CN111202738A

CN111202738A - Application of (5R) -5-hydroxyl triptolide in preparation of sepsis treatment medicine

Info

Publication number: CN111202738A
Application number: CN202010083829.8A
Authority: CN
Inventors: 宋振举; 杨依霖; 童朝阳; 孙湛; 邝中淑
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2020-02-10
Filing date: 2020-02-10
Publication date: 2020-05-29

Abstract

The invention discloses application of (5R) -5-hydroxyl triptolide in preparing a medicament for treating sepsis, reducing sepsis mortality and/or treating sepsis related diseases, wherein the medicament can relieve multiple organ organic injuries and/or abnormal inflammatory reactions caused by sepsis, effectively reduces the mortality of sepsis, and has a relatively high clinical application prospect.

Description

Application of (5R) -5-hydroxyl triptolide in preparation of sepsis treatment medicine

Technical Field

The invention belongs to the technical field of medicines, and relates to application of (5R) -5-hydroxy triptolide in preparation of a sepsis treatment medicine, in particular to application of (5R) -5-hydroxy triptolide in preparation of a medicine for treating sepsis, reducing sepsis mortality and/or treating sepsis-related diseases.

Background

Sepsis (sepsis) is a Systemic Inflammatory Response Syndrome (SIRS) caused by infection, and its clinical manifestations mainly include severe pulmonary infection, abdominal infection, urinary system infection, bloodstream infection, skin and soft tissue infection, etc. Severe sepsis, accompanied by organ dysfunction, insufficient tissue perfusion or hypotension, causes Multiple Organ Dysfunction Syndrome (MODS), even septic shock, which is one of the leading causes of death in clinically critical patients. According to reports, nearly 3150 thousands of sepsis patients exist in the world every year, the number of death patients reaches 530 thousands, the number of sepsis patients is estimated to be more than 500 thousands every year in China, the severe sepsis prevalence rate of surgical ICU and comprehensive ICU reaches 30-70%, and serious medical burden is caused to governments of various countries.

Currently, specific treatment measures are still lacking clinically, and treatment is carried out through a 'bundling' treatment scheme such as liquid resuscitation, vasoactive drugs, anti-infective drugs and glucocorticoids, but the death rate of sepsis remains high.

In addition, uncontrolled release of inflammatory mediators and inflammatory response disorders in the body during the development of sepsis are one of its important mechanisms. However, although immune-modulating drugs can suppress the degree of inflammation of patients to some extent by modulating immunity, anti-inflammatory action, etc., the therapeutic effect on organ failure is not so great, and even many drugs have been proven to cause MODS, and attempts to anti-endotoxin antibodies, antagonize a single inflammatory mediator, anti-TLR, etc. have failed. Therefore, the search for new targets and new therapeutic measures for preventing and treating sepsis is urgent.

Disclosure of Invention

In order to explore means and ways for treating and preventing sepsis, the inventor tries to treat multiple organ injuries caused by sepsis by using (5R) -5-hydroxyl triptolide, and unexpectedly finds that the compound has obvious treatment effect. Therefore, the invention aims to provide the application of (5R) -5-hydroxyl triptolide in preparing medicines for treating sepsis, reducing sepsis mortality and treating sepsis-related diseases.

In the medicine, the (5R) -5-hydroxy triptolide is one of the effective components or the only effective component.

Such sepsis-associated conditions include, but are not limited to, sepsis-induced multiple organ dysfunction syndrome, septic shock, systemic inflammatory response syndrome.

The above multiple organ dysfunction syndromes include, but are not limited to: liver injury, lung injury, kidney injury, spleen injury, intestinal injury, nervous system injury, circulatory system injury, or blood system injury.

In another embodiment, the damage to the blood system comprises a coagulation disorder.

In another embodiment, the liver injury comprises elevated liver enzymes, elevated bilirubin, or reduced albumin.

In another embodiment, the lung injury comprises acute lung injury, acute respiratory distress syndrome.

In another embodiment, the kidney injury comprises decreased urine volume and serum creatinine above normal standards.

In another embodiment, the spleen injury comprises a spleen imaging abnormality such as necrosis.

In another embodiment, the intestinal injury comprises bowel paralysis, intestinal flatulence, vomiting, and/or diarrhea.

In another embodiment, the nervous system injury comprises an acute disturbance of consciousness.

In another embodiment, the circulatory system injury comprises shock, elevated markers of myocardial injury (creatine kinase, troponin, etc.), myocardial contraction and/or relaxation dysfunction.

In another embodiment, the coagulation disorder comprises platelet decline, abnormal coagulation function index, and/or bleeding.

In another embodiment, the above medicament is useful for alleviating organic injury to multiple organs and/or abnormal inflammatory response due to sepsis.

In one embodiment, the medicament comprises a safe and effective amount of (5R) -5-hydroxytriptolide.

According to animal experiments, when administered to sepsis mice, (5R) -5-hydroxytriptolide is administered at a dose of 0.125-0.5mg/kg, preferably at a dose of about 0.25 mg/kg.

In another embodiment, the drug is administered in a dose of 0.25mg to 1 g/day, based on the total weight of the triptan-sulam tablet.

In another embodiment, the medicament is in a unit dosage form.

In another embodiment, the unit dosage form contains the active ingredient (5R) -5-hydroxytriptolide.

In another embodiment, the unit dosage form comprises a daily dosage of the unit dosage form.

In another embodiment, the medicament can be a pharmaceutical composition comprising (5R) -5-hydroxytriptolide and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier includes adjuvants, excipients, preservatives, absorption delaying agents, fillers, binders, adsorbents, pH buffering agents, disintegrants, solubilizers, flavoring agents, or combinations thereof.

In one embodiment, the above pharmaceutical composition may comprise at least one adjuvant therapeutic agent in addition to, or in combination with, the pharmaceutically active ingredient (5R) -5-hydroxytriptolide alcohol, for use in combination with (5R) -5-hydroxytriptolide alcohol.

In another embodiment, the combined administration comprises co-administration, or sequential administration.

In another embodiment, the sequential administration comprises sequential administration with a time interval.

The adjuvant therapeutic agent comprises one or more selected from antiinfective agent, vasoactive drug, anticoagulant, immunomodulator, and blood product. In another embodiment, the anti-infective drug may be selected from one or a combination of two or more of antibacterial, antifungal and antiviral drugs.

In another embodiment, the vasoactive agent can be selected from one or a combination of two or more of dopamine, norepinephrine, vasopressin, and other clinically commonly used vasoactive agents.

In another embodiment, the immunomodulator comprises a glucocorticoid, a protease inhibitor, an interferon, a thymosin peptide, and the like.

In another embodiment, the blood product comprises plasma, albumin, immunoglobulins, coagulation factors, apheresis platelets, whole blood, and the like.

The (5R) -5-hydroxyl triptolide has low toxicity, can be prepared into various preparations, and can be administered through multiple channels, thereby providing a new treatment means and approach for sepsis-related diseases.

In another embodiment, oral, injectable, intravenous, suppository, inhalant, or topical agents may be included.

In another embodiment, the oral dosage form comprises a tablet, capsule, syrup, orally disintegrating dosage form, or film.

In another embodiment, the oral dosage form comprises a sustained release, or controlled release, agent.

In another embodiment, the injectable agent comprises an intravenous agent, an intraperitoneal agent, an intraluminal agent, an irrigation agent, or a flushing agent.

In another embodiment, the inhalation comprises an aerosol, a powder, or a nasal spray.

In another embodiment, the topical agent comprises a patch, a paste, or a gel.

In another embodiment, the pharmaceutical dosage form is a tablet.

In another embodiment, the invention also provides a method of treating sepsis, reducing sepsis mortality, and/or treating a sepsis-associated condition, comprising the steps of:

administering a safe and effective amount of a medicament to a subject in need thereof, wherein the medicament comprises a safe and effective amount of (5R) -5-hydroxytriptolide.

In another preferred embodiment, the invention also provides a medicament for treating sepsis, reducing sepsis mortality, and/or treating sepsis-related conditions, wherein the medicament contains a safe and effective amount of (5R) -5-hydroxytriptolide.

In another embodiment, the subject in need thereof is a subject diagnosed with sepsis or presenting with one or more clinical manifestations of sepsis.

In another embodiment, the subject in need thereof is a mammal, e.g., a human, a mouse, a rabbit, a cat, a dog, a monkey, etc.

Drawings

FIG. 1 is a survival curve plotted using the survivor package, survivval package and ggplot2 package in the R language after different doses of (5R) -5-hydroxytriptolide alcohol were administered to a mouse model of sepsis. Wherein a is normal mice (blank control, n-10); group B was an unsupplemented sepsis mouse model group (n ═ 10); group C is a sepsis mouse model group administered with low dose (5R) -5-hydroxytriptolide (0.125mg/kg) (n ═ 10); group D is a sepsis mouse model group (n-10) to which medium dose (5R) -5-hydroxytriptolide (0.25mg/kg) was administered; group E is a sepsis mouse model group (n ═ 10) dosed with high dose (5R) -5-hydroxy triptolide (0.5 mg/kg).

FIG. 2 is a photomicrograph of a pathological examination of a mouse lung tissue section by HE staining. Wherein a is normal mice (blank control); b is an unsupplemented sepsis mouse model; and C is sepsis mouse (n is 10) dosed with medium dose (5R) -5-hydroxy triptolide (0.25 mg/kg).

FIG. 3 is a photomicrograph of a pathological examination of mouse kidney tissue sections by HE staining. Wherein a is normal mice (blank control); b is an unsupplemented sepsis mouse model; and C is sepsis mouse (n is 10) dosed with medium dose (5R) -5-hydroxy triptolide (0.25 mg/kg).

FIG. 4 is a photomicrograph of a pathological examination of mouse spleen tissue sections by HE staining. Wherein a is normal mice (blank control); b is an unsupplemented sepsis mouse model; and C is sepsis mouse (n is 10) dosed with medium dose (5R) -5-hydroxy triptolide (0.25 mg/kg).

FIG. 5 is a photomicrograph of a pathological examination of mouse intestinal tissue sections by HE staining. Wherein a is normal mice (blank control); b is an unsupplemented sepsis mouse model; and C is sepsis mouse (n is 10) dosed with medium dose (5R) -5-hydroxy triptolide (0.25 mg/kg).

FIG. 6 is a bar graph showing the result of conventional lymphocyte detection in peripheral blood of mice. Wherein a is normal mice (blank control); b is an unsupplemented sepsis mouse model; and C is sepsis mouse (n is 10) dosed with medium dose (5R) -5-hydroxy triptolide (0.25 mg/kg). *: p < 0.05; **: p < 0.01.

FIG. 7 is a bar graph showing the results of conventional neutrophil detection in peripheral blood of mice. Wherein a is normal mice (blank control); b is an unsupplemented sepsis mouse model; and C is sepsis mouse (n is 10) dosed with medium dose (5R) -5-hydroxy triptolide (0.25 mg/kg). *: p < 0.05; **: p < 0.01.

FIG. 8 is a bar graph showing the results of an Elisa assay of the inflammatory factor TNF- α in peripheral blood of mice, where A is normal mice (blank control), B is an unsupplemented sepsis mouse model, and C is a sepsis mouse dosed with a medium dose of (5R) -5-hydroxytriptolactone (0.25mg/kg) (n ═ 10): P < 0.05;: P < 0.01.

Detailed Description

Through extensive and intensive research, the inventor unexpectedly discovers that (5R) -5-hydroxy triptolide has excellent treatment effect on organ injury caused by sepsis. Experiments prove that when sepsis occurs, the (5R) -5-hydroxy triptolide can relieve organ injuries of lung, spleen, kidney, intestinal tissues and the like of a mouse, relieve inflammatory reaction, obviously reduce the death rate of the mouse caused by sepsis, does not generate obvious toxic or side effect, and has a higher clinical application prospect.

Term(s) for

Sepsis

Sepsis, as described herein, refers to a dysfunctional disease of the systemic organs caused by infection. The specific mechanism of sepsis is unknown, but during the development of sepsis it often leads to irreversible multiple organ failure due to the persistent presence of infectious agents and their toxins and subsequent uncontrolled release of inflammatory mediators in the body. However, neither anti-infective drugs nor known immunomodulators are effective in improving organ function, and even directly cause or aggravate MODS, for example, Activated Protein C (APC), anti-endotoxin antibodies, inflammatory factor antagonists, anti-TLR, etc. have been shown to aggravate mortality in septic patients.

The term "infection" refers to local tissue and systemic inflammatory response caused by invasion of pathogens such as bacteria, viruses, fungi, parasites, etc. into the human body. Often, the infectious condition is accompanied by the presence of infectious toxins and causes further tissue organ damage.

Multiple Organ Dysfunction Syndrome (MODS)

It refers to a clinical syndrome in which two or more organs or systems of the body are dysfunctional simultaneously or sequentially during acute diseases such as severe trauma, shock, infection and major surgical operations, so that homeostasis cannot be maintained.

In the clinical manifestations of sepsis, in addition to a well-defined focus of infection, there may be accompanying dysfunction of one or more systemic systems or organs. The dysfunction of the whole body system or a plurality of organs comprises a plurality of dysfunctions of the liver, the respiratory system, the kidney, the spleen, the gastrointestinal tract, the nervous system, the circulatory system, the blood system and the like. Specifically, typical clinical manifestations may include one or more of the following manifestations, such as the presence of fever or hypothermia, hyperglycemia, increased or non-increased inflammation indicators, difficult to correct hypotension, low oxygen hydration status, acute oliguria, increased blood creatinine, elevated markers of myocardial injury, blood coagulation dysfunction, and the like.

Systemic Inflammatory Response Syndrome (SIRS)

The endotoxin is a group of clinical symptoms which are finally expressed by the uncontrolled inflammatory response of an organism due to the systemic nonspecific inflammatory response generated by the serious injury of infectious or non-infectious factors such as infection, trauma, burn, operation, ischemia-reperfusion and the like, and the endotoxin is often a trigger of the systemic inflammatory response.

Septic shock (septic shock):

septic shock refers to shock due to sepsis and is defined as a condition of inadequate tissue perfusion in the patient, i.e., a persistent hypotensive state or blood lactate concentration of 4mmol/L or more after a volume test.

(5R) -5-Hydroxyperigerol ester alcohol

As used herein, "a compound of the present invention", "an active ingredient", "a tripterygium wilfordii derivative of the present invention", "triptolide", "T-8" and "LLDT-8" are used interchangeably and refer to (5R) -5-hydroxytriptolide alcohol having the following chemical structure:

in addition, the compounds of the present invention also include derivatives or equivalents of the compounds of the present invention that have been modified, altered, or substituted and have the same or substantially the same activity as the compounds of the present invention.

The invention proves that the compound has good sepsis treatment effect and effectively reduces the death rate of sepsis, and the compound CC of the invention₅₀256.6 +/-73.8 nM, very low toxicity and excellent clinical application foreground.

Medicine and administration dosage

The term "medicament" as used herein refers to a substance which contains the active ingredient of the present invention as the main active ingredient and has a therapeutic, ameliorating, palliative effect on sepsis and related conditions, or a reducing effect on sepsis mortality. Wherein the medicament contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by weight of the active ingredient of the present invention.

In another embodiment, the medicament further comprises a pharmaceutically acceptable carrier. In another embodiment, the medicament further comprises or may be administered in combination with one or more adjunctive therapeutic agents. Common adjunctive therapeutic agents include one or more of anti-infective agents, vasoactive drugs, anti-coagulants, immunomodulators, and blood products. The dosage administered may be an "effective amount" or a "safe and effective amount," and the terms "effective amount" and "safe and effective amount" are used interchangeably to refer to an amount that, upon administration, produces the desired therapeutic, palliative, ameliorating, palliative effect in the body of the subject (e.g., systemically, in the blood, or in a target organ) without unacceptable adverse effects in the subject. In the present invention, a safe and effective amount of (5R) -5-hydroxytriptolide alcohol can be obtained by conventional means, and can take the form of a single dose or multiple doses. For example, according to mouse experiments, the safe and effective amount may be 0.125-0.5mg/kg, 0.15-0.4mg/kg, 0.17-0.35mg/kg, 0.18-0.32mg/kg, or 0.2-0.3 mg/kg. Preferably, the dosage is about 0.25-0.5 mg/kg.

(5R) -5-hydroxytriptolide is typically administered to a septic patient in an amount that provides a therapeutically effective daily dose of the drug. It is known that an effective administration dose for a drug can be expressed as an effective administration dose per day or a daily dose.

As used herein, the term "daily dose" refers to the amount of (5R) -5-hydroxytriptolide given per day, regardless of the frequency of administration. For example, if a patient receives a unit dose of 10mg twice daily, the daily dose is 20 mg. It is to be understood that the use of the term "daily dose" does not imply that the specific dose amount must be administered once per day. However, in one particular embodiment, the frequency of administration may be once daily (q.d.), and the daily dose and unit dose are synonymous in this embodiment.

Specifically, the administration dose of the drug of the present invention is 0.01mg to 100mg per day, for example, 0.05 to 20mg per day, preferably 0.1mg to 10mg per day, and 0.2mg to 5mg per day, in terms of the active ingredient, and can be administered. The dosage of the drug of the invention is calculated by the commercially available commercial product of Ratengsh, and the dosage of the drug can be 0.25mg to 4 mg/day, such as 0.5mg to 2 mg/day and 1mg to 1.5 mg/day. Under the guidance of a skilled physician, the measurement can be adjusted according to the conditions of the disease condition, the characteristics, effectiveness, adverse reaction and the like of a patient.

Mode of administration

When the therapeutic medicine for treating the sepsis and the sepsis-related diseases, which takes (5R) -5-hydroxyl triptolide as an active ingredient, is applied to a patient, the therapeutic medicine can be orally taken or injected, and can also be applied by other routes. For example, the active ingredient or drug of the present invention can be used for the treatment of systemic or local organ dysfunction by intravenous administration, inhalation administration, intraperitoneal administration, intracranial administration, intraocular administration, subarachnoid administration, rectal administration.

The administration mode of the active ingredient or the medicament of the invention depends on the medicament dosage form, the constitution, the weight, the age, the focus part, the progress of the disease condition, the administration part, the administration amount and other treatment factors of the administration individual, and the effective administration dosage of the active ingredient (5R) -5-hydroxytriptolide alcohol in the medicament can be flexibly adjusted.

The active ingredient of the invention or the medicament of the invention can be administered at any stage of sepsis or at any stage of sepsis, for example when one or more organ dysfunctions occur, or one or more of the above-mentioned clinical symptoms occur, or in patients of mild, moderate, severe or critical severity. The time of administration may be determined by a clinician according to the improvement in clinical symptoms, e.g., 1-10 days, 20 days, 30 days, etc.

Furthermore, when the drug of the present invention contains a second active ingredient other than the active ingredient of the present invention as an adjunctive therapeutic agent for administration, it is understood that there is no drug interaction between the two active ingredients that would exacerbate the condition; when the agent of the present invention can be administered in combination with an adjunctive therapeutic agent, attention is also paid to the drug interaction between the agent of the present invention and the adjunctive therapeutic agent. Drug interactions can be determined by one skilled in the art using conventional pharmacodynamic, pharmacokinetic assays. Depending on the result of the drug interaction, a second active ingredient may be determined, or the active ingredients of the invention and adjunctive therapeutic agents may be selected for co-administration or sequential administration, e.g. administration of a drug of the invention before/after administration of the adjunctive therapeutic agent, at intervals of time, e.g. 15, 20, 30, 60 minutes or more, such as 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours later.

Dosage forms

The pharmaceutical agents of the present invention may be formulated into conventional dosage forms well known to those skilled in the art, and the method of preparation chosen will depend on the intended route of administration. For example, as a drug for oral administration, it may be in the form of tablets, capsules, microcapsules, pills, pellets, powders, sustained release formulations, solutions, suspensions; as a sterile solution, suspension or emulsion for parenteral injection; as a patch, ointment or cream for topical (e.g. dermal) administration or as a suppository for rectal administration; the dosage form of the invention also comprises intraocular dosage forms, such as gels and ointments; dosage forms of the invention also include liquid formulations (including pre-filled needles), powders and the like suitable for intravenous, intraperitoneal, local irrigation or lavage, or intracranial administration. In other embodiments, the medicament is in unit dosage form suitable for single administration of an accurate dose.

When the sepsis therapeutic agent of the present invention is a solid pharmaceutical composition, the pharmaceutical composition may include a pharmaceutically acceptable carrier. Among the acceptable carriers are adjuvants, excipients, preservatives, absorption delaying agents, fillers, binders, adsorbents, pH buffers, disintegrants, solubilizers, flavoring agents, other carriers, other inert ingredients, or combinations thereof. The particular type and amount of such pharmaceutically acceptable carriers can be selected by the skilled artisan based on common general knowledge in the art, or determined by simple experimentation. These pharmaceutically acceptable carriers should not affect the pharmacological activity of (5R) -5-hydroxytriptolide or cause adverse drug reactions.

When the medicament is an injection, the medicament can be prepared into solution for injection or freeze-dried powder injection. When the freeze-dried powder injection is prepared, a freeze-drying protective agent can be added, and the freeze-drying protective agent can be selected from maltose, trehalose, sucrose, mannitol, lactose, glucose, sorbitol, xylitol, erythritol, threonine or a mixture of more than two of the above.

The present invention may also provide controlled/sustained release pharmaceutical formulations containing a compound of the present invention as an active ingredient, wherein the rhythm and rate of release of the active ingredient is controlled and adjusted to allow less frequent administration or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

The medicaments of the invention are typically in a single dosage unit, i.e. a unit dose, whereas in other embodiments there may be a plurality of unit doses, e.g. the number of dosage units required for a particular regimen or clinical situation.

The present invention will be further described with reference to the following embodiments and the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the examples of the present invention, if no specific description is made about the experimental operating temperature, the temperature is usually room temperature (10 to 30 ℃).

The amount, content and concentration of various substances are referred to herein, wherein the percentages are by weight unless otherwise indicated.

As used herein in describing numerical features, the terms "about" or "approximately" mean that the number may be represented with a margin of error or variance of 10%, 9%, 8%, 7%, 6%, or 5%.

Unless otherwise defined, the recitation of ranges by numbers in this disclosure includes such numbers, as well as any number within the range.

The following examples were conducted under conditions not specified, and were selected in accordance with conventional methods and conditions, or in accordance with the specifications of commercial products. The invention mainly uses a histopathological analysis detection method to detect multiple organ injury indexes of the sepsis mouse.

Example 1 preparation of mouse sepsis model

A method for preparing a mouse model with sepsis by single intraperitoneal injection of Lipopolysaccharide (LPS) comprises the following specific steps:

1. grouping mice: 8-12 weeks old female C57 mice (weight of about 22-25g, provided by animal experiment center of Zhongshan Hospital of Fudan university) were randomly grouped according to their weights after adaptive feeding in SPF-grade animal room for one week, ensuring no statistical difference in the average weight of each group of 10 mice.

2. Preparing a sepsis mouse model by injecting lipopolysaccharide into the abdominal cavity: after dissolving lipopolysaccharide (purchased from Sigma) powder in Phosphate Buffered Saline (PBS) (purchased from Sigma), the powder was intraperitoneally injected into mice at a ratio of 6 mg/kg (mg/kg) of body weight to obtain a sepsis mouse model, and a control group was intraperitoneally injected with an equal volume of PBS as a control.

EXAMPLE 2 mortality study following administration of (5R) -5-Hydroxytriptolide to septic mice

Adopting a single administration mode of oral gavage to carry out administration on (5R) -5-hydroxy triptolide (after compound screening, the sepsis treatment effect of the (5R) -5-hydroxy triptolide is optimal and the toxicity to cells is minimum, the CC50 is 256.6 +/-73.8 nM, other derivatives of the tripterygium wilfordii are less than 10nM, the CC50 of the triptolide is 2.1 +/-0.2 nM, so the (5R) -5-hydroxy triptolide is selected as an active ingredient), and the specific steps are as follows:

1. grouping mice: 8-12 week-old female C57 mice (weight is about 22-25 g), after adaptive feeding in SPF level animal room for one week, are randomly divided into 5 groups according to the weight of the mice, namely a normal mouse (blank control, A) sepsis mouse model group (B), a sepsis mouse plus (5R) -5-hydroxy triptolide low dose (0.125mg/kg) group (C), a sepsis mouse plus (5R) -5-hydroxy triptolide medium dose (0.25mg/kg) group (D), a sepsis mouse plus (5R) -5-hydroxy triptolide high dose (0.5mg/kg) group (E), and the number of each group is 10.

2. Preparing (5R) -5-hydroxyl triptolide alcohol solution: dissolving (5R) -5-hydroxy triptolide powder in PBS containing 0.2% dimethyl sulfoxide (DMSO) to obtain 1mg/ml stock solution, and storing at-20 deg.C. Before use, stock solutions were diluted 10-fold with PBS to 0.1mg/ml, and diluted with appropriate amounts of PBS (0.1 ml per 10 g of body weight per mouse at doses of 0.125mg/kg, 0.25mg/kg, 0.5mg/kg), for example, 1.25 ml of a stock solution of (5R) -5-hydroxytriptolide would be diluted in 1.25 ml of PBS at a dose of 0.5mg/kg of (5R) -5-hydroxytriptolide for 10 mice with a body weight of 25 g.

3. Oral gavage administration of (5R) -5-hydroxytriptolide alcohol: for a sepsis mouse and (5R) -5-hydroxy triptolide low dose (0.125mg/kg) group, a sepsis mouse and (5R) -5-hydroxy triptolide medium dose (0.25mg/kg) group, a sepsis mouse and (5R) -5-hydroxy triptolide high dose (0.5mg/kg) group, the corresponding dose of (5R) -5-hydroxy triptolide is given in a single time by an oral gavage way, and a blank control group (normal mouse) and a sepsis mouse model group are given with PBS with equal volume.

4. Intraperitoneal injection of Lipopolysaccharide (LPS): after 30min, LPS was intraperitoneally injected at a ratio of 6 mg/kg (mg/kg) of body weight to sepsis mice plus (5R) -5-hydroxytriptolide (0.125mg/kg), sepsis mice plus medium (0.25mg/kg), sepsis mice plus (5R) -5-hydroxytriptolide (0.5mg/kg) and sepsis mouse model groups, respectively, according to the procedure of example 1, and a blank control group was intraperitoneally injected with an equal volume of PBS as a control.

5. The survival status of the mice was observed continuously over the following one week period and a survival curve was plotted, and the results are shown in fig. 1. The experimental result shows that the survival rate of the sepsis mouse is obviously improved after the (5R) -5-hydroxyl triptolide with each dose is given, and the (5R) -5-hydroxyl triptolide effectively improves the prognosis of the sepsis mouse. Among them, the protective effect of 0.25mg/kg is particularly excellent.

EXAMPLE 3 treatment of multiple organ injury in septic mice with (5R) -5-Hydroxytriptolide

Adopting a drug delivery mode of oral gavage to deliver the (5R) -5-hydroxyl triptolide (0.25mg/kg) dose to a sepsis mouse, dissecting a normal mouse and the sepsis mouse, taking specific tissues and organs of the normal mouse and the sepsis mouse, and carrying out HE staining pathological detection, which comprises the following specific steps:

1. grouping mice: 8-12 week-old female C57 mice (weight is about 22-25 g) are subjected to adaptive feeding in an SPF-level animal room for one week, and then are randomly divided into 3 groups according to the weight of the mice, wherein the 3 groups are respectively a blank control (normal mice) group, a sepsis mouse model group, a sepsis mouse and (5R) -5-hydroxytriptolide middle dose (0.25mg/kg) group, and the number of each group is 10.

2. Preparing (5R) -5-hydroxyl triptolide alcohol solution: dissolving (5R) -5-hydroxy triptolide powder in PBS containing 0.2% dimethyl sulfoxide (DMSO) to obtain 1mg/ml stock solution, and storing at-20 deg.C. Stock solutions were diluted 10-fold with PBS to 0.1mg/ml stock solution prior to use, and diluted with an appropriate amount of PBS at a dosage of 0.25mg/kg (0.1 ml per 10 g body weight per mouse), e.g., 0.625 ml of (5R) -5-hydroxytriptolide stock solution should be diluted in 1.875 ml PBS for 10 mice weighing 25 g.

3. Oral gavage administration of (5R) -5-hydroxytriptolide alcohol: for the sepsis mouse added (5R) -5-hydroxy triptolide middle dose (0.25mg/kg), the corresponding dose of (5R) -5-hydroxy triptolide is given in a single time in an oral gavage mode, and the blank control group and the sepsis mouse model group are given with PBS with equal volume.

4. Intraperitoneal injection of Lipopolysaccharide (LPS): after 30min, the sepsis mice were treated with LPS at a dose of 6 mg/kg (mg/kg) of body weight and the sepsis mice model group with the medium dose (0.25mg/kg) of (5R) -5-hydroxytriptolide, respectively, according to the procedure of example 1, and the blank control group was treated with PBS of the same volume as the control group.

5. Continuously observing the survival state of the mouse, after 24 hours, euthanizing the mouse, collecting right lung leaves, right kidney, spleen, liver middle leaves and intestinal tract jejunum segments, respectively adding 4 percent paraformaldehyde tissue fixing liquid with the volume being 10 times that of the right lung leaves, the right kidney, the spleen, the liver middle leaves and the intestinal tract jejunum segments, standing and fixing for 24 hours at room temperature, dehydrating by using gradient alcohol, embedding paraffin into tissue blocks, cutting paraffin sections with the thickness of 0.5mm by using a paraffin slicer, dewaxing, hydrating, dyeing, washing off excessive variegated colors, sealing and the like, and finally observing the pathological change of the intestinal tract tissue under a microscope, wherein the result is shown in a figure 2-5.

FIG. 2 shows the results of HE staining pathological detection of lung tissue, the normal morphology of lung tissue of sepsis mouse is obviously destroyed, the alveoli are ruptured, inflammatory reactions such as alveolar wall thickening, cell nucleus aggregation and the like and pathological changes such as pulmonary congestion and edema and the like are obviously generated, and the (5R) -5-hydroxy triptolide effectively relieves lung tissue damage caused by sepsis.

FIG. 3 shows the results of HE staining pathological detection of kidney tissue, a great amount of punctate and granular inflammatory cell infiltration appears in kidney tissue of sepsis mice, vacuole degeneration of renal tubular epithelial cells is obviously increased, and (5R) -5-hydroxy triptolide effectively relieves kidney tissue damage caused by sepsis.

FIG. 4 shows the results of pathological detection of HE staining of spleen tissues, and compared with the blank control group of mice, the spleen tissues of sepsis mice have increased white marrow quantity and more hair growth centers, and the (5R) -5-hydroxy triptolide effectively reduces spleen tissue damage caused by sepsis.

FIG. 5 shows the results of HE staining pathology of intestinal tissue; the villi in the intestinal tissues of sepsis mice are seriously damaged, and the (5R) -5-hydroxyl triptolide is administered to effectively relieve the intestinal tissue damage caused by sepsis.

The experimental results show that (5R) -5-hydroxyl triptolide can improve the pathological conditions of lung, kidney, spleen and intestinal tract tissues of a sepsis mouse, and is used for treating and/or preventing and protecting the damage of multiple organs of the sepsis mouse.

Example 4 improvement of abnormal inflammatory response in septic mice Using (5R) -5-Hydroxytriptolide

The method comprises the following steps of (5R) -5-hydroxy triptolide (0.25mg/kg) administration to a sepsis mouse by adopting an oral gavage administration mode, taking peripheral blood to detect the blood conventional blood cell level (including lymphocyte, neutrophil and the like), and measuring the level of inflammatory factor cell necrosis factor (TNF- α) in the peripheral blood of the mouse by using an Elisa method, wherein the specific steps are as follows:

5. The survival status of the mice was continuously observed: after 24 hours, the eye picking method collects peripheral blood of the mouse, immediately takes 10 microliter of whole blood, and detects blood routine by using a full-automatic blood cell analyzer. The analyzer used in the invention is a V-53 full-automatic blood cell analyzer for animals, and reagents using matched reagent packages of the analyzer are purchased from Shenzhen Merrill biomedical electronics GmbH. The results are shown in FIGS. 6-7.

6. Peripheral blood was collected using a heparin anticoagulant tube containing a small amount of liquid, the whole blood obtained was mixed well with the anticoagulant, and the upper plasma sample was obtained by centrifugal force at 3000rpm for 10 minutes, and the TNF- α Elisa kit (cat No. 1217202, Dayou) was used to measure the level of TNF- α in plasma according to the instructions, the results are shown in FIG. 8.

FIG. 6 shows the results of measurement of lymphocyte levels in peripheral blood routine. Compared with a blank control group, the proportion of the lymphocytes of the sepsis mice is obviously reduced; and the administration of (5R) -5-hydroxy triptolide effectively increased the level of lymphocytes, indicating that the inflammatory response caused by sepsis was alleviated.

FIG. 7 shows the results of measurement of the neutrophil level in the peripheral blood routine. Compared with a blank control group, the proportion of the neutral particles in the sepsis mouse is obviously increased; while administration of (5R) -5-hydroxytriptolide effectively reduced neutrophil levels, indicating that the inflammatory response caused by sepsis was alleviated.

FIG. 8 shows the result of detecting inflammatory factor TNF- α in plasma, compared with the blank control group, the expression level of TNF- α in sepsis mice is obviously increased, and the administration of (5R) -5-hydroxyl triptolide effectively reduces the level of TNF- α, which indicates that the inflammatory reaction caused by sepsis is relieved.

The experimental results show that (5R) -5-hydroxyl triptolide can relieve abnormal inflammatory reaction caused by sepsis. Furthermore, in the above experiments, no serious drug-related adverse reactions were observed.

Claims

Use of (5R) -5-hydroxytriptolide in the manufacture of a medicament for the treatment of sepsis, for reducing sepsis mortality, and/or for the treatment of sepsis-related conditions.
2. Use according to claim 1, wherein the sepsis associated state comprises sepsis-induced multiple organ dysfunction syndrome, septic shock, systemic inflammatory response syndrome.
3. The use of claim 2, wherein said multiple organ dysfunction syndrome comprises one or more conditions selected from the group consisting of: liver injury, lung injury, kidney injury, spleen injury, intestinal injury, nervous system injury, circulatory system injury, and blood system injury.
4. Use according to claim 1, wherein the medicament is for the reduction of sepsis-induced organic injury of multiple organs and/or abnormal inflammatory responses.
5. The use of claim 1, wherein said medicament comprises a safe and effective amount of (5R) -5-hydroxytriptolide.
6. The use of claim 1, wherein the medicament is a pharmaceutical composition comprising (5R) -5-hydroxytriptolide and a pharmaceutically acceptable carrier.
7. The use of claim 6, wherein the pharmaceutically acceptable carrier comprises adjuvants, excipients, preservatives, absorption delaying agents, fillers, binders, adsorbents, pH buffers, disintegrants, solubilizers, flavorants, or combinations thereof.
8. The use according to claim 1, wherein the medicament is a pharmaceutical composition comprising, in addition to the pharmaceutically active ingredient (5R) -5-hydroxytriptolide, at least one adjunctive therapeutic agent; or (5R) -5-hydroxytriptolide in combination with at least one adjunctive therapeutic agent.
9. The use of claim 8, wherein the adjunctive therapeutic agent comprises one or more selected from anti-infective agents, vasoactive drugs, anti-coagulants, immunomodulators, and blood products.
10. The use of claim 1, wherein the medicament is in a dosage form comprising oral, injectable, intravenous, suppository, inhalant, or topical.