KR20210103848A - Pharmaceutical composition for alleviating or treating sepsis comprising hydroxyurea - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for alleviating or treating sepsis comprising hydroxyurea as an active component. Hydroxyurea can effectively inhibit the increase of neutrophils caused by viruses or bacteria that have penetrated into the individual from the outside, thereby protecting the individual from side effects that can be caused by bacterial LPS or viruses. Therefore, when the pharmaceutical composition of the present invention is administered to an individual suffering from sepsis, a survival rate of the individual can be significantly increased. Therefore, the pharmaceutical composition according to the present invention can be usefully used for alleviation or treatment of sepsis.

Description

Pharmaceutical composition for alleviating or treating sepsis comprising hydroxyurea {PHARMACEUTICAL COMPOSITION FOR ALLEVIATING OR TREATING SEPSIS COMPRISING HYDROXYUREA}

The present invention relates to a pharmaceutical composition for prophylactic inhibition of sepsis progression, alleviation of sepsis or treatment comprising hydroxyurea as an active ingredient.

Sepsis refers to a disease in which a serious inflammatory reaction occurs throughout the body due to infection with microorganisms. Fever of 38℃ or higher, hypothermia or lower of 36℃, respiratory rate of 24 or more per minute (tachypnea), heart rate of 90 or more per minute (tachycardia), increase or decrease in the number of white blood cells in the blood It is defined as sepsis when it is caused by Sepsis is a disease with a mortality rate of up to 30%, and 30 million cases occur worldwide every year.

The definitions of sepsis and septic shock are constantly changing. As the pathophysiological understanding of sepsis deepens (decreased function of normal organs, biochemistry, immunology, circulatory system, etc.), the definition of sepsis was renewed in 2016 (2016, JAMA). It is a major organ dysfunction that threatens to cause an organ failure, and the most representative organ failure is evaluated with a Sepsis-related Organ Failure Assessment (SOFA) score of 2 or more.

For the treatment of sepsis, an appropriate antibiotic is prescribed after finding an infected part of the body that causes sepsis through physical examination, blood test, and imaging test. In addition, in the treatment of sepsis, drug treatment for maintaining various homeostasis in the body, including antibiotics, and supportive therapy for overcoming dysfunction of each organ are concurrently used. However, in order to find out the causative bacteria of sepsis, it takes at least 3 to 5 days to collect the patient's blood and culture the bacteria, and it is difficult to treat it because it can die within a short time after the onset of sepsis. In addition, a fundamental treatment for sepsis has not yet been developed.

Mervyn Singer et al., Clinical Review & Education 2016 JAMA

Although research has been conducted to treat sepsis, the developed drug has insufficient therapeutic effect or side effects, so there is a need to develop a safe and effective sepsis treatment agent. Accordingly, an object of the present invention is to provide a pharmaceutical composition for prophylactic inhibition of sepsis progression, alleviation of sepsis, and treatment of sepsis.

In order to solve the above problems, one aspect of the present invention provides a pharmaceutical composition for alleviating or treating sepsis comprising hydroxyurea as an active ingredient.

Another aspect of the present invention provides a method of alleviating or treating sepsis comprising administering the pharmaceutical composition to a subject.

Hydroxyurea, an active ingredient of the pharmaceutical composition of the present invention, is produced in an emergency without changes in the granulocytes of the peripheral blood during the so-called “emergency granulopoiesis” process by severe pathogens. By regulating the production of granulocytes, it can suppress the progression of sepsis. That is, hydroxyurea can alleviate or treat sepsis by inhibiting the increase in neutrophils caused by viruses and/or bacteria. In addition, it is possible to protect the subject by alleviating or treating sepsis that has already been generated. Therefore, when the pharmaceutical composition of the present invention is administered to a subject suffering from sepsis, the survival rate of the subject can be significantly increased. Therefore, the pharmaceutical composition according to the present invention can be usefully used for prophylactic inhibition of sepsis progression, alleviation or treatment of sepsis.

1 shows an experimental schedule for confirming the effect of hydroxyurea (HU) in a bacterial induced sepsis model.
Figure 2 confirms the survival rate according to the HU treatment in mice induced sepsis using lipopolysaccharide (LPS). At this time, HU indicates a group administered once a day HU, HU'' indicates a group administered HU twice a day.
Figure 3 confirms the survival rate according to the HU treatment in mice induced sepsis using LPS. In this case, HU'' represents a group administered with HU twice a day.
Figure 4 confirms the effect of increasing the survival rate of mice according to the HU treatment when the Western Reserve vaccinia virus (Vaccinia virus Western Reserve strain, WR virus) infecting mice.
Figure 5 shows the number of virus particles in the blood of the mouse according to the HU treatment when the Western Reserve vaccinia virus infects the mouse.
Figure 6 confirms the effect of increasing the survival rate of the mouse according to the HU treatment when the Western Reserve vaccinia virus infects the mouse.
7 is an observation of morphological changes in lung and liver tissues after administration of wild-type WR vaccinia virus in a normal mouse model.
Figure 8 shows the experimental schedule for confirming the effect of HU in the influenza virus (Influenza virus) induced sepsis model.
Figure 9 confirms the effect of increasing the survival rate of the mouse according to the HU treatment when the influenza virus infects the mouse.
10 is an observation of the change in body weight of the mouse according to the HU treatment when the influenza virus infects the mouse.
11 shows the experimental results to determine whether HU has an antiviral effect. At this time, CC ₅₀ represents a concentration at which about 50% of the cytotoxic reaction occurs, and EC ₅₀ represents a concentration at which about 50% of the effect of the drug appears.
Figure 12 confirms the effect of reducing the occurrence of skin rash and pustules after administration of Vaccinia virus Wyeth and HU in a monkey model.
13 shows the results of observing changes in body temperature and body weight of monkeys after administration of Wyeth and HU.
14 shows the measurement of the number of virus particles in the blood on the 8th day of administration of Wyeth alone and Wyeth and HU in combination.
15 shows the effect of reducing skin rash and pustules after administration of Vaccinia virus Wyeth and HU in a monkey model at 2 days, 4 days, 6 days, and 8 days.
16 shows the changes in absolute neutrophil count (ANC), whole blood cell (WBC), and absolute lymphocyte count (ALC) in the monkey model.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a pharmaceutical composition for alleviating or treating sepsis comprising hydroxyurea as an active ingredient.

The term "hydroxyurea" used in the present invention is a compound having the following formula.

[Formula 1]

Although the exact mechanism of the hydroxyurea is not known, it is known as an anticancer agent that inhibits DNA synthesis. In addition, the hydroxyurea may be included in the pharmaceutical composition in the form of a commercialized drug containing hydroxyurea. The commercialized drug containing the hydroxyurea component may be Hydroxyurea ^® , Hydrea ^® , Droxia ^TM , Mylocel ^TM , Siklos ^® hydrin capsules, but is not limited thereto.

In addition, the hydroxyurea may be administered at a dose of 0.1 mg/kg/day to 90 mg/kg/day. Specifically, the dosage of the hydroxyurea is 0.1 mg/kg/day to 90 mg/kg/day, 1 mg/kg/day to 80 mg/kg/day, 5 mg/kg/day to 70 mg/kg /day, 10 mg/kg/day to 60 mg/kg/day, or 20 mg/kg/day to 50 mg/kg/day may be administered. In this case, the hydroxyurea may be administered orally or in the form of an injection. In addition, hydroxyurea may be administered once a day to multiple doses. The number of administration may be administered twice a day, three times a day, four times a day, or five times a day. In one embodiment, it was administered twice a day.

As used herein, the term “septicemia” refers to a disease in which a severe inflammatory reaction occurs throughout the body. In particular, sepsis can cause serious organ damage in an individual due to an abnormality of the individual's immune system due to microbial infection. Specifically, according to a study on the pathophysiology of sepsis, in sepsis, after infection by microorganisms, immune cells such as neutrophils, macrophages, and monocytes are activated. Due to the activation of these immune cells, the secretion of cytokines such as IL-1, IL-6, IL-8, TNF-α is increased, and the transcription factor NF-κB present in the cells is activated, thereby causing an inflammatory response throughout the body. happens In addition, septic shock is a condition in which the death rate is increased due to the occurrence of circulatory, cellular, and metabolic abnormalities due to worsening of sepsis symptoms. If timely and appropriate treatment is not given, it can cause shock or death.

Specifically, sepsis refers to a case where the diagnostic criteria for systemic inflammatory response syndrome are satisfied in a patient with suspected or proven infection. Systemic inflammatory response syndrome is characterized by body temperature >38°C or <36°C, heart rate >90 beats/min, respiration rate >20 beats/min or PaCO ₂ <32 mmHg, and white blood cell count >12,000 cells/mm ³ or <4,000 cells It is defined as a case where two or more of /mm ^{3 or undifferentiated form >10% are satisfied.}

In this case, the sepsis may be induced by infection with a microorganism, and the microorganism may be a bacterium, a fungus or a virus. In this case, the microorganism that can cause sepsis may be a microorganism of the genus Streptococcus species and Enterococcus species belonging to Gram-positive bacteria, but is not limited thereto. Specifically, the microorganism causing sepsis may be Staphylococcus pneumoniae or Staphylococcus aureus. In addition, it may be a genus Klebsiella species, Pseudomonas species, and Enterobacter species belonging to Gram-negative bacteria, but is not limited thereto. Specifically, the microorganism causing sepsis may be Escherichia coli, Vibrio vulnificus, or Hemophilus influenzae. In particular, the sepsis may be caused by lipopolysaccharides present in microorganisms.

In addition, viruses include influenza virus, adenovirus, herpes simplex virus, measles virus, lentivirus, retrovirus, and cytomegalovirus. Cytomegalovirus, baculovirus, Reovirus, Adeno-associated virus, Myxoma virus, Vesicular stomatitis virus, Poliovirus, It may be any one of Newcastle disease virus, Parvovirus, Coxsackie virus, Senecavirus, Vaccinia virus, or Poxvirus. In this case, the virus may be in a wild-type or mutated form.

The pharmaceutical composition comprising the hydroxyurea of the present invention as an active ingredient can be effectively used for sepsis as well as septic shock.

Unless otherwise specified herein, 　 "active ingredient" refers to a component that exhibits activity alone or together with an adjuvant (carrier) that has no activity by itself.

In addition, the pharmaceutical composition may further include an antibiotic. The antibiotics are doripenem, cefepime, imipenem, meropenem, ceftazidime, ceftaroline fosamil, ceftriaxone, It may be imipenem, vancomycin, teicoplanin, cefotaxime, metronidazole, or an aminoglycoside antibiotic, but is not limited thereto.

The pharmaceutical composition may be administered in an amount of 0.1 mg/kg to 200 mg/kg of antibiotics once based on the body weight of the subject to be administered. Specifically, the pharmaceutical composition is 0.1 mg/kg to 200 mg/kg, 1 mg/kg to 190 mg/kg, 5 mg/kg to 180 mg/kg, 10 mg/kg to 170 mg/kg 20 mg/kg Antibiotics in an amount of from to 160 mg/kg or from 40 mg/kg to 150 mg/kg may be administered. That is, it is most preferable from a pharmacological point of view that the pharmaceutical composition is administered with an antibiotic in an amount in the above range based on the body weight of an individual with sepsis.

The pharmaceutical composition may be administered to a human or other mammal suspected of having sepsis or suffering from sepsis. In addition, the pharmaceutical composition may be an injection that can be administered intravenously, intramuscularly or subcutaneously, preferably an injection preparation.

The pharmaceutical composition may be prepared as an injection that is physically and chemically very stable by adjusting the pH using a buffer solution such as an aqueous acid solution or phosphate that can be used as an injection in order to ensure product stability according to the distribution of the injection prescription. Specifically, the pharmaceutical composition may include water for injection. The water for injection means distilled water made to dissolve the solid injection or to dilute the water-soluble injection.

The pharmaceutical composition may include a stabilizer or a solubilizing agent. For example, the stabilizing agent may be sodium pyrosulfite or ethylenediaminetetraacetic acid, and the solubilizing agent may be hydrochloric acid, acetic acid, sodium hydroxide, sodium hydrogen carbonate, sodium carbonate or potassium hydroxide.

Another aspect of the present invention provides a kit for alleviating or treating sepsis comprising a first composition containing hydroxyurea as an active ingredient and a second composition containing an antibiotic as an active ingredient.

The first composition and the second composition may be administered concurrently, sequentially, or in reverse order. Specifically, the first composition and the second composition may be administered simultaneously. In addition, the first composition may be administered first, and then the second composition may be administered. Furthermore, the first composition may be administered after first administering the second composition. In addition, the second composition may be administered first, then the first composition may be administered, and then the second composition may be administered again.

The first composition and the second composition may be administered to a human or other mammal having sepsis suspected or suffering from sepsis. In addition, the first composition and the second composition may be an injection that can be administered intravenously, intramuscularly or subcutaneously, and preferably an injection preparation.

The first composition and the second composition are physically and chemically very stable injections by adjusting the pH by using a buffer solution such as an aqueous acid solution or phosphate that can be used for injection in order to ensure product stability according to the distribution of the injection prescription. can be manufactured. Specifically, the first composition and the second composition may include water for injection. The water for injection means distilled water made to dissolve the solid injection or to dilute the water-soluble injection.

The first composition and the second composition may include a stabilizer or a dissolution aid. For example, the stabilizing agent may be sodium pyrosulfite or ethylenediaminetetraacetic acid, and the solubilizing agent may be hydrochloric acid, acetic acid, sodium hydroxide, sodium hydrogen carbonate, sodium carbonate or potassium hydroxide.

Another aspect of the present invention provides a method for alleviating or treating sepsis comprising administering to a subject the above-described pharmaceutical composition. Here, the subject may be a mammal, preferably a human.

In this case, the administration may be intravenous (intra-venous), intra-muscular (intra-muscular) or intra-dermal (intra-dermal) administration. Through this, when the pharmaceutical composition according to the present invention is administered to an individual suffering from sepsis, it can be used for alleviating or treating sepsis by regulating immune cells.

In this case, the step of administering an antibiotic may be further included. In this case, the antibiotic used and the administration method are as described above.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Experimental Example 1. Effect of increasing survival rate on bacterial induced sepsis

An animal experiment was performed to see if the survival rate could be improved through an immune response by treatment with hydroxyurea in a mouse model induced by sepsis due to LPS (Sigma, Lipopolysaccharides from Escherichia coli O55: B5L2880). For each group, there were 7 mice, and LPS was administered intraperitoneally at 10 mg/kg on day 0, and hydroxyurea was administered intraperitoneally to both groups except for the control group at 30 mg/kg intraperitoneally for 5 days/week from one day before LPS treatment. 1 day or 2 times/day (FIG. 1). The control group administered with LPS alone died on the second day, whereas the group administered with hydroxyurea once/day survived until day 12, and the group administered twice/day with three animals until day 12 It survived and showed a survival rate of 43% ( FIGS. 2 and 3 ).

Experimental Example 2. Effect of increasing survival rate upon infection with Western Reserve virus

Experimental Example 2.1. Confirmation of treatment effect of viral sepsis by HU

Vaccinia virus Western Reserve species (Western Reserve, WR) is known as a virulent virus with a high rate of lethality due to good virus propagation in a syngeneic mouse model. In order to verify that HU enhances the survival rate due to lethal virus infection, vaccinia virus WR species virus (1 x 10 ⁵ pfu) was administered to the nasal mucosa and infected, followed by animal experiments. Mice were divided into control group, g-CSF (25 ug/kg) administration group, and HU (50 mg/kg) administration group. The G-CSF administration group is to induce an increase in neutrophils, and this is to compare the HU administration group with the immunomodulatory effect due to a decrease in neutrophils (FIG. 4).

As a result of the experiment, the HU administration group showed a 40% survival rate until the 21st day, and showed a significant difference from the other two groups (p=0.01). In the case of the control group and the g-CSF administration group, all mice died before 2 weeks after virus administration, and there was no difference between the groups ( FIGS. 5 and 6 ). The number of virus particles in the three groups was compared in blood to determine whether this difference in survival rate was a difference in virus-induced toxicity. As a result, the number of virus particles was the highest in the HU-administered group. Although HU administration reduced the number of neutrophils and showed the highest survival rate even though virus proliferation was promoted, it was confirmed that the survival rate due to WR virus infection can be increased due to immune regulation, not reducing the toxicity caused by the virus itself.

In this case, the WR virus used above is a recombinant vaccinia virus in which the thymidine kinase (TK) gene is deleted. For its preparation, wild-type vaccinia virus Wyeth spp. (NYC Department of Health) and Western Reserve spp. were purchased from the American Type Culture Collection (ATCC). For recombination, the TK region of wild-type vaccinia virus was substituted with a shuttle plasmid with a firefly luciferase reporter (p7.5 promoter) gene or a shuttle plasmid with a GFP gene using a vector.

Experimental Example 2.2. Identifying Changes HU Affects an Individual's Immune System

The relationship between virus proliferation and neutrophil infiltration in relation to mouse death was confirmed through histological analysis. The mouse group was divided into a group administered with WR vaccinia virus alone, a group administered with G-CSF to increase absolute neutrophil count (ANC), and a group administered with HU to suppress ANC, and 1 x 10 ⁷ pfu WR vaccinia virus was administered to each group in the nasal mucosa. After administration, the tissue was collected by sacrificing death or just before death. A normal mouse to which the virus was not administered was also sacrificed as a negative control.

As a result, the G-CSF administration group had the shortest survival period, and the HU administration group significantly increased the survival period. As a result of tissue staining (H&E), there was some transudate and exudate in the alveoli in all experimental groups except the normal group, and Zone 3 ischemic changes in the liver were observed (FIG. 7). These results suggest that the acute lethal toxicity caused by the wild-type WR vaccinia virus was more likely to be caused by a hemodynamic change such as a decrease in blood flow caused by neutrophils in the blood rather than the cytotoxicity of the virus proliferation itself. It explains well why it is important for acute immunotoxicity of viruses.

Experimental Example 3. Effect of increasing survival rate during influenza virus infection

Experimental Example 3.1. Confirmation of treatment effect of viral sepsis by HU

42 healthy mice (male, Balb/c) were divided into 6 groups (n=7/group), and H1N1 influenza A (swine flu, PR8 strain, 3xMLD ₅₀ ) virus was intranasally infected in groups except for the control group (day). 0). Three groups infected with the virus were treated with Tamiflu (oseltamivir) and hydroxyurea, known as representative antiviral agents, alone or in combination. Two concentrations of Tamiflu 0.1 mg/kg and 10 mg/kg were used, and 50 mg/kg of hydroxyurea was used (FIG. 8).

As a result, all mice in the virus-treated group died on day 11, whereas all mice in the three groups treated with Tamiflu survived regardless of the concentration. In the group treated with hydroxyurea alone (HD group), two mice died on the 9th and 10th days, respectively, but the remaining 5 mice survived ( FIGS. 9 and 10 ).

From the above results, it was confirmed that HR (95% CI) was 0.44 (0.06-0.64). That is, in the survival rate graph, the hazard ratio HR (hazard ratio) of the group treated with HU alone was statistically lower by 0.44 compared to the group treated with only virus (confidence interval 95%). can

Experimental Example 3.2. Confirmation of the mechanism of action of HU

In order to investigate the mechanism by which the effect of hydroxyurea to increase the survival rate against viral infection or how it differs from the mechanism of antiviral drugs, the antiviral effects after cell culture with each drug were compared in vitro. In addition to hydroxyurea, three influenza viruses (H1N1 PR8, H3N2 HongKong, Lee) and three different antiviral agents (ribavirin, amantadine, and oseltamivir varboxylate) were used in the analysis.

After culturing MDCK cells in 96 wells, three influenza virus strains (H1N1 PR8, H3N2 HongKong, Lee) were each infected at 0.001 pfu/cell, and then cultured at 33°C to 35°C under serum-free conditions. After one hour, after washing with PBS, MEM and TPCK-trypsin (2 ug/ml, Sigma) were added, and then, 72 hours later, the cell viability by the virus was measured by MTT assay.

As a result, the CC ₅₀ of hydroxyurea was determined to be 1,561 ug/ml, but hydroxyurea had an antiviral effect on influenza A (H1N1 PR8, H3N2 HongKong) and B (Lee) viruses up to a concentration of 1,561 ug/ml. was confirmed not to appear at all. On the other hand, Tamiflu (OSV-C) showed a specific antiviral effect on all three types of viruses, and the degree was shown in the order of ribavirin and AMT.

This result means that the effect of increasing the survival rate on influenza virus infection by hydroxyurea administration is not due to virus infection inhibition. That is, it can be seen that it is different from the mechanism of increasing the survival rate of antiviral agents such as Tamiflu. Mice treated with hydroxyurea alone also showed a decrease in body weight until the 9th day, similar to mice treated with virus alone. However, after that, all the surviving mice recovered rapidly and recovered to the same healthy state as the mice treated with Tamiflu on the 18th day. This confirmed that the lethality due to viral infection was reduced by neutrophil cell regulation of hydroxyurea (FIG. 11).

Experimental Example 4. Confirmation of systemic inflammatory response inhibition by HU in monkey model

^{Vaccinia Virus (Wyeth) (1 x 10 8} pfu) or Wyeth and HU (WR: 1 x 10 ⁸ pfu, HU - 80 mg/) used above in male cynomologus monkeys (n=3) aged 45-55 months. kg and 30 mg/kg/d) were evaluated for toxicity after systemic administration. Compared to the Wyeth alone group, the HU and WR combination administration group significantly weakened systemic inflammation, and skin redness lesions such as pustules were suppressed ( FIGS. 12 and 15 ). In addition, there was a tendency that the body temperature increased in the combined administration group compared to the virus alone administration group was suppressed (FIG. 13). There was no difference in weight loss between the two groups, and although it was not very clear, neutrophil cell proliferation was suppressed in the HU group. There was no significant difference between the groups in the number of virus particles in the blood on the 8th day after virus administration ( FIG. 14 ). In addition, as a result of confirming changes in absolute neutrophil count (ANC), whole blood cell (WBC), and absolute lymphocyte count (ALC) in the monkey model, all of these values decreased on average and were stable. was confirmed to be maintained (FIG. 16).

Claims (11)

A pharmaceutical composition for alleviating or treating sepsis comprising hydroxyurea as an active ingredient. According to claim 1,
The sepsis is caused by bacteria or viruses, sepsis relief or pharmaceutical composition for treatment. According to claim 1,
A pharmaceutical composition for relieving or treating sepsis, further comprising an antibiotic. 4. The method of claim 3,
The antibiotics are doripenem, cefepime, imipenem, meropenem, ceftazidime, ceftaroline fosamil, ceftriaxone, Imipenem (imipenem), vancomycin (vancomycin), teicoplanin (teicoplanin), cefotaxime (cefotaxime), metronidazole (metronidazole) or aminoglycoside (aminoglycoside) of the series antibiotics, sepsis relief or therapeutic pharmaceutical composition. 5. The method according to any one of claims 1 to 4,
The pharmaceutical composition is a pharmaceutical composition for alleviating or treating sepsis, characterized in that it can inhibit the increase in neutrophils caused by bacteria or viruses. 6. The method of claim 5,
The neutrophil increase is characterized in that caused by bacterial lipopolysaccharide, sepsis relief or therapeutic pharmaceutical composition. 5. The method according to any one of claims 1 to 4,
The pharmaceutical composition is a pharmaceutical composition for alleviating or treating sepsis, characterized in that it is for intravenous administration. A kit for alleviating or treating sepsis comprising a first composition comprising hydroxyurea as an active ingredient and a second composition comprising an antibiotic as an active ingredient. 9. The method of claim 8,
The antibiotic is ceftriaxone, imipenem, vancomycin, teicoplanin, cefotaxime, ceftazidime, metronidazole or aminoglycoside ( aminoglycoside) series antibiotics, sepsis relief or treatment kit. A method of alleviating or treating sepsis comprising administering hydroxyurea to a subject. 11. The method of claim 10,
The method of alleviating or treating sepsis, further comprising the step of administering an antibiotic.

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