Nitazoxanide for the treatment of sepsis

Abstract

The present invention relates to a compound selected from nitazoxanide, tizoxanide and tizoxanide glucuronide, for use in a method of treating sepsis in a subject in need thereof.

Description

[0001] This invention relates to nitazoxanide, tizoxanide, or tizoxanide glucoside for the treatment or prevention of sepsis. Background Technology

[0002] Sepsis is a dysregulated immune response to infection that leads to organ dysfunction. Its development results from a complex dysregulation of the host's response to infection, most often bacterial. This host response dysregulation is characterized not only by increased inflammation but also by immunosuppression. This inadequate response to infection leads to cellular dysfunction and ultimately organ failure. Single-organ dysfunction is rare in sepsis; it usually involves several organs. Mortality in sepsis patients is related to the number of organs affected.

[0003] Many patients with sepsis develop circulatory failure, leading to abnormal cellular oxygen metabolism. This abnormality manifests as elevated blood lactate levels, typically >2 mEq / L. Patients who require vasopressors to maintain a minimum mean arterial pressure despite adequate volume resuscitation and have elevated blood lactate levels are clinically diagnosed with septic shock.

[0004] Sepsis is a systemic inflammatory response to infection, characterized by two or more criteria defining a systemic inflammatory response syndrome. Severe sepsis is a condition complicated by organ dysfunction and septic shock (hypotension despite adequate fluid resuscitation). The end of this spectrum is multiple organ dysfunction syndrome, defined as altered organ function in acutely ill patients who are unable to maintain homeostasis without intervention.

[0005] Sepsis and the resulting multiple organ failure are the most common causes of death in many intensive care units. An estimated 750,000 cases of severe sepsis occur annually in the United States, with a high mortality rate. Sepsis is now the 12th leading cause of death in the United States. In fact, the definition of sepsis as "a systemic inflammatory response syndrome caused by infection" reflects the concept that sepsis is a result of an uncontrolled inflammatory cascade.

[0006] Increasing evidence now suggests that widespread apoptotic death leads to the depletion of immune cells and may impair a patient's ability to fight off infection.

[0007] Apoptosis represents the execution of an ATP-dependent death program, often initiated by death receptor linkages, leading to a caspase activation cascade, including activation of caspase-9 and subsequent activation of effector caspases. Once activated, caspase-9 can directly cleave and activate caspase-3 and caspase-7.

[0008] The caspase gene family consists of 15 mammalian members, classified based on the structure and function of their prodomains. The caspase family can be further divided into two functional subgroups. Inflammatory caspases (caspase-1, -4, -5, 11, -12, -13, and -14) play roles in cytokine maturation and inflammatory responses. Apoptosis-involved caspases are further divided into two functional subgroups: apoptosis-inducing caspases (caspase-2, -8, -9, -10, and -15) and effector caspases (caspase-3, 6, and 7).

[0009] Effector caspases are responsible for initiating markers of the degradation phase of apoptosis, including DNA fragmentation, cell contraction, and cell membrane bubbling.

[0010] Furthermore, serum caspase-3 levels at the time of diagnosis of severe sepsis in patients with sepsis were associated with mortality. Therefore, serum caspase-3 levels can be used as a prognostic biomarker. Increased caspase-3 activity was found in various body sites in animal models of sepsis. Additionally, caspase-3 activity in lymphocytes of sepsis patients was higher than in healthy controls, and caspase-3 activity in the spleen of sepsis patients was higher than in non-sepsis patients.

[0011] Current treatment for sepsis aims to limit the progression of organ dysfunction by providing rapid infection control, stabilizing hemodynamics, and providing as much organ support as possible to ensure organ function recovery. However, the treatment of sepsis and septic shock remains a substantial unmet medical need.

[0012] NTZ (nitrozonide, [2-[(5-nitro-1,3-thiazolyl)carbamoyl]phenyl]acetate), first described in 1975, has shown high efficacy against anaerobic protozoa, worms, and a broad spectrum of microorganisms, including both anaerobic and aerobic bacteria. NTZ is a drug approved in the United States for the treatment of diarrhea caused by the protozoan parasites Cryptosporidium parvum and Giardia intestinalis.

[0013] NTZ can also confer antiviral activity and has also shown broad anticancer properties by interfering with key metabolic signaling pathways and pro-death signaling pathways.

[0014] This surprisingly demonstrates that NTZ can be used to treat sepsis in those in need. Summary of the Invention

[0015] This invention stems from a surprising observation that NTZ improved survival in a preclinical sepsis model. The inventors have also demonstrated that the active metabolite of NTZ, tezolinide (TZ), directly protects hepatocytes from cytokine-induced cell death by inhibiting caspase activity.

[0016] Therefore, the present invention relates to NTZ, TZ, TZ glucoside (TZG) or pharmaceutically acceptable salts thereof, in a method for treating sepsis.

[0017] The present invention relates more particularly to a compound selected from NTZ, TZ, and TZG, used in a method of treating sepsis in a subject of need. In one particular embodiment, the compound is NTZ.

[0018] In one particular implementation, the sepsis is caused by a bacterial infection.

[0019] In another specific embodiment, the compound is used to protect vital organs by inhibiting cytokine-induced apoptosis that occurs during sepsis. In yet another embodiment, the compound is used to defend against cytokine-induced cell death by inhibiting caspase activity.

[0020] In one particular implementation, the subject suffers from sepsis with multiple organ failure or is at risk of sepsis with multiple organ failure. In another implementation, the subject suffers from septic shock or is at risk of septic shock.

[0021] In another embodiment, the compound is used to slow or stop the progression of sepsis.

[0022] In yet another embodiment, the compound is used as a single active agent in the method. Alternatively, in another embodiment, the compound is used in combination with an antimicrobial agent such as an antibiotic in the method. In a particular embodiment, the antimicrobial agent is a carbapenem antibiotic, such as ertapenem. Attached Figure Description

[0023] Figure 1 NTZ treatment improves survival rate after CLP surgery

[0024] Survival curves of mice treated with NTZ and untreated (medium) after CLP surgery

[0025] The survival curves between NTZ and the media group were compared using the log-rank Mantel-Cox test, and the result was P = 0.07.

[0026] Figure 2 TZ inhibits TNFα-induced caspase 3 / 7 activity in HepG2.

[0027] For multiple comparisons using ANOVA and Fisher's LSD test to compare TNFα or astrococcal sulfadiazine with untreated (A) and TZ with media (B), *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively.

[0028] ### indicates that the Student's T-test was used and p < 0.001

[0029] Figure 3 TZ pretreatment inhibits astrosarcin-induced caspase 3 / 7 activity in HepG2 cells.

[0030] A. Effect of astrocytocin on caspase 3 / 7 activity in HepG2 cells (n=24). Student's t-test was used to assess statistical significance. ***p<0.001

[0031] B. Effect of TZ pretreatment on asteroidin-induced apoptosis in HepG2 cells (n = 8 to 24). Cells were pretreated with TZ for 16 hours before the addition of asteroidin. Statistical significance was assessed using one-way ANOVA and Dunnett's test (comparison of TZ-treated cells to untreated cells). ***p < 0.001

[0032] Figure 4 Treatment with TZ and NTZ, accompanied by astrocytocin, inhibited caspase 3 / 7 activity in HepG2 cells.

[0033] The effect of A.TZ on asteroidin-induced apoptosis in HepG2 cells (n=6). Simultaneous addition of TZ and asteroidin was performed, followed by measurement of caspase 3 / 7 activity.

[0034] The effect of B. NTZ on asteroidin-induced apoptosis in HepG2 cells (n=6). NTZ and asteroidin were added simultaneously, and then caspase 3 / 7 activity was measured.

[0035] For A and B, multiple tests were performed using one-way ANOVA and Dunnett's test to assess statistical significance (comparison of TZ or NTZ-treated cells with untreated cells). **p<0.01, ***p<0.001

[0036] Figure 5 NTZ with or without pretreatment improves survival after CLP-induced sepsis.

[0037] A. Study Summary - White squares represent untreated (control mice), black triangles represent mice treated with NTZ BID.

[0038] B. Survival curves of control mice, mice receiving NTZ with 3 days of pretreatment, and mice receiving NTZ from the day of CLP surgery. Survival curves were compared between the Gehan-Breslow-Wilcoxon comparison groups. **p<0.01, ***p<0.001

[0039] Figure 6 NTZ with or without pretreatment improves 7-day survival after CLP-induced sepsis.

[0040] At the end of the study, the survival rate of mice treated with medium, NTZ pretreatment for 3 days, or NTZ treatment was [data missing].

[0041] Figure 7 NTZ improves the health score of CLP-induced septic mice.

[0042] In mice treated with media, NTZ for 3 days pretreatment, or NTZ treatment, the evolution of six independent parameters was considered to assess animal health 4 days post-CLP surgery. Severity was assessed from 0 (no signs) to 3 (more severe).

[0043] Figure 8 NTZ administration after sepsis induction effectively improves survival rate.

[0044] A. Study Summary. White squares represent untreated (control mice), and triangles represent mice treated with NTZ (1 triangle = once daily or QD, two triangles = twice daily or BID).

[0045] B. Survival curves of control mice, mice that received NTZ BID 1 hour before and 3.5 hours after CLP, and mice that received NTZ only 3.5 hours after CLP. Survival curves between the Gehan-Breslow-Wilcoxon comparison groups were used. **p<0.01, ***p<0.001

[0046] C. Survival rates of control mice, mice that received NTZ BID 1 hour before and 3.5 hours after CLP, and mice that received NTZ only 3.5 hours after CLP surgery at the end of the study.

[0047] Figure 9 NTZ administration after sepsis induction effectively improves health scores.

[0048] Animal health was assessed for evolution of six independent parameters during the 4-day period following sepsis induction in control mice, mice that received NTZ BID 1 hour before and 3.5 hours after CLP, and mice that received NTZ only 3.5 hours after CLP. Severity was assessed from 0 (no signs) to 3 (most severe). Detailed Implementation

[0049] This invention relates to NTZ or TZ(G) for the treatment or prevention of sepsis.

[0050] The term “object” or “patient” as used in this article refers to mammals, preferably humans.

[0051] As stated above, the term "sepsis" as used herein refers to a harmful systemic inflammatory response to infection, formally defined as the presence of infection along with its systemic manifestations. The term sepsis as used herein includes sepsis of any severity and its complications, such as sepsis with multiple organ failure and septic shock.

[0052] In one particular embodiment of the invention, the subject suffers from sepsis or its complications or is at risk of sepsis or its complications.

[0053] In another specific embodiment, the subject suffers from sepsis caused by one or more microbial species. In particular, the subject may suffer from sepsis caused by bacterial, fungal, or viral infection. In yet another embodiment, the sepsis is caused by a bacterial infection.

[0054] In one particular implementation, the treatment or prevention method consists of the application of NTZ, TZ, or TZG as a single active ingredient.

[0055] The term "treatment" as used herein encompasses both therapeutic and preventative measures, where the goal is to prevent or mitigate (alleviate) undesirable physiological changes or disturbances. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, stabilization of the pathological state (specifically, non-deterioration), slowing or halting disease progression, and improving or alleviating pathology. In particular, for the purposes of this invention, treatment aims to slow the progression of sepsis and reduce the risk of further complications. It may also involve prolonging survival compared to the expected survival without treatment. In one particular embodiment, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof is used to reduce sepsis-related mortality. NTZ, TZ(G), or a pharmaceutically acceptable salt thereof may also be used to slow or halt the progression of sepsis. In particular, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof is used to prevent the progression of sepsis in subjects with sepsis, especially to prevent the progression of sepsis to septic shock. In another embodiment, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof is used to prevent organ failure, especially multiple organ failure, in subjects with sepsis.

[0056] In the context of this invention, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof are administered to the subject in a therapeutically effective amount. In one particular embodiment, NTZ or TZ, or a pharmaceutically acceptable salt thereof, is administered. In a further embodiment, NTZ, or a pharmaceutically acceptable salt thereof, particularly NTZ, is administered to the subject.

[0057] "Therapeutic effective dose" refers to the amount of medicine that effectively achieves the desired therapeutic effect. Therapeutic effective doses of a medicine can vary depending on factors such as an individual's disease state, age, sex, weight, and the medicine's ability to elicit the expected response in the individual. Therapeutic effective dose is also the amount by which the beneficial therapeutic effect of the medicine outweighs any toxic or harmful effects. The effective dose and dosage regimen of a medicine depend on the disease or condition to be treated and can be determined by someone skilled in the art. A physician with ordinary skill in the art can readily determine and prescribe the effective dose of the desired pharmaceutical composition. For example, a physician can start with a dose of the medicine used in the pharmaceutical composition below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, the appropriate dose of the composition of the present invention is the amount of the minimum effective dose of compound that produces a therapeutic effect according to a specific dosage regimen. Such an effective dose will generally depend on the factors described above.

[0058] NTZ, TZ(G), or pharmaceutically acceptable salts thereof can be formulated into pharmaceutical compositions, the pharmaceutical compositions further comprising one or more pharmaceutically acceptable excipients or media (e.g., saline solution, physiological solution, isotonic solution, etc.), said excipients or media being compatible with the intended use of the drug and being known to those skilled in the art.

[0059] These compositions may further comprise one or more agents or media selected from dispersants, solubilizers, stabilizers, preservatives, etc. Agents or media that can be used in these formulations (liquid and / or injectable and / or solid) include, in particular, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, gum arabic, liposomes, etc.

[0060] These compositions can be formulated into injectable suspensions, syrups, gels, oils, ointments, pills, tablets, suppositories, powders, gel capsules, capsules, aerosols, etc., ultimately using a galen form or device to ensure prolonged and / or slow release. For such formulations, agents such as cellulose, carbonates, or starch can be advantageously used.

[0061] NTZ or TZ(G) can be in the form of pharmaceutically acceptable salts, particularly acid or base salts compatible with pharmaceutical use. Salts of NTZ and TZ(G) include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium salts, and alkylated ammonium salts. These salts can be obtained during the final purification step of the compound, or by incorporating the salt into a previously purified compound.

[0062] NTZ, TZ(G), or pharmaceutically acceptable salts thereof can be administered in various forms via different routes. For example, the compounds can be administered systemically, orally, parenterally, by inhalation, by nasal spray, by nasal instillation, or by injection (e.g., intravenous injection), by intramuscular route, by subcutaneous route, by transdermal route, by local route, by intra-arterial route, etc. Of course, the route of administration will be adapted to the form of the drug according to procedures known to those skilled in the art.

[0063] In one particular embodiment, the compound is formulated as a tablet. In another particular embodiment, the compound is administered orally.

[0064] The frequency and / or dosage of administration can be adjusted by those skilled in the art based on the patient's function, pathology, and form of administration. Typically, NTZ or TZ(G) can be administered at doses between 0.01 mg / day and 4000 mg / day, for example, from 50 mg / day to 2000 mg / day, or from 100 mg / day to 2000 mg / day; particularly from 100 mg / day to 1000 mg / day. In a particular embodiment, the NTZ, TZ(G), or a pharmaceutically acceptable salt thereof is administered at a dose of about 1000 mg / day, particularly 1000 mg / day. In a particular embodiment, the NTZ, TZ(G), or a pharmaceutically acceptable salt thereof is administered orally at a dose of about 1000 mg / day, particularly 1000 mg / day, particularly as tablets. If necessary, it can be administered once daily or even several times daily. In one embodiment, the compound is administered at least once daily, such as once daily, twice daily, or three times daily. In a particular embodiment, the compound is administered once or twice daily. In particular, oral administration can be performed once daily during meals, such as breakfast, lunch, or dinner, by taking a tablet containing a dose of about 1000 mg, particularly 1000 mg. In another embodiment, the tablet is administered orally twice daily, for example by taking a first tablet containing a dose of about 400 mg, about 500 mg, or about 600 mg, particularly 500 mg, of the compound during a meal, and a second tablet containing a dose of about 500 mg, particularly 500 mg, of the compound during another meal on the same day.

[0065] In another specific embodiment, NTZ or TZ(G) is administered in combination with another active ingredient, preferably an antimicrobial agent such as an antibiotic, antifungal agent, or antiviral agent. Of course, as is known in the art, the most suitable antimicrobial agent will be selected based on the organism or virus causing the infection. In one specific embodiment, sepsis is caused by a bacterial infection, and the antimicrobial agent is an antibiotic. Antibiotics that can be used to treat bacterial infections are known in the art. Illustrative families of antibiotics include, but are not limited to, β-lactam antibiotics (e.g., penicillins), tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulfonamides, glycopeptides, aminoglycosides, and carbapenems. In one specific embodiment, NTZ or TZ(G) may be combined with a carbapenem family such as ertapenem.

[0066] NTZ or TZ(G) and the antimicrobial agent can be administered to the target in the same or separate pharmaceutical compositions. In one particular embodiment, the present invention provides a pharmaceutical composition comprising NTZ or TZ(G), an antimicrobial agent, and a pharmaceutically acceptable excipient. This pharmaceutical composition can be used in the method of the present invention to treat or prevent sepsis. In another embodiment, the present invention provides a method wherein...

[0067] A first pharmaceutical composition comprising NTZ or TZ(G) and a pharmaceutically acceptable excipient; and

[0068] A second pharmaceutical composition comprising an antimicrobial agent;

[0069] All were applied to the subjects to treat or prevent sepsis.

[0070] The first and second pharmaceutical compositions can be used simultaneously, separately, or sequentially (i.e., the first pharmaceutical composition can be administered before or after the second pharmaceutical composition). Therefore, the present invention also provides a kit-of-parts comprising:

[0071] A first pharmaceutical composition comprising NTZ or TZ(G) and a pharmaceutically acceptable excipient; and

[0072] A second pharmaceutical composition comprising an antimicrobial agent;

[0073] It can be used simultaneously, separately, or sequentially in the treatment or prevention of sepsis.

[0074] The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0075] Example

[0076] Example 1: NTZ improves survival in a preclinical model of sepsis

[0077] Multimicrobial sepsis induced by cecal ligation and puncture (CLP) is characterized by systemic inflammatory dysregulation followed by immunosuppression. The mouse CLP model mimics the progression and characteristics of human sepsis and can therefore be used to determine the effectiveness of drugs in preventing the transition from sepsis to septic shock.

[0078] This study aimed to investigate the potency of NTZ against male C57BL6J(BL6) mice in a CLP model. The potency of the test compound was evaluated based on animal survival rates during the study period.

[0079] To minimize stress, the handling of animals was conducted with care. All experiments were conducted in accordance with the French Ministry of Agriculture's guidelines on experiments using laboratory animals (Law No. 87-848). The research was carried out in accordance with the Animal Health Regulation (Council Directive No. 2010 / 63 / UE of September 22, 2010 on animal protection and French Decree No. 2013-118 of February 1, 2013).

[0080] Cecal ligation and puncture surgery

[0081] Nine-week-old male C57BL6J mice (supplier Janvier-France) weighing 23-25g upon arrival were anesthetized intraperitoneally with 250 μL of a cerazine / ketamine solution (ketamine (Imalgene, Boehringer, Germany) 6.75 mg / kg and cerazine (Rompund 2%, Bayer, Germany) 2.5 mg / kg). A 1-1.5 cm incision was made along the midline of the abdomen, the cecum was located, and tightly ligated with 4-0 silk sutures (mild grade) at half the distance between the distal end and the base of the cecum. After the midline ligation, the cecum was penetrated once from the mesentery in the opposite direction to the mesentery using a 21-gauge needle. A small amount of feces was expelled to ensure the wound remained open. The cecum was then returned to its original position within the abdomen, and the abdomen was closed with sutures and wound edge clamps. The mice's weight evolution and mortality were monitored until day 7.

[0082] Solution

[0083] NTZ (Interchim, France) was administered orally via tube feeding at a dose of 50 mg / kg twice daily. NTZ treatment began 3 days prior to CLP. On the day of surgery, NTZ (50 mg / kg) was administered once an hour before CLP, followed by a second dose of NTZ (50 mg / kg) when the animals recovered from anesthesia. BID treatment was then continued daily until the end of the study (n = 15). Mice receiving NTZ mediator (carboxymethyl cellulose (#C4888, Sigma-Aldrich, Germany) via BID served as controls (n = 10).

[0084] Ertapenem 10 mg / kg (ORB134782 / PO8952, Interchim / Biorbyt) was used as a pharmacological reference control and was administered 1 hour before surgery on day 0 and continued daily after CLP surgery (n=10).

[0085] result

[0086] In the group of mice that received only the aforementioned medium, CLP caused a 100% mortality rate 3 days post-surgery. Figure 1 Conversely, 47% of the mice treated with NTZ remained alive 3 days after surgery, and even 33% remained alive 7 days after intervention. Notably, NTZ improved survival even better than pharmacologically proven ertapenem, which saved only 10% of the mice by the end of the study.

[0087] In conclusion, NTZ has a beneficial effect on the survival rate of mice with CLP-induced multimicrobial sepsis.

[0088] Example 2: NTZ protects hepatocytes from cytokine-induced apoptosis

[0089] An uncontrolled cytokine storm that occurs during the transition from sepsis to septic shock leads to cell death in various tissues, potentially endangering the function of vital organs such as the liver.

[0090] This study aims to investigate the efficacy of NTZ in protecting hepatocytes from cell damage, particularly cytokine-induced apoptosis.

[0091] Evaluation of TNFα-induced apoptosis in human hepatocytes

[0092] To evaluate the effect of NTZ on human hepatocytes subjected to cytokine-induced cellular stress, the human hepatoblastoma-derived HepG2 cell line (#85011430, ECACC, UK) was cultured in high-glucose DMEM medium (#41965, Gibco, France) at 37°C in a 5% CO2 incubator with or without the active metabolite TZ. The high-glucose DMEM medium was supplemented with 10% fetal bovine serum (FBS, #10270, Gibco), 1% penicillin / streptomycin (#15140, Gibco), 1% sodium pyruvate (#11360, Gibco), and 1% MEM non-essential amino acids (#11140, Gibco).

[0093] To evaluate the activity of caspase 3 / 7, a surrogate marker of apoptosis, 5 × 10⁻⁶ g of caspase 3 / 7 was used. 4Cells were seeded in 96-well plates (Thermo Fischer, Germany). After cell adhesion (8 hours), cells were pretreated for 16 hours in FBS-deprived cell culture medium with TZ (Interchim, France) at doses ranging from 0.3 to 3 μM. Subsequently, tumor necrosis factor α (TNFα) (#C6378, Promocell, Germany) was added to the wells at doses of 10 or 30 ng / ml for another 24 hours. Astrococcus (10 μM) (#19-123 MG, Sigma-Aldrich, Germany) was used as a reference for inducing apoptosis. Cells were incubated with astrococcus for 3 hours, and then caspase activity was measured.

[0094] Caspase 3 / 7 activity was measured using the Caspase Glow™ 3 / 7 assay (#G8093, Promega, USA). Luminescence was measured using a Spark microplate reader (#30086376, Tecan, USA). Luminescence intensity (RLU) was directly correlated with caspase 3 / 7 activity.

[0095] result

[0096] HepG2 incubation with TNFα induces apoptosis, as shown by the 1.5-fold increase in caspase 3 / 7 activity at 10 ng / ml TNFα and the 1.7-fold increase at 30 ng / ml, an effect comparable in magnitude to that of the apoptosis inducer astrococcus. Figure 2 A). In the presence of 10 ng / ml TNFα, treatment with TZ significantly reduced caspase activity in a dose-responsive manner. Figure 2 B), at a TZ dose of 3 μM, achieved 40% inhibition. Notably, this effect was confirmed with higher doses of TNFα. Figure 2 C). These results indicate that TZ directly protects hepatocytes from cell death by inhibiting caspase activity.

[0097] Example 3: The direct and rapid effect of NTZ on hepatocyte apoptosis

[0098] This study aimed to investigate the efficacy of NTZ and its active metabolite TZ in protecting hepatocytes from cell damage induced by a potent apoptosis inducer, astrosaponin (a protein kinase inhibitor that activates caspase), with or without pretreatment.

[0099] plan

[0100] The HepG2 cell line derived from human hepatoblastoma was cultured as described in Example 2.

[0101] 1.5 × 10⁻⁶ cells were laid in a 384-well plate (#781080, Greiner, France). 4Caspase 3 / 7 activity was assessed in individual cells. After cell adhesion (8 hours), cells were serum starved for 16 hours with or without the NTZ metabolite TZ. Cells were then treated for 4 hours with a high dose of astrococcal agent (30 μM, #569397, Sigma-Aldrich, Germany) supplemented with 0.1–10 μM TZ (#RP253, Interchim) or 1–6 μM NTZ (#RQ550, Interchim, France), followed by cell lysis and caspase activity measurement. Caspase 3 / 7 activity was measured as previously described.

[0102] result

[0103] HepG2 cells incubated with astrococcus strongly induced apoptosis, as evidenced by an 11-fold increase in caspase 3 / 7 activity. Figure 3 A). When used as a pretreatment, TZ significantly reduced astrocytocin-induced caspase activity in a dose-dependent manner, achieving 82% inhibition at a dose of 6 μM TZ. Figure 3 B). Interestingly, the simultaneous addition of astrocytocin and 6 μM TZ without TZ pretreatment also reduced caspase activity by 64% ( Figure 4 A). Under these conditions, NTZ showed a similar effect, with an inhibition of 78% of caspase activity (A). Figure 4 B). These results indicate that NTZ and its active metabolite TZ are effective inhibitors of apoptosis, protecting hepatocytes from significant cellular damage that occurs during sepsis.

[0104] Example 4: NTZ improves the survival rate of CLP mice without NTZ pretreatment.

[0105] Given the rapid effects of NTZ observed in vitro, we investigated the efficacy of NTZ in protecting against sepsis in a CLP model under two curative settings.

[0106] As described in Example 1, multimicrobial sepsis was induced in C57BL6J mice via CLP surgery. NTZ was prepared as previously described, and as... Figure 5 As shown in A, oral administration at 100 mg / kg / day BID was initiated 3 days prior to CLP (3-day pretreatment) or on the same day as the CLP surgery (without pretreatment). Male C57BL / 6J mice (8 weeks old, Janvier, France) were divided into 3 groups of 24 mice each. After 7 days of environmental acclimatization:

[0107] - Group 1 received mediators for 3 days before CLP surgery and for 6 days after surgery.

[0108] - Group 2 received nitrozonidine (NTZ) for 3 days before CLP surgery and nitrozonidine for 6 days after surgery (pretreatment).

[0109] - Group 3 received mediators for 3 days before CLP surgery and NTZ for 6 days after surgery (without pretreatment).

[0110] NTZ treatment was performed twice daily at 9:00 AM and 5:00 PM. On the day of CLP surgery (day 0), mice received either NTZ or the mediator (groups 2 and 3) one hour before anesthesia.

[0111] A health scoring system for sepsis severity in a mouse sepsis model has been published to harmonize with human endpoints and normalize the scores observed across animals throughout the experiment (Shrum B, Anantha RV, Xu SX, et al., Arobust scoring system to evaluate sepsis severity in an animal model. BMC Res Notes 2014; 7:233). Animals were observed individually, changes were recorded, and scores were assigned based on the intensity of the changes. Observations included changes in appearance, activity, responsiveness to stimuli, eye opening, respiratory quality, and weight changes. For each of these clinical signs, severity was measured on a scale of 0 to 3. Evolution of severity was tracked, average scores at each time point were calculated, and plotted graphically. Arbitrarily, mice that died or were euthanized received a score of 4.

[0112] result

[0113] NTZ treatment (with or without 3-day pretreatment) significantly improved survival rates after CLP-induced sepsis. Figure 5 B). Although the mortality rate in the control group reached 60% at 55 hours post-surgery, the mortality rates in NTZ-treated mice with and without pretreatment were only 25% and 17%, respectively. At the end of the study, 45.8% of the NTZ-pretreated mice and 58.3% of the NTZ-treated mice survived, compared to only 12.5% ​​of the untreated mice. Figure 6 ).

[0114] The evolution of sepsis severity was assessed using health scores. Across all observational criteria, NTZ-treated mice (with or without pretreatment) scored lower than control mice, suggesting that NTZ treatment resulted in lower overall sepsis severity and improved animal health. Figure 7 ).

[0115] Example 5: NTZ is a highly effective treatment for sepsis in curative settings.

[0116] As previously described, multimicrobial sepsis was induced in C57BL6J mice via CLP surgery, and NTZ was prepared and administered orally. To investigate the rapid effect of NTZ against sepsis, NTZ was administered post-surgery, i.e., when bacterial leakage of the intestinal microbiota occurred in the peritoneum. Figure 8 As shown in A, male C57BL / 6J mice (8 weeks old, Janvier, France) were divided into 3 groups, with 20 mice in each group:

[0117] - Group 1 received the medium for the second time 1 hour before surgery, followed by a second application after surgery and twice daily for 6 days after CLP surgery.

[0118] - Group 2 received the first dose of nitrozonidine (NTZ) 1 hour before surgery, followed by a second dose of NTZ (2 times, 50 mg / kg) 3.5 hours after surgery, and then twice daily for 6 days after CLP surgery.

[0119] - Group 3 received the first dose of NTZ (100 mg / kg) only 3.5 hours after surgery, followed by 50 mg / kg twice daily for 6 days after CLP surgery.

[0120] During the 6-day period following the surgery, mice were treated twice daily at 9:00 AM and 5:00 PM with NTZ at a dose of 100 mg / kg / day (oral, BID). Control mice received the medium in the same manner to avoid any bias between control and treated mice.

[0121] result

[0122] NTZ treatment starting on the day of CLP surgery, whether before and after surgery (BID T-1h / T+3.5h) or only after surgery (QD T+3.5h), has a significant beneficial effect on survival. Figure 8 B). Although the mortality rate in the control group reached 55% at 55 hours post-surgery, the mortality rates in mice treated with NTZ BID T-1h / T+3.5h and QD T+3.5h were only 5% and 15%, respectively. At the end of the study (day 7), 80% and 70% of the mice treated with NTZ BID T-1h / T+3.5h and QD T+3.5h survived sepsis, respectively, compared to only 20% of the untreated mice. Figure 8 C). As mentioned above, the evolution of sepsis severity was also assessed using health scores. Mice treated with QD T+3.5h NTZ showed improved scores on all observation criteria, suggesting lower overall sepsis severity and significant health improvement after sepsis induction. Figure 9 ).

[0123] in conclusion

[0124] In summary, these results demonstrate that NTZ is a very rapid and effective compound that protects against cell death and improves health and survival in sepsis.

Claims

1. Use of a compound selected from nitrozonide (NTZ), tezozonide (TZ), and TZ glucoside (TZG) in the preparation of a medicament for treating sepsis in patients of need.

2. The use according to claim 1, wherein the sepsis is caused by a bacterial infection.

3. The use according to claim 1 or 2, wherein the compound is used to protect vital organs by inhibiting cytokine-induced apoptosis that occurs during the transition from sepsis to septic shock.

4. The use according to claim 1 or 2, wherein the compound is used to defend against cytokine-induced cell death by inhibiting caspase activity.

5. The use according to claim 1 or 2, wherein the subject suffers from sepsis with multiple organ failure or is at risk of sepsis with multiple organ failure.

6. The use according to claim 1 or 2, wherein the subject suffers from septic shock or is at risk of septic shock.

7. The use according to claim 1 or 2, wherein the compound is used to slow or stop the progression of sepsis.

8. The use according to claim 1 or 2, wherein the compound is used as a single active agent in the method.

9. The use according to claim 1 or 2, wherein the compound is used in combination with an antimicrobial agent in the method.

10. The use according to claim 9, wherein the antimicrobial agent is an antibiotic.

11. The use according to claim 10, wherein the antimicrobial agent is a carbapenem antibiotic.

12. The use according to claim 10, wherein the antimicrobial agent is ertapenem.

13. The use according to claim 1 or 2, wherein the compound is NTZ.

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