Peptides for the treatment of cytokine storm syndrome

Abstract

A pharmaceutical composition comprising the APL peptide identified as SEQ ID NO: 1 is disclosed as an inhibitor of a hyperinflammatory state characterized by an increase in cytokines or interleukins and inflammatory molecules, the concentrations of which increase until a condition defined as a "cytokine storm" is reached. The concentration of sucrose in the composition increases the biological activity of the peptide and facilitates its intravenous administration. The peptide in the pharmaceutical composition is useful for the manufacture of a medicament for treating diseases associated with hyperinflammation, such as COVID-19, dengue fever, macrophage activation syndrome, respiratory sepsis, and diseases associated with chimeric antigen receptor T-cell therapy, and acute respiratory distress syndrome. Also disclosed is a method for treating such diseases by administering a therapeutically effective amount of the composition.

Description

[Technical Field]

[0001] The present invention relates to the fields of biomedical and pharmaceutical industry, in particular to a pharmaceutical composition for the treatment and prevention of cytokine storm syndrome.

[0002] The composition comprises the peptide identified as SEQ ID NO: 1 and can be used to treat diseases such as acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis, dengue fever, hepatic encephalopathy and pericarditis. [Background technology]

[0003] It has been reported that a subgroup of individuals infected with the SARS-CoV-2 virus and developing the infectious disease Covid-19 develop a hyperinflammatory state. In these patients, a "cytokine storm" of interleukins (ILs) characterized by increases in IL-2, IL-17, IL-6, and TNFα, among others, has been described. This hyperinflammatory state can lead to the patient's death (Mehta, et al. (2020). The Lancet, 395:1033-1034).

[0004] Meanwhile, a retrospective study of 150 confirmed cases of Covid-19 in Wuhan, China, showed a significant increase in ferritin concentrations in patients who did not survive (1297.6 ng / ml) compared to levels of this protein in patients who survived (614 ng / ml) (Ruan et al. (2020). Intensive Care Med; https: / / doi.org / 10.1007 / s00134-020-05991-x).

[0005] Severe and critically ill patients affected by COVID-19 develop acute respiratory distress syndrome (ARDS). This syndrome is characterized by the presence of alveolar inflammatory events that are propagated by the systemic circulation. During this process, pulmonary endothelial involvement occurs, vascular permeability increases, and cells and macromolecules enter the alveolar space, where they inactivate pulmonary surfactant and establish a typical hyaline membrane (Janz and Ware (2014). Clin Chest Med;35:685-96). The epithelial barrier also changes, and the inflammatory response is exacerbated by the secretion of inflammatory ILs such as TNFα, IL-1β, and IL-6, as well as other inflammatory mediators. This in turn leads to the activation of neutrophils, monocytes, and macrophages, and the migration of these molecules and cells into the alveolar space, further amplifying the initial lesion. This hyperinflammatory process also occurs in other diseases, such as respiratory sepsis, macrophage activation syndrome, and sepsis in adults and neonates.

[0006] Currently, there is no effective treatment for patients with Covid-19 disease. Approved treatments are used to treat other diseases, including monoclonal antibodies against IL-1 (anakinra), IL-6 (tocilizumab), and Janus kinase protein inhibitors. However, these drugs are not particularly effective and have immunosuppressive effects. Therefore, their use in the treatment of Covid-19 can lead to a deterioration in the patient's overall condition, as the virus persists throughout the course of the disease and even after clinical recovery. Furthermore, there are no effective and safe treatments for respiratory sepsis, macrophage activation syndrome, neonatal sepsis, or respiratory distress. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is great interest in obtaining effective drugs to treat the hyperinflammation and "cytokine storm" that characterize these pathologies. [Means for solving the problem]

[0008] Description of the invention The present invention solves the above-mentioned problems by providing a pharmaceutical composition for the treatment and prevention of cytokine storm syndrome, comprising a peptide identified as SEQ ID NO: 1 at a concentration of 1.8-3.6 mg / mL and a sucrose concentration between 20-30 mg / mL, which surprisingly synergistically enhances the biological activity of the peptide, as well as being a determinant of the induction of regulatory T cells that control the magnitude of inflammation. The composition of the present invention is capable of reducing the hyperinflammation that characterizes a subgroup of Covid-19 patients who progress to a severe stage characterized by respiratory sepsis, macrophage activation syndrome, and respiratory distress, such as occurs in neonatal and adult sepsis. Administration of the peptide identified as SEQ ID NO: 1 reduces the levels of inflammatory cytokines in patients with Covid-19 disease without causing immunosuppression or serious side effects associated with peptide therapy. DETAILED DESCRIPTION OF THE INVENTION

[0009] The peptide identified as SEQ ID NO: 1 is derived from the region of the HSP60 protein consisting of amino acids 83 to 109. HSP60 is highly immunogenic in both healthy individuals and patients with autoimmune diseases. However, the levels of antibodies and T cells that this protein can induce are higher in patients with autoimmune diseases (de Jong et al. (2009). Arthritis Rheum. 7(60):1966-1976).

[0010] This protein has a great potential to stimulate the inflammatory response of the innate immune system. Furthermore, HSP60 has been shown to induce the expression of IL-12 and IL-15 genes, cytokines involved in the induction of the Th1 phenotype. These findings suggest that HSP60 functions as a danger signal for the innate immune system (Habich et al. (2005). J. Immunol. 3(174):1298-1305). On the other hand, peptides derived from HSP60 may also constitute danger signals (Quintana and Cohen (2011). Trends Immunol. 2(32):89-95).

[0011] The peptide of SEQ ID NO: 1 contains several overlapping T cell epitopes. It inhibits CD4+CD25 T cells in ex vivo assays using peripheral blood mononuclear cells from rheumatoid arthritis patients. high It increases the frequency of Treg cells with a Foxp3+ phenotype, but this does not occur in cells from healthy donors (Barbera et al. (2016). Cell Stress. Chaperons. 4(21):735-744).

[0012] International Patent Applications Nos. PCT / CU2005 / 000008 and PCT / CU2009 / 000009 claim a peptide identified as SEQ ID NO: 1 and its use in the treatment of rheumatoid arthritis, as well as Crohn's disease, ulcerative colitis, and type I diabetes, respectively. All of these conditions are non-infectious diseases. Furthermore, Patent Application WO2019 / 129315 describes a highly stable pharmaceutical composition comprising the peptide of SEQ ID NO: 1, the administration of which causes a decrease in antibodies against the citrullinated peptide and other parameters closely related to protein citrullination.

[0013] The peptide identified as SEQ ID NO: 1 is obtained by chemical synthesis. Through the experiments described in this invention, the immunological mechanisms induced by the peptide that contribute to the control of excessive inflammation can be evaluated. This pharmaceutical composition was well tolerated with no evidence of immunosuppression and a high safety profile. No serious adverse events were observed in any patient.

[0014] For the purposes of this invention, cytokine storm syndrome is understood as an increase in inflammatory cytokine levels that reaches concentrations above normal. These inflammatory cytokines include IL-6, TNFα, IL-1, and IL-17. It also refers to an increase in other cytokines, such as IL-10, IL-8, and IL-5. It also includes an increase in inflammatory biomarkers, such as C-reactive protein, fibrinogen, D-dimer, liver transaminases, lactate dehydrogenase, and ferritin. Increases in these molecules associated with cytokine storm occur in patients suffering from inflammatory conditions, such as acute respiratory distress, macrophage activation syndrome, respiratory sepsis, dengue fever, hepatic encephalopathy, and pericarditis. Clinical evaluation may reveal persistent fever, weakness, fatigue, and nausea in patients.

[0015] In another aspect, the present invention discloses the use of a pharmaceutical composition comprising a peptide identified as SEQ ID NO: 1 in the manufacture of a medicament for the treatment and prevention of cytokine storm syndrome. Surprisingly, treatment with a composition comprising the peptide improved interstitial pneumonia, reduced acute-phase inflammatory reactants between 24 and 48 hours, and reduced inflammatory biomarkers, allowing for the withdrawal of mechanical ventilation in patients with Covid-19. The rapid decrease in C-reactive protein after only 24 hours was an unexpected result. Previously, patients with rheumatoid arthritis treated with this peptide failed to demonstrate a significant decrease in C-reactive protein (Prada, et al. (2018). J Clin Trials 1(8):1-11).

[0016] The present invention demonstrates the ability of intravenously administered peptide identified as SEQ ID NO: 1 to reduce dyspnea, hyperinflammation, and levels of inflammatory acute phase reactants and inflammatory cytokines in patients with Covid-19. Surprisingly, these results were replicated in patients with respiratory sepsis who presented with respiratory distress syndrome.

[0017] In a specific example, a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 is used in the manufacture of a medicament for the treatment and prevention of acute respiratory distress syndrome, macrophage activation syndrome, adult or neonatal respiratory sepsis, systemic inflammatory response syndrome, cytokine storm syndrome, which manifests as venous or arterial thromboembolism, a sudden increase in ferritin levels, severe pericarditis, a sudden increase in liver transaminases causing hepatic encephalopathy, or diseases associated with chimeric antigen receptor T-cell therapy. Among the conditions treated with a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 is cytokine storm syndrome, which underlies the systemic inflammatory response syndrome that occurs in patients after burns or severe trauma or in acute inflammatory diseases. In one embodiment of the invention, the agent is administered intravenously.

[0018] In one embodiment of the invention, the medicament is used for the treatment and prevention of cytokine storm syndrome caused by an infectious disease, in a particular embodiment, the infectious disease is Covid-19 or dengue fever.

[0019] An object of the present invention is a method for the treatment or prevention of cytokine storm syndrome, comprising administering to a patient in need thereof a pharmaceutically effective amount of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1. In particular embodiments, cytokine storm syndrome manifests as acute respiratory distress syndrome, macrophage activation syndrome, adult or neonatal respiratory sepsis, systemic inflammatory response syndrome, venous or arterial thromboembolism, a sudden rise in ferritin, severe pericarditis, a sudden rise in liver transaminases causing hepatic encephalopathy, or diseases associated with chimeric antigen receptor T cell therapy. In one embodiment, the cytokine storm (treated or prevented by the method of the present invention) is caused by an infection.

[0020] In one embodiment, the method of the present invention is useful for treating an infectious disease such as Covid-19 or dengue fever. In another embodiment of the present invention, the method comprises administering the composition intravenously.

[0021] Administration of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 to patients induced a reduction in events associated with dyspnea, which requires the patient to:

[0022] - Oxygen therapy to maintain oxygen saturation (SO2) above 93% - Oxygen saturation > 3 percentage points or arterial oxygen pressure (PaO2) > 10% with stable fraction of inspired oxygen (FiO2) in the past 24 hours - Increased FiO2 or other need for mechanical ventilation to maintain stable SO2 in the past 24 hours - Increase in the number and / or extent of areas of lung consolidation.

[0023] Furthermore, administration of the compositions of the present invention is useful for treating patients suffering from the infectious disease Covid-19, when said patients require oxygen therapy of 6 L / min or more, plus any of the following conditions:

[0024] - Gasping or choppy speech - Oxygen therapy 6L / min and respiratory rate >22 breaths / min - PaO2: less than 65mmHg - Deterioration of X-ray images - Fever above 39°C - Decreased baseline values ​​of hemoglobin, platelets, and white blood cells (hemoglobin ≤ 9.2 gm / dl, white blood cells ≤ 5000 / mm 3 , platelets ≦110,000 / mm - A decrease in erythrocyte sedimentation rate inconsistent with an increase or no change in C-reactive protein - Increased baseline triglyceride levels of more than 3 mmol / L - Baseline ferritin level increased from 500ng / ml or absolute ferritin level ≥ 2000ng / ml - Aspartate aminotransferase transaminase ≥ 30 IU / L - Increased D-dimer - Fibrinogen less than 2.5g / L - Appearance of neurological symptoms

[0025] The compositions of the present invention are also useful for treating patients with Covid-19 who enter a critical state without requiring oxygen therapy, but who present with an initial increase in ferritin from 500 ng / ml or absolute ferritin levels of 2000 ng / ml or greater, and progressive radiological signs of multifocal interstitial pneumonia.

[0026] The compositions of the present invention are also useful in treating diseases characterized by a state of acute hyperinflammation, such as those that develop in adults or neonates as respiratory sepsis; and diseases that develop respiratory distress syndrome, as well as those syndromes caused by mechanical and physical causes. [Brief explanation of the drawings]

[0027] [Figure 1A]Frequency of T cells expressing the CD4+Foxp3+ phenotype isolated from the spleens of BALB / c mice after inoculation with a pharmaceutical composition of the peptide identified as SEQ ID NO: 1 when sucrose was added at concentrations of 10 mg / mL (10), 30 mg / mL (30), and 80 mg / mL (80), as quantified by flow cytometry. Data analysis was performed using the Kruskal-Wallis and Dunn statistical tests. Different letters indicate statistically significant differences (P<0.05). [Figure 1B] Frequency of T cells expressing the CD4+Foxp3+ phenotype isolated from the spleens of BALB / c mice after inoculation with a pharmaceutical composition of the peptide identified as SEQ ID NO: 1 when sucrose was added at concentrations of 20 mg / mL (20), 30 mg / mL (30), and 40 mg / mL (40), as quantified by flow cytometry. Data analysis was performed using the Kruskal-Wallis and Dunn statistical tests. Different letters indicate statistically significant differences (P<0.05). [Example]

[0028] Detailed explanation of examples / practical examples Example 1. Treatment of patients with Covid-19 disease with a pharmaceutical composition comprising the peptide identified as SEQ ID NO:1. This study presents 10 patients admitted to the intensive care unit of the Luis Díaz Soto Military Hospital with the infectious disease Covid-19. These patients received medical treatment in accordance with the protocol established by the Cuban Ministry of Public Health for the treatment of this disease. The patients developed acute respiratory distress syndrome and were administered mechanical ventilation / airway pressure relief ventilation (VAM APRV) on the day of presentation. After receiving informed consent as established by Cuban regulatory authorities and the hospital's ethical review committee, the patients were administered a pharmaceutical composition containing a peptide identified as SEQ ID NO: 1. The peptide was obtained by synthetic chemistry using methods well known in the art.

[0029] The pharmaceutical composition was administered intravenously once every 12 hours. Eight patients received 1 mg of peptide per patient per application. Patients in codes 5 and 6 received 2 mg every 12 hours. These patients had cancer among their comorbidities. Patient demographic characteristics and days of VAM APRV and peptide administration are reflected in Table 1.

[0030] [Table 1]

[0031] As can be seen from the table, the 10 patients with Covid-19 who received the peptide had risk factors for the disease becoming severe or critical. Only one patient had no comorbidities, but advanced age (80 years old) was a risk factor. Notably, patient No. 5 presented with deep vein thrombosis. However, the clinical course of the 10 patients who received the peptide was favorable. It is believed that the acute respiratory distress syndrome (ARDS) had resolved by the time of extubation. These results indicate that peptide treatment is effective in treating ARDS. The disappearance of this syndrome in the patients coincided with their clinical and radiological improvement. The patient with DVT also had a favorable course. These patients did not show symptoms of immunosuppression. Before treatment, they presented with lymphopenia, a characteristic sign in Covid-19 patients. As a result of treatment with this peptide, circulating lymphocyte levels gradually returned to normal.

[0032] Expert literature reports that 85% to 90% of patients with acute respiratory distress syndrome do not survive. In Cuba, among patients confirmed with SAR-COV-2 who progressed to a severe or critical stage, there was a 68.6% fatality rate among individuals not treated with a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1. In contrast, among patients confirmed with SAR-COV-2 who were severely or critically ill and treated with a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1, there was a 13.0% fatality rate.

[0033] Example 2. Administration of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 to prevent the establishment of respiratory distress syndrome. Severely ill and critically ill patients with Covid-19 may develop respiratory distress syndrome, which can lead to death. Surprisingly, administration of a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 prevented the onset of acute respiratory distress syndrome. The peptide was administered at an early stage, after the first signs (progressive radiological indicators of multifocal interstitial pneumonia, elevated ferritin (2x normal), and pulse oximetry showing oxygen saturation below 94). Therefore, administration of this peptide avoided the need for mechanical ventilation. In this way, the high incidence of nosocomial sepsis was avoided.

[0034] This example involves a 42-year-old female patient with Covid-19 disease admitted to the intermediate care unit of the Pedro Kouri Tropical Medicine Institute Hospital. The patient received medical treatment according to the protocol established by the Cuban Ministry of Public Health for the treatment of this disease, but suddenly developed respiratory distress accompanied by a fever of 39°C and a ferritin level of 700 ng / mL. With informed consent, a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 was administered intravenously at a dose of 1 mg every 12 hours. After two hours, the respiratory distress disappeared and the temperature decreased. The patient did not develop acute respiratory distress syndrome, and after 72 hours, the ferritin level had decreased to less than 500 ng / mL.

[0035] Additionally, we treated three other patients in critical condition. Their demographic characteristics and comorbidities are shown in Table 2. These patients were admitted to the intermediate care unit at the Luis Diaz Soto Military Hospital.

[0036] [Table 2]

[0037] These patients also presented with initial warning signs, such as persistent fever above 38°C and respiratory distress requiring oxygen therapy via mask or nasal prong to maintain oxygen saturation above 93%. These patients also presented with tachypnea of ​​above 25 breaths per minute, requiring oxygen therapy via mask or nasal prong to maintain oxygen saturation above 93%.

[0038] A pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 was administered intravenously at a dose of 1 mg every 24 hours. Two hours after administration, respiratory distress disappeared and body temperature decreased. The patient did not develop acute respiratory distress syndrome. Ferritin levels decreased to less than 200 ng / mL after 72 hours.

[0039] These results demonstrate that treatment of patients with Covid-19 disease with a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 avoids the use of mechanical ventilation in these patients before they develop acute respiratory distress syndrome.

[0040] Example 3. Reduction of C-reactive protein in patients with Covid-19 disease treated with a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1. C-reactive protein is one of the most commonly used markers of the inflammatory process, and it is considered one of the acute-phase reactants of inflammation. A pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 was administered intravenously. Eight of the ten patients suffering from Covid-19, whose treatment is described in Example 1, received 1 mg of the peptide every 12 hours. Patients with codes 5 and 6, who had cancer among their comorbidities, received 2 mg of the peptide every 12 hours. Unexpectedly, treatment with the peptide of SEQ ID NO: 1 significantly reduced C-reactive protein levels in the treated patients. These results are shown in Table 3.

[0041] [Table 3]

[0042] The decrease in C-reactive protein during treatment of these patients is a direct indication that the peptide of sequence number 1 suppresses inflammation, and this result is consistent with the resolution of acute respiratory distress syndrome and the clinical and radiological improvement of the patients. The results obtained in this study are surprising: during clinical trials in patients with rheumatoid arthritis treated with the same peptide, this reduction in the reactant was not found during the acute phase of inflammation.

[0043] Example 4. Reduction of inflammatory cytokines in patients with Covid-19 disease treated with a pharmaceutical composition comprising the peptide identified as SEQ ID NO:1. In this study, levels of the inflammatory cytokines IL-6, IL-17, IL-1, IFNγ, and TNFα were measured in patients with Covid-19 disease who were treated with a composition comprising the peptide identified as SEQ ID NO: 1. The patients were in a critical condition. The treatment of these patients is described in Example 1.

[0044] The cytokines were quantified in the serum of patients identified by numbers 1, 2, 3, 4, 7, and 8 in Table 1, Example 1. For cytokine measurement in serum samples, the virus was inactivated under strict biosecurity measures. Quantification was performed immediately before administering the first dose of peptide, and 48, 72, and 96 hours after starting treatment with the peptide.

[0045] All cytokine measurements were performed using specific enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer's recommendations (Quantikine® R&D Systems, USA). Briefly, 50 μL of the designated dilution of each cytokine was added to each well, followed by 200 μL of either the standard curve, sample, or control. The plate was incubated at room temperature for 2 hours, after which the contents of each well were discarded. The plate was then washed three times with 1X washing solution, and 200 μL of conjugated antibody was added to each well and incubated at room temperature for 1 hour. This wash was repeated, and 200 μL of substrate solution was added to each well. The plate was incubated at room temperature for 20 minutes in the dark, after which 50 μL of stop solution was added to each well, and the optical density was measured at 450 nm using a microplate reader (Thermo Scientific Multiskan Go, Finland). A second reading was taken at 570 nm, and the resulting value was subtracted from the 450 nm reading to correct for optical imperfections on the plate. The concentration of each cytokine was measured in triplicate. The results obtained for IL-6 are shown in Table 4.

[0046] [Table 4]

[0047] As can be seen in the table, when critically ill patients received the peptide of SEQ ID NO: 1, there was a decrease in the levels of the cytokine IL-6, a cytokine that is significantly increased in the inflammatory process. It has been described that patients with Covid-19 disease who progress to the critical stage have elevated levels of this cytokine, in contrast to patients with milder symptoms of the disease. Similarly, a decrease in IL-17, IL-1, IFNγ, and TNFα was observed in these patients.

[0048] These results confirm that administration of the peptide suppresses the hyperinflammation and cytokine storm that develops in patients with Covid-19 disease progressing to the severe stage.

[0049] Example 5. Treatment of a patient with Covid-19 disease who presented with high ferritin levels without respiratory distress with a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1. The patient was a 66-year-old woman with a history of type 2 diabetes, exogenous obesity, and grade 4 fatty liver disease. She was hospitalized with a diagnosis of COVID-19 and presented with gastrointestinal symptoms, including dyspepsia, nausea, and occasional diarrhea. She was prescribed prokinetic drugs (prokineticins) and antiemetics. She also had a high ferritin level (>1500 ng / mL) and an elevated erythrocyte sedimentation rate with decreased hemoglobin.

[0050] For this clinical condition, the specialist prescribed a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1, administered at a dose of 1 mg every 12 hours. Surprisingly, 96 hours after treatment, ferritin levels had decreased to a concentration of 350 ng / mL.

[0051] Treatment with the pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 was continued for an additional 72 hours.

[0052] After treatment ended, ferritin levels normalized and the patient became symptom-free.

[0053] These results indicate that treatment with a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 can reduce ferritin levels when they are elevated by viral infection.

[0054] Example 6. Treatment of patients with pericarditis of viral etiology with a pharmaceutical composition comprising the peptide identified as SEQ ID NO:1. The treated patient, a 48-year-old man, was admitted to Holguin Military Hospital with confirmed Covid-19. Two days after admission, the patient presented with a fever of 38°C and two bouts of diarrhea. He was diagnosed with pericarditis secondary to myocarditis of viral etiology (Covid-19), but without hemodynamic effects. The patient did not experience respiratory distress. Treatment was initiated with a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 at a dose of 1 mg every 12 hours.

[0055] Surprisingly, electrocardiograms improved 24 hours after administration, and the patient became asymptomatic and showed significant clinical improvement 96 hours after administration. These results confirmed that a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 reduces inflammation occurring in conditions such as pericarditis secondary to myocarditis, without hemodynamic repercussions due to viral etiology.

[0056] Example 7. Treatment of patients with Covid-19 hepatic encephalopathy with a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1. The patient was a 55-year-old white woman with a history of arterial hypertension. She was admitted to the Luis Díaz Soto Hospital, isolation room, internal medicine service after testing positive for the coronavirus that causes COVID-19 disease. Treatment for this disease was initiated according to established national protocols. During her hospitalization, she remained asymptomatic until the 10th day. On that day, she reported episodes of anxiety and tension accompanied by high blood pressure (160 / 90 mmHg). She was examined by a psychiatrist, and complementary laboratory tests were performed. The patient did not exhibit shortness of breath. The psychiatrist diagnosed an anxiety syndrome with psychotic elements. On the 11th day, the patient's clinical symptoms worsened, presenting with vomiting, high blood pressure, a tendency toward tachycardia, and no sphincter relaxation. She was transferred to the intensive care unit. A blood count confirmed the presence of severe hyponatremia (Na concentration 105 mmol / L). Computed tomography showed no craniocerebral changes, and complementary testing revealed markedly elevated liver enzymes and leukocytosis. Based on these results, the patient was diagnosed with hepatic encephalopathy. The patient was discontinued from all hepatotoxic medications, dietary modifications were implemented, and gradual sodium supplementation was continued. Treatment with a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 was initiated intravenously at a rate of 1 mg every 12 hours. Daily clinical follow-up with complementary laboratory tests was performed. Surprisingly, after 72 hours of treatment with the pharmaceutical composition containing the peptide identified as SEQ ID NO: 1, the patient's progress was favorable, with a decrease in liver enzymes, progressive reduction in creatine kinase and blood nitrogen compounds, and normalization of sodium levels. After 96 hours of treatment, clinical features consistent with hepatic encephalopathy were completely resolved, and administration of the pharmaceutical composition containing the peptide was continued for an additional 3 days. These results confirm that administration of the pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 reduces inflammation associated with conditions such as hepatic encephalopathy.

[0057] Example 8. Treatment of dengue patients with a pharmaceutical composition comprising the peptide identified as SEQ ID NO:1. A 19-year-old female patient visited the Luis Díaz Soto Hospital with a fever of 38.5°C (98.5°F), a fever that had persisted for more than 72 hours, and a severe generalized headache. She also had posterior eye pain associated with conjunctival injection. Based on these symptoms, the patient was admitted with suspected dengue fever.

[0058] Physical examination of the patient revealed a hyperthermia of 39.9°C, a blood pressure of 90 / 60 mmHg, tachycardia, and mild distal coolness. Skin inspection by the physician revealed petechiae in the forearm and abdominal areas, and a loop test was positive. The patient was treated with volume support of 30 mL per kg of body weight for 24 hours.

[0059] Twenty-four hours after admission, the patient developed abdominal pain and vomiting. Complementary testing revealed leukopenia (total white blood cell count 2.9 x 10), erythrocyte sedimentation rate 95 mm / h, C-reactive protein 150 mg / mL, lactate dehydrogenase 918 U / L, glutamic oxalacetic transaminase 93 IU / L, and glutamic pyruvic transaminase 58 IU / L. Abdominal ultrasound confirmed the presence of moderately intense free fluid in all four gallbladder angles. The gallbladder exhibited a thickened wall and cystic edema without calcification. The patient received supportive care, including crystalloid solutions administered at a rate of 35 to 45 mL per kg of body weight over 24 hours.

[0060] Due to the patient's indolent progress and poor response to intravenous therapy, the medical team determined that the patient had developed macrophage activation syndrome secondary to infection with the virus that causes dengue fever. This diagnosis was confirmed by the laboratory parameters associated with this syndrome: ferritin: 1893 mg / L, fibrinogen: 2.31 g / L, C-reactive protein: 150 mg / mL, and erythrocyte sedimentation rate: 53 mm / h.

[0061] Based on these results, the patient was transferred to the intensive care unit and administered a pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 intravenously at a rate of 1 mg every 12 hours. The administration of the composition aimed to modulate the immune response and thus control the cytokine storm that had developed in the patient, according to clinical characteristics and laboratory parameters. After 48 hours of treatment, the patient showed significant improvement, with the disappearance of abdominal pain, arterial hypotension, and distal cold sensation. The patient resumed eating. After 72 hours, the patient's clinical and radiological improvement continued, and the administration frequency of the pharmaceutical composition containing the peptide was then reduced to 1 mg every 24 hours. From the seventh day of administration onward, the patient became clinically and radiologically asymptomatic.

[0062] Dengue virus infection was confirmed by a positive IgM serum test. All laboratory results returned to normal, and the patient was discharged.

[0063] These results confirm those outlined in the previous examples, which confirm that a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 controls the cytokine storm that may be associated with hemophagocytic syndrome, which may be associated with various pathologies such as dengue fever progressing to a severe stage of the disease.

[0064] Example 9. Effect of sucrose concentration on the pharmacological potentiation of peptide of SEQ ID NO: 1 for reducing inflammation. To use the peptide identified as SEQ ID NO: 1 as an inducer of molecular mechanisms that reduce excessive inflammation in patients with COVID-19 and other pathologies, the effect of the sugar sucrose on the composition was examined over a range of concentrations to select the optimal concentration of the sugar sucrose associated with the induction of regulatory T cells when the formulation was administered intravenously. The composition was prepared using 1.8 mg / mL to 3.5 mg / mL of the peptide identified as SEQ ID NO: 1, dissolved in sodium acetate buffer, pH 4, to which sucrose was added at different concentrations as described below.

[0065] Male BALB / c mice were randomly divided into three groups of 10. Mice in each group were intravenously inoculated as follows:

[0066] - Group 1: Pharmaceutical composition of the peptide identified as SEQ ID NO: 1 to which sucrose was added at a concentration of 10 mg / mL. - Group 2: Pharmaceutical composition of the peptide identified as SEQ ID NO: 1 to which sucrose was added at a concentration of 30 mg / mL. - Group 3: Pharmaceutical composition of the peptide identified as SEQ ID NO: 1 to which sucrose was added at a concentration of 80 mg / mL.

[0067] Four days later, mice received a second dose under the same conditions. Ten animals per group were sacrificed eight days after the first inoculation. Mouse spleen cells were preincubated with Mab 2.4G2 (eBiosciences, USA) to block nonspecific binding to Fc receptors. Cells were then labeled in PBS containing 3% calf serum and saturating amounts of the following mAbs (eBiosciences, USA): APC-Cy7-anti-CD4 conjugate (clone RM4-5) and APC-anti-CD25 conjugate (PC61). Foxp3 transcription factor activity was assessed using PE-conjugated anti-Foxp3 mAb (FJK-16s; eBioscience, USA) according to the manufacturer's recommendations. An isotype-irrelevant mAb (rat PE-Cy5 IgG2a; eBioscience, USA) was used as a control. The area corresponding to lymphocytes was boxed according to the distribution characteristics of lymphocytes in the flow cytometry histogram of the LSR-II™ cytometer. Analysis was performed using the FlowJo® program (Tree Star).

[0068] As can be seen in Figure 1A, mice inoculated with a formulation containing the peptide identified as SEQ ID NO: 1 and sucrose at a concentration of 30 mg / mL had a significant increase in the proportion of regulatory T cells, indicating a synergistic effect between the components of the composition.

[0069] To determine whether this concentration of sucrose has a decisive effect on the induction of regulatory T cell frequency, we performed an experiment under similar conditions to the previous one, but using a narrower range of sucrose concentrations.

[0070] Mice were randomly divided into three groups of 10. Mice in each group were intravenously inoculated as follows:

[0071] - Group 1: Pharmaceutical composition of the peptide identified as SEQ ID NO: 1 to which sucrose was added at a concentration of 20 mg / mL. - Group 2: Pharmaceutical composition of the peptide identified as SEQ ID NO: 1 to which sucrose was added at a concentration of 30 mg / mL. - Group 3: Pharmaceutical composition of the peptide identified as SEQ ID NO: 1 to which sucrose was added at a concentration of 40 mg / mL.

[0072] Surprisingly, mice treated with these concentrations of sucrose, 20-40 mg / mL, showed a significant increase in the percentage of regulatory T cells compared to the group treated with 10-80 mg / mL in the previous experiment, observing a synergistic effect between the components of the composition, with a greater effect in the group of mice treated with the pharmaceutical composition of the peptide identified as SEQ ID NO: 1, to which sucrose was added at concentrations of 20 and 30 mg / mL (Figure 1B).

[0073] From these results, it can be concluded that the addition of sucrose at concentrations ranging from 20 to 30 mg / mL is a determinant for the induction of regulatory T cells that control the magnitude of inflammation, and that this preparation, administered intravenously, has the ability to control pathologies characterized by excessive inflammation. [Sequence List Free Text]

[0074] SEQ ID NO: 1 <223> Description of artificial sequences: peptide-type APL

Claims

1. A pharmaceutical composition for the treatment or prevention of cytokine storm syndrome, comprising the peptide identified as SEQ ID NO: 1 and a pharmaceutically acceptable excipient.

2. 10. The pharmaceutical composition of claim 1, wherein the peptide identified as SEQ ID NO: 1 is at a concentration ranging between 1.8 mg / mL and 3.6 mg / mL and the sucrose is at a concentration between 20 and 30 mg / mL.

3. 2. The pharmaceutical composition according to claim 1, characterized in that it is applied by the intravenous route.

4. The pharmaceutical composition according to claim 1 for the treatment or prevention of cytokine storm syndrome.

5. 5. The pharmaceutical composition of claim 4, wherein the cytokine storm syndrome is selected from the group of diseases consisting of acute respiratory distress syndrome, macrophage activation syndrome, adult or neonatal respiratory sepsis, systemic inflammatory response syndrome, venous or arterial thromboembolism, a sudden increase in ferritin levels, severe pericarditis, a sudden increase in liver transaminases causing hepatic encephalopathy, or a disease associated with chimeric antigen receptor T-cell therapy.

6. The pharmaceutical composition of claim 4, which is administered by intravenous route.

7. The pharmaceutical composition according to claim 4, wherein the cytokine storm syndrome is caused by an infectious disease.

8. The pharmaceutical composition of claim 7, wherein the infectious disease is Covid-19 or dengue fever.

Images