Peptide combination for the control of aldosterone synthesis in the function of pressure control and disorders due to infectious and other diseases

Abstract

The present Invention relates to a newly designed product, i.e. substance (B+C) that combines two different mechanisms of action that allow the control of aldosterone synthesis and secretion without compromising the metabolism of cortisol (corticosterone In rats) in the.tone glomerulosa of the adrenal gland. The innovative design of the described molecule enables ths individual use of substance (B+C) in the treatment of diseases such as hyper and hypo aldosteronism, Conn's syndrome and cytokine storm caused by Covid infection or as co-therapy with corticosteroids in the treatment of a whole range of immunological diseases. The innovative design of Substance (8+C) has an additional practical and economic advantage because the patient receives both components of substance (B+C) simultaneously with just one application, regardless of the medicine formulation. This not only simplifies the medicine synthesis, dosing and clinical research, but also the application of the medicine in clinical practice. For successful and selective control of aldosterone synthesis, which in nature also means selective control of blood pressure, it was necessary to combine the two substances described in order to ensure action in patients with elevated and / or towered blood pressure with one application of Substance (B+C). This feature of Substance (B+C) is important, especially in the treatment of patients with permanently impaired aldosterone synthesis, such as Conn's disease and similar autoimmune diseases, or in combmatton with corticosteroids where prolonged or repeated corticosteroid therapy is indicated. Finally, this invention is extremely important because It enables the safe treatment of a large group of patients with hypertension who also have secondary diseases of the immune system, various inflammations or viral Infections such as Covid where corticosteroid therapy is indicated, but difficult to implement in practice due to the negative effect of corticosteroids on blood pressure.

Description

[0001] PEPTIDE COMBINATION FOR THE CONTROL OF ALDOSTERONE SYNTHESIS IN THE FUNCTION

[0002] OF PRESSURE CONTROL AND DISORDERS DUE TO INFECTIOUS AND OTHER DISEASES

[0003] 1. TECHNICAL FIELD

[0004] The invention relates to a combination of designed synthetic peptides, for which this invention assumes in principle and proves their ability to control aldosterone synthesis, which essentially represents the area of peptide application in the pharmaceutical industry and medicine.

[0005] The invention also relates to the effects and possibilities of application of the mutual connection and combination of two peptides, as follows.

[0006] According to the International Patent Classification (IPC), the invention belongs to the field of:

[0007] A - daily necessities, class A61 - medical sciences, subclass A61K - preparations for medical purposes and A61P - therapeutic action of medical preparations, area C chemistry and subclass C07K peptides.

[0008] 2. TECHNICAL PROBLEM

[0009] This invention solves two important problems:

[0010] 2a) Hypertension / hypotension, conditioned by weak or difficult control of aldosterone synthesis, i.e. conditioned by chronic hypo or hyper-aldosteronism with disruption of the renin-angiotensin connection, including certain pathological acute conditions, conditioned by exacerbations of various diseases or diseases treated with corticosteroids or

[0011] 2b) In the case of respiratory function disorders in viral and other allergic diseases, in which the renin - angiotensin connection additionally complicates the respiratory function caused by the malfunction of the synthesis and secretion of aldosterone, such as in acute Covid 19 infection / inflammation with lung fibrosis, but also in other possible pathological conditions.

[0012] 3. BACKGROUND OF THE VEIMTION

[0013] 3.1. In the existing state of the art, there is a similar combination which, io addition to the structural difference in the first peptide (substance), has a different purpose because it is used for the treatment of multiple sclerosis. The aforementioned similar combination does not solve the problem of aldosterone synthesis, as is the case in this application. This application is a solution to the problem arising from a disorder of aldosterone synthesis, which represents a new Indication. In the current state of medical and pharmaceutical technology, the problem of aldosterone synthesis has not been successfully solved by existing similar substances, because it could not be predicted on the basis of scientific knowledge that the existing substances could have an obvious effect on the regulation of aldosterone synthesis, which is undoubtedly proven in this application. 3.2. Similar existing substances, individually or in combination, have never before been described, used or proven as inhibitors and selective regulators of aldosterone synthesis.

[0014] 4. TECHNICAL BACKGROUND

[0015] The essence of the invention contains the idea in which the joint combination of the substances 8 and C described below, as (B*C), in the target ratio of 3B:1C to 5B:1C, can selectively control ths synthesis of aldosterone, and also the pressure, not allowing pressure oscillations in certain situations when,, for example, therapeutic applications of medicines lead to an increase / decrease in pressure, it is also similar in certain pathological conditions when it is necessary to control blood pressure through the renin-angiotensin coupling. By applying the aforementioned combination, the synthesis and secretion of aldosterone is stabilized, which is extremely important in order to protect the function of the heart, lungs, kidneys and other vital organs. By applying the new generation of pressure regulators described below, unwanted side effects associated with the use of most of the aldosterone regulators known so far are eliminated. Examples of negative pressure disorders are described in patients treated with corticosteroids as the main therapy for more severe acute inflammations, Immunological diseases, but also in specific chronic diseases and inflammations caused by bacteria and viruses. The use of the peptide combination described above can be very useful in the treatment of well-known diseases caused by hyperaldosteronism, such as Conn’s syndrome, where the main pathology is caused by a life- threatening uncontrolled increase in pressure, but also by other infections, such as infections caused by viruses, including Covid 19, the current cause of the current pandemic.

[0016] This invention shows that the combination and different formulations of the substances {B and C) described below can fulfill the conceptual task of regulating high blood pressure that occurs as a result of disorders in the synthesis and secretion of aldosterone. An adequate combination and formulation of the substances (B and Q regulates systolic pressure by the mechanism of controlling the synthesis of aldosterone and maintaining its secretion at approximately physiological values, and thus also maintains the physiological balance of the renin-angiotensin connection. All of the above in its entirety points to completely new possibilities for the application of the combination of these two peptides, for a new indication that has not been known as a therapeutic option until now. These are the substances, as follows:

[0017] 4.1 Pentapeptide as a sequence of the following amino adds: (Met-Phe-Gly-Gly-Tyr) or MEGGY and Its analogues, with and without polar ends, in the continuation of the text as Substance B. The inventors put. the idea of applying the peptide MEGGY in that configuration so that, all the connections and effects of this peptide in the Innovative combination of 8-t-C are realized with Methionine as the starting amino add, ail with the aim of achieving the expected faster effect and bettor physiological effect, which was confirmed in experiments on animal models described below. It is essential for the success and achievement of the results shown in the experiment on animal models to specify the quality of substance B, which is done in Table 1: Specifications of the quality of substance B. Although it can be considered an endogenous peptide, like metenkephalin, in this invention a different peptide sequence of amino acids is used, as a different substance (8) because then, in that case, the beginning of the peptide is the amino add methionine, with which, in principle, the endogenous synthesis of all proteins begins, so it is more natural that this amino add sequence is also applied to substance B, whereby then, the sequence and quantification of the other amino adds in substance B is identical to that in ordinal number 4, Table 1. Quality specifications of substance 8. Substance 8 can also have tyrosine as the starting amino acid, although it is not preferred in this invention, but then at ordinal number 4, Table 1. Quality specifications of substance 8 the sequence of the other amino acids is GGFM or Gly, Gly, Rhe, Met, with unchanged quantification of each individual amino add.

[0018] 4.2 ACTH 1-13. As a sequence of 13 following amino adds: (Ser-Tyr-Ser-Met-Glu-Hls-Phe-Arg- Trp- Glylys-Pro-Val) or STSMEHFRWGKPV and its analogs VPKGWRFHEMSYS with and without polar ends as Substance C.

[0019] 43 Here, the essence of the invention is in the combination (B-t-C), as a new substance that can be in a unique lyophilizate for injection, granulate as a mixture for tablets and capsules, or a powder mixture for nasal or inhalation use as technical possibilities of the invention.

[0020] Substances under 4.1 and 4.2 can be mixed and linked in various ways and in different ratios, but the most suitable mutual ratio is 5B:1C to at least 3B:1C in one dose for application, regardless of the size of the individual dose, as long as those links between substances B and C can be a simple mixture, a mixture in a common lyophilizate in a prepared syringe for subcutaneous (subcutaneous) application or REG (Pegylated), i.e. a mutual link realized with Poly-Ethylene-Glycol of long chains from 2 to 100 kDa. If several individual doses with different lengths of the mutual PEG bond are pegylated in one dose so that those PEG bonds between substance B and substance C have PEG values from 2 to 100 kDa, it is possible to obtain a single dose with a prolonged effect due to the different release time of the active substances (Bs-C).

[0021] The most suitable sites for PEG attachment are the corresponding polar ends of both substances B and C, but if they are synthesized without polar ends and none are present (as a possible variant without polar ends), then mutual attachment at the amino acid Lysine is possible.

[0022] The basis of ths idea was verified by an experiment / model of the control of aldosterone synthesis on four groups of rats., so that the mutual combination of substances iB-rC? has a direct effect on the synthesis of aldosterone, consequently also in a correlational action with the effects on cytochromes CYP1181 and CYP11B2. The aim is to inhibit the synthesis of aldosterone and its variations / osdllations (above and below physiological values) and consequently to affect pressure changes, without the unwanted side effects of existing medicines developed with the idea and task of controlling aldosterone synthesis and regulation of the renin-angiotensin coupling.

[0023] In the development of this invention and testing the hypothesis, it was necessary to use a substance that will be designated as substance A in the detailed description, and it refers to dexamethasone, which is the most commonly used corticosteroid in medical practice and as such was used in this study only for the induction of aldosterone synthesis, as stated in the detailed description of the invention below.

[0024] 5. RESEARCH ON THE RAT MODEL, RESULTS AND FIGURE DESCRIPTION

[0025] 5.1 Details of experiments on the rat model:

[0026] To prove the effect of peptide combinations., female rats divided into 4 experimental groups with 7 female rats in each group were used. All rats received a dose of 2 mg / kg of saline or substance by subcutaneous application 1 hour before taking the serum fur analysis. The duration of the experiment was 5 consecutive days, with identical daily dosing and sampling. Substance A, dexamethasone was expectedly used to induce aldosterone synthesis.

[0027] 1) The first group was the control group, which received physiological solution (2 mg / kg) and served as a neutral control

[0028] 2) The second group received substances B and C together, as (B+C) in the ratio (SB :1C), as a mixture in a unique lyophilizate, 2 mg / kg mixture.

[0029] 3) The third group received substance (A) - dexamethasone to initiate the synthesis of aldosterone 2.mg / kg.

[0030] 4) The fourth group received all three substances together, as Ae (B+Q 2 mg / kg,

[0031] On the fifth day of the experiment, one hour after the application of the tested substances, the rats were sacrificed and the adrenal glands were collected and prepared for immunohistochemical analysis of CypllB2 and Disabled-2 enzymes, while the rat sera collected during the experiment were analyzed for aldosterone and corticosterone content in plasma.

[0032] (The experiment was performed in a certified laboratory: Solder Bio PATH, Pre-Cim / caJ Pharmacology & Pathology CRO; 5541 Central Ave., Suite 260, Boulder, CO 80301; USA).

[0033] 5,2 Test results end picture descriptton:

[0034] Figure 1. Schematic representation of the synthesis of aldosterone and cortisol in the adrenal gland

[0035]

[0036] Figure 2. Mean values ± standard error of aldosterone in the serum of rets on the first day of the experiment and 1 hour after the administration of the saline solution {Control! with the substance (B+C). Values are expressed in picograms per milliliter (pg / mi).

[0037] 1, Control 2. 84-C

[0038] Figure 3. Mean values ± standard error of aldosterone in rat. serum on the first day of the experiment and one hour after dosing with saline (Control), substance (IHC), substance A and the combination of substances (B-rC) and substance A. Values are expressed in picograms per milliliter (pg / ml).

[0039] Figure 4. Mean values t standard error of aldosterone hi rat serum on the Sth day of the experiment after treatment of rats with saline (Control), substance (B-rC), substance A and the combination of substances (B-s-C) and substance A. Measurements were performed 1 hour after dosing. Values are expressed in picograms per milliliter (pg / ml).

[0040] In order to definitively confirm that Substance (B+C) is active in the adrenal cortex, specifically in the zona giomeruiosa where aldosterone is synthesized, we applied extremely sensitive and specific immunohistochemical methods to identify the CypllB2 enzyme end the D«sabled-2tw* enzyme.

[0041] Figure 5. Immunolocalization of CypllB2 enzyme In the outer part of the adrenal cortex of rats on the Sth day of the experiment after treatment of rats with saline (Control), substance (B<-C), substance A and a combination of substances (B*C) and substance A. Arrows indicate the ^ona giomerulosa in the adrenal cortex of rats selectively stained for visualization of ths CypllB2 enzyme. Significant thickening of the xona giomerulosa is evident in rats treated with substance A (dexamethasone), which is direct evidence of increased cellular activity in that zone and increased aldosterone synthesis. Microscopic magnification xlO in the first row of images and x20 In the second row.

[0042] Figure 6. Immunolocalization of the enzyme Disabled-2 in the outer part of the rat adrenal cortex on the Sth day of the experiment after treating rats with saline (Control), substance (B*C), substance A and a combination of substances (B-s-C) and substance A. Arrows indicate the zona giomerulosa in the rat adrenal cortex selectively stained for visualization of Cytochrome P-45021-Hydroxylase.

[0043] Significant thickening of the glomerular zones is evident in rats treated with substance A (dexamethasone), which is diced: evidence of increased cellular activity in that zone and increased aldosterone synthesis. Microscopic magnification xlO in the first row of images and x20 in the second row. Figure 7. Mean values ± standard error of corticosterone in rat serum on the 5th day of the experiment after treatment of rats with saline (Control), substance (B+-C), substance A and the combination of substances (B+C) and substance A. Measurements were performed 1 hour after dosing. Values are expressed in picograms per milliliter (pg / mi).

[0044] Figure No. 8. Exampie from the description of the formulation of substances (B*C) in a gastro-resistant tablet, as a form of oral administration of the invention, contained in Table No. 1, under c|

[0045] Figure No. 9.. Example from the description and formulation of substances (BK) in a gastro- resistant capsule and two ways of producing the finished preparation for oral administration of the Invention, contained in Table No. 2.

[0046] Figure 1. shows a schematic representation of the synthesis of aldosterone and cortisol in the adrenal cortex of humans.

[0047] Figure 2. shows that substances B and Cfwhen sublimated together, as (B+C) in the target ratio and administered to rat group 2, have no effect on the reduction or increase of aldosterone, Le.fthe change is so insignificant that it is within the standard deviation / statistkal error.

[0048] 9 Figure 3. shows the mean values of aldosterone in serum, 1 hour after administration, by groups: Controls after dosing with saline solution (Control; Group 1), Substance (B-t-C) - (Group 2), Substance A - (Group 3) and the combination of Substance A with Substances (B+C) - (Group 4).

[0049] The figure shows that Substances (B-rC) as a mixture in a unique lyophilizate, only slightly reduce the level of aldosterone (within the limit of statistical error) in the serum, while substance A - dexamethasone, causes a significant and strong drop in aldosterone in the serum 1 hour after application / inocuiation.

[0050] Figure 4 shows the mean value of aldosterone after 5 days of dosing in rats, one hour after dosing. An increase in the level of aldosterone was evident in the third group that received substance A, i.e. dexamethasone.

[0051] After a certain time, substance A ™ dexamethasone, causes an increase in aldosterone above physiological values, while in group 2, which received substances (Bs-C), there are no such oscillations. Aldosterone oscillation was significantly reduced in group 4 in which (B-rC) was applied together with substance (A). This result is unequivocal proof that the combination of substances (B-rC) regulates and maintains the level of aldosterone within approximately physiological limits. In conclusion, substance (B-rC) regulates the synthesis and secretion of aldosterone, which automatically reduces blood pressure oscillations.

[0052] The combination of substances (B-tC) prevents the variations (decrease and increase) of aldosterone in serum caused by dexamethasone, but similar changes also occur in diseases such as hypo- and hyper- aldosteronism, which are caused by disorders of the synthesis and secretion of aldosterone in the adrenal cortex. An experiment on a rat model has unequivocally proven that the combination of substances (B+C) does not allow the substance (A), the corticosteroid dexamethasone, to cause oscillations in the form of a sudden decrease and then an increase in aldosterone above physiological values. Even when substance A is administered simultaneously with substance (B-t-C), substances (Bs-C), sublimated and administered together, have the potential to inhibit aldosterone oscillations and autonomous regulation of aldosterone and thus blood pressure.

[0053] Figure 5. shows histochemical findings showing immunolocalixation of CypllB2 in the adrenal cortex of control rats or control group (1), rats treated with Substance (B+C) group {2}, rats treated with Substance A group (3) and rats treated with Substance A-HB+C), group (4). The enzyme Cyp21B2 (aldosterone synthase) is a protein that is only present in the zona glomerulosa of the adrenal cortex and is primarily regulated by the renin-angiotensin system. It is the only enzyme in the human body that is able to synthesize aldosterone in humans and as such plays a key role in maintaining electrolyte balance and regulating blood pressure.

[0054] Immunohistology of CypllSZ clearly shows Increased staining intensity and enlargement of the glomerular zone In rats treated with Substance A (3). Substance B^C has no effect on the activity of the CypllB2 enzyme, group

[0055] (2). The combination of Substance A+(B+C) shows normal levels of the CypllB2 enzyme group (4). Arrows indicate localization of CypllB2 in the glomerular zone and brackets indicate enlargement of the glomerular zone in rats in group 3 receiving Substance A (dexamethasone).

[0056] Figure 5. shows histochemical findings showing immunoiocalixation of the enzyme disabied- 2 in the adrenal cortex of control rats or the control group (1), rats treated with Substance (B*C) group (2), rats treated with Substance A group (3) and rats treated with Substance A*(B*C), group (4). The enzyme disabled-2 is only present in the zona glomeruiosa of the adrenal cortex of rats and is primarily responsible for the regulation of aldosterone secretion.

[0057] Immuno-histology of disabled-2 dearly shows increased staining intensity and enlargement of the glomerular zone in rats treated with Substance A (3). Substance (B*C) has no effect on the activity of the CypllB2 enzyme, group

[0058] (2). The combination of Substance A*(B*C) shows normal levels of the CypllB2 enzyme group (4). Arrows indicate localization of CypllB2 in the glomerular zone and brackets indicate enlargement of the glomerular zone in rats in group 3 receiving Substance A (dexamethasone).

[0059] Figure 7. shows that substances B and C as a unique lyophilization mixture (B*C) in combination have no effect on the level of corticosterone in the serum of rets. A significant decrease In corticosterone levels (significance level *p<0.001) is also evident in rats treated with substance (A).

[0060] Figure §. shows an example from the description of the formulation of substances (B-rC) in a gastro-resistant tablet, as a form of oral application of the invention, contained in Table Number 1.

[0061] Figure 9. shows an example from the description and formulation of substances (B*C) in a gastro-resistant capsule and two methods of production of the finished preparation for oral application of the invention, contained in Table Number 2.

[0062] 6, DETAILED DESCRIPTION OF THE INVENTION

[0063] One of the most important tasks of aldosterone in the body is the activation of the mineralocorticoid receptor (MR) in the kidney and the promotion of sodium (Na) retention along with potassium (K)uiexcretion. An increase in aldosterone in the blood causes an increase in blood pressure and promotes other diseases of the heart, circulatory system and kidneys. Activation of the renin-angiotensin system (RAS) also causes an increase in aldosterone in the blood.. Accordingly, medicines that block the mineralocorticoid receptor (MR antagonists) are in clinical use to prevent heart diseases as well as diseases of the circulatory system Although RAS system inhibitors and MR antagonists have shown positive results in clinical application in terms of reducing the pathological effects of elevated aldosterone on the heart and vascular system, these medicines also cause unwanted consequences including side effects such as gynecomastia, changes in the menstrual cycle, impotence, hyperkalemia and elevated aldosterone values According to the mentioned facts, there was a need to develop medicines that directly prevent the synthesis of aldosterone in the glomerular zone of the adrenal gland’*3*. Aldosterone in humans is synthesized from deoxycorticosterone in ths zona glomerulosa of the adrenal giand by the enzymatic action of cytochrome 450 - aldosterone synthase (CYP11B2) (Figure inhibition of cytochrome CYP11B2 results in a decrease in the synthesis and concentration of aldosterone in the blood., which causes a decreased activity of aldosterone at the Intracellular mineralocorticoid receptor in the kidney., but also at receptors in the heart and blood vessels*uLin humans, cytochrome CYP11B2 shares similar homology, 93% similarity, with cytochrome CYP11B1 (1.1- Hydroxylase) which has an important role in cortisol synthesis^4', Considering that cortisol, as the main glucocorticoid in mammals, is critical for the regulation of stress and the body's Immune response11**, the selectivity of medicines against cytochromes CYP11.B1 and CYP11B2 is of crucial importance for the successful development of medicines that should inhibit, the synthesis of aldosterone, but not cortisol. Unlike humans, rats lack cytochrome CYP17 in the adrenal cortex^^cConsequently, rats create a very small amount of cortisol, so instead they create corticosterone, which is the main glucocorticoid in rats, and is also a precursor for the synthesis of aldosterone.

[0064] Aldosterone is a steroid hormone that, in a dos® higher than physiological, causes an increase in blood pressure, enhances the inflammatory reaction, causes fibrotic changes and damage to organs such as the heart, kidneys and liverilG'5'?s. As mentioned at the beginning, the last three steps in the synthesis of aldosterone are catalysed by mitochondrial cytochrome P450, by the enzyme CYP11B2 (aldosterone synthesis) which controls the amount of synthesized aldosterone (Figure 1).

[0065] Consequently, activating or blocking CYP11B2 causes an increase, or decrease, in the amount of aldosterone circulating in the serum. A group of medicines that block CYP11B2 were developed with the idea of selectively blocking aldosterone synthesis in the zona glomerulosa of the adrenal giand in order to prevent a whole range of diseases caused by an increase or decrease in circulating aldosterone, including a decrease or increase in blood pressure and heart failureu*'i#*. There are also other cytochromes that has#® a structure similar to that of CYP11B2, including CYP11B1, but also CYP17, CYP19 and CYP21. CYPs 11 B catalyze the biosynthesis of cortisol, which regulates glucose metabolism, while CYPs 17, 19 and 21 catalyze the synthesis of hormones such as estrone and testosterone. In order to ensure the safe use of CYP11B2 inhibitors in clinical practice, the newly synthesized substances or their combinations used to formulate new medicines, as described below, must be highly effective selective CYP11B2 inhibitors, in order not to cause side effects associated with inhibition of cytochrome CYP11B1 and cortisol synthesis. Likewise, an ideal medicine should not cause a complete blockade of aldosterone synthesis and / or have a rebound effect that would result in an uncontrolled increase or decrease in heart pressure caused by a variation of aldosterone in the serum.

[0066] 6.1 Clinical application results of experiments related to effects on aldosterone

[0067] From the attached results of the described experiment on female rats, we see that substances B and C, sublimated into a mixture (S-sC) in a unique lyophilizate, act to regulate aldosterone synthesis in the glomerutesa zone of the adrenal giand, so substance (B-sC) has the potential to treat, prevent, and / or reduce or improv® the symptoms of diseases caused by elevated aldosterone or variations in aldosterone synthesis, including diseases such as hypertension, cardiac arrest congestion, primary aldosteronism, Cushing's syndrome, Corm’s disease and chronic kidney failure, fibrosis, and some other diseases. Therefore, this described result of the experiment on female rats is important for the conclusion that the combination (BK| is very effective as a regulator of aldosterone synthesis in cases where aldosterone must be controlled.

[0068] Substances B and C, or their sublimed bond in the mixture (B-s-C), represent a safer alternative to previously known inhibitors of aldosterone synthesis by acting on cytochrome CYP11B2 because:

[0069] 1| It regulates, Le. does not cause complete blockade, of aldosterone synthesis, thus preventing large oscillations of blood pressure and maintaining aldosterone within physiological limits, graphical representation (Figure 2) and

[0070] 2) It selectively inhibits only CYP11B2 (Figures 3 to 6) while having no effect on corticosterone synthesis, which is controlled by cytochrome CYP11B1, as can be seen in the graphical representation (Figure 7),

[0071] 3) In conditions of acute conditions and pulse therapy with corticosteroids, substance (B+C) should be a mandatory addition to corticosteroids in order to prevent variations in aldosterone and blood pressure, which would at the same time allow for prolonged treatment with corticosteroids even in patients with hypertension,

[0072] 4) Substance (€), which is part of substance (B-vC), has additional analgesic effects, so this positive component would also be helpful in the treatment of patients treated with corticosteroids for pain caused by inflammation.

[0073] 6.2 Clinical application of test results related to effects on corticosterone

[0074] One of the important tasks of the experiment on rats was to demonstrate the lack of effect of substance (BtC) on corticosterone and in order to obtain indisputable evidence of the selective effect / action of the combination of substances (B-rCl on the synthesis and secretion of aldosterone, but not corticosterone. The experiment undoubtedly confirmed that substances B and C together, or substance (B*C), have no effect on the synthesis and / or secretion of corticosterone.

[0075] Dexamethasone (as substance A| was used in the experiment because it is one of the most commonly used corticosteroids in the clinic but also because it has a well-known effect on the synthesis and secretion of cortisone in humans, or corticosterone in rats.

[0076] Substances B and C, as a single lyophilization mixture (B*C) in combination have no effect on the level of corticosterone in serum, white substance (A) causes a significant decrease in corticosterone (*p<0.001) in the serum of rats compared to control animals, graphical representation Figure 7.

[0077] In accordance with the above, the addition of Substance (8*C) to Substance (A) does not prevent the decrease in corticosterone caused by Substance (A), so that in the newly created combination A*(B*C) we achieve a selective effect of Substances (B*C) on aldosterone synthesis without causing changes in the level of corticosterone in the serum of rats. The graphic presented in Figure 7 shows the mean values of corticosterone in the serum of control rats after dosing with saline solution (Control; Group 1), Substance (B+C) (Group 2), Substance A (Group 3) and the combination Substances A + (B+C) (Group 4). From Figure 7, it is visible on the graph that Substance (8-rC) has no effect on the level of corticosterone while dexamethasone causes a significant, decrease in corticosterone in serum 1 hour after dosing. The addition of Substances (B*C) to Substances A does not prevent the decrease In corticosterone caused by Substance (A), which is why the action of (B*C) Is selective, even in conditions when used together with Substance (A), so that Substance (B+C) can rightly be considered a selective regulator of aldosterone synthesis, as seen in Figures 3 and 4. Also, the combination of Substances (B*C) is a selective inhibitor / regulator of aldosterone synthesis in and in situations where hypo- and hyper-aldosteronism is present.

[0078] The test results prove that the substance (B+C) proved to be a regulator of aldosterone synthesis, even in conditions when it is used together with dexamethasone, he. substance (A), as A^B-frC), which is the justification for formulating (B-rC) as an effective regulator of aldosterone synthesis into acceptable pharmaceutical preparations for use in indications where selective control of aldosterone synthesis is necessary.

[0079] 6,3 Possible different pharmaceutical forms in the application of the combination of substances (B+Q

[0080] The mixture (B-tC) In a common lyophilisate., powder or solution ready for injection intramuscular or subcutaneous administration, or administration in the form of a gastro- resistant tablet or capsule, sublingual tablet and possibly other ways of administration, is possible in addition to the existing described (B-f-C) in which the first amino add is methionine in substance B and in combinations (B-rQ, in which substance B is also synthesized as VGGFM. Also, substance C can be synthesized as VPKGWBFHEM5Y5 in addition to the already described sequence and as such combined in (B+C) with both forms of pentapeptide in which the first amino add is methionine but also in the variant described above in which the first, amino add is tyrosine. Nevertheless, in the Invention, the synthesis of pentapeptide with the first amino add methionine is preferred, due to the proven effect and faster action, as described in the presented experiment, on four groups of female rats.

[0081] 6.3,1 Formulation of substances B+C for administration in the form of a coated gastro- resistant tablet

[0082] 14

[0083] Procedure: a) Tablet cores are made by granulating the active substance with a filter and moistening with an aqueous solution of soluble starch and glycerin. After drying, the granulate is mixed with magnesium stearate and tableted into tablet cores. b) Tablet cures can also be made by direct compression of active substances B and C with spray-dried lactose or microcrystalline cellulose with the addition of magnesium stearate in an amount of 1%, and the cores thus obtained are coated with a targeted copolymer coating. Ethanol and isopropanol evaporate during the coating process. cj A combination is also possible, where the active substance, for example (B) is made by the method of wet granulation and tableted as one layer, in a two-layer tablet, while the other active substance C Is mixed separately with microcrystaliine cellulose or spray dried lactose and with the addition of magnesium stearate is tableted as a second layer in the tablet core, after which the three-layer core is coated with a copolymer of methacrylic add as an envelope

[0084] Ethanol and isopropanol evaporate during the coating process, and methacrylic add copolymer (Number 14 in Table Number 1| is responsible for creating a film (coating) on the tablet that is responsible for protecting the active substances (8 + C) until delivery in the large or small intestine, depending on the desired goal A preferred variant of this formulation is the creation of a PEG interconnection of active substances B-rC into a unique lyophilizate, which is coated with a copolymer coating as described under point b).

[0085] 63.2 Formulation of substances (B+C) for administration in the form of a gastro-resistant capsub

[0086] Example 1:

[0087] The mass of the substances in Table Na. 2 as a granulate of a common mixture (B*C) is used to fill gastro-resistant capsules that are designed to be disintegrated in the large or small intestine with a targeted delay, depending on the goal and the required length of resistance, but also on the place where we want the capsules to start acting (Figure 9; Variant 1). Additionally, the lyophilizate of substances 8 and C can be packaged in separate, standard capsules in the prescribed quantities as shown in Table No. 2, and then packaged together in a gastro-resistant capsule, as shown in Figure 9; Variant 2.

[0088] Example 2: a) A granulate Is prepared by direct tableting of the active substances separately or together with spray-dried lactose or microerystaliine cellulose with the addition of magnesium stearate, the same as the tablet core in Table No. 1, from which a briquette is formed. After the briquette with a diameter larger than the tablet is produced, it is ground in a dry granulator, sieved and coated with a gastro- resista nt coating and, after drying, filled into empty gastro-resistant capsules, as in Figure 9; Variant 1.

[0089] By gastro-resistant capsules, we mean those that are made so that their shell breaks down into a column, the so-called “Encode technology” and are available on the free market, here we consider it essential, due to the sensitivity of substances (8) and (C) to the stomach barrier, that the granulate of substance (Bt-Q is also temporarily covered with a gastro- resistant shell before filling the gastro-resistant capsule. b| A technical version is also possible in which the substances (B and CJ are each separately packed in their own ordinary / standard capsule, and then assembled, as primarily packed in mini capsules in the main, gastro-resistant capsule, which contains other supplements with active substances as in Figure 9 (Variant 2).

[0090] - Process / or redudnp water fn substances 8 and C, or f8*C / / or encapsu / ated dosage / arms

[0091] If necessary, ethanol Is added to precipitate substances (B and C) from the aqueous solution. The addition of absolute ethanol to dry peptides results in the binding of water into a eutectic mixture with ethanol where the percentage of water in ethanol is 3 to 4%. Water removal from the finished powder is possible by adding absolute ethanol in the amount required to achieve an ethanol-water ratio of, for example, 96:4, where water loss occurs from substances (B and C) or (S-s-C) in the joint lyophilizate.

[0092] After in-process checks and filtration through a bacteriological filter with a pore size of 0.22 pm, the solution of substances (B and C) Is treated with absolute alcohol, slowly added with vigorous stirring until the precipitate is separated. The content of the remaining substance in the solution is checked. The resulting precipitate is separated and washed with absolute ethanol at least three times. After washing, it is dried in a vacuum to a constant mass and the water content is determined.

[0093] The procedure is controlled in the same way and possibly repeated, until a water result of zero is achieved. Water content ~ 0.0 to 1.0% is necessary when making formulations that require a very low water content, such as in tablets or capsules, and is directly related to the shelf life or stability of the product, as a finished medicine. The peptide precipitation process can be carried out after lyophilisation or decantation of the precipitated dry peptide 8 or C, including (B*C) together, as the basis for formulations in tablets and capsules, as shown in Tables No. 1 and No. 2.

[0094] 633 Formulation of substances (B+C) for the production of inhalation and nasal spray

[0095] Procedure:

[0096] Active substances B and C together with polysaccharide (such as chitin or similar available polymer) are dissolved in the prescribed amount and a powder with a partide size of about 5 micrometers is made using a suitable technique (spraying with drying). After that, it is necessary to coat such powder particles with additives that will increase lipophilicity and solubility, that is, facilitate its passage through the intranasal route. The primary packaging of the powder obtained In this way is done in a suitable apparatus (pressurized device) so that it can be applied as medicine to the lungs or in a blister pack that is applied nasally.

[0097] Possible procedures for coating active substances 8 and C for inhalation and nasal spray are as follows: a) Procedure for coating substances (B and C) for the B*C powder mixture with oleic add

[0098] Appropriate amounts of active substances B and € are weighed and dissolved in water for injection. The content of the substances is checked, the solution is filtered through a bacteriological filter with a pore size of 0.22 pm, then the solution is treated with absolute alcohol, slowly added with vigorous stirring until the precipitate is separated. The content of the remaining substance in the solution is checked. The resulting precipitate is separated and washed with absolute ethanol at least three times. After that, the polypeptide mixture is washed with a S% solution of oleic add in ethanol, with continuous rotation of the powder during the moisture removal process. A free-flowing powder is obtained that is "liquid", i.e. has properties similar to liquids. During the production process, the particle size and oleic add content are determined. The substances prepared in this way with increased lipophilicity and solubility are used for further processing in the process of manufacturing a pharmaceutical form, i.e. a spray for inhalation or nasal administration. b) Procedure for coating substances B and C for the mixture (Bs-C) in powder form with chitin-based polysaccharides

[0099] Chitin-based polysaccharides are incorporated into particles of active substances in two ways:

[0100] The chitin solution, together with the active substances B and C, is sprayed, similar to the oleic acid described under point a), except that instead of the oleic add, together with B and C, chitin is sprayed. The rest of the procedure is identical as described under point a).Comblnations of degradable starch, combinations of starch and dextran, hyaluronic add esters, modified gelatin, oleic add and other fatty adds, bile salts, alpha amino boric add, aprotinin, phospholipids, and other substances that can serve this purpose can also be used as penetration enhancers.

[0101] 6.3.4 Formulation of substances (B-rCj for administration In the form of buccal or sublingual tablets

[0102] Procedure:

[0103] The active substances are homogenized with all the additives described in Table IMo. 4 in powder, or if they are in a common lyophilizate, they are also homogenized and then pressed with pressing tools. The tablets are pressed under a pressure of 6-12 N, which depends on the size of the buccal or sublingual dose and the amount of total substances in the mixture.

[0104] 6.3.5 Formulation of substances (B-rC) for use in the form of injection application

[0105] Substances (B+C) can be formulated into a joint lyophilizate by dissolving both substances in distilled water individually and mixing them in the required or targeted ratio and quantity. The dissolved mixture (B-rC) is then filled into vials / contalners and then subjected to the lyophilization process, resulting in a unique lyophilizate {Bs-C) as an application dose In vials ready for application. Reconstitution of the dose in the lyophilizate into the solution for the syringe is performed with 1 - 2 mi of distilled water or physiological solution, which is injected into a container or vial with the lyophilizate, drawn into the syringe and then applied subcutaneously or intramuscularly.

[0106] 63.6 Formulation of substances (B+C) for administration in the form of suppositories and anal suppositories

[0107] *Polybase is a mixture for suppositories that consists of polyethylene glycol of different molecular weights or like a ready-made mass with a different release rate, such as aerosols of vegetable fats and the like.

[0108] ** Cocoa butter and its substitutes based on fat bases and the like.

[0109] Procedure:

[0110] Substances (B) and (C) are mixed in (BK) in the prescribed quantities, after which they are added to one part of the melted base or polybase (about 50% of the base or polybase), everything is homogenized by mixing, and after homogenization, the rest of the base is added to the mixture, homogenized again, and the entire resulting mixture is poured into molds (torpedo shape) for targeted suppositories.

[0111] LIST OF USED LABELS

[0112] - Lyophilizate - Is a powder obtained by a special process of extracting water at very low temperatures in a vacuum, from a liquid substance or mixture.

[0113] - HPLC is an abbreviation for high-performance liquid chromatography control analytical method

[0114] (high performance liquid chromatography).

[0115] - RP HPLC is an abbreviation for reversed phase high-performance liquid chromatography - (Reversed phase High-performance liquid chromatography)

[0116] - 3D (HPLC) is an abbreviation for 3D control analytical method of high performance liquid chromatography or

[0117] Liquid Chromatograph equipped with a Diode Array controlled by the HPLC (3 Dimensions)

[0118] - (AAA) is the abbreviation for amino add analysis

[0119] - e.u. / ml is the abbreviation for the number of international units - Cfu / g is the unit for determining bacterial colonies (colony forming unit) (GC) is the abbreviation for gas chromatography

[0120] - (LAL) is the abbreviation for Limulus amebocyte lysate - a test for the presence of endotoxin

[0121] - IM is the abbreviation for the unit of pressure (Newton), when pressing tablets or tablet cores

[0122] 7. APPLICATION METHODS

[0123] The main goal of the application of this invention is to control and regulate the synthesis and secretion of aldosterone in pathological conditions when aldosterone in the serum oscillates uncontrollably (rises or falls), which results in pressure oscillations (hypertension and hypotension) in patients with various diseases, but also in patients who receive therapies that, as an unwanted side effect, can cause oscillations in the synthesis and secretion of aldosterone and thus blood pressure.

[0124] During the targeted research, the selective effect of the combmation of substances (B-t-C) as a selective regulator of aldosterone synthesis and secretion in the zona gtomeruiosa of the adrenal cortex was observed and undoubtedly proven, and it was proven (Figures 3, 4, S and 6) that thanks to the newly observed and proven effect of this combination, it can be applied in all therapies in which corticosteroids or glucocorticoids are used, which have been proven to have an undesirable effect on the synthesis and secretion of aldosterone, and thus on blood pressure.

[0125] The combination of substances (B-rC) in the various forms of administration described here should be, or at least preferred, as a mandatory adjuvant therapy in the therapeutic use of corticosteroids / giucocarticolds in: a) AH patients suffering from high blood pressure, caused by acute or chronic hypo- or hyper-aldosteronism. b) In patients suffering from Conn’s disease, which is the result of hyperaldosteronism and other diseases conditioned and caused by hyper or hypo aldosteronism c) In viral diseases where inflammatory reactions ere accompanied by a sadden increase In pressure, such as in the case of a cytokine storm m patients with Covid- 19 infection where as a result of viral inflammation we have pulmonary flbrosis accompanied by hypertension (angiotensin pathway).

[0126] The statement under point c) is of particular importance in the current Covid-19 situation and requires additional explanation. The pathobiology of Covid-19 infection requires the binding of the SARS-CoV-2 virus to the angiotensin converting enzyme 2 (ACE2) receptor, through which the virus enters the body's cells, where viral replication occurs, resulting in generalized infection and systemic diseases<4A.The ACE2 enzyme is most present in the lungs, heart and blood vessels, and Is a key part of the renin-angiotensin system (RAS), which is also extremely important in the development of cardiovascular diseases (CVD). Consequently, patients with heart disease have a significantly increased risk of fatal Covid -19 Infecttan^lCVD can be a primary consequence of infection with the Covid-19 virus, but also a secondary consequence of acute lung damage due to cytokine storm, increased heart rate, hypertension and inflammatory changes, each of which individually and / or together can cause a fatal outcome^^.Finally, it is assumed that antiviral medicines as well as medicines used to control hypertension such as RAS inhibitors can also further damage the heart and vascular system****. Although it is not recommended to discontinue therapy wsth RAS inhibitors in patients with elevated blood pressure, additional control of unwanted consequences of the mentioned therapies is strictly recommendedud). In conclusion, substance therapy (B*C) may be critical in the fight against CVD induced by Covid-19, antiviral medicines and / or RAS inhibitors.

Claims

PATENT CLAIMS1. Combination of peptide substances, dwacterixed in that, it consists of a common combination of substances B*C, which are further defined undec paragraph 4} items 4.1 and 4.2., with a description under item 4.3, with a description of the results under item 5), in the manner as presented m the corresponding figures numbers 2 to 7, with a detailed description under item 6) and a description of the corresponding methods, formulation of the substances into various production forms for the administration of the product in the application of the invention, contained in tables number 1 to number 5 and described in the descriptions of the production processes from item 6.3,1 to item 6.3.6.

2. Combination of peptide substances according to claim 1, characterised In that, it is used in the treatment of diseases described under item 7).