Methods of treating sepsis

Abstract

Disclosed are methods for treating sepsis in a mammalian patient in need of such treatment that rely on apheresis to achieve selective removal of galectin-3 to control or reduce the level of galectin-3, which has been demonstrated to be a driver of the cascade cycle of inflammation in humans. The data and discovery are based on human body tests, pig tests, and rat tests. Acute kidney injuries, typically associated with sepsis, in mammals are also demonstrated to be effectively treated by the disclosed and claimed methods. The selective removal of Gal-3 by apheresis is also shown to be effective for the treatment of AKI irrelevant to sepsis.

Description

[0001] Priority data and incorporation by reference

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 546,447, filed October 30, 2023. The entire disclosure of that application is incorporated herein by reference without limitation. Background of the Invention Technical Field

[0004] This invention relates to the treatment of sepsis. Sepsis is a life-threatening condition caused by a dysregulated inflammatory response to infection. Despite accounting for nearly twenty percent (20%) of all deaths worldwide, there are no safe and effective targeted therapies for sepsis in the clinical setting. Here, we disclose a method for treating sepsis in mammals. The treatment described and disclosed herein, while based on a three-part study evaluating the association between galactolectin-3 (Gal-3) and sepsis, sepsis-related mortality, and acute kidney injury (AKI), is in fact a complete treatment. Validation and studies are described below.

[0005] As previously stated, the research and validation relied upon in this article are based on three parts. First, in a human cohort of patients admitted to the Intensive Care Unit (ICU) for sepsis, mean serum Gal-3 concentrations were significantly higher than in healthy controls. Within the sepsis patient group, Gal-3 concentrations increased significantly over time compared to survivors. Elevated Gal-3 was also associated with acute kidney injury (AKI).

[0006] Secondly, in a rat sepsis model, Gal-3 removal via apheresis significantly improved survival. Treatment details are described below, but the significant increase in survival marks the first treatment to characterize mammalian patients in this way.

[0007] Third, survival was significantly improved in the swine sepsis model, and sepsis severity was significantly reduced in pigs undergoing Gal-3 depletion, as evidenced by significantly lower extravascular lung water index (ELWI), lactate levels, and significantly lower use of vasopressors and intravenous fluids compared to controls. Furthermore, Gal-3 depletion resulted in reduced histopathology in the lung, heart, kidney, and liver tissues. Our findings demonstrate, as disclosed herein, that Gal-3 depletion provides a targeted therapy for the management of sepsis, sepsis-related organ damage (multiple organ dysfunction syndrome (MODS), and sepsis-associated acute kidney injury (SA-AKI)). Background Technology

[0008] Sepsis is a life-threatening condition caused by a dysregulated inflammatory response to infection and is associated with a significant global disease burden, causing approximately 11 million (11 million) deaths annually, accounting for about 20 percent (20 percent) of all deaths worldwide. Sepsis can lead to septic shock and multiple organ dysfunction syndromes (MODS), including acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), and liver dysfunction. Sepsis-associated AKI is a common complication of sepsis, accounting for 50 percent (50 percent) of AKI cases in intensive care units (ICUs) and is associated with a higher risk of long-term kidney disease and mortality compared to other types of AKI.

[0009] Currently, sepsis management primarily involves intravenous antibiotic treatment of infection, intravenous fluid resuscitation, and additional organ support (12-14). Given the high incidence of sepsis-related morbidity and mortality, novel therapies targeting the underlying pathophysiology of sepsis are extremely important. Considering the central role of the inflammatory response in sepsis, immune system modulation is a key target for potential therapies. Although several therapies have been investigated, including statins, activated C proteins, and monoclonal antibodies, there is currently no approved, safe, and effective targeted therapy for sepsis in a clinical setting.

[0010] Gal-3 is a glycan-binding protein belonging to the lectin family, secreted by monocytes, macrophages, and epithelial cells in response to cellular and tissue damage and inflammation. Gal-3 participates in the regulation of inflammation, immune responses, tissue repair, and cell death, and influences the activation and release of pro-inflammatory cytokines, including interleukin (IL)-1, IL-6, nuclear factor κ-light chain enhancer (NFκB) for activated B cells, and tumor necrosis factor-α (TNF-α).

[0011] A substantial body of evidence indicates that Gal-3 is a key driver of the cytokine storm during sepsis, leading to immune dysregulation through endothelial cell activation, resulting in vascular leakage and organ dysfunction. In sepsis patients, elevated serum Gal-3 concentrations are associated with poorer outcomes, including higher mortality. Furthermore, Gal-3 directly induces renal tubular cell apoptosis, causing kidney inflammation and damage, and is an important independent predictor of SA-AKI. Lower Gal-3 activity in animal models is associated with reduced sepsis rates and improved sepsis-related outcomes, as well as reduced AKI severity and incidence. Moreover, compared to controls, Gal-3 inhibition was associated with significantly reduced mortality and AKI incidence in a sepsis rat model. Elevated serum Gal-3 concentrations have been shown to precede increases in serum IL-6 and creatinine, highlighting Gal-3 as an early biomarker of sepsis-associated AKI and a driver of the inflammatory cascade.

[0012] Plasma adsorption utilizes an adsorption column to selectively remove and thereby eliminate potentially harmful molecules, such as cytokines and other inflammatory mediators, from plasma. Plasma adsorption has been previously evaluated in sepsis models and inflammatory conditions. We previously developed a pheromone apheresis method using selective removal of Gal-3 for plasma adsorption, designed to reduce Gal-3, which effectively lowered serum Gal-3 concentrations. See For example, U.S. Patent No. 10,953,148.

[0013] In this paper, we describe the relationship between serum Gal-3 concentration, sepsis-related organ damage, and mortality in a cohort of sepsis patients and demonstrate the role of Gal-3 adsorption columns in two animal models of sepsis. Our study demonstrates that the same approach, selective withdrawal of Gal-3 via apheresis, is an effective treatment for both sepsis-related acute kidney injury (AKI) and sepsis-independent AKI.

[0014] This invention provides a treatment for sepsis, a devastating disease frequently encountered in places where other illnesses are treated—hospitals, clinics, etc. Regardless of where it occurs, this invention stipulates that, as an initial step, a mammalian patient (typically a human) is first identified as having sepsis. As will be appreciated by those skilled in the art, sepsis is best diagnosed using a holistic approach.

[0015] To effectively identify mammalian patients requiring treatment for sepsis, the most effective approach is to use definitions that combine clinical criteria and diagnostic biomarkers. These will be elaborated upon below.

[0016] A) Clinical signs and symptoms:

[0017] Fever (high body temperature) or low body temperature.

[0018] Tachycardia or bradycardia depends on age and baseline heart rate.

[0019] Rapid breathing or age-appropriate changes in respiratory rate.

[0020] The mental state changes, ranging from confusion to coma.

[0021] Signs of hypotension or peripheral shutdown For example (Cold extremities, prolonged capillary refill time) indicate impending or existing shock.

[0022] B) Laboratory indicators:

[0023] Elevated inflammatory markers, such as CRP or procalcitonin.

[0024] Evidence of organ dysfunction, such as elevated serum creatinine, abnormal liver function tests, thrombocytopenia, or coagulation.

[0025] Lactic acidosis or elevated serum lactate levels indicate insufficient tissue perfusion.

[0026] C) Evidence of infection:

[0027] Positive cultures or other evidence of infection from blood or other sterile sites.

[0028] Clinical evidence suggesting a high probability of infection (e.g., purulent discharge, imaging findings).

[0029] D) Risk factors:

[0030] Patients with chronic conditions such as diabetes, immunosuppression, cancer, chronic organ dysfunction, or those who have recently undergone surgery.

[0031] Newborns and the elderly, due to their vulnerable immune status.

[0032] Recent exposure to invasive procedures, indwelling devices, or hospitalized patients increases the risk of healthcare-associated infections.

[0033] E) Immediate treatment requirements:

[0034] Antimicrobial therapy needs to be initiated rapidly within the first few hours of identification. Fluid resuscitation and hemodynamic support should be performed as directed by clinical assessment. This should be accompanied by monitoring and support of organ function in an intensive care setting, if necessary and as required.

[0035] No single factor is decisive in identifying patients with sepsis, but sepsis is neither newly discovered nor undocumented. This invention begins with identifying mammalian patients, which can be humans, who require treatment for sepsis. Those skilled in the art are familiar with the indications and identification of sepsis. What has been lacking previously is an effective treatment for sepsis. Summary of the Invention

[0036] The applicant has determined that sepsis in mammals, including humans, can be effectively treated by selectively removing Gal-3 via apheresis. This ex vivo treatment of blood to reduce the concentration of galactolectin-3 (GAL-3) has been proven effective in treating and curing sepsis in established animal models and in human cohorts. Given that sepsis is frequently contracted and spread in hospital settings, apheresis treatment does not present problems when bringing patients to treatment centers.

[0037] To effectively treat sepsis, apheresis is used to treat patients. Numerous known apheresis protocols and treatments exist for various purposes. To effectively treat a patient to overcome sepsis, apheresis can be performed on blood (plasma) separated into individual portions to remove one or more of white blood cells, platelets, red blood cells, and plasma. The details and protocols of apheresis vary considerably, but are concisely summarized and detailed in U.S. Patent No. 10,953,148, assigned to Eliaz Therapeutics, Inc., California. Recently, the same researchers have made advances in the use of whole blood apheresis for treatment, as detailed in U.S. Patent Application Serial No. 17 / 964,644. Therefore, whole blood apheresis can now also be used to treat certain cases, depending on the goals and conditions. The disclosures of U.S. Patent No. 10,953,148 and U.S. Patent Application Serial No. 17 / 964,644 are both incorporated herein by reference in their entirety.

[0038] Apheresis is a technique developed to treat a variety of conditions and pathologies. Many of these treatments are achieved by removing a type, an agent, or a component of the blood and returning the remainder to the patient. Whether it is apheresis of whole blood or more conventional post-absorption treatment, the target for treating sepsis in this invention is galactolectin-3, referred to herein as Gal-3.

[0039] Gal-3 has been demonstrated to be an upstream mediator in the potential inflammatory cascade of sepsis. Treatment of sepsis patients with interventions that reduce blood Gal-3 levels by at least approximately 12% has been shown to be effective in reversing their condition, as demonstrated in this application. This process of removing blood components (in this case, Gal-3) using apheresis has gradually been referred to as the “selective withdrawal” of the target.

[0040] The average blood level of Gal-3 in healthy adults typically ranges from 8 to 15 ng / ml. In patients with sepsis, Gal-3 levels are usually significantly elevated beyond this range. The effectiveness of apheresis in reducing Gal-3 levels in patients with sepsis can vary; while selective withdrawal of galactolectin-3 via apheresis will result in a significant reduction in the entire column, the reduction in blood Gal-3 levels may not be reflected in the initial and acute phases, as patients may continue to produce excess Gal-3 during the initial phase of their disease. Typically, Gal-3 apheresis treatment in sepsis should initially be administered continuously for 1 to 3 days, preferably 1 to 5 days, depending on the patient's Gal-3 levels and clinical outcomes after initial treatment. Additional treatment may be considered based on the patient's Gal-3 levels and clinical response after the initial treatment series. This invention has demonstrated significant efficacy in some patients following single-day treatment.

[0041] The primary focus of this invention is the successful treatment of sepsis by reducing sepsis-associated gal-3 levels in the patient's blood. As discussed throughout this application, acute kidney injury, or AKI, is closely associated with and occurs concurrently with sepsis. Effects and events associated with AKI caused by sepsis can also be effectively treated by apheresis to reduce gal-3 levels. Therefore, while the invention disclosed herein is sometimes referred to as an effective treatment for sepsis and in other paragraphs as an effective solution to damage caused by AKI, it is an effective treatment—relying on reducing gal-3 levels by selectively withdrawing gal-3 via apheresis, which is also an effective treatment for sepsis-associated AKI. Selective withdrawal is achieved by allowing the patient's blood to flow through an array of binders fixed to the inside of the apheresis device. These binders are typically natural or recombinant antibodies that effectively bind galectin-3. The thus bound gal-3 remains in the apheresis vessel as the blood flows through and is subsequently returned to the patient. If the binding coefficient is sufficiently high, the binder can be a natural protein, etc.

[0042] The effectiveness of this invention has been repeatedly demonstrated. The applicant has demonstrated the invention using rat models, in pig experiments, and finally in human cohort trials. Human trials always have the greatest impact and require the greatest attention—the human trials are described below. Experiments using a well-established rat sepsis model are then discussed in detail. Finally, a detailed study of a pig sepsis model is presented. The combination of human, rat, and pig experiments provides a compelling demonstration of the effectiveness and value of the claimed invention. Attached Figure Description

[0043] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate exemplary embodiments of the invention and, together with the general description given above and the detailed description given below, serve to explain the features of the invention.

[0044] Figure 1 Serum levels of IL-6, Gal-3, and creatinine were measured in patients with sepsis and controls.

[0045] Figure 2 Serum concentrations of IL-6, Gal-3, and creatinine (Cr) were shown in sepsis survivors compared to non-survivors.

[0046] Figure 3 Serum concentrations of IL-6, Gal-3, and Cr on days 1, 2, and 3 are shown in sepsis patients with AKI (n=33) and without AKI (n=54).

[0047] Figure 4 The percentage of survival in rat sepsis CLP models with and without Gal-3 apheresis is shown (n=10 and 9, respectively).

[0048] Figure 5 The values ​​of extravascular lung water index (ELWI) over time are shown in a porcine sepsis lipopolysaccharide injection (LPS) model undergoing Gal-3 apheresis, compared to the control.

[0049] Figure 6 The results show the lactate concentration in a porcine sepsis model that underwent Gal-3 apheresis (treatment group) compared to the control group.

[0050] Figure 7 Serum Gal-3 concentrations in the treatment group and the sham surgery (control) group were shown at baseline, after LPS, and at 1, 2, and 3 hours after treatment.

[0051] Figure 8 Serum IL-6 concentrations (pg / ml) in the treatment group and sham-operated control group at baseline, 0 hours, 1 hour, 2 hours and 3 hours after LPS are shown.

[0052] Figure 9 The percentage of survival is shown in porcine sepsis LPS models with and without Gal-3 apheresis.

[0053] Figure 10 AL shows comparative histological samples among four different tissue types. Detailed Implementation

[0054] As stated above, this invention is disclosed based on results obtained from three distinct and mutually supporting experiments. The applicant's invention is illustrated through the details of numerous human cohort trials. Since human trials always receive the most specific and precise attention, these trials are reported below. Following the human cohort trials, we report trials involving a well-established rat model. The third trial relied upon and reported herein is a porcine sepsis model trial. Further discussion details of the invention are provided following the report of the porcine model trial.

[0055] Part 1: Human Queues

[0056] This study included a total of 114 (114) participants, comprising 87 (87) patients with sepsis and 27 (27) healthy controls. Compared with the control group, the sepsis group showed significantly elevated serum concentrations of Gal-3 and IL-6 at all time points, and remained significant after excluding diabetic patients (p<0.001) (Table 1; Figure 1 (A and 1C). Compared with the mean concentration of 9.66 ng / ml in the control group, sepsis patients showed mean serum Gal-3 concentrations of 20.68 ng / ml, 15.49 ng / ml, and 15.13 ng / ml on days 1 (the day of ICU admission), 2, and 3, respectively (P<0.001) (Table 1; Figure 1 (A and 1B). At all time points, there were no significant differences in serum creatinine concentrations between the sepsis group and the control group (Table 1; Figure 1 C; All P > 0.05).

[0057] Table 1. Characteristics of patients in the control group and the sepsis group.

[0058]

[0059] Note: Data is displayed as median values, with interquartile range spanning from the 25th to the 75th percentile.

[0060] In survivors, mean serum Gal-3 concentrations decreased on days 2 and 3, while in non-survivors, mean serum Gal-3 concentrations increased on day 3. Figure 2 On day 3, the mean serum Gal-3 concentration was significantly higher in the non-survivor group compared with the survivors (P=0.007) (Table 1; Figure 2 B). Urine analysis of Gal-3 demonstrated no significant difference in urinary Gal-3 excretion between survivors and non-survivors, and Gal-3 excretion did not change significantly over time in any group.

[0061] The incidence of (all-cause) AKI was similar between survivors and non-survivors (44% vs. 35%) (Table 2). Furthermore, there were no significant differences in mean serum creatinine, IL-6 and lactate concentrations, SOFA scores, or APACHE II scores between survivors and non-survivors. (Table 2) Figure 2 B and 2F).

[0062] Table 2: Characteristics of surviving and non-surviving sepsis patients.

[0063]

[0064] Note: Data is displayed as median values, with interquartile range spanning from the 25th to the 75th percentile.

[0065] Compared with patients without sepsis-associated AKI, patients with sepsis and sepsis-associated AKI had significantly higher APACHE II scores, SOFA scores, and serum lactate (P<0.01; Table 3). Patients with sepsis-associated AKI showed significantly higher serum Gal-3 concentrations on days 2 (P=0.01) and 3 (P=0.005) than patients without sepsis-associated AKI; (Table 3; Figure 3 (C and 3D). At any time point, there was no significant difference in serum IL-6 concentrations between patients with and without sepsis-associated AKI, although levels decreased over time (Table 3;). Figure 3 (A and 3B).

[0066] Table 3: Characteristics of sepsis patients with and without AKI.

[0067]

[0068] Note: Data are presented as median values, with interquartile ranges spanning the 25th to 75th percentiles. Cr = creatinine, Gal-3 = galactoglobulin 3, IL6 = interleukin-6, LAC = lactate, SOFA = sequential organ failure assessment, APACHE II = acute physiology and chronic health evaluation II.

[0069] Experiment 2: Rat sepsis CLP model and Gal-3 apheresis

[0070] Nineteen rats underwent CLP (cecal ligation and puncture). One hour after the CLP procedure, ten animals underwent a two-hour apheresis to remove Gal-3, while nine animals underwent a two-hour sham apheresis.

[0071] Nine out of ten animals in the single-apheresis treatment group survived to the end of the study on the seventh day, while eight out of nine animals in the control group died on the fourth day (P<0.001). Figure 4 ).

[0072] Part 3: LPS Model of Swine Sepsis and Gal-3 Apheresis

[0073] Following the results from the rat model, we investigated the effect of selective Gal-3 withdrawal apheresis in a septic porcine LPS model that more closely mimics human sepsis (41). Prior to LPS-mediated sepsis induction, there was no significant difference in ELWI between the treatment and control groups. Following LPS induction, pigs treated with selective Gal-3 withdrawal apheresis showed minimal increases in ELWI over time relative to baseline throughout the three-hour experimental period. In contrast, in the sham apheresis group, ELWI began to increase significantly over time after LPS infusion (P=0.021) and continued to increase significantly at 1, 2, and 3 hours after sepsis induction (P<0.001). Figure 5 Starting 1 hour after LPS infusion, the Gal-3 selective withdrawal apheresis group showed significantly lower ELWI compared to the sham operation group, and remained significantly lower at 2 and 3 hours after LPS (1 hour: P = 0.002; 2 hours: P = 0.007; 3 hours: P = 0.002). Figure 5 ).

[0074] Prior to LPS infusion, there was no significant difference in serum lactate concentration between the treatment and control groups. Following sepsis induction, serum lactate concentrations increased in both groups, with a greater increase in the control group. Figure 6 From the time of LPS infusion, the mean serum lactate concentration in the Gal-3 apheresis group was significantly lower than that in the sham surgery group at all time points (P<0.05). Figure 6 ).

[0075] At baseline, there was no significant difference in mean serum Gal-3 concentration between the Gal-3 selective withdrawal group and the control group. After LPS-induced sepsis, the mean serum Gal-3 concentration in the Gal-3 withdrawal group was significantly lower than that in the control group (P=0.001). Figure 7 At 1, 2, and 3 hours after sepsis induction, serum Gal-3 concentrations in the Gal-3 apheresis group remained significantly lower than in the control group (1 hour: P = 0.009; 2 hours: P = 0.005; 3 hours: P = 0.003). Figure 7 ).

[0076] Before LPS infusion, there was no significant difference in serum IL-6 concentration between the treatment and control groups. Following LPS-mediated sepsis induction, IL-6 concentrations gradually increased in both groups, with a significantly faster increase in the control group. Figure 8 At 3 hours after sepsis induction, IL-6 levels in the treatment group were significantly lower than in the control group (P=0.0076). Figure 8 Furthermore, the total fluid requirement (P<0.001) and cumulative norepinephrine dose (P=0.0025) in the control group were significantly higher than those in the Gal-3 apheresis group.

[0077] During the 24-hour study period, the survival rate of pigs in the Gal-3 apheresis group (68.8%) was significantly higher than that in the control group (26.7%) (P=0.004). The survival rate in the control group declined rapidly after LPS-mediated sepsis induction, reaching 33.3% at 6 hours after LPS infusion and 26.7% at 12 hours after LPS infusion. Figure 9 In the treatment group, the corresponding survival rates were 87.5% and 68.8%, respectively. Figure 9 ).

[0078] Compared with the control group, histological analysis of tissues from the treatment group showed a reduction in LPS-mediated post-septic pathological changes. Figure 10 Table 4). Impaired structural integrity, necrosis, and immune cell infiltration were observed in all organ and tissue samples from the control group. Figure 10 (Table 4), while necrosis and cell infiltration were not present in most organ and tissue samples from the Gal-3 single-apheresis group (Table 4).

[0079] Table 4: Key findings from histological analysis of tissues from the treatment and control groups of pigs.

[0080]

[0081] discuss

[0082] Here we describe the results of three distinct but complementary studies: 1) a cohort of sepsis patients where we examined the associations between clinical variables, Gal-3, and other inflammatory markers; 2) a rat sepsis model of cecal ligation and puncture treated with Gal-3 adsorption columns (with sham surgery); and 3) a porcine sepsis LPS model treated with Gal-3 adsorption columns (with sham surgery). We found that in the sepsis patient cohort, persistently elevated serum Gal-3 concentrations were associated with sepsis progression (or delayed remission), sepsis-related acute kidney injury, and sepsis-related mortality. In the animal models of sepsis, Gal-3 removal using selective withdrawal apheresis significantly reduced sepsis severity, decreased associated mortality, and reduced pathological changes on histological assessment.

[0083] Our cohort study results are consistent with previous studies exploring the role of Gal-3 in sepsis pathogenesis, sepsis-related acute kidney injury (AKI), and sepsis-related mortality (21, 24, 30, 31, 33). Compared to controls, patients admitted to the ICU for sepsis had elevated serum Gal-3 and IL-6 concentrations. Among sepsis non-survivors, serum Gal-3 concentrations increased over time and were significantly higher on day 3 than in survivors. These findings demonstrate an association between elevated Gal-3 and sepsis, sepsis mortality, and AKI.

[0084] In a validated porcine model of sepsis induced by LPS, we found significantly reduced Gal-3 levels at all time points after apheresis compared to the control group, demonstrating successful Gal-3 removal (42). As demonstrated by ELWI and serum lactate concentrations, Gal-3 removal resulted in a significant reduction in sepsis severity compared to the control group. Notably, ELWI did not significantly increase at any time point after both LPS infusion and Gal-3 apheresis, while ELWI in the control group continued to worsen at every time point after LPS infusion. Similarly, Gal-3 removal attenuated the increase in serum lactate in the Gal-3 apheresis group compared to the control. Notably, fluid and vasopressor requirements were also significantly greater in the control group than in the Gal-3 apheresis group. These findings demonstrate that Gal-3 removal using apheresis in porcine models significantly reduces sepsis severity, as demonstrated by both clinical measurements and biomarkers.

[0085] Recent studies have highlighted the role of Gal-3 in the inflammatory response (21, 24, 31, 33). Gal-3 regulates immune cells and pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), leading to the internalization of endotoxins such as LPS and directly inducing renal tubular cell apoptosis (23, 25, 26, 31, 32). We found that Gal-3 removal resulted in a significant decrease in IL-6 3 hours after sepsis induction compared to controls, consistent with previous findings in animal models treated with Gal-3 inhibitors (23, 33). The delayed trend in IL-6 reduction after Gal-3 removal, combined with the rapid reduction in sepsis severity, suggests the role of Gal-3 as an upstream mediator in the potential inflammatory cascade of sepsis (33).

[0086] In this study, organ tissue histopathology in the Gal-3 apheresis group showed significantly reduced tissue necrosis and improved structural integrity compared to the control group (43). In the control group, lung, heart, kidney, and liver tissues showed impaired structural integrity and necrosis, with evidence of extensive lung necrosis and frequent alveolar dilatation, cardiomyocyte disorder and deformity, extensive renal tubular epithelial cell necrosis with severe interstitial hemorrhage, and extensive hepatocyte deformity, among other findings. In contrast, in the Gal-3 apheresis group, only occasional or mild necrosis was observed in the lung and kidney tissues, liver congestion with sinusoidal dilatation, and normal cardiomyocyte morphology. The reduction in histopathology corresponds to the decrease in sepsis severity observed in the Gal-3 apheresis group. Although histopathological analysis during the study and postmortem limited follow-up assessment in the pig model, these findings suggest that Gal-3 removal not only improves sepsis severity but may also reduce chronic morbidity and organ dysfunction following sepsis (30,44).

[0087] In rat and porcine sepsis models, selective removal of Gal-3 via adsorption column resulted in significantly improved survival compared to controls. Our findings confirm and extend previously published findings by demonstrating that reducing serum Gal-3 concentrations via column adsorption attenuates many of the adverse effects of sepsis on organ structure and function, indicating the role of Gal-3 in sepsis mortality and other complications. Combined with observational data from humans, selective removal of Gal-3 via apheresis may represent a novel in vitro approach for managing sepsis and related complications (including AKI) in critically ill patients.

[0088] Given the pathophysiology of sepsis, novel therapies targeting immune dysregulation require further exploration. As demonstrated by previous studies and our current findings, Gal-3 presents a unique target for sepsis treatment (15, 16, 21, 32, 34, 45). We have demonstrated that Gal-3 can be effectively removed using apheresis in a porcine sepsis model. Following our pig studies, we were able to significantly improve the efficiency and capacity of Gal-3 reduction in XGAL-3 columns. We anticipate this improvement will lead to better clinical outcomes. Gal-3 removal significantly reduced sepsis severity, organ damage, and mortality. Based on these findings, Gal-3 removal using apheresis represents a potential future therapy for sepsis treatment. Future research is necessary to further explore the mechanisms by which Gal-3 plays a role in sepsis pathogenesis and to refine the approach of Gal-3 removal as a potential targeted therapy in sepsis management.

[0089] Typically, patents and patent applications do not faithfully reproduce all the details and information regarding the methods used to implement the disclosed invention. Because the treatment of sepsis and sepsis-related AKI in this invention is based on three distinct and intensified studies and the method steps employed therein, we hereby provide the methods used in detail. Detailed methodological information allows those skilled in the art to compare and contrast the methods employed and the results obtained, thereby allowing for independent verification of the results and their implications.

[0090] Human queue

[0091] Patient recruitment and survival

[0092] Between August 2020 and December 2022, we recruited patients diagnosed with septic shock (n=87) from the Intensive Care Unit (ICU) of Zhongnan Hospital, Wuhan University, Hubei Province, China. We also recruited healthy volunteers from the general population in Hubei Province, China (n=27). Our study examined patients identified as male and female and analyzed the distribution among patients with different clinical outcomes. The diagnosis of sepsis was based on the Third International Consensus (Sepsis-3) Definitions (4). The presence of acute kidney injury (AKI) was determined according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria (46). We obtained information on patients' past medical conditions, including diabetes, chronic lung disease, oncology, cardiovascular disease, hypertension, cerebrovascular disease, chronic liver disease, and chronic kidney disease. We excluded patients with a history of substance abuse, recurrent infections, immunodeficiency, pregnancy, renal replacement therapy, discharge from the ICU in the previous year, and organ transplantation. We calculated survival and AKI from the day of ICU admission until death or discharge, up to 28 days. All participants or their representatives provided written informed consent, and patient data was de-identified and stored on a password-protected research computer.

[0093] Biochemical measurements

[0094] We collected blood samples for biochemical analysis from 87 patients with septic shock within 6 hours of ICU admission. Baseline samples were obtained on day 1 of ICU admission, with additional samples obtained on days 2 and 3. Baseline blood samples were collected only from healthy volunteers. Blood samples were centrifuged at 1,000 x g for 15 minutes at 2–8 °C, and plasma supernatants were collected and stored at -80 °C. Serum Gal-3 (R&D Systems, Minneapolis, MN, USA) and serum IL-6 (Elabscience Biotech, Wuhan, Hubei, China) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Serum creatinine (Cr) was measured using a Cr assay kit (Cat. No. E-BC-K139-S, Elabscience, Wuhan, China). Urine samples were collected from 54 (62%) randomly selected sepsis patients to analyze urinary Gal-3 concentrations.

[0095] Sepsis-related parameters

[0096] To assess disease severity and predict patient prognosis, we measured the Sequential Organ Failure Assessment (SOFA) score upon ICU admission (Seymour et al., 2016). Additionally, we calculated the Acute Physiology and Chronic Health Evaluation II (APACHE II) scores within 24 hours of ICU admission (47, 48). We measured point-of-care serum lactate concentrations at the bedside using a fingertip device (StatStrip® Lactate, Nova Biomedical Corporation, Waltham, MA, USA), reporting them as mmol / L.

[0097] Animal research

[0098] 1. Rat sepsis model of cecal ligation and perforation (CLP) and single apheresis

[0099] Monoclonal antibodies targeting rat Gal-3 demonstrated high specificity and affinity, as validated by the Biacore technique for detailed affinity and kinetic analyses and by in vitro attenuation of rat Gal-3. The proprietary antibody (Eliaz Therapeutics Inc., Santa Rosa, California, 95401) was selected through a meticulous development process involving the isolation and purification of various clones from rabbits immunized against Gal-3, each showing promising initial binding characteristics. The best-performing monoclonal antibody was then expressed and generated using an established Chinese hamster ovary (CHO) cell line (49). The purified antibody was then ligated to agarose beads and loaded into a column through which whole blood could be passed using a connection compatible with standard apheresis equipment. An empty column was used as a sham for control.

[0100] Sepsis induction using cecal ligation and perforation

[0101] Standardized induction of sepsis in animal models can be achieved via cecal ligation-perforation or lipopolysaccharide (LPS) injection (34, 42, 50–52). Nineteen male adult Sprague-Dawley rats (400–600 gr) were purchased from the Animal Care and Use Committee of Wuhan University. We induced sepsis in all rats using cecal ligation-perforation (CLP) (52), by ligating 25% of the cecum and then performing two punctures below the ileocecal valve using a 20-gauge needle. After abdominal wall suturing, the rats were subcutaneously resuscitated with 20 mL / kg preheated saline.

[0102] Gal-3 was removed using a single-agent technique.

[0103] A rat CLP model was used as a preliminary study to demonstrate the use of an adsorption column for selectively withdrawn Gal-3 removal in a sepsis model. The study used ten rats in the treatment group and nine rats in the control group. All rats (n=19) were anesthetized with isoflurane. Blood was drawn from the right femoral vein of the rats and allowed to flow through a rat Gal-3 apheresis column (treatment group) or an empty sham-operated column (control group), then returned via the jugular vein. The blood flow rate was maintained at 0.8–1.0 mL / min using a micropump. Plasma separation was not feasible due to the inherently low plasma volume in rats. As a result, whole blood apheresis was used. It began 60 minutes after CLP and continued for two hours.

[0104] Survival measurement

[0105] Survival rates were recorded daily for 7 days following CLP in both the Gal-3 apheresis and sham-operated groups. Measurements were repeated promptly and periodically, depending on the study size and complexity.

[0106] 2. LPS model of swine sepsis and single-donor apheresis

[0107] Preparation of a plasma adsorption column for Gal-3 removal in pigs. The apheresis separation column for selective removal of Gal-3 was prepared in a manner and design no different from the other columns discussed herein.

[0108] XGAL-3 technology aims to provide a single-absorption adsorbent for selectively reducing both bound and unbound circulating Gal-3. Eliaz Therapeutics Inc. has developed an ex vivo immunoadsorption column utilizing a human anti-Gal-3 antibody developed using a CHO expression system based on the CHO-K1 cell line. The antibody was 100% pure by size exclusion-high performance liquid chromatography (SEC-HPLC). The antibody's affinity for porcine Gal-3 was demonstrated in ex vivo and in vivo studies. The purified antibody was ligated to agarose beads and loaded onto the column (XGAL-3®, Eliaz Therapeutics Inc., Santa Rosa, California, 95401).

[0109] Using lipopolysaccharide to induce sepsis

[0110] Lipopolysaccharide (LPS) is an endotoxin and extracellular component of Gram-negative bacteria that activates an acute systemic inflammatory response by triggering the release of inflammatory cytokines and immune mediators (42, 50); these substances are commonly used to generate sepsis-like physiology in the absence of infection (34), as demonstrated in porcine models via intravenous infusion (41). LPS-mediated sepsis induction was performed in 31 male Bama miniature pigs (Sus scrofa domestica; Hubei Yizhicheng Biotechnology Co., Ltd.) weighing approximately 30 kg (41). On the day of the procedure, animals underwent intubation, anesthesia, and surgical preparation, including insertion of a central venous catheter, a Swan Ganz catheter, and femoral and internal jugular vein catheters. A 60-minute stabilization and recovery period followed. After this, an intravenous LPS infusion of 2 µg / kg / h was initiated, increased to 4 µg / kg / h after 10 minutes, and maintained for another 20 minutes, for a total of 30 minutes. A 30-minute recovery period followed this step. The animals then began an accelerated LPS infusion period, during which the LPS infusion rate was restored to 6 µg / kg / h for 10 minutes, followed by an increase to 12 µg / kg / h for 10 minutes, and then to 24 µg / kg / h for another 25 minutes, for a total of 45 minutes. The LPS infusion was then stopped, and the animals were given fluid resuscitation and vasopressor support as directed.

[0111] Gal-3 was removed using a single-agent technique.

[0112] A double-lumen dialysis catheter was inserted into the femoral vein. Ten minutes after the start of the acceleration phase, the dialysis catheter was opened and plasma adsorption was initiated using the Spectra Optia hematologic separation system, with the plasma flow rate controlled at 25 mL / min. Sixteen animals (treatment group) underwent selective Gal-3 depletion via therapeutic selective apheresis using an XGAL-3 column loaded with monoclonal anti-Gal-3 antibodies. Figure 1 Fifteen animals (control group) underwent sham apheresis via plasma separation without column filtration. Figure 1Apheresis treatment was initiated 10 minutes after the start of the acceleration phase (LPS dose of 12 mcg / kg / h) and continued for 3 hours after the final LPS dose, for a total of 3 hours and 35 minutes. During resuscitation and supportive care, fluid supplementation for the animals consisted of 0.9% NaCl solution, dextran-40, and 5% glucose solution. Fluid intake during the experiment, along with the use of norepinephrine as a vasopressor, was responsible for normalizing the animals' blood pressure levels. Blood samples were collected every 60 minutes throughout the LPS induction and apheresis. To support standard care principles, the pigs were provided with ventilation support, vasopressors (e.g., norepinephrine) (53), and fluid resuscitation for the first 6 hours. The animals were then transported to an animal facility and provided with regular water and food for the remainder of the study once they were awake, extubated, and hemodynamically stable. Heart rate (HR), blood pressure (BP), mean arterial pressure (MAP), and cardiac index (CI) were measured throughout the experiment. Mortality was recorded at 3, 4, 5, 6, 12 and 24 hours. All surviving pigs were then euthanized, and tissue samples were collected from the kidneys, heart, liver and lungs for comparative histological analysis.

[0113] Sepsis-related measurements

[0114] The extravascular lung water index (ELWI) is an important indicator of inflammation associated with systemic sepsis and is used to quantify the amount of extravascular fluid in the lungs (54, 55). Lactate, a major biomarker of sepsis severity, was measured using blood samples (56). Gal-3 and IL-6 concentrations were determined from blood samples. Furthermore, we assessed survival within 24 hours after sepsis induction. Post-treatment samples of lung, heart, liver, and kidney tissue were obtained and histologically examined using H&E staining. Biopsy interpretation was provided by histopathologists unaware of the treatment group.

[0115] statistics

[0116] We expressed normally distributed continuous variables as mean ± SEM and analyzed differences between groups using one-way ANOVA or independent samples t-tests. We compared skewed continuous variables using the Mann-Whitney U test, and expressed them as median + interquartile range (IQR) after confirmation by the Shapiro-Wilk test. A p-value of 0.05 or less was considered statistically significant.

[0117] We adjusted for potential confounding effects, including age and pre-existing conditions, using generalized linear models (GLMs) with gamma distributions. We examined the relationship between Gal-3 and the likelihood of mortality and AKI using binomial GLMs with binary outcomes (mortality and AKI). Furthermore, we examined the relationship between Gal-3 and continuous variables using the Spearman rank correlation coefficient and used the Mann-Whitney U test to examine differences in the distributions of continuous variables.

[0118] We plotted the survival rates of the sham surgery group and the treatment group using the Kaplan-Meier multiplication limit method and compared them using the log-rank test. We considered a two-sided p < 0.05 to be statistically significant. All statistical analyses were performed using R software version 4.2.3 (57).

[0119] These studies, along with previous tests, focus on the selective withdrawal or selective removal of Gal-3. As shown, this is achieved via a primary apheresis column. Multiple columns can be used, as disclosed in previous patents, including U.S. Patent No. 10,953,148. These secondary columns provide the opportunity to “fine-tune” treatment and to advance and support treatment through selective withdrawal (the terms selective withdrawal and selective removal are used interchangeably herein to refer to the method employed in which apheresis is used to reduce the level of Gal-3 or other targets). Therefore, in addition to the selective withdrawal / removal of Gal-3, the present invention further includes the selective withdrawal of other targets, including inflammatory compounds, cytokines, and LPS, via whole blood or plasma apheresis. In addition to the removal of Gal-3, this invention specifically considers the selective removal of LPS, CRP, various cytokines, and microbial agents (whole blood), including the removal of specific targets such as lipopolysaccharide (LPS), C-reactive protein, IL-4, IL-6, IL-1β, IL-10, TNF-α, NF-κB, High Mobility Group Protein B1 (HMGB1), and combinations thereof. More generally, other cytokines can also be treated through selective removal, including various chemokines, interferons, and other interleukins. These include, but are not limited to, interleukin-1 (IL-1), interleukin-10 (IL-10), interleukin-8 (IL-8), interleukin-12 (IL-12), interferon-γ (IFN-γ), monocyte chemoattractant protein-1 (MCP-1 / CCL2), transforming growth factor-β (TGF-β), and interleukin-17 (IL-17). These and similar media are discussed at www.sinobiological.com / resource / cytokines / role-of-cytokines-in-sepsis.

[0120] As described in similar texts such as U.S. Patent No. 10,953,148 and many others, effective treatment may not only rely on the selective removal of gal-3 and other blood mediators from the blood, but may also rely on the administration or addition of therapeutic or beneficial elements before the blood is returned to the mammal. Typically, adding agents to the plasma is an effective and efficient means of administering treatments such as drugs, vitamins, or similar components. Therefore, while not necessary for the selective removal of gal-3, the methods described in this disclosure involve administering multiple agents to the patient, which can be effectively achieved by adding them to the plasma and / or whole blood during the apheresis procedure. Examples include various homeopathic medicines, metabolic modulators, immune response modulators, drugs, and various cytotoxic compounds, antimicrobial agents, chelating agents, which may be targeted to aid in treatment.

[0121] Acute kidney injury (AKI) is characterized by the sudden inability of a mammal's kidneys to filter waste products from the blood. When the kidneys are unable to filter waste, it can gradually progress to harmful levels. The chemical composition of the blood may become unbalanced. Acute kidney injury was formerly known as acute kidney failure. Acute kidney injury is most common in hospitalized individuals, primarily those requiring intensive care. Acute kidney injury ranges from mild to severe. If severe, persistent, and untreated, it can be fatal. However, as disclosed in this article, AKI can be reversed. Individuals in good health may regain normal or near-normal kidney function.

[0122] The research detailed in this paper demonstrates the effectiveness of treatment for previously difficult-to-treat or untreatable conditions. This paper shows that apheresis, designed to achieve selective removal of Gal-3, is an effective treatment for sepsis. Given the frequent occurrence of sepsis in hospital or outpatient settings, the ability to provide apheresis to sepsis patients complements the effective treatment described herein. Furthermore, our research supports the effective treatment of sepsis-related acute kidney injury (AKI). AKI is frequently encountered in sepsis patients, complicating treatment and effective recovery. Sepsis and sepsis-induced AKI-related conditions can be treated by selectively withdrawing Gal-3 to reduce or at least control serum Gal-3 levels. Beyond the disease and treatment based on sepsis, this application demonstrates an effective treatment for AKI itself, unrelated to sepsis-related complications. The treatment of AKI, with or without sepsis-related problems and complications, provides another opportunity for effective treatment using selective Gal-3 withdrawal achieved through apheresis.

[0123] References

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[0188] Various implementation schemes have been described in detail with reference to the accompanying drawings. The documents submitted herein... Figure 1-10 Detailed information is provided regarding the three trials omitted or described in this article. We have made every effort, whenever possible, to avoid duplication or overlap between one set of results and another.

[0189] While the invention has been disclosed both generally and with reference to specific alternatives, those alternatives are not intended to be limiting unless reflected in the claims set forth below. As those skilled in the art to which this application is directed will recognize, the invention is limited only by the terms of the claims and their equivalents.

Claims

1. Methods for treating sepsis in mammals, including: a) Identifying mammalian patients requiring treatment for sepsis. b) Repeatedly administer selective withdrawal apheresis containing galactolectin-3 (Gal-3) to the mammalian patients thus identified, for a period of time sufficient to reduce the Gal-3 level in the blood of the mammalian patients.

2. The method of claim 1, wherein the time period is at least three days, and wherein the patient receives the apheresis treatment at least once a day.

3. The method according to claim 2, wherein the time period is at least five days.

4. The method of claim 1, wherein the mammalian patient exhibits acute kidney injury (AKI) associated with the sepsis.

5. Methods for treating acute kidney injury (AKI) in mammals with this need, including: a) Identify mammalian patients who require treatment for AKI. b) Repeatedly administer selective withdrawal apheresis containing galactolectin-3 (Gal-3) to the mammalian patients thus identified, for a period of time sufficient to reduce the Gal-3 level in the blood of the mammalian patients.

6. The method of claim 1, wherein the treatment of the patient further comprises administering at least one agent to the patient, wherein the agent is selected from the group consisting of metabolic modulators, immune response modulators, drugs, cytotoxic compounds, antimicrobial agents, chelating agents, and combinations thereof.

7. The method of claim 5, wherein the treatment of the patient further comprises administering a pharmaceutical agent to the patient, wherein the pharmaceutical agent is selected from the group consisting of metabolic modulators, immune response modulators, pharmaceuticals, cytotoxic compounds, antimicrobial agents, chelating agents, and combinations thereof.

8. The method of claim 5, wherein the treatment of the patient further comprises selectively removing at least one additional blood component selected from the group consisting of lipopolysaccharide (LPS), C-reactive protein, IL-4, IL-6, IL-1B, IL-10, TNFα, NF-κB, and combinations thereof.

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