of the total axon length in the field of view. Incubation with cisplatin for 72 hours reduced the total axon length in the field of view to approximately 4046 μm. This decrease in axon network density by cisplatin was statistically significant (-24%, p <0.001) compared to vehicle.

Incubation with 10 ng / ml NGF for 72 hours prevented cisplatin-induced axon loss and caused a significant increase in axon length compared to cultures incubated only in medium.

Incubation with 1 nM 7β-OH-EPIA protected the sensory neurons from cisplatin-induced axon loss after 72 hours. The indicated effect was statistically significant. The total axon length was 4948 μm after incubation for 72 hours with 1 nM 7β-OH-EPIA, which corresponds to a decrease of 70.8% in cisplatin-induced toxicity.

Table 9 The effect of filler (0.1% DMSO), NGF (10 ng / ml) and 7β-OH-EPIA (1 nM, 10 nM and 100 nM) on the length of axons of sensory neurons after incubation for 48 hours in the presence of cisplatin (3 μg / ml) Impact Concentration Average axon length (μm) Standard error of the mean n % compared to control culture without cisplatin p (compared with vehicle + cisplatin) Filler (0.1% DMSO) - 5459.72 184 12 one hundred p <0.001 Filler intoxication (0.1% DMSO) with 3 μg / ml cisplatin - 4585.52 133 12 84 - NGF intoxication with 3 μg / ml cisplatin 10 ng / ml 6362.44 297 12 117 p <0.001 Intoxication of 7β-OH-EPIA with 3 μg / ml cisplatin 100 nM
10 nM
1 nM 4442.51
5549.41
5377.89 125
146
106 12
12
12 81
102
99 p> 0.05
p <0.001
p <0.001

Table 10 The effect of filler (0.1% DMSO), NGF (10 ng / ml) and 7β-OH-EPIA (1 nM, 10 nM and 100 nM) on the length of axons of sensory neurons after incubation for 72 hours in the presence of cisplatin (3 μg / ml) Impact Concentration Average axon length (μm) Standard error of the mean n % compared to control culture without cisplatin p (compared with filler + cisplatin) Filler (0.1% DMSO) - 5320.43 156.74 12 one hundred p <0.001 Filler intoxication (0.1% DMSO) with 3 μg / ml cisplatin - 4046.14 138.52 12 76 - NGF intoxication with 3 μg / ml cisplatin 10 ng / ml 6000.30 221.31 12 113 p <0.001 Intoxication of 7β-OH-EPIA with 3 μg / ml cisplatin 100 nM
10 nM
1 nM 4080.89
4275.04
4947,99 172.07
125.14
130.81 12
12
12 77
80
93 p> 0.05
p> 0.05
p <0.001

Protection provided by 7β-OH-EPIA from cisplatin-induced neuronal death (loss of cell bodies of neurons)

Incubation with 3 μg / ml cisplatin for 48 hours

After incubation in a medium with “filler” without cisplatin for 48 hours, an average of 61 sensory neurons were detected in the field of view. Incubation with 3 μg / ml cisplatin reduced the number of neurons on average to 41 sensory neurons in the field of view. The loss of cell bodies of neurons under the influence of cisplatin was statistically significant (-33%, p <0.001) compared with the number of cell bodies detected after incubation for 48 hours without cisplatin, that is, in a medium containing only filler.

Incubation with 10 ng / ml NGF for 48 hours almost completely prevented cisplatin-induced cell loss.

Incubation with 1 nM, 10 nM and 100 nM 7β-OH-EPIA protected the sensory neurons from cisplatin-induced cell death after 48 hours. The indicated effect was statistically significant. The total number of sensory neurons in the field of view was 51, 45, and 50 after incubation for 48 hours, respectively, with 1 nM, 10 nM, and 100 nM 7β-OH-EPIA, which corresponds to a decrease in cisplatin-induced cell death by 53.1%, respectively. 18.7% and 43.8%.

Incubation with 3 μg / ml cisplatin for 72 hours

After incubation in a medium with “filler” without cisplatin for 72 hours, an average of 54 sensory neurons were detected in the field of view. Incubation with 3 μg / ml cisplatin reduced the number of neurons on average to 33 sensory neurons in the field of view. The loss of cell bodies of neurons under the influence of cisplatin was statistically significant (-39%, p <0.001) compared with the number of cell bodies detected after incubation for 72 hours without cisplatin, that is, in a medium containing only filler.

Incubation with 10 ng / ml NGF for 48 hours almost completely prevented cisplatin-induced cell loss.

Incubation with 1 nM, 10 nM and 100 nM 7β-OH-EPIA almost completely protected sensory neurons from cisplatin-induced cell death after 72 hours. The indicated effect was statistically significant. The total number of sensory neurons in the field of view was 52, 55, and 52 after incubation for 72 hours with 1 nM, 10 nM, and 100 nM 7β-OH-EPIA, respectively, which corresponds to a decrease in cisplatin-induced cell death by 93%, 104%, respectively and 93%.

Table 11 The effect of filler (0.1% DMSO), NGF (10 ng / ml) and 7β-OH-EPIA (1 nM, 10 nM and 100 nM) on the number of cell bodies in the field of view after incubation with cisplatin (3 μg / ml) within 48 hours Impact Concentration Average value Standard error of the mean n % compared to control culture without cisplatin p (compared with vehicle + cisplatin) Filler (0.1% DMSO) - 60.83 1,59 12 one hundred p <0.001 Filler intoxication (0.1% DMSO) with 3 μg / ml cisplatin - 40.75 1.88 12 67 - NGF intoxication with 3 μg / ml cisplatin 10 ng / ml 58.33 1.38 12 96 p <0.001 Intoxication of 7β-OH-EPIA with 3 μg / ml cisplatin 100 nM
10 nM
1 nM 49.58
45.50
51,42 1.12
1.26
2.11 12
12
12 82
75
85 p <0.001
p <0.05
p <0.001

Table 12 The effect of filler (0.1% DMSO), NGF (10 ng / ml) and 7β-OH-EPIA (1 nM, 10 nM and 100 nM) on the number of cell bodies in the field of view after incubation with cisplatin (3 μg / ml) within 72 hours Impact Concentration Average value Standard error of the mean n % compared to control culture without cisplatin p (compared with vehicle + cisplatin) Filler (0.1% DMSO) - 53.75 1.29 12 one hundred p <0.001 Filler intoxication (0.1% DMSO) with 3 μg / ml cisplatin - 32.83 1.47 12 61 - NGF intoxication with 3 μg / ml cisplatin 10 ng / ml 54.92 1.41 12 1,2 p <0.001 Intoxication of 7β-OH-EPIA with 3 μg / ml cisplatin 100 nM
10 nM
1 nM 52.25
54.58
52,42 1.24
1.37
1.65 12
12
12 97
1,2
98 p <0.001
p <0.001
p <0.001

Example 7

The present study evaluated the stimulatory effect of 7β-OH-EPIA on axon growth in dissociated spinal motor and sensory neurons in primary cultures. Neurons were stained with anti-β-tubulin antibody and the total length of neurons was determined in an In Cell Analyzer. Neuron cultures were incubated with 10 ^-9 M - 10 ^-4 M 7β-OH-EPIA and the density of the neuron network was determined after incubation for 6 hours, 12 hours and 24 hours. Nerve growth factor (BDNF) isolated from the brain, specific growth factor for motor neurons and nerve growth factor (NGF), specific growth factor for sensory neurons were used as reference compounds. The data obtained show that 7β-OH-EPIA has a significant neurotrophic effect on both motor neurons and sensory neurons. These effects, which were comparable to the effects of BDNF and NGF, were especially pronounced at nanomolar concentrations of hydroxysteroid. In general, the results confirm the possibility of using 7-hydroxysteroids to stimulate axonal proliferation and treat peripheral neuropathy.

1. Materials and methods

1.1. Obtaining cultures of rat motor spinal cord neurons

The motor neurons of rat spinal cord were obtained by the method described in the publication Martinou et al., 1992 (Martinou JC, Martinou I, Kato AC, Cholinergic differentiation factor (CDF / LIF) promotes survival of isolated rat embryonic motoneurons in vitro. Neuron. 1992 Apr .; 8 (4): 737-44). Pregnant female Wistar rats with a 15-day gestation were killed by cervical displacement and fetuses removed from the uterus. The spinal cord was removed and placed in ice-cooled Leibovitz medium (L15, Invitrogen, 11415-049) containing 1% bovine serum albumin (BSA without fatty acid, Eurobio, Les Ulis, France, GXXBSA01-65). The meninges were carefully removed.

Spinal motor neurons were dissociated by trypsin treatment for 20 minutes at 37 ° C (trypsin EDTA, 10-fold, Invitrogen 15400054), diluted in PBS without calcium and magnesium (Invitrogen 2007-03). The reaction was terminated by adding Dulbecco's Modified Eagle Medium (DMEM, Invitrogen 21969-035) containing grade II DNase I (0.1 mg / ml, Roche diagnostic 104159) and 10% fetal calf serum (FCS, Invitrogen 10270098). The cell suspension was mechanically dissociated by passing through a 10-ml pipette 3 times. Then the cells were centrifuged with an acceleration of 580 g for 10 minutes at room temperature. The debris of the dissociated cells was resuspended in L15 medium and the resulting suspension was enriched in motor neurons by centrifugation with 180 × g acceleration for 10 minutes at room temperature on a layer of BSA solution (3.5%) in L15 medium. The supernatant was removed and debris resuspended in L15 medium containing DNase I (1%). Then the suspension was placed on an optiprep gradient (density: 1.06 g / ml; Abcys, Paris, France; 1030061) and centrifuged with an acceleration of 335 × g for 15 minutes at room temperature. The upper phase containing purified motor neurons was collected, resuspended in L15 medium, and centrifuged at 800 × g for 10 minutes at room temperature. Then the debris was resuspended in a culture medium of a certain composition, consisting of a neurobasal medium containing 2% B27, 2 mM L-glutamine and penicillin 50 units / ml - streptomycin 50 μg / ml. Viable cells were counted on a Neubauer cytometer using a trypan blue exclusion test (Sigma T8154), then seeded at 30,000 cells / well on 96-well plates (pre-sensitized with poly-L-lysine; Nunclon, Invitrogen P5899) and cultured with 37 ° C in a humid chamber in an atmosphere consisting of air (95%) and CO ₂ (5%).

1.2. Obtaining cultures of rat sensory neurons

Rat sensory neurons were obtained by the method described in a publication by Hall et al 1997 (Hall AK, Ai X, Hickman GE, MacPhedran SE, Nduaguba CO, Robertson CP. The generation of neuronal heterogeneity in a rat sensory ganglion. J. Neurosci. 1997 , Apr 15; 17 (8): 2775-84). Female rats (15 days gestation) were euthanized by displacement of the cervical spine (Rats Wistar; Janvier, Le Genest-St-Isle, France) and fetuses removed from the uterus. The spinal cord with the posterior root ganglion (DRG) was removed and placed in ice-cooled Leibovitz medium (L15, Fisher 11415-049) containing penicillin 50 units / ml - streptomycin 50 μg / ml (PS, 1%) and bovine serum albumin ( BSA 1%, Sigma A6003). DRG was isolated and dissociated by trypsin treatment for 20 minutes at 37 ° C (trypsin EDTA, 10-fold, 10%, Fisher 15400054), diluted in PBS without calcium and magnesium (Fisher 2007-03). The reaction was terminated by adding Dulbecco's Modified Eagle Medium (DMEM, Fisher 21969-035) containing grade II DNase I (0.1 mg / ml, Roche diagnostic 104159) and fetal bovine serum (FBS 10%, Fisher 10270-098) . The cell suspension was triturated with a 10 ml pipette and centrifuged at 350 × g for 10 minutes at room temperature. The debris of dissociated cells was resuspended in a culture medium of a specific composition.

Viable cells were counted on a Neubauer cytometer using a trypan blue exclusion test (Sigma) and plated at 25,000 cells / well in 96-well plates (Nunc). Wells were pre-sensitized with poly-L-lysine (10 μg / ml, Sigma P2636) in ultrapure sterile water (Merck Eurolab 60759.01).

Cells were left for adhesion for 2 hours and kept in a humid chamber at 37 ° C in an atmosphere of 5% CO ₂ /95% air.

1.3. Incubation of cultures of motor neurons of the spinal cord with active compounds

The culture medium was replaced with a culture medium of a certain composition after incubation for 12 hours in accordance with the conditions described below:

control culture (vehicle, 0.1% DMSO);

7β-OH-EPIA 10 ^-4 M, 10 ^-5 M, 10 ^-6 M, 10 ^-7 M, 10 ^-8 M and 10 ^-9 M (in 0.1% DMSO);

BDNF 50 ng / ml and 10 ng / ml (in 0.1% DMSO).

After incubation for 6 hours, 12 hours and 24 hours, the cells were fixed for 5 minutes in a solution of ethanol / acetic acid (95% / 5%) at -20 ° C and washed three times in PBS.

1.4. Incubation of cultures of sensory neurons with active compounds

The culture medium was replaced with a culture medium of a certain composition after incubation for 12 hours in accordance with the conditions described below:

control culture (vehicle, 0.1% DMSO);

7β-OH-EPIA 10 ^-4 M, 10 ^-5 M, 10 ^-6 M, 10 ^-7 M, 10 ^-8 M and 10 ^-9 M (in 0.1% DMSO);

NGF 50 ng / ml and 10 ng / ml (in 0.1% DMSO).

After incubation with 7β-OH-EPIA for 6 hours, 12 hours and 24 hours, the cells were fixed for 5 minutes in a solution of ethanol / acetic acid (95% / 5%) at

-20 ° C and washed three times in PBS.

1.5. Axon Growth Analysis

Motor and sensory neurons were labeled with a monoclonal anti-β-tubulin antibody (Sigma T8660) diluted 1: 400 in an incubation solution (PBS with 5% FCS and 0.1% Samonin, Sigma S-7900). The specified antibody specifically labels cell bodies and axons of neurons.

After an incubation of 2 hours, the cells were washed in PBS and incubated with goat IgG against mouse Alexa Fluor 488 (Molecular Probes A11001) diluted in a ratio of 1: 300 in the incubation solution to detect anti-β-tubulin III antibodies.

The analysis of the total length of neurons labeled with antibodies against β-tubulin (neurons) was performed using the In Cell Abalyser 1000 3.2 workstation software.

Results are expressed as a percentage compared to vehicle. Groups were compared using the unpaired Student t-test.

2. Results

2.1. The effect of drug administration on the axon length of motor neurons

Analysis of the total cell elongation indicates the neurotrophic properties of the test compounds.

a) After incubation for 6 hours

The results are shown in table 13. The introduction of BDNF in the amount of 50 ng / ml and 10 ng / ml significantly increased the density of the axon network formed by motor neurons, respectively, by 180% and 193% compared with the filler (p <0.001).

Incubation with 10 ^-8 M and 10 ^-9 M 7β-OH-EPIA for 6 hours significantly increased the density of the axon network of motor neurons by 150% and 188%, respectively, compared with the filler (p <0.001).

b) After incubation for 12 hours

The results are shown in table 14. The introduction of 50 ng / ml and 10 ng / ml BDNF significantly increased the density of the axon network formed by motor neurons, respectively, by 152% and 173% compared with the filler (p <0.001).

Incubation from 10 ^-4 M - 10 ^-9 M 7β-OH-EPIA for 12 hours significantly increased the density of the axon network by 226% -137% compared with the filler.

c) After incubation for 24 hours

The results are shown in Table 15. Incubation with 10 ng / ml BDNF for 24 hours significantly increased axon network density compared to vehicle (170%, p <0.01). However, incubation with 50 ng / ml BDNF did not cause a significant change in axon network density.

Incubation with 10 ^-8 M 7β-OH-EPIA for 24 hours increased the density of the axon network by 174% compared to the vehicle (p <0.005).

2.2. The effect of drug administration on axon length of sensory neurons

a) After incubation for 6 hours

The results are shown in table 16. The introduction of 50 ng / ml and 10 ng / ml NGF for 6 hours significantly increased the density of the axon network formed by sensory neurons, respectively, by 273% (p <0.001) and 191% (p <0.01) .

Incubation with 10 ^-7 M, 10 ^-8 M and 10 ^-9 M of 7β-OH-EPIA for 6 hours increased the network density of axons of sensory neurons by 171% (p <0.001), 176% (p <0.005) and 149, respectively % (p <0.05) compared with the control cell culture.

b) After incubation for 12 hours

The results are shown in table 17. Incubation with 50 ng / ml and 10 ng / ml NGF significantly increased the density of the axon network formed by sensory neurons compared with the filler, respectively, by 216% (p <0.001) and 128% (p <0.05 )

Incubation with 10 ^-9 M and 10 ^-7 M of 7β-OH-EPIA significantly increased the density of the axon network compared to the excipient by 145% (p <0.001) and 134% (p <0.05), respectively.

c) After incubation for 24 hours

The results are shown in table 18. After incubation for 24 hours with 50 ng / ml and 10 ng / ml NGF, the density of the axon network formed by sensory neurons significantly increased by 309% and 371%, respectively, compared with the filler (p <0.001).

After incubation with 10 ^-7 M, 10 ^-8 M and 10 ^-9 M 7β-OH-EPIA for 24 hours, the axon network density increased by 173% (p <0.005), 174% (p <0 , 01) and 147% (p <0.05).

Table 13 Motor neurons - The length of the axons in the field of view after incubation for 6 hours Impact Concentration The average value (microns) Standard error of the mean n % compared to the filler R Filler (0.1% DMSO) - 254.3 19.0 12 one hundred - Bdnf 50 ng / ml
10 ng / ml 457.2
489.5 19,4
19.0 12
12 180
193 p <0.001
p <0.001 7β-OH-EPIA 10 ^-4 M
10 ^-5 M
10 ^-6 M
10 ^-7 M
10 ^-8 M
10 ^-9 M 315.9
293.3
354.5
350.5
382.4
479.3 18.9
22.8
33.7
34.3
25.8
40.8 12
12
12
12
12
12 124
115
139
138
150
188 p <0.05
p <0.05
p <0.05
p <0.05
p <0.001
p <0.001

Table 14 Motor neurons - The length of the axons in the field of view after incubation for 12 hours Impact Concentration The average value (microns) Standard error of the mean n % compared to the filler R Filler (0.1% DMSO) - 423.8 38.3 12 one hundred - Bdnf 50 ng / ml
10 ng / ml 643.7
731.3 34.1
34.8 12
12 152
173 p <0.001
p <0.001 7β-OH-EPIA 10 ^-4 M
10 ^-5 M
10 ^-6 M
10 ^-7 M
10 ^-8 M
10 ^-9 M 602,4
680.9
580.3
956.7
759.0
764.9 24.8
30.8
55.5
54.6
61.6
59.9 12
12
12
12
12
12 142
161
137
137
179
180 p <0.001
p <0.001
p <0.05
p <0.001
p <0.001
p <0.001

Table 15 Motor neurons - Axon length in the field of view after incubation for 24 hours Impact Concentration The average value (microns) Standard error of the mean n % compared to the filler R Filler (0.1% DMSO) - 215.8 39.6 12 one hundred - Bdnf 50 ng / ml
10 ng / ml 293.6
366.1 20.1
29.6 12
12 136
170 p <0.001
p <0.001 7β-OH-EPIA 10 ^-4 M
10 ^-5 M
10 ^-6 M
10 ^-7 M
10 ^-8 M
10 ^-9 M 188.4
200.9
224.8
271.7
374.6
265.9 23.3
20.7
36.7
34.0
22.3
26.2 12
12
12
12
12
12 87
93
104
126
174
123 p <0.05
p <0.05
p <0.05
p <0.05
p <0.005
p <0.05

Table 16 Sensory neurons - Axon length in the field of view after incubation for 6 hours Impact Concentration The average value (microns) Standard error of the mean n % compared to the filler R Filler (0.1% DMSO) - 102,2 14.1 12 one hundred - NGF 50 ng / ml
10 ng / ml 279.1
195.2 31.8
28.8 12
12 273
191 p <0.001
p <0.01 7β-OH-EPIA 10 ^-4 M
10 ^-5 M
10 ^-6 M
10 ^-7 M
10 ^-8 M
10 ^-9 M 103,2
114.8
97.7
151.8
179.3
174.2 10.7
8.2
11,4
11.7
16,0
9.1 12
12
12
12
12
12 101
112
96
149
176
171 p <0.05
p <0.05
p <0.05
p <0.05
p <0.005
p <0.001

Table 17 Sensory neurons - Axon length in the field of view after incubation for 12 hours Impact Concentration The average value (microns) Standard error of the mean n % compared to the filler R Filler (0.1% DMSO) - 298.4 12.8 12 one hundred - NGF 50 ng / ml
10 ng / ml 644.3
381.3 40,0
31,4 12
12 216
128 p <0.001
p <0.05 7β-OH-EPIA 10 ^-4 M
10 ^-5 M
10 ^-6 M
10 ^-7 M
10 ^-8 M
10 ^-9 M 290.3
321.0
287.0
398.4
368.3
431.8 32,3
18,4
24.3
35.8
32,2
24.2 12
12
12
12
12
12 97
108
96
134
123
145 p <0.05
p <0.05
p <0.05
p <0.05
p <0.05
p <0.001

Table 18 Sensory neurons - Axon length in the field of view after incubation for 24 hours Impact Concentration The average value (microns) Standard error of the mean n % compared to the filler R Filler (0.1% DMSO) - 220.7 12,4 12 one hundred - NGF 50 ng / ml
10 ng / ml 682.1
819.9 64,2
61.5 12
12 309
371 p <0.001
p <0.001 7β-OH-EPIA 10 ^-4 M
10 ^-5 M
10 ^-6 M
10 ^-7 M
10 ^-8 M
10 ^-9 M 228.6
244.8
189.9
380.8
384.3
324.9 38.8
28,2
24.4
45.8
52,2
45.1 12
12
12
12
12
12 104
111
86
173
174
147 p <0.05
p <0.05
p <0.05
p <0.005
p <0.01
p <0.05

Claims (5)

1. The use of a compound representing 7α-hydroxyepiandrosterone, or 7β-hydroxyepiandrosterone, or a pharmaceutically acceptable salt or ester thereof, for the manufacture of a medicament for the treatment or prevention of an inflammatory condition selected from the group consisting of: inflammation, inflammatory disease peripheral organs selected from a liver or kidney, inflammatory respiratory disease, diabetes mellitus and its complications, pain associated with inflammation, inflammation -negative skin diseases, multiple sclerosis, peripheral arterial diseases and their complications, coronary artery disease and its complications of atherosclerotic renal artery stenosis, rheumatoid arthritis, primary and secondary osteoarthritis and their complications, inflammatory diseases characterized by degeneration of articular cartilage, acromegaly and wound healing. 2. The use according to claim 1 for the preparation of a medicinal product intended for the treatment or prevention of pain associated with inflammation, diseases of peripheral arteries and their complications, diseases of the coronary artery and its complications, inflammatory diseases of the respiratory tract, inflammatory diseases characterized by degeneration of articular cartilage. 3. The use according to claim 1 or 2, where the inflammatory disease of the respiratory tract includes asthma, rhinitis, bronchitis or chronic obstructive pulmonary disease. 4. The use according to claim 1 for the preparation of a medicament intended for the treatment or prevention of pain associated with inflammation, atherosclerotic stenosis of the renal artery, type 2 diabetes and its complications, asthma and chronic obstructive pulmonary disease, rheumatoid arthritis, primary and secondary osteoarthritis, and their complications. 5. The use according to claim 1 or 2, wherein said compound is 7α- or 7β-hydroxyepiandrosterone or an ester thereof.

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