WO2022002060A1

WO2022002060A1 - Method for antiviral and cytokine storm treatment

Info

Publication number: WO2022002060A1
Application number: PCT/CN2021/103142
Authority: WO
Inventors: Daniel Gligorov; Valentinovich TULIN DMITRY; Ivanovna SHEVCHENKO OLGA; Sergeevna CHERNYSH NATALYA
Original assignee: Vamos Biotech (Shanghai) Co., Ltd
Priority date: 2020-06-29
Filing date: 2021-06-29
Publication date: 2022-01-06
Also published as: CN116249543A

Abstract

The present invention provides a new application of Allostatin, for treating a viral infection, cytokine storm or fever. In some embodiments, Allostatin is also used in prevention and treatment of cytokine storm or fever caused by viral infection and other conditions. In some embodiments, the virus is a virus associated with respiratory infection or respiratory virus, for example, corona virus.

Description

[Title established by the ISA under Rule 37.2] METHOD FOR ANTIVIRAL AND CYTOKINE STORM TREATMENT

FIELD OF THE INVENTION

The invention relates to the field of immunotherapy and new application of Allostatin. The priority area of application of the invention is the treatment of virosis of humans and other warm-blooded animals and the prevention and suppression of cytokine storm.

BACKGROUND OF THE INVENTION

For at least 300 years the immune system has been targeted to improve human health. As is well known in recent years, the modulation and exploiting of immune system have great potential in the treatment of malignancy and infectious disease. Studies on the activity of vaccines, the role of new checkpoint molecules, new pathways for stimulation of innate responses, and even the genetic determinants of response will all inform both basic immune mechanisms and have applications in the generation of effective immunity to pathogens. (Samantha L. Bucktrout, etc., Recent advances in immunotherapies: from infection and autoimmunity, to cancer, and back again, Genome Medicine, 2018) .

Alloferon is a group of bioactive, slightly cationic peptides, with immunomodulatory properties. It is isolated from infected insects. In vitro experiments reveal that the synthetic version of Alloferon has stimulatory activities on natural killer lymphocytes, whereas in vivo trials indicate induction of IFN production in mice after treatments with synthetic Alloferon. Additional in vivo experiments in mice indicate that Alloferon has antiviral and antitumor capabilities (Sergey Chernysh, Antiviral and antitumor peptides from insects, PNAS, 2002) . At present, an injectable formulation of Alloferon has been registered in Russia as an antiviral drug. However, in vitro experiments have shown that depending on the concentration in culture media, Alloferon may both inhibit (at high concentrations) and stimulate (at low concentrations) proliferation of tumor cells, thus, the possibility of using Alloferon for tumor therapy is restricted.

To further explore the potential of immunomodulating peptides, a new family of peptides, named Allostatin was recently developed (WO2005/068491) . Unlike Alloferon, Allostatin, at both high and low concentrations, exhibits a reduced growth-stimulating activity and enhanced antiproliferative and cytotoxic activities towards tumor cells. It’s already known that, Allostatin is capable of activating cytotoxic effect of NK cells, as well as increasing and maintaining the population of NK and T lymphocytes producing IFNγ and increasing their sensitivity to the activating signals of IL-2 and IL-12. However, it’s hard to say whether and how Allostatin may work clinically. It’s also not easy to tell, with all these non-specific immune activation functions above, for which diseases and conditions Allostatin could play a role in the treatment.

SUMMARY OF THE INVENTION

In this invention, a method of the antiviral and anti-cytokine storm or fever of Allostatin is developed. Thus, provided in the present invention is a method for treating a viral infection, comprising administering to a subject an effective amount of Allostatin or the preparation thereof.

Specifically, provided herein are:

1. A method for treating a viral infection, comprising administering to a subject an effective amount of a peptide comprising an amino acid sequence as shown by SEQ ID NO: 1.

2. The method of item 1, wherin the virus is associated with respiratory infection.

3. The method of item 2, wherin the virus is corona virus or influenza virus.

4. The method of item 3, wherin the corona virus is COVID19 virus.

5. The method of any one of items 1-4, wherein the subject infected with virus has fever.

6. The method of any one of items 1-5, wherein the subject infected with virus has cytokine storm.

7. The method of any one of items 1-6, wherein the peptide improves or alleviates one of more symptoms selected from the group consisting of fever, fatigue, dyspnea, and hypoxemia (low oxygen saturation of blood) .

8. The method of item 1, wherin the virus is herpes simplex virus or genitalia mucous herpes.

9. A method to prevent or suppress cytokine storm, comprising administering to a subject an effective amount of a peptide comprising an amino acid sequence as shown by SEQ ID NO: 1.

10. The method of item 9, wherein the peptide improves or alleviates the symptoms caused by cytokine strom.

11. The method of item 10, wherein the symptoms caused by cytokine strom is selected from the group consisting of fever, fatigue, dyspnea, and hypoxemia (low oxygen saturation of blood) .

12. A method of any one of items 1-11, wherein the peptide activates cytotoxic NK cells and/or inhibits proimflammatory NK cells.

13. The method of item 12, wherein the cytotoxic NK cells are CD16+CD56 ^dim NK Cells.

14. The method of any one of items 12-13, wheren the peptide inhibits proimflammatory NK cells expressing KIR2DL4.

15. The method of any one of items 12-14, wherein the peptide promotes the survival, proliferation or development of T cells.

16. A method for treating fever, comprising administering to a subject an effective amount of a peptide comprising an amino acid sequence as shown by SEQ ID NO: 1.

17. The method of item 16, where the peptide alleviates or reduces fever of the subject.

The present invention also provides a use of the peptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the manufacture or preparation of medicines for the treatment of viral infection. In some embodiments, the virus is associated with respiratory infection. In some embodiments, the virus is corona virus or influenza virus. In some embodiments, the corona virus is COVID19 virus. In some embodiments, the subject infected with virus has fever. In some embodiments, the subject infected with virus has cytokine storm. In some embodiments, the peptide improves or alleviates one of more symptoms selected from the group consisting of fever, fatigue, dyspnea, and hypoxemia (low oxygen saturation of blood) . In some embodiments, the virus is herpes simplex virus or genitalia mucous herpes.

The present invention also provides a use of the peptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the manufacture or preparation of medicines for preventing or suppressing cytokine storm, comprising administering to a subject an effective amount of a peptide comprising an amino acid sequence as shown by SEQ ID NO: 1. In some embodiments, the peptide improves or alleviates the symptoms caused by cytokine strom. In some embodiments, the symptoms caused by cytokine strom is selected from the group consisting of fever, fatigue, dyspnea, and hypoxemia (low oxygen saturation of blood) . In some embodiments, the peptide activates cytotoxic NK cells and/or inhibits proimflammatory NK cells. In some embodiments, the cytotoxic NK cells are CD16+CD56 ^dim NK Cells. In some embodiments, the peptide inhibits proimflammatory NK cells expressing KIR2DL4. In some embodiments, the peptide promotes the survival, proliferation or development of T cells.

Also provided herein is a use of the peptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the manufacture or preparation of medicines for treating fever, comprising administering to a subject an effective amount of a peptide comprising an amino acid sequence as shown by SEQ ID NO: 1. In some embodiments, the peptide alleviates or reduces fever of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1E. show the increase of NK cells expressing activation markers (CD69 in Fig. 1A, CD25 in Fig. 1B, NKp80 in Fig. 1C, CD244 in Fig. 1D) and the decrease of NK cells expressing receptors that mediate inhibition (KIR2DL4 in Fig. 1E) of NK cells.

Figures 2A-2C. show the increase of T cells expressing activation markers (CD25 in Fig. 2A, CD127 in Fig. 2B, CD27 in Fig. 2C) .

Figure 3. shows the increase of cytotoxic NK cells (CD3-/CD16+/CD56 ^dim) expressing activation makers (CD244 and NKG2A) .

Figure 4. shows the decrease of cytokine producing-NK cells (CD3-/CD16-/CD56 ^bright) which express proinflammatory receptors (KIR2DL4) .

DETAIL DESCRIPTION OF THE INVENTION

Infections caused by herpes simplex virus 1 (HSV-1) , HSV-2, varicella zoster virus (VZV) , Epstein-Barr virus (EBV) , cytomegalovirus (CMV) , human papillomaviruses (HPVs) , and molluscum contagiosum virus (MCV) are common, and their incidence continues to grow despite a wide range of available and experimental therapies (Jashin J. Wu, etc., Advances in Antiviral Therapy, Dermatologic Clinics, 2005) . Besides, coronaviruses are pathogens with a serious impact on human and animal health. Coronaviruses mostly cause enteric or respiratory disease, which can be severe and life threatening, e.g., in the case of the zoonotic coronaviruses causing severe acute respiratory syndrome (SARS) , coronavirus disease 2019 (COVID-19) , and Middle East Respiratory Syndrome (MERS) in humans. Despite the economic and societal impact of such coronavirus infections, and the likelihood of future outbreaks of additional pathogenic coronaviruses, our options to prevent or treat coronavirus infections remain very limited (Adriaan H de Wilde, Host Factors in Coronavirus Replication, Curr Top Microbiol Immunol. 2018) . At present, it is known that the modulation and exploiting of immune system can be a promising method for treating such infectious diseases.

Allostatin (a peptide with the amino acid sequence: His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly (SEQ ID No. 1) was generated in 2004, and its antitumor characters have been relatively carefully explored. Allostatin is reported to have antiproliferative and immunomodulatory activity to tumor cells both in vivo and in vitro (WO2005/068491) . However, as a cytokine-like effector, the antiviral activities and other application in disease treatment of Allostatin have been poorly researched. In the following examples in this article, the authors tested Allostatin both in vivo and in vitro, and also on the patients. The results present the potential of Allostatin in adaptive immunity enhancement, cytotoxic NK cell activation, and imflammation control. In addition, the patients’ reactions to Allostatin support the application of Allostatin in the treatment of virus infection and the suppression and prevention of cytokine storm.

It’s known that current therapies for viral infections of the skin can reduce or suppress symptoms, but there is no known cure and no available method to reduce the frequency of outbreaks after antiviral drug cessation (Jashin J. Wu, etc., Advances in Antiviral Therapy, Dermatologic Clinics, 2005) . While in the exemplary examples in this invention, Allostatin is applied on the surface of patients’ skin with a proper dosage and frequencies, showing a better efficacy than existing therapies. As shown in example 1, it was obvious that the efficacy difference between Acyclovir and Allomedin (a product containing Allostatin as the only active ingredient) groups was dominated at the beginning and the development stage of the symptoms (when the virus is active) rather than the end stage.

Besides, as is known, a new coronavirus named SARS-CoV-2 was identified as the causative agent of a several acute respiratory infection named COVID-19. COVID-19 can be confirmed based on the patient's history, clinical manifestations, imaging characteristics, and laboratory tests. Chest CT examination plays an important role in the initial diagnosis of the novel coronavirus pneumonia. Multiple patchy ground glass opacities in bilateral multiple lobular with periphery distribution are typical chest CT imaging features of the COVID-19 pneumonia (Xu et al., Imaging and clinical features of patients with 2019 novel coronavirus SARS-CoV-2, Eur J Nucl Med Mol Imaging., 2020) . SARS-CoV-2 showed phylogenetic similarities to both SARS-CoV and MERS-CoV viruses, and some of the clinical features are shared between COVID-19 and previously identified beta-coronavirus infections, however there are still many unresolved questions regarding the pathogenesis of this disease (Coperchini et al. The cytokine storm in COVID-19: An overview of the involvement of the chemokine/chemokine-receptor system, Cytokine &Growth Factor Reviews, June 2020) . Despide of this, in example 2 and 3, the signs of cytokine storm, high uncontrolled hyperthermia, ache, difficulty in breath, in patients infected with COVID-19 in some way went away after been given one injection of allostatin (1 mg, subcutaneously) .

According to the facts above, provided in this invention is a method for treating a viral infection or for suppressing or preventing cytokine storm in a proper way. In some embodiments, the method is for treating a virosis, comprising administering to a subject an effective amount of a compound or the preparation thereof, wherein the compound comprises the amino acid sequence shown as His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly (SEQ ID NO: 1) . In some embodiments, the compound is a peptide. In some embodiments, the compound is a part of a chemical that is not a natural protein or peptide.

According to the method, in some embodiments, the subject treated with is infected with virus that led to rash, wart, or lesion on skin or mucosa. In some embodiments, the subject is infected with virus that led to inflammatory response of skin or mucosa. In some embodiments, the virus is selected from human papillomavirus (HPV) , herpes simplex virus (HSV) , molluscum contagiosum virus (MCV) and varicella zoster virus (VZV) . Example 1 proves the antiviral efficacy of Allostatin when it’s applied to the lesions on the skin or on the mucosa, thus, in some embodiments, the compound or the preparation thereof is applied topically. In some embodiments, the compound or the preparation thereof is applied externally. In some embodiments, the compound or the preparation thereof is applied cutaneously. In some embodiments, the compound or the preparation thereof is applied subcutaneously. In some embodiments, the compound or the preparation thereof is applied to the mucosa. In some embodiments, the compound or the preparation thereof is applied directly to the lesions.

According to the method, in some embodiments, the subject is infected with virus that led to cytokine storm. In some embodiments, the subject is infected with virus that led to systemic inflammatory response. In some embodiments, the virus is selected from corona virus, cytomegalovirus, Epstein-Barr virus, influenza virus, or variola virus. In some embodiments, the virus is avian H5N1 influenza virus, severe acute respiratory syndrome coronavirus (SARS-CoV) , or SARS- COV-2 virus. According to Example 2, both subcutaneous injection and intravenous injection of Allostatin results in relieve in symptoms of patients infected with viruses that led to inflammatory response or rash, wart or lesion in skin or mucosa. Thus, in some embodiments, the compound or the preparation thereof is applied subcutaneously. In some embodiments, the compound or the preparation thereof is applied intravenously. In some embodiments, the compound or the preparation thereof is injected or infiltrated into the tissues comprising the virus infected cells.

According to the method, the compound or the preparation thereof is also applied concomitantly with IFN-γ, IL-2, and/or IL12 in the treatment of viral infection.

Besides, the data in Table 1 shows that absolute majority of patients with skin herpes and genitalia mucous tunic herpes (labial and genital herpes, correspondingly) whose symptoms were scarcely improved when treated with conventional medications were effectively treated by Allomedin. And according to Table 2 and 3, compared to the standard acyclovir treatment, Allomedin rapidly eliminates inflammation symptoms like itch, burning, oedema and the duration of the symptoms decreased about 10 times. Thus the provided method in some embodiments, Allostatin or the preparation thereof is utilized in treating patients who scarcely response to the traditional medications. And in some specific embodiments, comparing to that of conventional treatment, the duration of symptoms decreases about 10 times when patients are treated with Allostatin or the preparation thereof.

In Example 3, Allostatin is tested both in vitro on cytokine storm models and in clinical treatment of patients having systemic inflammatory response or the signs of cytokine storm. The result clearly shows the effectiveness of Allostatin application in preventing and suppressing cytokine storm in both infectious diseases and non-infectious diseases. The term cytokine storm refers to the general concept of an excessive or uncontrolled release of proinflammatory cytokines. The use of the term in infectious disease research began in early 2000 in reports on cytomegalovirus, Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis, group A streptococcus, influenza virus, variola virus, and severe acute respiratory syndrome coronavirus (SARS-CoV) . The term appears to have first been applied in the context of avian H5N1 influenza virus infection in 2005, after which it began to appear more frequently in the scientific literature (Tisoncik et al., Into the Eye of the Cytokine Storm, Microbiology and Molecular Biology Reviews) . Cytokine storms are associated with a wide variety of infectious and noninfectious diseases and have even been the unfortunate consequence of attempts at therapeutic intervention. Previous reviews have centered on the advent of the concept or its role in graft-versus-host disease, multiple sclerosis, pancreatitis, or multiple organ dysfunction syndrome (Tisoncik et al., Into the Eye of the Cytokine Storm, Microbiology and Molecular Biology Reviews) .

Accordingly, provided in this invention is also a method to prevent or suppress cytokine storm comprising administering to a subject an effective amount of a compound or the preparation thereof, wherein the compound comprises the amino acid sequence as shown as His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly (SEQ ID NO: 1) . In some embodiments, the compound is a peptide. In some embodiments, the compound is a part of a chemical that is not is a natural protein or peptide.

According to the method to prevent or suppress cytokine storm, in some embodiments, the subject is infected with virus that causes cytokine storm. In some embodiments, the virus is selected from the group comprising corona virus, cytomegalovirus, Epstein-Barr virus, influenza virus, and variola virus. In some embodiments, the subject is suffering graft-versus-host disease, group A streptococcus infection, multiple sclerosis, pancreatitis or multiple organ dysfunction syndrome. In some embodiments, the compound or the preparation thereof is applied subcutaneously. In some embodiments, the compound or the preparation thereof is applied intravenously.

According to all the clinical results shown in the examples, Allostatin starts to exert an antiviral activity at an early stage of virus infection and play a role in the suppression of cytokine storm. It’s known that, at the early stage of virus infection, immune system is activated and involves immune cells including NK cells, and then at the later stage, T cells are activated for adaptive immunity. Besides, cytokine storm can result from a systemic inflammatory reaction led by infectious diseases, and "turning off" (block, deactivate) NK cells at the initial stage of the infectious process causes systemic inflammatory response. The result of intensive research on infectious diseases of viral etiology was the formation of a clear understanding of the critical role of NK cells, which, as it turned out, are the link between innate and adaptive human immunity (A. Moretta et al., 2008) . Thus, we focus on NK cells and T cells, trying to find a reason for the anti-viral and anti-cytokine storm function of Allostatin.

Tests of Allostatin in Example 4 indicate the functions of Allostatin in the activation of cytotoxic NK cells, the initiation of adaptive immune response, and the inhibition of proimflammatory NK cells. Thus, provided in this invention is also a method to activate cytotoxic NK cells, to promote adaptive immune response, and/or to inhibit proimflammatory NK cells by administering to a subject an effective amount of a compound or the preparation thereof, wherein the compound comprises the amino acide asequence of His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly (SEQ ID NO: 1) . In some embodiments, the compound is a peptide. In some embodiments, the compound is a part of a chemical that is not is a natural protein or peptide. In some embodiments, the cytotoxic NK cells are CD16 ⁺CD56 ^dim NK Cells. In some embodiments, to activate cytotoxic NK cells is to increase the activation markers on NK cells. In some embodiments, to activate cytotoxic NK cells is to derease the inhibition receptors on the cytotoxic NK cells. In some embodiments, to activate cytotoxic NK cells is to increase CD69, CD25, NKp80 and/or CD244 expressing on the NK cells. In some embodiments, to activate cytotoxic NK cells is to decreas KIR2DL4 expressing on the NK cells. In some embodiments, to promote adaptive immune response is to promote the proliferation and/or development of T cells. In some embodiments, to promote adaptive immune response is to promote the differentiation and clonal expansion of T cells. In some embodiments, to promote adaptive immune response is to improve the survival of cytotoxic T cells. In some embodiments, to promote adaptive immune response is to maintain and/or increase memory T cells. In some embodiments, to promote adaptive immune response is to increase the activation markers on T cells. In some embodiments, to promote adaptive immune response is to increase CD25, CD127 and/or CD27 expressing on the T cells. In some embodiments, to inhibit proimflammatory NK cells is to reduce the CD56 ^bright NK cells which express KIR2DL4.

EXAMPLES

Example 1. The clinical efficacy of Allomedin in the treatment of Herpes simplex virus (HSV) infections

Allomedin containing Allostatin (a peptide with the sequence: His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly (SEQ ID No. 1) ) and execipients (carbopol gel (base) , Allantoin, phenoxyethanol, ethylhexylglycerin, sodium hydroxide) commonly used in dermatological and cosmetic formulationswas applied on face and mucosa of patients with skin herpes and genitalia mucous herpes daily. The patients were clinically evaluated in specialized clinics, hospitals and medical centers as follows:

1. Russian medical academy of postgraduate education, St. Petersburg (dermatology and cosmetology department, stomatology department)

2. St. Petersburg city center for prevention of infectious diseases

3. Pasteur research institute, St. Petersburg

4. Center for preventive Medicine, St. Petersburg

5. Center of clinical immunology, St. Petersburg

6. Republican clinical hospital for infectious diseases

7. First St. Petersburg medical university (department of otorhinolaryngology)

8. District antenatal clinic №1, St. Petersburg

9. District antenatal clinic №2, St. Petersburg

10. Medical university, Petrozavodsk

Allostatin treatment for skin herpes and genitalia mucous herpes

Clinical data characterizing Allomedin therapeutic efficacy in patients infected with Herpes simplex virus included 104 patients (81 females, 23 males) experiencing relapsing herpes of different location more than 4 years before the study. The recorded medical history of these patients showed little improvement of the symptoms when treated with conventional medications. Age of the patients was varied from 18 to 60 years.

Allomedin was applied onto the surface of herpetic lesions 2 to 3 times a day during 3 to 5 days. In each application, the affected area was gelled by Allomedin. Patients were determined to have positive responses when anyone of the following happened:

the symptoms relieved, e.g. the area of the herpetic lesion narrowed, the number of herpetic lesions decreased, the duration of symptoms decreased, and/or reduced itching, pain, swelling, and/or oedema, etc.;

the frequency of relapses decreased;

the symptoms in following relapses were milder; or

the relapse stopped;

Results were independently collected from different medical facilities listed above and summarized in Table 1.

Table 1. Allomedin clinical efficacy in the therapy of herpes relapses

Data of Table 1 demonstrate that absolute majority of patients with skin herpes and genitalia mucous tunic herpes (labial and genital herpes, correspondingly) can be effectively treated by Allomedin.

Comparison of the efficacies of Allomedin and Acyclovir on herpes

Clinical studies of Allomedin efficacy in labial and genital herpes demonstrated also extraordinary fast relief of herpes symptoms elimination compared to the treatment with Acyclovir, a standard antiviral medication, according to the labeled instructions.

Symptom of patients were recorded from the treatment of Allomedin or Acyclovir and till the end of a relapse. Patients were interviewed in each visit and their subjective feelings were recorded in their medical history. Data of patients subjective feelings were summarized in Tables 2 and 3 which illustrated the fact that Allomedin rapidly eliminates inflammation symptoms like itch, burning, oedema so that duration of the symptoms decreased about 10 times compared to the standard acyclovir treatment. Besides, it was obvious that the efficacy difference between the Acyclovir and Allomedin groups was dominated at the beginning and the development stage of the symptoms (when the virus is active) rather than the end stage. This demonstrated the antiviral effect of Allostatin.

Table 2. Efficacy comparison of Allomedin and acyclovir in the reduction of cold sores symptoms (labial herpes) (data from the Center of clinical immunology, St. Petersburg) .

Table 3. Efficacy comparison of Allomedin and acyclovir in the elimination of genital herpes symptoms (data from the Pasteur research institute, St. Petersburg) .

Example 2. The clinical efficacy of Allostatin in the treatment of Corvid-19

A 35 years old man got COVID-19 like symptoms as follows: hard to breath and weakness, but no fever. Computed Tomography (CT) of the chest organs was typical for COVID-19 associated bilateral pneumonia with the defeat of at least 50%of the pulmonary parenchyma. CT2 (moderate) . Besides, all other family members of the man were infected and were tested positive for COVID19.

After the conclusion of CT, the man was treated with 1mg Allostatin subcutaneously for once, and no other treatments were administered.

As a result, in 1-2 hours after the injection, the symptoms relieved, breathing returned to normal and the man felt better.

More casese of patients with COVID-19 are shown in Example 3, demonstrating the excellent efficacy of Allostatin in the treatment of COVID-19.

Example 3. Cytokine storm prevention and suppression by Allostatin

In vitro tests

Human keratinocytes in medium (RPMI 1640 with 10%FBS) were exposed to UVB (110 J/m ²) , then add with 2 mg/ml of Allostatin. Medium supernatants were measured by ELISA 24h later for IL- 1 α/β, IL-6 and IL-18. Results are representative of three independent experiments. Treatment with allostatin led to a statistically significant decrease in the production of pro-inflammatory cytokines (table 4) .

Table 4. In vitro Test of Allostatin in suppressing excessive cytokine production

Production of IL-6 by human keratinocytes after ultraviolet treatment positively correlates with degree of p38MAPK phosphorylation. That’s to say p38MAPK inhibitor can be a good suppressor of IL-6 production. According to table 5, Allostatin plays a similar role as the p38MAPK inhibitor in the suppressiong of excessive IL-6.

Table 5. Comparison of Allostatin and p38MAPK inhibitor in suppressing excessive IL-6 production

*indicates pyridinyl imidazole

**indicates P < 0.001

Clinical trials

To determine the effect of Allostatin in treatment of hyperthermia, a sign of cytokine storm, a clinical study was conducted within 15 patients.

All patients experienced hyperthermia within the range between 37.5 and 39.3℃. Average temperature measured across the group was 38.5 ℃.

With exception of one patient suspected of COVID19 who received Allostatin 2 hours after he experienced temperature higher than normal, Allostatin was administered at least 6 hours after the start of hyperthermia. In cases of Pneumonia patients, Allostatin was administered 3 days later.

The record of patients symptems, treatment and therapeutic effect of each patients are shown as below:

CASE 1

Condition: Hyperthermia

GENDER: male

AGE: 55

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? 2005

2. What was the highest temperature you had? 38.

3. How long did the fever last? first day

4. Did you experience any of these following symptoms?

● excessive sweating. -no

● exhaustion. -no

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -yes

● headache or mild light-headedness. -yes

● nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

7. When did you receive Allostatin injection? - 6 hours after the onset of hyperthermia

8. Did fever reduce 1 hour after the first Allostatin injection? - fever stopped

9. How many Allostatin injections did you receive and in what time period? - 1 (one)

10. Besides Allostatin, did you receive any other medication for the treatment? - no

11. How long did it take for the symptoms of the viral infection to disappear? - after overnight

12. Were you tested for the existence of the virus after the treatment? If yes, when was this done? - no

13. How happy are you with the use of Allostatin in your treatment? - completely healthy

14. Will you recommend Allostatin in treatment of hyperthermia to others? - definitely yes

15. Do you suffer from any serious diseases, or conditions, including chronic diseases and conditions? If yes, please specify. - diabetes

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 2

Condition: Hyperthermia

GENDER: male

AGE: 12

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? June of 2020

2. What was the highest temperature you had? 39.4

3. How long did the fever last? One day

4. Did you experience any of these following symptoms?

● excessive sweating. –yes

● exhaustion. -yes

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -yes

● headache or mild light-headedness. -yes

● nausea. -yes

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

7. When did you receive Allostatin injection? - 8 hours after the onset of hyperthermia

8. Did fever reduce 1 hour after the first Allostatin injection? - no fever.

11. How long did it take for the symptoms of the viral infection to disappear? - overnight

13. How happy are you with the use of Allostatin in your treatment? - (full recovery)

15. Do you suffer from any serious diseases, or conditions, including chronic diseases and conditions? If yes, please specify. - no

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 3

Condition: Hyperthermia

GENDER: female

AGE: 9

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? June of 2020

2. What was the highest temperature you had? 39.2

3. How long did the fever last? A day

4. Did you experience any of these following symptoms?

● excessive sweating. -no

● exhaustion. -yes

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -yes

● headache or mild light-headedness. -yes

● nausea. -yes

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

7. When did you receive Allostatin injection? - 9 hours after the onset of hyperthermia

8. Did fever reduce 1 hour after the first Allostatin injection? - the fever stopped

13. How happy are you with the use of Allostatin in your treatment? - completely healthy (full recovery)

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 4

Condition: Hyperthermia, pneumonia

GENDER: female

AGE: 48

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? July of 2019

2. What was the highest temperature you had? 38.7

3. How long did the fever last? few day

4. Did you experience any of these following symptoms?

● excessive sweating. -yes

● exhaustion. -no

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -yes

● headache or mild light-headedness. -yes

● nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -yes, pneumonia

6. Did you take a laboratory test? - yes

7. When did you receive Allostatin injection? - 3 days after the onset of hyperthermia

8. Did fever reduce 1 hour after the first Allostatin injection? yes

9. How many Allostatin injections did you receive and in what time period? - 3 (three times every other day)

10. Besides Allostatin, did you receive any other medication for the treatment? - yes; antibiotics

11. How long did it take for the symptoms of the viral infection to disappear? - after second injection relief came

13. How happy are you with the use of Allostatin in your treatment? - full recovery

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 5

Condition: Hyperthermia

GENDER: male

AGE: 49

NAME OF VIRAL INFECTION: flu-like syndrome, systemic inflammatory reaction after severe sunburn

1. When did high fever start? 2010, 2013, august of 2019

2. What was the highest temperature you had? > 39

3. How long did the fever last? three day

4. Did you experience any of these following symptoms?

● excessive sweating. -yes

● exhaustion. -yes

● flushed or red skin. -yes

● muscle cramps, spasm, and pain. -yes

● headache or mild light-headedness. -yes

● nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

7. When did you receive Allostatin injection? - 2 days after the onset of hyperthermia

8. Did fever reduce 1 hour after the first Allostatin injection? Yes,

9. How many Allostatin injections did you receive and in what time period? - 2 (two)

11. How long did it take for the symptoms of the viral infection to disappear? - after second injection

13. How happy are you with the use of Allostatin in your treatment? - symptom relief; decrease in body temperature

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 6

Condition: Hyperthermia

GENDER: male

AGE: 34

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? 2018

2. What was the highest temperature you had? 38.8

3. How long did the fever last? first day

4. Did you experience any of these following symptoms?

● excessive sweating. -no

● exhaustion. -yes

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -yes

● headache or mild light-headedness. -yes

● nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

7. When did you receive Allostatin injection? - 12 hours after the onset of hyperthermia

8. Did fever reduce 1 hour after the first Allostatin injection? no fever

13. How happy are you with the use of Allostatin in your treatment? - very happy

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 7

Condition: Hyperthermia

GENDER: male

AGE: 51

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? 2008

2. What was the highest temperature you had? 39

3. How long did the fever last? first day

4. Did you experience any of these following symptoms?

● excessive sweating. -no

● exhaustion. -yes

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -no

● headache or mild light-headedness. -yes

● nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

8. Did fever reduce 1 hour after the first Allostatin injection? Yes.

13. How happy are you with the use of Allostatin in your treatment? - happy

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 8

Condition: Hyperthermia

GENDER: male

AGE: 48

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? March 2020

2. What was the highest temperature you had? 38.7

3. How long did the fever last? first day

4. Did you experience any of these following symptoms?

● excessive sweating. -no

● exhaustion. -no

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -no

● headache or mild light-headedness. -yes

● nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

7. When did you receive Allostatin injection? - first day of hyperthermia

8. Did fever reduce 1 hour after the first Allostatin injection? no fever

9. How many Allostatin injections did you receive and in what time period? - 3

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 9

Condition: Hyperthermia

GENDER: male

AGE: 32

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? 2015

2. What was the highest temperature you had? High, 39

3. How long did the fever last? first day

4. Did you experience any of these following symptoms?

● excessive sweating. -no

● exhaustion. -yes

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -no

● headache or mild light-headedness. -yes

● nausea. -yes

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

8. Did fever reduce 1 hour after the first Allostatin injection? yes

13. How happy are you with the use of Allostatin in your treatment? - happy

14. Will you recommend Allostatin in treatment of hyperthermia to others? - yes

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 10

GENDER: female

AGE: 34

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? In the evening (May, 2020)

2. What was the highest temperature you had? 37.6

3. How long did the fever last? first day

4. Did you experience any of these following symptoms?

● excessive sweating. -no

● exhaustion. -no

● flushed or red skin. -no

● muscle cramps, spasm, and pain. -no

● headache or mild light-headedness. -yes

● nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

7. When did you receive Allostatin injection? - 2 hours after the onset of hyperthermia

8. Did fever reduce 1 hour after the first Allostatin injection? yes

11. How long did it take for the symptoms of the viral infection to disappear? - after injection and night, I felt absolutely good

13. How happy are you with the use of Allostatin in your treatment? yes

15. Do you suffer from any serious diseases, or conditions, including chronic diseases and conditions? If yes, please specify. - nothing

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 11

GENDER: female

AGE: 38

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? In the evening (February of 2020)

2. What was the highest temperature you had? 38++

3. How long did the fever last? first day

4. Did you experience any of these following symptoms?

a) excessive sweating. -no

b) exhaustion. -no

c) flushed or red skin. -no

d) muscle cramps, spasm, and pain. -yes

e) headache or mild light-headedness. -no

f) nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

7. When did you receive Allostatin injection? - 4 hours after the onset of hyperthermia

8. Please describe what happened 1 hours after you took the first Allostatin injection? - after 1 hour nothing happened because I went to sleep immediately

11. How long did it take for the symptoms of the viral infection to disappear? - in the morning I felt good, 36.6 and without any flu-like syndrome

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 12

GENDER: female

AGE: 64

NAME OF VIRAL INFECTION: flu-like syndrome

1. When did high fever start? Middle of the day (May of 2020)

2. What was the highest temperature you had? 38

3. How long did the fever last? first day

4. Did you experience any of these following symptoms?

g) excessive sweating. -yes

h) exhaustion. -no

i) flushed or red skin. -no

j) muscle cramps, spasm, and pain. -yes

k) headache or mild light-headedness. -yes

l) nausea. -no

5. Did you seek a medical help and if yes, what diagnosis were given? -no

6. Did you take a laboratory test? - no

8. Please describe what happened 1 hours after you took the first Allostatin injection? - after 1 hour I felt better and in the evening, I had 36.5

11. How long did it take for the symptoms of the viral infection to disappear? - next morning after injection I felt better, had 36.6, but continuer to felt flu-like syndrome. After 2 days after injection I felt absolutely healthy

13. How happy are you with the use of Allostatin in your treatment? - achieved full recovery

15. Do you suffer from any serious diseases, or conditions, including chronic diseases and conditions? If yes, please specify. - cancer in remission

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 13

Condition: pneumonia

GENDER: male

AGE: 35

NAME OF VIRAL INFECTION: COVID19-like syndrome

1. When did high fever start? HAVE NO FEVER

2. What was the highest temperature you had? 36.8

3. How long did the fever last? 0

4. Did you experience any of these following symptoms?

m) excessive sweating. -yes

n) exhaustion. -no

o) flushed or red skin. -no

p) muscle cramps, spasm, and pain. -NO

q) headache or mild light-headedness. -NO

r) nausea. –no

MY SYMPTOMS WAS - hard to breath, weakness, no temperature

5. Did you seek a medical help and if yes, what diagnosis were given? -yes,

Conclusion based on the results of Computed Tomography of the chest organs: CT picture is typical for COVID-19 associated bilateral pneumonia with the defeat of at least 50%of the pulmonary parenchyma. CT2 (moderate) .

6. Did you take a laboratory test? - NO

7. When did you receive Allostatin injection? - once, after receipt of the Conclusion

8. Please describe what happened 1 hours after you took the first Allostatin injection? - I just felt better

9. How many Allostatin injections did you receive and in what time period? - 1 (ONE TIME)

11. How long did it take for the symptoms of the viral infection to disappear? - Relief came after first injection in 1-2 hours. This evening I felt better after injections. I started breathe normally.

12. Were you tested for the existence of the virus after the treatment? If yes, when was this done? - yes. After one day while I made injections specialists come to my home and gave the COVID test. But results were negative.

14. Will you recommend Allostatin in treatment of viral infections hyperthermia to others? -definitely yes

16. Did you have any surgery in the past 3 years? If yes, please specify - no

CASE 14:

NAME OF THE PATIENT OR INITIALS: ENK

GENDER: female

AGE: 48

NAME OF VIRAL INFECTION: COVID19 LIKE

1. When was the first time you started experiencing symptoms of the viral infection? 13.05.2020

2. Did you experience the following symptoms?

a) Most common symptoms: Fever, dry cough, tiredness? –Tiredness

b) Less common symptoms: Aches and pains, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, a rash on skin, or discoloration of fingers or toes

c) Serious symptoms:

Difficulty breathing or shortness of breath, chest pain or pressure, loss of speech or movement/No

3. Did you seek a medical help? /No.

4. Did you take a laboratory test for the viral infection? If yes, what is the name of the viral infection? No.

5. When did you receive Allostatin injection? -On the 1st day, before injection temperature was about 37.5℃.

6. Please describe what happened 1 hours after you took the first Allostatin injection? Temperature dropped to 36.5℃.

7. How many Allostatin injections did you receive and in what time period? /

2 injection

8. Besides Allostatin, did you receive any other medication for the treatment? No.

9. How long did it take for the symptoms of the viral infection to disappear? In 2 days

10. Were you tested for the existence of the virus after the treatment? If yes, when was this done? /No. There were contacts with the coronavirus in the family

11. How happy are you with the use of Allostatin in your treatment? /I was pleased and happy.

12. Will you recommend Allostatin in treatment of viral infections to others? Absolutely yes.

13. Do you suffer from any serious diseases, or conditions, including chronic diseases and conditions? If yes, please specify. /No

14. Did you have any surgery in the past 3 years? If yes, please specify/No

CASE 15:

NAME OF THE PATIENT OR INITIALS: OVV

GENDER: male

AGE: 17

NAME OF VIRAL INFECTION: COVID19 LIKE

1. When was the first time you started experiencing symptoms of the viral infection? - 02.02.2020

2. Did you experience the following symptoms?

a) Most common symptoms: Fever, dry cough, tiredness? –Yes. All this symptoms.

c) Serious symptoms:

3. Did you seek a medical help? /Yes.

4. Did you take a laboratory test for the viral infection? If yes, what is the name of the viral infection? /No.

5. When did you receive Allostatin injection? -On the fifth day, but before injection temperature was about 38.5℃.

6. Please describe what happened 1 hours after you took the first Allostatin injection? Temperature dropped to 37℃ and headache decreased.

7. How many Allostatin injections did you receive and in what time period? /

Only 1 injection

8. Besides Allostatin, did you receive any other medication for the treatment? /No.

9. How long did it take for the symptoms of the viral infection to disappear? Half a day (after I slept a night)

10. Were you tested for the existence of the virus after the treatment? If yes, when was this done? /No.

11. How happy are you with the use of Allostatin in your treatment? -I was pleased and excited.

13. Do you suffer from any serious diseases, or conditions, including chronic diseases and conditions? If yes, please specify. /No.

14. Did you have any surgery in the past 3 years? If yes, please specify /No.

The two tables below is the conclusion of the information of patients above.

Table 6 Reduction of temperature after administration of Allostatin regardless of the condition:

Table 7 presents the recovery time of the patients treated with Allostatin based on the conditions:

The data above show the effect of Allostatin in relieving systemic symptoms and inhibiting the cytokine storm.

Example 4. in vivo effects of Allostatin on lymphocytes

Material and methods

The Allostatin peptide was administered subcutaneously to clinically healthy volunteers at a dose of 3 mg once. Venous blood was collected immediately before the administration of allostatin (point "0 h" ) and 36 hours (point "36 h" ) after injection.

The mononuclear blood fraction was isolated using BD CPT (Cat. № 362753) and stained with a mixture of monoclonal antibodies. The analysis was performed on a BD FACS Aria III cytometer.

Results

In healthy volunteers, 36 hours after injection, there was a statistically significant (p <0.001) increase of NK cells (Fig. 1A-1D) and T cells (Fig. 2) expressing activation markers and decrease of NK cells expressing inhibitory receptors (Fig. 1E) . In addition, as shown in Fig. 3, activated cytotoxic NK cells are significantly increased. While NK cells with imflammation mediating-receptors decreased (Fig. 4) . These results indicates that in vivo application of Allostatin on one hand enhances adaptive immunity and activates cytotoxic NK cells, and on the other hand helps in controlling imflammation.

Claims

A method for treating a viral infection, comprising administering to a subject an effective amount of a peptide comprising an amino acid sequence as shown by SEQ ID NO: 1.
The method of claim 1, wherin the virus is associated with respiratory infection.
The method of claim 2, wherin the virus is corona virus or influenza virus.
The method of claim 3, wherin the corona virus is COVID19 virus.
The method of any one of claims 1-4, wherein the subject infected with virus has fever.
The method of any one of claims 1-5, wherein the subject infected with virus has cytokine storm.
The method of any one of claims 1-6, wherein the peptide improves or alleviates one of more symptoms selected from the group consisting of fever, fatigue, dyspnea, and hypoxemia (low oxygen saturation of blood) .
The method of claim 1, wherin the virus is herpes simplex virus or genitalia mucous herpes.
A method to prevent or suppress cytokine storm, comprising administering to a subject an effective amount of a peptide comprising an amino acid sequence as shown by SEQ ID NO: 1.
The method of claim 9, wherein the peptide improves or alleviates the symptoms caused by cytokine strom.
The method of claim 10, wherein the symptoms caused by cytokine strom is selected from the group consisting of fever, fatigue, dyspnea, and hypoxemia (low oxygen saturation of blood) .
A method of any one of claims 1-11, wherein the peptide activates cytotoxic NK cells and/or inhibits proimflammatory NK cells.
The method of claim 12, wherein the cytotoxic NK cells are CD16 ⁺CD56 ^dim NK Cells.
The method of any one of claims 12-13, wheren the peptide inhibits proimflammatory NK cells expressing KIR2DL4.
The method of any one of claims 12-14, wherein the peptide promotes the survival, proliferation or development of T cells.
A method for treating fever, comprising administering to a subject an effective amount of a peptide comprising an amino acid sequence as shown by SEQ ID NO: 1.
The method of claim 16, where the peptide alleviates or reduces fever of the subject.