Synthetic peptide is217 with dual antimicrobial and immunomodulatory activity for treatment of sepsis

Abstract

The present invention relates to synthetic peptide IS217 or peptide variant thereof and formulations, combinations and / or compositions of said synthetic peptide IS217 or peptide variant thereof. The present invention provides antimicrobial, immunomodulatory and anti-inflammatory effects of the synthetic peptide IS217 or peptide variant thereof, and formulations, combinations and / or compositions thereof for use in treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.

Description

Synthetic peptide IS217 with dual antimicrobial and immunomodulatory activity for treatment of sepsisField of inventionThe present invention relates to synthetic peptide IS217 and formulations, combinations and / or compositions thereof. The present invention relates to antimicrobial, immunomodulatory and anti-inflammatory effects of synthetic peptide IS217, and formulations, combinations and / or compositions thereof. The formulations, combinations and / or compositions of synthetic peptide IS217 demonstrate significant reduction in mortality, improved organ function, and protection against sepsis-induced damage. Accordingly, the present invention provides the synthetic peptide IS217 and the formulations, combinations and / or compositions thereof as potential therapeutic for sepsis.Background of inventionSepsis is a complex disorder characterized by disrupted inflammatory balance, often triggered by infection. It poses a significant global health concern due to its high mortality rate and costly treatment. Infections or traumas introduce pathogenic microorganisms or toxins into the bloodstream, causing sepsis or systemic inflammatory response syndrome. This can lead to multiple organ failure and a high risk of death. The pathogenic process involves dysregulation of the inflammatory response, coagulation cascade activation, thrombosis, endothelial disruption, tissue and cellular hypoxia, resulting in multiple organ dysfunction and irreversible shock. Figure 1 illustrates pathogenic mechanisms from infection to septic shock, modified from Jon A Buras et al., 2005.Early recognition and management of sepsis are crucial to prevent septic shock, associated with higher mortality. Timing of clinical intervention is vital, and early, appropriate treatment significantly increases survival chances. Despite aggressive care, 20- 30% of septic shock patients may die if treatment is delayed.The incidence of sepsis has risen over the past 30 years, with a global mortality rate of one person every 2.8 seconds. Around 85% of sepsis cases and related deaths occur in low- and middle-income countries, showcasing significant regional variations (News and opinions 2022; Sweden: Pemilla’s 8-day old daughter died from sepsis-caused by resistant bacteria Klebsiella).The COVID- 19 pandemic has further impacted sepsis epidemiology, with 32.5% of 200 COVID- 19 hospitalizations manifesting sepsis. COVID- 19 patients often experience adepressed immune response and liver injury, contributing to sepsis (WHO corona virus disease dash broad, 2020; WHO Coronavirus Disease (COVID- 19) Dashboard. Available from: https: / / covidl9.who.int. Accessed 24 October 2020).Bacterial infections, particularly gram-negative, are the primary cause of sepsis, accounting for over 90% of cases. Gram-negative infections (62%) are more frequent than gram-positive (47%). Key culprits include Staphylococcus aureus (30%, with 14% methicillin-resistant), Pseudomonas spp. (14%), Escherichia coli (13%), and Candida species (19%). pneumoniapneumoniainfluenzaA simplified overview of current notions regarding the pathogenesis of sepsis is shown in Figure2 [adopted and modified from NielsC. et al., 2003, Ignacio Rubio, et al., 2019, William C. Aird et al., 2003, FloreaLupu et al., 2014 and MichelaGiustozzi a et al., 2021].Human sepsis unfolds in two stages: an initial hyperinflammatory phase (SIRS) lasting days, followed by a prolonged immunosuppressive phase (CARS). These phases, linked to increased mortality, may overlap. Death distribution shows two peaks: one in the early phase, to a lesser extent, and a second peak after 2-3 months that rises over the next 3 years. The hyper-inflammatory phase, marked by a "Cytokine storm," plays a vital role in severe infections, contributing to inflammation, tissue damage, and conditions like acute lung injury, influenza, and severe COVID- 19. This phase triggers an inflammatory state, activating immune pathways and causing endothelial, cellular, and cardiovascular dysfunction characterizing sepsis. Mortality in the later period results from protracted immunosuppression, featuring anergy, lymphopenia, and secondary infections, leading to organ injury / failure.The host inflammatory response can be viewed as a balanced response between pro-inflammatory mediators (referred to as SIRS) and anti-inflammatory mediators (referred to as the CARS) [Jon A Buras et al., 2002]. Figure3 shows the SIRS and CARS in sepsis. Figured illustrates two stages of sepsis. A hyper-inflammatory phase with immunosuppression and multiple organ dysfunctions occurs after infection. This may also resolve, particularly with appropriate support, but it frequently results in death [JamesD. Faix et al., 2013].Diagnosing sepsis is challenging due to nonspecific signs and symptoms. Early diagnosis is crucial, requiring identification of a focus of infection accompanied by at least two signs of systemic inflammatory response syndrome for a standard clinical diagnosis of sepsis.Complications associated with septic shock include delirium in the brain, liver dysfunction, acute hepatitis, acute renal failure, proteinuria, decrease in arterial oxygenlevels, acute respiratory distress syndrome, tachypnea, arterial hypotension, cardiomyopathy, impaired organ perfusion, gastrointestinal symptoms (nausea, vomiting, diarrhea, peritonitis, ileus), splenomegaly, and skin manifestations such as mottling, increased capillary refill time, purpuras, erythema, and necrosis (ecthyma gangrenosum) [Timothy W. Evans et al., 2002; Bone, R. C. et al., 1992; Blanco J, 2008]. Figure 5 shows clinical presentation of sepsis.Biomarkers play a crucial role in sepsis diagnosis, aiding in distinguishing between the early hyperinflammatory and later immunosuppressive phases, monitoring treatment, and identifying the infection source (bacterial, fungal, viral) for effective antibiotic therapy [Savitri Kibe et al., 2011].A multi-marker approach, analyzing both hyperinflammatory and anti-inflammatory cytokines, aids in identifying patients transitioning between sepsis states [S. Gibotet al., 2012]. This approach is crucial for predicting severe sepsis onset and determining patients suitable for novel immune-restoring therapies [K. Reinhart et al., 2012]. Several biomarkers used in sepsis diagnosis are outlined in Table 1.Routine clinical investigations, including C-reactive protein (CRP), procalcitonin (PCT), and Interleukin-6 (IL-6), along with laboratory tests, are essential for ongoing monitoring from study day 1 until ICU discharge or death [S. Bhattacharya et al., 2005; Guido Marcello M et al., 2016; K AKirkebpen et al., 1999].Table 1: Cytokine biomarkers for sepsisTable 2: Laboratory clinical and biochemical biomarker investigationsGuidelines prioritize three components in sepsis management: (1) immediate patient stabilization, focusing on reversing life-threatening anomalies and monitoring vital signs; (2) infection control, involving prompt reduction of infection burden through source control measures and early antibiotic treatment; (3) septic response regulation, which includes strategies to modulate the host response, such as decreasing coagulation [Nguyen HB et al., 2006],The treatment plan encompasses both anti-microbial and non-anti-microbial therapies. Key interventions include early goal-directed therapy, appropriate antimicrobials, source control, corticosteroids, recombinant human activated protein C, and low tidal volume mechanical ventilation [Nguyen HB et al., 2006].Anti-sepsis strategies target the body's excessive inflammatory response, a leading cause of mortality even with successful antibiotic treatment [Hosny et al., 2013]. Prominent approaches include: Anti-Inflammatory Drugs:• Immunomodulatory Agents: Such as hydrocortisone, IVIG, or MSCs, aim to modulate the hyperinflammatory response [Niels C Riedemann et al., 2003 and 2015],Cardiovascular Drugs: Include short-acting beta-blockers, selepressin, or levosimendan, focusing on the cardiovascular system [Niels C Riedemann et al., 2003 and 2015],Corticosteroids:• Hydrocortisone: Administered in physiological doses at the onset of sepsis, proves effective in restoring hemodynamic stability. The optimal dosing and patient selection for corticosteroids remain subjects of investigation [Chao Ren et al., 2017],Anti-Endotoxin Strategies:• Anti-Endotoxin Antibodies: Studies suggest limited efficacy, while novel compounds like lipopolyamines and BPI show promise but await human sepsis trial validation [Elisabeth C. van der Slikkeet al., 2021].Anti-Cvtokine Therapies:• Anti-TNF Agents: Trials have shown a slight reduction in mortality, although these agents, standard in autoimmune disorders, are not yet established in sepsis treatment [Bradley D Freeman et al., 2000; Ali, T., Kaitha, S. et al., 2013].Anti-Inflammatorv Therapies:• IL-ip Antagonists: Initial promise in sepsis animal models, Anakinra (IL- IRA) Phase II trials showed mortality reduction, but Phase III studies did not confirm the benefit.• IL-6 Receptor Antibodies: Tocilizumab and sarilumab, though not evaluated for sepsis, improved survival in severe COVID-19 cases [Sara La Manna et al., 2018].• Other Pro-Inflammatory Cytokines: Clinical trials target specific host mediators (e.g., TNF, interleukins), showing marginal effectiveness. Ongoing research explores additional anti-inflammatory strategies.• Heparin: Widely used anticoagulant, crucial for survival in sepsis-induced DIC.• Activated Protein C (Drotrecogin Alfa): FDA-approved with immunomodulatory properties but later withdrawn due to questionable efficacy.• Selepressin: Phase III trials terminated due to futility results [ClinicalTrials.gov identifier: NCT02508649].• Imatinib: Tyrosine kinase inhibitor attenuates thrombin-induced endothelial dysfunction but has potential side effects [Bakhtiari, K et al., 2004].• Thrombomodulin (ART 123): Awaiting approvals after phase III RCT completion [ClinicalTrials.gov identifier: NCT01598831].• Cilengitide (InnovoSep): Represents a non-antibiotic approach, acting as prophylactic treatment to prevent sepsis progression [Medha Baranwal, 2019].Anti-Nitric Oxide Therapy:• NOS Inhibitors: Varied effects; non- selective inhibitors increased mortality in septic shock.546C88: Isoform-unselective NOS inhibitor significantly reduced survival in septic shock patients [Schwartz S et al., 1997].Pipeline of Sepsis Drugs:• Ongoing research emphasizes new antibiotics, anti-inflammatory agents, and anticoagulant drugs. Pan-specific vaccines and cytoprotective treatments for microvascular endothelium show promise.COVID- 19 increases sepsis risk, demanding diagnostic solutions. Hospitalized COVID- 19 patients are prone to coinfections, with over 50% having secondary infections, nearly 100% experiencing sepsis, and around 70% facing septic shock.Existing tests take up to 72 hours; the market sees a demand for rapid diagnostic tests for early sepsis detection [Grand View Research Report, Sepsis Diagnostics Market Size, Share & Trends Analysis Report by Product (Assay Kits & Reagents, Blood Culture Media), By Technology (Microbiology, Immunoassays), By Pathogen, By Method, By End-user and Segment Forecasts, 2022 - 2030. Report ID: GVR-1-68038-352-2],Sepsis has been a challenge for pharmaceutical companies in clinical trials, but new research offers hope for successful therapeutic techniques. Limited sepsis-specific therapy choices; current interventions involve monitoring, pathogen-targeting treatments, supportive care, and symptomatic treatment. Antibiotics help reduce mortality, but their limitations and patient variability suggest the need for combination therapies or dedicated drugs based on individual immune responses [Safiah Mai et al., 2012]. No immunomodulatory drugs have proven better efficacy than placebo in large trials, highlighting safety and tolerability challenges. Ongoing research aims to discover new treatment targets, with peptide anti-infective therapies showing promise in infection treatment.Key therapies in severe sepsis / septic shock aim at reversing immune response and cytokine-related pathogenic mechanisms [Brett I Kaplan et al., 2007]. Additional inflammatory mediators include complement C5 / C5a, HMGB1, adenosine A2 receptor, pro-apoptotic molecules, and TLRs [Meaad A. et al., 2021]. Anticytokine treatments facechallenges due to the complexity of sepsis and diverse patient populations. Advances in understanding innate immunity provide opportunities for therapeutic development, focusing on cytokine effects [Heming N, et al., 2016].Novel paradigms explore host-directed immunomodulatory treatments, leveraging natural host mechanisms to enhance therapeutic benefits. Innate defence regulator peptides and agonists of innate immunity components are considered potential immune modulators. Immunomodulation as adjunctive therapy supports antibiotics and antivirals, offering advantages by targeting the host rather than the pathogen [Robert E W Hancock et al., 2016],Reducing costs and increasing success rates are critical for industry engagement in sepsis therapeutic development. Ongoing preclinical and clinical assessments explore novel antisepsis strategies for more effective future treatments [Angus DC et al., 2001].The strategies mentioned fall into two categories: monotherapy and combination therapy. Combination therapy, due to its high delivery efficiency, local concentration, synergistic effect, and low resistance frequency, is more effective. The surge in antibiotic resistance presents a global public health threat, pushing humanity into a "post-antibiotic" era. The misuse of broad- spectrum antibiotics hastens resistance and disrupts vital gut flora [Bingqing Yang el al., 2021 and John C. Marshall et al., 2022]. Addressing antimicrobial resistance requires innovative antibacterial pipelines, including narrow-spectrum agents, probiotics, nanotechnology, phage therapy, and CRISPR-Cas9 technologies. Antibody generation against infection-causing pathogens, like Staphylococci and P. aeruginosa, is also explored [Amit Pant et al., 2021]. Combination therapy is favored for high efficiency, local concentration, synergistic effects, and low resistance frequency.Narrow-Spectrum Antimicrobial Agents: Peptides as Therapeutic AgentsBroad- spectrum agents, while effective against mixed infections, indiscriminately kill bacteria, disrupting microbiota balance and inducing superinfection. Novel antibiotics are sought to address multi-drug resistance and provide selective and innovative antiinflammatory therapies. Narrow- spectrum antimicrobial medicines, such as antibiotics, antimicrobial peptides (AMPs), and lysins, offer targeted therapy solutions. AMPs, cationic and amphipathic molecules are promising alternatives due to diverse action modes. Despite challenges like toxicity, recent designs, known as designed antimicrobial peptides (dAMP), show increased potency, efficacy, specificity, and reduced toxicity [Hancock RE, Rozek et al., 2000 and 2002].Antimicrobial Peptides (AMPs):AMPs serve as host defense against infections, representing a potential alternative to antibiotics.Cationic and amphipathic, AMPs interact with cell membranes, demonstrating potent antimicrobial, anti-biofilm, and immunomodulatory properties. Naturally occurring and synthetic AMPs show varied effectiveness, with designed antimicrobial peptides (dAMP) exhibiting increased potency and reduced toxicity [Chou et al., 2019]. Innovative approaches, like bacteria-targeting peptides nanoparticle - aided systems, magnetic nanoparticles (MNPs), and lipid-based nanocarriers (NLCs), enhance transport efficacy, plasma half-life, and treatment outcomes [Hussain et al., 2018; Kuo et al., 2016; Chia-Chih Liao et al., 2021].The pursuit of novel antibiotics and targeted therapies demonstrates a critical response to the growing antimicrobial resistance crisis. These innovative approaches aim to overcome challenges and provide effective solutions for the treatment of bacterial infections.Designed Antimicrobial Peptides (DAPs) encompass synthesized peptides showcasing extensive antibacterial activity, while those with synthetic peptides exhibiting immunomodulatory action are termed Host Defense Peptides (HDPs) [Bommarius B, Jenssen H et al., 2010 and Nicole J. Afacan et al., 2012]. Cationic HDPs, characterized by positively charged and hydrophobic residues, demonstrate broad-spectrum antimicrobial and immunomodulatory activities, making them potential candidates for innovative anti- infective therapies. LL-37 and defensins, for instance, influence innate immune cell functions, promoting chemokine and cytokine production, immune cell chemoattraction, angiogenesis, and wound healing, ultimately enhancing the survival of septic mice [Braff MH, Hawkins MA et al., 2005; Oren Z, Lerman JC et al., 1999 andTjabringaa GS et al., 2006],A notable synthetic peptide, clavanin-MO, displays potent antimicrobial and immunomodulatory properties both in vitro and in vivo [Silva, O. N et al., 2016]. Extensive research on the immunomodulatory aspects of HDPs has led to the development of synthetic peptides with enhanced immunomodulatory activities, often referred to as Innate Defense Regulator (IDR) peptides [Nijnik A, Yang D, Chertov O et al., 2001 andMookherjee N et al., 2007]. The immunomodulatory activities of HDPs and IDR peptides elucidate their effectiveness in treating microbial infections. The multifaceted biological activity of anti-infective peptides stems from their capacity to manipulate immune-cell function, exert direct antimicrobial activities, or a combination of both [Jesse M. Jaynes et al., 2012; and Mohamed F. Mohamed et al., 2016].Physical Characteristics and Therapeutic Potential of Host Defense Peptides (HDPs):Physical Characteristics:Host Defense Peptides (HDPs) typically range from 12 to 50 amino acids, featuring a prevalence of basic over acidic amino acids, resulting in a net positive charge of +2 to +9. About 50% of the amino acids are hydrophobic. The presence of basic and hydrophobic amino acids facilitates the folding of linear HDPs into amphipathic secondary structures upon interaction with lipid bilayers. There are four structural classes of HDPs based on their secondary structures:1. P-sheet (e.g., defensins)2. a-helical (e.g., cathelicidins, magainin)3. Looped peptides (e.g., bactenecin)4. Extended structures rich in arginine, glycine, histidine, proline, and / or tryptophan. The first two classes are more common than the latter two [Yang D et al., 2001].Therapeutic Potential:1. Anti-microbial Activity:HDPs exhibit broad-spectrum anti-infective activity through direct microbial killing.They neutralize endotoxins, recruit immune cells, modulate pro- cytokine / chemokine production, suppress harmful inflammation, induce cell differentiation, and enhance adaptive immune responses, cell survival, wound healing, and angiogenesis [Hancock RE, et al., 2006].2. Anti-endotoxin Activity:HDPs can reduce pro -inflammatory mediators such as tumor necrosis factoralpha (TNF-a) by inhibiting or regulating toll-like receptor (TLR) signaling pathways. Direct binding to lipopolysaccharide (LPS) also contributes to potent anti-inflammatory activity.3. Immunomodulatory Activities:HDPs enhance cell survival and polarize the adaptive immune system, demonstrating adjuvant activity.Selective immunomodulatory capabilities act against multi-drug resistant infections by boosting protective immunity while modulating excessive inflammation.Key signaling pathways involved in HDP immunomodulation include mitogen-activated protein kinases (MAPK) p38, JNK, ERK1 / 2, Src-family kinases, NF-KB, and PI3 kinase pathways [Opal SM, 2010].4. Attributes of Synthetic HDP Derivatives:Small size makes them potential prototypes for anti-infective therapeutics against multidrug-resistant (MDR) pathogens. Enhance chemokine production, induce chemotaxis, and block endotoxin responses via the innate immune system, reducing the likelihood of resistance.The multifunctional nature of these peptides makes them excellent candidates, especially in combination with other antimicrobial therapies, representing a new approach for clinical success as novel immunomodulatory drugs.In the subsequent sections, HDPs in preclinical development, clinical trials, and peptides identified as potential lead compounds are explored [Table 3].Table 3: Host Defense Peptides in clinical trials and potential lead peptides with described activityGaps and Challenges in Sepsis Research:I. Sepsis Therapy:1. Multi-Drag Resistance:Ongoing challenge in sepsis treatment due to increasing resistance among pathogens.2. Controversies in Pharmacologic Interventions:Existing therapies (Corticosteroids, Anti -endotoxin, pro-inflammatory, and Anticoagulant strategies) have controversies, reflecting treatment heterogeneity or unclear biologic redundancy.Targeting individual inflammatory mediators lacks proven efficacy, showing potential harm in septic patients [John C. Marshall et al., 2022],II. Sepsis Clinical Trials:1- Lack of Replication in Efficacy:Early efficacy of anti-sepsis strategies not consistently replicated in subsequent trials, highlighting challenges in improving clinical outcomes. Past trial failures attributed to the inclusion of non-selective patient groups; efforts needed for defining homogeneous septic patient groups.TIL Sepsis Preclinical Trials: Why Challenges Persist:1. Flaws in Preclinical Studies:Successful preclinical candidates for sepsis drugs fail to show efficiency in humans. Issues include inadequacies in animal models not mimicking human disease and flaws in experimental design and reporting.2- Heterogeneity in Animal Models:Animal models often lack representation of co-morbidities, appropriate insult types, duration, and supportive therapy .Differences in antibiotic administration in animal models compared to human sepsis add to disparities. Ideal preclinical models should standardize to mimic severe and chronic sepsis syndromes, considering clinical scenarios [Marcin F. Osuchowskieta / ., 2018].3. Current Animal Models :Toxaemia models (e.g., EPS injection), surgical models (e.g., CLP, CASP), and exogenous bacteria infusion. Lack of a single satisfactory preclinical model mimicking clinical complexity necessitates the use of two animal models according to regulatory guidelines. Standardization is crucial, with debates on the validity of LPS models and acknowledgment of CLP as a more appropriate, gold standard model.Addressing these gaps requires focused efforts in refining therapeutic strategies, enhancing trial methodologies, and standardizing preclinical models for better translation to clinical success [Stevens RD et al, 2009].As sepsis involves inflammation, research targets the innate immune response. Host defense peptides (HDPs) offer a novel anti-infective therapy by enhancing infectionresolving immunity, modulating innate immune responses, and dampening pro- inflammatory reactions in vitro and in vivo. The evaluation of novel synthetic HDPs for antimicrobial and immunomodulatory properties signifies a new approach to combat antibiotic-resistant infections. Sepsis outcomes and therapy responsiveness hinge on the complex interplay between host response and pathogen dynamics. Specific strategies, beyond antibiotics and standard support, are crucial. Immunomodulating agents, especially host defense peptides (HDPs), hold promise. Candidate treatments must undergo screening with appropriate animal models for translational success.Considering the challenges known in field for known treatment strategies of sepsis and identified research gaps, the present inventionprovides synthetic HDPs / IDR peptides which demonstrate antimicrobial and immunomodulatory properties, making them potential candidates for anti-infective therapeutics in sepsis treatment.Summary of inventionThe present invention studies anti-microbial, immunomodulatory and antiinflammatory activities of synthetic peptide IS217.The present invention aims to establish the mechanistic pathway of synthetic peptide IS217 for the treatment of sepsis in animal models that mimic human sepsis.Accordingly, the present invention provides in vitro activity of peptide IS217 for cytotoxicity and anti-inflammatory activity on murine macrophage RAW 264.7 cell lines stimulated with LPS.In another embodiment the present invention provides anti-toxicity and antiimmunogenicity of the test peptide IS217.In still another embodiment the present invention provides in vitro activity of test peptide IS217 for anti-microbial activity and time kill assays against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae and the inhibition of biofilm formation against S. aureus.In yet another embodiment the present invention provides confirmatory studies for in vitro and in vivo anti-inflammatory activity of test peptide IS 217 on murine macrophages stimulated with LPS and carrageenan paw edema model in mice respectively.In another embodiment the present invention provides in vivo efficacy activity of test peptide (IS217) using E. coli induced peritonitis animal model and Caecal Ligation Puncture (CLP) animal model.The inventor of the present invention conducted the following studies:1. Peptide Characterization:• Confirmation of mass and purity for three synthetic peptides using HRMS and HPLC.2. In vitro Studies:• Evaluation of cytotoxicity, selectivity index, and toxicity profiles through MTT assay.• Anti-inflammatory activity on RAW 264.7 macrophages stimulated with LPS, analyzedvza ELISA, western blotting, and RT qPCR.3. Safety and Immunogenicity Assessment:• Acute toxicity and immunogenicity studies on test peptide IS217.4. Confirmatory Anti-microbial and Anti-inflammatory Studies:• In vitro antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae using microplate broth dilution method.• Time kill kinetic studies for bactericidal effects and inhibition of biofilm formation against S. aureus.• In vitro anti-inflammatory studies on murine macrophages and carrageenan- induced paw edema in mice.5. In vivo Proof of Concept (PoC) Efficacy:• Tested peptide IS217, particularly the peptide (IS 217) in E. co / z-induced bacterial load and Cecal Ligation Puncture (CLP) animal models.• Administered two doses via subcutaneous (S.C) and intravenous (IV) routes.• Assessed survival rates and therapeutic efficacy through biochemical and clinical biomarker estimations in serum, peritoneal lavage fluid, and BALF using ELISA, western blotting, and histology (H&E staining).The present invention provides decapeptide IS217 of SEQ. ID NO 1 or peptide variant thereof for treatment, prevention, prophylaxis and / or amelioration of sepsis, one ormore symptoms and / or conditions associated with sepsis. The IS217 is a non-biologic, nonsteroidal and synthetic small peptide of 10 amino acids that is capable of exhibiting antimicrobial, immunomodulatory and anti-inflammatory activities. Studies have been conducted, and experiments have been performed as mentioned in the foregoing which establish that the decapeptide has antimicrobial, immunomodulatory and anti-inflammatory properties.This invention therefore, relates to IS217 (SEQ. ID NO 1) or peptide variant thereof and pharmaceutical formulation and combination comprising therapeutically effective amount of IS217 (SEQ. ID NO 1) or peptide variant thereof as a drug for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis. The present invention further relates to use of IS217 (SEQ. ID NO 1) or peptide variant thereof, formulations and / or combinations comprising therapeutically effective amount of IS217 (SEQ. ID NO 1) or peptide variant thereof for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.The IS217 (SEQ. ID NO 1) or peptide variant thereof and pharmaceutical formulation and combination comprising therapeutically effective amount of IS217 (SEQ. ID NO 1) or peptide variant thereof of the present invention are suitable as injectables, preferably intravenous or subcutaneous, more preferably as subcutaneous mode of administration. The formulations and / or combinations of the invention are easy to prepare and cost-effective.The formulations and / or combinations of present invention can include variant of IS217 peptide. The variant is a functionally active variant and may be obtained by changing sequence of IS217 and is characterized by having a biological activity similar to that displayed by IS217 of SEQ. ID NO.l from which the variant is derived. The variant includes anti-microbial, immunomodulatory and anti-inflammatory abilities of IS217. The functionally active variant of IS217 protein or domains thereof may be obtained by sequence alterations in sequence of IS217, wherein the peptide with the sequence alterations retains function of unaltered peptide. Such sequence alterations can include, but are not limited to, (conservative) substitutions, deletions, mutations and insertions. The variant can comprise at least 80% of the sequence of IS217, preferably at least 85%, still more preferably at least 90%, even more preferably at least 95% and most preferably at least 97%, 98% or 99%. The variant is derived from the IS217 by at least one amino acid substitution and / or deletion, wherein the functionally active variant has a sequence identityto IS217 of at least 80%, more preferably at least 85%, still more preferably at least 90%, even more preferably at least 95% and most preferably at least 97%, 98% or 99%. The variant of IS217 is functionally active in the context of the present invention, if the activity of the variant amounts to at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, most preferably at least 99% of the activity of IS217 without sequence alteration. The activity of the variant may be determined or measured as described in the examples and then compared to that obtained for IS217 of the present invention.In an embodiment the present invention provides a peptide of SEQ. ID NO 1 or peptide variant thereof for use in treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.In another embodiment the present invention provides an antimicrobial, immunomodulatory and anti-inflammatory pharmaceutical formulation for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis, said formulation comprising a peptide of SEQ. ID NO 1 or peptide variant thereof in an amount of from 0.01 pg / ml to 1000 pg / ml, preferably 0.5 pg / ml to 500 pg / ml and one or more suitable pharmaceutically acceptable excipients.In a still another embodiment the present invention provides that the formulation comprises one or more suitable pharmaceutically acceptable excipients are selected from the group consisting of suitable carriers, diluents, vehicles, disintegrants, swelling agents, antioxidants, buffering agents, bacteriostatic agents, emollients, emulsifiers, plasticizers, penetration enhancers, preservatives, cryoprotectants, neutralizers, fragrance additives, dispersants, surfactants, binders and lubricants.In a yet another embodiment the present invention provides that peptide variant of IS217 is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the SEQ. ID NO 1.In further embodiment the present invention provides that the formulation is an injectable formulation, preferably for subcutaneous or intravenous route of administration, and optionally wherein said one or more symptoms of sepsis are fever, low body temperature, chills, shivering, rapid breathing, difficulty in breathing, confusion and / or change in mental status, tachycardia, low blood pressure, skin rash, pain or discomfort, sweating, clammy skin, and optionally wherein said conditions associated with sepsis are acute lung injury, septic shock, acute kidney injury, sepsis-induced cardiomyopathy,weakened immune system, sepsis-induced thrombocytopenia, hyperglycemia, hypoglycaemia, sepsis-induced coagulopathy, acute respiratory distress syndrome and sepsis-induced hypoperfusion.In still further embodiment the present invention provides a formulation for treatment, prevention, prophylaxis and / or amelioration of inflammation in a subject in need thereof, comprising a therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof, wherein said formulation:(i) inhibits or reduces expression of one or more of inflammatory markers selected from p38 kinases, IL-ip, IL-6, IL-12, TNF-a, IL-12P40, CCL2, VEGF, TLR4, IL-12p70, NO, TLR7, TLR8, TLR9 and GAPDH reduction in phosphorylation of ERK1 / 2;(ii) is capable of one or more of antimicrobial activity;(iii) completely or partially reduces release of at least one pro-inflammatory cytokines;(iv) downregulates phosphorylation of p38 mitogen activated protein kinase (MAPK); and / or(v) completely or partially increases release of at least one anti-inflammatory marker.In yet further embodiment the present invention provides an antimicrobial, immunomodulatory, anti-inflammatory pharmaceutical combination for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis, said combination comprising a therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof in an amount from 0.01 pg / ml to 1000 pg / ml, preferably 0.5 pg / ml to 500 pg / ml and one or more additional active agent, and wherein said additional active agent is selected from one or more of antibiotic agent, one or more of anti-inflammatory agent and one or more of immunosuppressant agent, preferably wherein said other active agent is selected from dexamethasone, lipopolysaccharide, ciproflaxin, penicillin, streptomycin or cefoxitin; and wherein said one or more additional active agent is in an amount of 0.01 pg / ml to 1000 pg / ml.In another embodiment the present invention provides that a method for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject comprising administering to the subject in need thereof a therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof of present invention.In yet another embodiment the present invention provides that the peptide or theformulation of the present invention may comprise a peptide variant which is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the SEQ. ID NO 1.In still another embodiment the present invention provides that a subject is administered from 0.1 pg / ml to 1000 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereof.In further embodiment the present invention provides that a subject is administered from 0.5 pg / ml to 500 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereofIn another embodiment the present invention provides that one or more symptoms of sepsis are fever, low body temperature, chills, shivering, rapid breathing, difficulty in breathing, confusion and / or change in mental status, tachycardia, low blood pressure, skin rash, pain or discomfort, sweating and clammy skin; and wherein conditions associated with sepsis are acute lung injury, septic shock, acute kidney injury, sepsis-induced cardiomyopathy, weakened immune system, sepsis-induced thrombocytopenia, hyperglycemia, hypoglycaemia, sepsis-induced coagulopathy, acute respiratory distress syndrome and sepsis-induced hypoperfusion.In an embodiment the present invention provides that peptide of SEQ. ID NO 1 or peptide variant thereof is suitable as an injectable, preferably by subcutaneous or intravenous route of administration.In further embodiment the present invention provides that the peptide of SEQ. ID NO 1 or peptide variant thereof is administered to the subject one to three times per day, one to three times per week or one to three times per month.In still further embodiment the present invention provides use of a peptide of SEQ. ID NO 1 or peptide variant thereof for manufacture of a medicament for the treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.In another embodiment the present invention provides a kit for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, said kit comprising a peptide of claim 1, and one or more of additional therapeutically active agent, wherein the additional therapeutically active agent is at least one antibiotic agent, at least one immunomodulatory agent and at least one anti-inflammatory agent.In yet another embodiment the present invention provides a product comprising a peptide of present invention, and one or more of additional therapeutically active agent, as acombined preparation for separate, simultaneous or sequential use in the treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, wherein the additional therapeutically active agent is at least one antibiotic agent, at least one immunomodulatory agent and at least one anti-inflammatory agent.In still another embodiment the present invention provides a method comprising administering a therapeutic formulation comprising therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof to a subject, wherein said subject is suffering from sepsis, one or more symptoms or conditions associated with sepsis, and wherein administering said therapeutic formulation reduces one or more symptoms of sepsis or conditions associated with sepsis.In further embodiment the present invention provides that administering a therapeutic formulation comprising therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof to a subject reduces or inhibits release of at least one pro- inflammatory cytokine; reduces or inhibits expression of at least one inflammatory marker; downregulates phosphorylation of p38 mitogen activated protein kinase (MAPK); exhibits antimicrobial effect; and / or increases release of at least one anti-inflammatory marker in said subject.In still further embodiment the present invention provides use of a peptide of SEQ. ID NO 1 or peptide variant thereof for manufacture of a medicament for the treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.In yet further embodiment the present invention provides use of formulation of present invention for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, comprising administering to said subject therapeutically effective amount of said formulation.In another embodiment the present invention provides use of a peptide of SEQ. ID NO 1 or peptide variant thereof in a method for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, wherein the method comprises administering to the subject in need thereof therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof.In a further embodiment the present invention provides a method of treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, comprising administering to said subjecttherapeutically effective amount of formulation of present invention.In a still further embodiment the present invention provides that in the method of treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, the subject is administered from 0.1 pg / ml to 1000 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereof.In a yet further embodiment the present invention provides that in the method of treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, the subject is administered from 0.5 pg / ml to 500 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereof.In another embodiment the present invention provides that in the method of treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, said one or more symptoms of sepsis are fever, low body temperature, chills, shivering, rapid breathing, difficulty in breathing, confusion and / or change in mental status, tachycardia, low blood pressure, skin rash, pain or discomfort, sweating and clammy skin; and wherein conditions associated with sepsis are acute lung injury, septic shock, acute kidney injury, sepsis-induced cardiomyopathy, weakened immune system, sepsis-induced thrombocytopenia, hyperglycemia, hypoglycaemia, sepsis-induced coagulopathy, acute respiratory distress syndrome and sepsis-induced hypoperfusion.In yet another embodiment the present invention provides that in the method of treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, said peptide of SEQ. ID NO 1 or peptide variant thereof is suitable as an injectable, preferably by subcutaneous or intravenous route of administration.In still another embodiment the present invention provides that in the method of treatment, prevention, prophylaxis and / or amelioration of sepsis one or more symptoms and / or conditions associated with sepsis in a subject, the peptide of SEQ. ID NO 1 or peptide variant thereof is administered to the subject one to three times per day, one to three times per week or one to three times per month.In an additional embodiment the present invention provides a method of treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, comprising administering to said subject therapeutically effective amount of formulation of the present invention.Description of the drawingsFigure 1 illustrates pathogenic mechanisms from infection to septic shock, modified from Jon A Buras et al., 2005.Figure 2 illustrates pathophysiological changes of sepsis due to infection.Figure 3 illustrates SIRS and CARS in sepsis.Figure 4 illustrates two stages of sepsis.Figure 5 illustrates clinical presentation of sepsis.Figure 6 (a,b) illustrates step wise procedure followed in studies conducted for testing in vitro activity.Figure 7 illustrates picture representation of microdilution assay.Figure 8 illustrates collection of peritoneal macrophages.Figure 9 illustrates schematic representation of two most used sepsis models.Figure 10 illustrates step-wise procedure of the Cecum Ligation and Puncture (CLP) model, (a) Setup of the surgery table; (b) IP injection of anesthesia; (c) confirmation of anesthesia by touch; (d) shaving the surgery part of the mice; (e) placing the mice on the surgery table and disinfecting the surgical part of the mice; (f) skin incision; (g) locating and exposing the cecum; (h) and (i) ligated the cecum; (j) puncturing the cecum with a 20- gauge needle; (k) extruding the cecal matter / suspension; (1) replacing the cecum back into peritoneal sac of the mice; (m) and (n) skin closing suturing steps; (o) after the suturing; (p) applying the betadine to the surgical part of the mice; (q) and (r) placing the mice on the heating pad for maintaining the body temperature and recovery of the anesthesia; and (s) post operated surgical mice after recovery from anesthesia.Figure 11 illustrates study design and treatment regimen in CLP modelFigure 12 (A-C) illustrates schematic representation of three trials and inter-trial intervals in NORT.Figure 13 illustrates collection of peritoneal lavages from the mice [Meurer SK,2016].Figure 14 illustrates confirmation of presence of E. coli strain on a UTI (Urinary Tract Infection) chromogenic agar plate. E. coli strain was used for causing sepsis symtoms in animal studies. E. coli formed pink to red colonies due to fermentation of lactose and production of P-glucuronidase.Figure 15 illustrates study design and treatment regimen in E. co / z-induced septic peritonitis.Figure 16 illustrates HRMS chromatogram of peptide IS 217.Figure 17 illustrates HPLC chromatogram of peptide IS 217.Figure 18 illustrates 3D Structure of test peptide IS 217.Figure 19 illustrates chemical structure of test peptide IS217.Figure 20 illustrates morphological change in macrophage RAW 264.7 cells, (a -f) LPS- treated with test peptide IS 217 (3.12, 6.25, 12.5,25,50 and 100 pg / mL, respectively; (g) control / untreated; (h) LPS-(1 pg / mL) treated only; and (i) LPS-treated with dexamethasone (500 pg / mL).Figure 21 (a, b) illustrates IL-ip (a) and IL-6 (b) secretion measured using ELISA. Data is presented as fold change to LPS stimulated and un-stimulated (control) cells. Data were shown as the means ± SD. Each experiment was repeated in triplicate. ****p < 0.001, ***p< 0.01, **p < 0.05, in comparison to LPS group. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 22 (a, b) illustrates TNF-a (a) and IL- 10 (b) secretion measured using ELISA.Data is presented as fold change to LPS stimulated and un-stimulated (control) cells. Data were shown as the means ± SD. Each experiment was repeated in triplicate. ****p < 0.001, ***p< 0.01, **p < 0.05, in comparison to LPS group. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 23 (a, b, c) illustrates protein levels of IL- 10 (a, c), and TNF- a (b, c) evaluated by western blot.Figure 24 (a.b, c) illustrates protein levels of TLR -4 (a, c) and VEGF (b, c) evaluated by western blot.Figure 25 (a, b) illustrates protein levels of P38 (a) and ERK1 / 2 (b) evaluated by western blot. Data is presented as fold change to LPS stimulated and un-stimulated (control) cells. Data were shown as the means ± SD. Each experiment was repeated in triplicate. ****p < 0.001, ***p < 0.01, **p < 0.05, in comparison to LPS group. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 26 illustrates effect of test peptide IS 217 on TNF- a mRNA expression detected by RT-qPCR.Figure 27(a, b) illustrates effect of test peptide (IS 217) on IL-6 (a) and IL-ip (b) mRNA expression detected by RT-qPCR. Data is presented as fold change to LPS stimulated and un-stimulated (control) cells. Data were shown as the means ± SD. Each experiment was repeated in triplicate. ****p < 0.001, ***p < 0.01, **p < 0.05, in comparison to LPS group. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 28 illustrates effect of test peptide (IS 217) on CCL2 mRNA expression detected by RT-qPCR. The test peptide inhibits LPS-induced pro-inflammatory cytokine expression at the mRNA level. Data is presented as fold change to LPS stimulation and unstimulated (control) cells. Data were shown as the means ± SD. Each experiment was repeated in triplicate. ****p < 0.001, ***p < 0.01, **p < 0.05, in comparison to LPS group. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 29 illustrates HRMS chromatogram of peptide: IS 217 +0.9% NaCl solution at 0 h.Figure 30 illustrates HRMS chromatogram of peptide: IS 217 +0.9% NaCl solution at 2- 8° after 24h.Figure 31 illustrates HRMS chromatogram of peptide: IS 217 +0.9% NaCl solution at room temperature after 24h.Figure 32 illustrates IL-ip (a) and TNF-a (b) cytokines level detection in vivo. BALB / C mice were treated with IS217 peptide at the dose of 0.1ml per animal (corresponding to 0.6 mg / kg and 1.2 mg / kg, respectively). Serum samples were collected 48 h after IV injections and applied to the ELISA assay. p< 0.01 and denoted *** as data were considered statistically significant.Figure 33 illustrates time-kill kinetics of IS 217 against S. aureus. Results shown in Table 38 are Mean + SEM obtained from triplicate experiments where P < 0.005, P< 0.05 and denoted as *** &** and ### & ## when compared with standard drug and vehicle control groups, respectively.Figure 34 illustrates time-kill kinetics of IS 217 against P.aeruginosa .Results shown in the Table 38 are Mean + SEM obtained from triplicate experiments where P < 0.005, P< 0.05 and denoted as *** &** and ### & ## when compared with standard drug and vehicle control groups, respectively.Figure 35 illustrates time-kill kinetics of IS 217 against E. co / z.Results shown in the Table 38 are Mean + SEM obtained from triplicate experiments where P < 0.005, P< 0.05 and denoted as *** &** and ### & ## when compared with standard drug and vehicle control groups, respectively.Figure 36 illustrates time-kill kinetics of IS 217 against K.pneumonia. Results shown in the Table 38 are Mean + SEM obtained from triplicate experiments where P < 0.005, P< 0.05 and denoted as *** &** and ### & ## when compared with standard drug and vehicle control groups, respectively.Figure 37 illustrats effect of IS 217 on 48 h old biofilms of S. aureus. The effect of peptide IS 217and antibiotic (Ciprofloxacin) on 48 h old biofilms of S. aureus. Results shown in the table are Mean ± SEM obtained from triplicate experiments where P < 0.005, P< 0.05 and denoted as *** &** and ### & ## when compared with standard drug and vehicle control groups, respectively.Figure 38 illustrates IL- 10 cytokines level detection in vitro in mice macrophages. The values represent the means of at least three independent experiments performed in triplicate (mean ± SEM). A significant difference from the control (LPS alone) was indicated as P< 0.001, P< 0.01 and denoted as **** &***, respectively.Figure 39 (a, b, c and d) illustrates TNF-a (a), IL-6 (b,) IL-ip (c) and IL-12p70 (d) cytokines level detection in vitro in mice macrophages.Figure 40 illustrates production of NO levels in vitro in mice macrophages. Effects of IS 217 on NO levels in LPS -stimulated RAW264.7 macrophages. The nitrite concentration was analysed using Griess reagent. The values represent the means of at least three independent experiments performed in triplicate (mean ± SEM). A significant difference from the control (LPS alone) was indicated as P< 0.001, P< 0.01 and denoted as ***&**, respectively.Figure 41 (a,b,c and d) illustrates effect of test peptide (IS 217) on TNF-a (a), IL-6, (b) IL-ip (c) and IL-12p70 (d) cytokines expression was detected by RT-qPCR. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM). A significant difference from the control (LPS alone) was indicated as P< 0.001, P< 0.01 and denoted as *** &**, respectively. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 42 illustrates effect of test peptide (IS 217) on IL- 10 cytokine expression was detected by RT-qPCR.Figure 43 illustrates effects of IS 217 on iNOS expression.Expression of the iNOS protein was determined by Western blotting analysis. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM). A significant difference from the control (LPS alone) was indicated as P< 0.001, P< 0.01 and denoted as *** &**, respectively. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 44 illustrates effects of IS 217 on the expression of p-Erk in LPS -stimulated RAW264.7 cells. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM). A significant difference from the control (LPSalone) was indicated as P< 0.001, P< 0.01 and denoted as *** &**, respectively. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 45 illustrates effects of IS 217 on the expression of p-p38 in LPS- stimulated RAW264.7 cells. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM). A significant difference from the control (LPS alone) was indicated as P< 0.001, P< 0.01 and denoted as *** &**, respectively. Bars indicate means and vertical lines standard error of mean of three independent experiments analyzed in duplicate.Figure 46 illustrates representative photographs of carrageenan-induced inflamed mice paws treated with test peptide IS 217 of two doses 0.6 and 1.2 mg / kg and with dexamethasone (5 mg / kg body weight) at 18 h: (a) control, (b) Disease control, (c) IS 217 - 0.6mg / kg, (d) IS 217 -1.2 mg / kg, (e) Dex-5mg / kg.Figure 47(a) illustrates % inhibition of paw edema measured by the volume displacement in carrageenan-induced inflamed mice paws. Figure 47(b) illustrates % increase in paw volume in carrageenan-induced inflamed mice paws treated with test peptide IS 217 of two doses 0.6 and 1.2 mg / kg and with dexamethasone (5 mg / kg body weight). Data was expressed as mean ± SEM (n=6). A significant difference was indicated as P< 0.01, P< 0.05 and denoted as *** &**, respectively, when compared with standard drug dexamethasone at different time intervals.Figure 48 illustrates that IS 217 administrations protect against established CLP sepsis. BALB / c mice were subjected to sepsis (CLP) and 2 h later treated with IS 217 0.6 mg / kg and 1.2 mg / kg respectively. Survival was monitored for up to 5 days. The results are the mean ± SD of (n=5) animals *P <0.02 and *** P < 0.05 compared to sham control group.Figure 49(a,b,c and d) illustrates quantification of IL-ip (a) , IL-6 (b) ,TNF-a (c) and IL- 12 (d) in serum was performed 24 h after the CLP.Figure 50 illustrates quantification of IL- 10 in serum performed 24 h after the CLP.The results were expressed as the mean +SEM (n=4-5 animals / group). P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 51(a,b) illustrates therapeutic administration of IS 217, IV, improves survival in septic mice. Survival was monitored at regular intervals until 18h(a) and 10 d (b) and survival curves were plotted (a and b).Data are shown as mean ± SEM of 5-6 mice in each group. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted 1as ***, **&*anj ###, ##&# compared to disease control and sham control groups, respectively.Figure 52(a,b) illustrates therapeutic administration of IS 217, SC, improves survival in septic mice. Survival was monitored at regular intervals until 18h (a)and 10 d (b)and survival curves were plotted (a and b). Data are shown as mean ± SEM of 5-6 mice in each group. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&*anj ###, ##&# compared to disease control and sham control groups, respectively.Figure 53 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) administration on liver and kidney functions of mice after CLP surgery. The results represent means ± SEM of 5-6 mice / group. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 54 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -SC) administrations on liver functions of mice after CLP surgery. Treatment with IS 217 ameliorates organ injury induced by CLP. IS 217-0.6 & 1.2 mg / kg, SC was injected into mice 2 h after CLP. At 18 h after CLP, mice were killed, and the amounts of AST, and ALT in sera were measured.Figure 55 illustrates serum lactate levels after the treatment with IS 217 -0.6 and 1.2 mg / kg -IV at 18h of post CLP. Data are shown as mean ± SEM of 5-6 mice in each group. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ### ,##&# compared to disease control and sham control groups respectively.Figure 56 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on serum TNF-a levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 4h, 18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 57 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -SC) on serum TNF-a level in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h and 10 days post-CLP challenge via SC route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 58 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on serum IL-6 levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 4h, 18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001,P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 59 illustrates effect of IS 217 (0.6and 1.2 mg / kg -SC) on serum IL-6 levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h and 10 days post-CLP challenge via SC route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 60(a) illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on serum IL-ip levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at (a) 4h, 18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively. Figure 60(b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg - SC) on serum IL-ip levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h and 10 days post-CLP challenge via SC route. Data are expressed as mean ± SE M. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 61 illustrates effect of IS 217 (0.6 and 1.2 mg / kg-IV) on serum IL- 12 levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 4h, 18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 62 illustrates effect of IS 217 (0.6 and 1.2 mg / kg-SC) on serum IL- 12 levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h and 10 days post-CLP challenge via SC route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 63 illustrates effect of IS 217 (0.6 and 1.2 mg / kg-IV) on serum IL- 10 levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 4h, 18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 &p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 64 illustrates effect of IS 217 (0.6 and 1.2 mg / kg-SC) on serum IL- 10 levels in CLP induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h and 10days post-CLP challenge via SC route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 65(a) illustrates effect of IS 217 (0.6 and 1.2 mg / kg-IV) on serum VEGF levels in CLP induced sepsis. Mice (n =5- 6 / group) and VEGF levels estimated at 4h, 18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively. Figure 65(b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg- SC) on serum VEGF levels in CLP induced sepsis. Mice (n =5- 6 / group) and VEGF levels were estimated at 18 h post-CLP challenge via SC route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 66 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on TNF-a levels in the bronchoalveolar lavage fluid. Mice (n =5- 6 / group) and TNF-a levels were estimated at 4h and 18 h post-CLP challenge via IV route.Figure 67 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on IL-6 levels in the bronchoalveolar lavage fluid. Mice (n =5- 6 / group) and IL-6 levels were estimated at 4h and 18 h post-CLP challenge via IV route.Figure 68 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on VEGF levels in the bronchoalveolar lavage fluid. Mice (n =5- 6 / group) and VEGF levels were estimated at 4h and 18 h post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 69 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -SC) on IL- 10 levels in the bronchoalveolar lavage fluid. Mice (n =5- 6 / group) and IL- 10 levels were estimated at 4h and 18 h post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 70 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -SC) on IL-6, TNF-a and IL- 10 levels in the bronchoalveolar lavage fluid. Mice (n =5- 6 / group) and IL-6, TNF-a and IL- 10 levels were estimated at 18 h post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant anddenoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 71(a, b) illustrates peptide IS 217 (0.6 and 1.2 mg / kg via IV (a) and SC (b) treatment on the lung wet-to-dry weight ratio and protein concentration in the BALF of CLP -induced sepsis mice. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 72(a, b) illustrates peptide IS 217 (0.6 and 1.2 mg / kg) via IV (a) and SC (b) treatment on sepsis induced pulmonary MPO activity. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 73 illustrates protein expression of total and phosphor ERK * and p38 in lung tissue. The data was repeated by duplicate independent experiments. The results are pooled samples of each group.Figure 74 illustrates macroscopic pictures of spleen of all groups of mice -18h after CLP surgery.Figure 75 illustrates effects of IS 217 (0.6 and 1.2 mg / kg -IV) treatments on changes in spleen relative organ weight of mice-18h after CLP surgery. The results represent means ± SEM of 5-6 mice / group. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 76 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on TNF-a levels in the spleen homogenate. Mice (n =5- 6 / group) and TNF-a levels were estimated at 4 h,18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 77 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on IL-ip levels in the spleen homogenate. Mice (n =5- 6 / group) and IL-ip levels were estimated at 4 h,18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 78 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on IL-6 levels in the spleen homogenate. Mice (n =5- 6 / group) and IL-6 levels were estimated at 4 h,18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 &p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 79 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on TNF -a levels in the peritoneal fluid of CLP induced sepsis. Mice (n =5- 6 / group) and TNF -a levels were estimated at 4 h,18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 80 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on IL-6 levels in the peritoneal fluid of CLP induced sepsis. Mice (n =5- 6 / group) and IL-6 levels were estimated at 4 h,18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 81 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on IL-ip levels in the peritoneal fluid of CLP induced sepsis. Mice (n =5- 6 / group) and IL-ip levels were estimated at 4 h,18 h and 10 days post-CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 82 illustrates effect of IS 217 (0.6 and 1.2 mg / kg-SC) on IL-6, TNF -a and IL- 10 levels in the peritoneal fluid of CLP induced sepsis. Mice (n =5- 6 / group) and IL-6, TNF - a and IL- 10 levels were estimated at 18 h post-CLP challenge via SC route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 83 (a,b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg-IV (a) and SC(b) on procalcitonin levels in the peritoneal fluid of CLP induced sepsis. Mice (n =5- 6 / group) and procalcitonin levels were estimated at 18 h post-CLP challenge via IV and SC route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&#compared to disease control and sham control groups, respectively.Figure 84 illustrates total and differential blood cell counts from mice treated with IS 217 (0.6 and 1.2 mg / kg-IV) of CLP induced sepsis and from sham and CLP induced mice. Mice(n =5- 6 / group) and total and differential blood cell counts were estimated at 18 h post- CLP challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* and ###, ##&# compared to disease control and sham control groups, respectively.Figure 85 illustrates photographs of representative sections of liver (a) and lungs (b) sections were prepared and stained with H&E, visualized at 200X magnification are shown. -IV route -18 h of post CLP.Figure 86 (a,b,c&d) illustrates production of cytokines. Quantification of TNF-a (a), IL-6 (b), IL-ip (c) and IL- 10 (d) in serum was performed 24 h after the E. coli infection. The results were expressed as the mean +SEM (n=4-5 animals / group).Figure 87 illustrates experimental mice shows signs of infection at 18 h post E. coli ATCC 8739™ (5.0 X108E. coli CFU / per mouse).Figure 88 (a,b) illustrates therapeutic administration of IS 217, IV, improves survival in septic mice. Survival was monitored at regular intervals until 18h (a) and 7 d (b) and survival curves were plotted. Data are shown as mean ± SEM of 5-6 mice in each group. Significance in differences of survival percentages were assessed by a log rank test for Q trend. A significant difference from the disease control (E. coli'. 5X10 CFU / per mouse) was indicated as *P< 0.05, **P< 0.01, or ***P< 0.001. Figure 88(c) illustrates therapeutic administration of IS 217, IV, improves survival in septic mice in comparison with standard drug (Cefoxitin). Survival was monitored at regular intervals until 18h and survival curves were plotted. Data are shown as mean ± SEM of 5-6 mice in each group. Significance in differences of survival percentages were assessed by a log rank test for trend. A significantQ difference from the disease control (E. coli: 5X10 CFU / per mouse) was indicated as *P< 0.05, **P< 0.01, or ***P< 0.001.Figure 89 (a,b) illustrates therapeutic administration of IS 217, SC, improves survival in septic mice. Survival was monitored at regular intervals until 18h (a) and 7 days (b) survival curves were plotted. Data are shown as mean ± SEM of 5-6 mice in each group. Significance in differences of survival percentages were assessed by a log rank test for Q trend. A significant difference from the disease control (E. coli: 5X10 CFU / per mouse) was indicated as *P< 0.05, **P< 0.01, or ***P< 0.001.Figure 90 (a,b,c&d) illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV and SC) administration on liver (a,b) and kidney functions (c,d)of mice after E.coli infection. At 18 h after infection induction, mice were killed, and the amounts of AST, ALT, Bil, and creatinine, in sera were measured by both routes of administration (IV&SC). The resultsrepresent means ± SEM of 5-6 mice / group. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control groups, respectively.Figure 91 (a,b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV(a) and SC(b) administration on BUN levels of mice after E.coli infection. At 18 h after infection induction, mice were killed, and the amounts of BUN in sera were measured. The results represent means ± SE of 5-6 mice / group. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control 1 groups, respectively.Figure 92 (a,b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV (a) and SC(b)) administration on serum CRP levels of mice after E.coli infection. The results represent means ± SE of 5-6 mice / group. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group, respectively.Figure 93 illustrates effect of IS 217 (0.6 and 1.2 mg / kg-IV) on serum TNF-a levels in E. coli induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h and 7 days post-E. coli infection via IV route and data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 94 (a,b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg-IV) on serum IL-6 (a) and IL-ip (b) levels in E.coli induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h and 7 days post- E. coli infection via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 95 (a,b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg-IV) on serum IL- 12 (a) and IL- 10 (b) levels in E.coli induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h and 7 days post-E. coli infection via IV route. Data are expressed as mean ± SEM. P <0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 96 (a,b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg -SC) on serum IL-6, IL-ip , TNF-a (a)and IL-10 (b) levels in E.coli induced sepsis. Mice (n =5- 6 / group) and cytokine levels were estimated at 18 h post- E. coli infection via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 97 illustrates effect of IS 217 (0.6 and 1.2 mg / kg) via IV route on TNF-a levels in the bronchoalveolar lavage fluid. Mice (n =5- 6 / group) and TNF-a levels were stimated at 4 h and 18 h post-E. coli infection.Figure 98 (a,b) illustrates effect of IS 217 (0.6 and 1.2 mg / kg) via IV route on IL-6 and IL- 10 levels in bronchoalveolar lavage fluid. Mice (n =5- 6 / group) and IL-6 and IL- 10 levels were estimated at 4 h and 18 h post-E. coli infection. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group, respectively.Figure 99 illustrates effect of IS 217 (0.6 and 1.2 mg / kg) via SC route on TNF-a, IL-6 and IL- 10 levels in the bronchoalveolar lavage fluid. Mice (n =5- 6 / group) and TNF-a, IL-6 and IL- 10 levels were estimated at 18 h post-E. coli infection. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group, respectively.Figure 100 illustrates peptide IS 217 (0.6 and 1.2 mg / kg) via IV treatment on the lung wet- to-dry weight ratio and protein concentration in the BALF of E. coli infection -induced sepsis mice. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 101 illustrates effect of peptide IS 217 (0.6 and 1.2 mg / kg) via IV treatment on sepsis induced pulmonary myeloperoxidase (MPO) activity. The MPO activity was estimated in lung homogenates from sepsis mice in the different treatment groups. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 102 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on TNF -a, IL-6 and IL-ip levels in the peritoneal fluid of E. coli infection induced sepsis. Mice (n =5- 6 / group) and TNF -a, IL-6 and IL-ip levels were estimated at 18 h post- infection challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P,<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 103 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on IL- 10 levels in the peritoneal fluid of E. coli infection induced sepsis. Mice (n =5- 6 / group) and IL- 10 levels were estimated at 18 h post-infection challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 104 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -SC) on TNF -a, IL-6 and IL-P levels in the peritoneal fluid of E. coli induced sepsis. Mice (n =5- 6 / group) and TNF -a, IL-6 and IL-P levels were estimated at 18 h post-CLP challenge via SC route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 105 illustrates effect of IS 217 (0.6 and 1.2 mg / kg -IV) on procalcitonin levels in the peritoneal fluid of E. coli induced sepsis. Mice (n =5- 6 / group) and procalcitonin levels were estimated at 18 h post-E. coli infection challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P<0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 106 illustrates total and differential blood cell counts from mice treated with IS 217 (0.6 and 1.2 mg / kg) of E. coli - induced sepsis. Mice (n =5- 6 / group) and total and differential blood cell counts were estimated at 18 h post-E. coli infection challenge via IV route. Data are expressed as mean ± SEM. P < 0.001, P,0.01 & p< 0.05 was considered as statistically significant and denoted as ***, **&* compared to disease control group.Figure 107 illustrates photographs of representative sections of liver (a) and lungs (b) sections were prepared and stained with H&E; visualized at 200X magnification are shown. Data shown in mean +SEM from 3-4 mice of all groups of E. coli induced sepsis, treatments, and control animals-IV route -18 h.Figure 108(a) illustrates various mechanisms of action of antimicrobial peptides [adapted from Ulm, H et al., 2012]. MN: polymorphonuclear neutrophils; ADP: adenosidediphosphate; ATP: adenoside triphosphate. Figure 108(b,c) illustrates proposed mechanism of action of anti-microbial activity of peptide IS 217. Figure 108(d) illustrates proposed mechanisms of peptide IS 217 by activating the MAPK signaling pathway, decreasing / inhibiting TNF-a, IL-ip and IL-6 production. TLR, toll like receptor; ERK, extracellular signal-related kinases; p38, p38 mitogen-activated protein kinase.Figure 109(a,b) illustrates proposed mechanism / effect of IS 217 on cytokine storm and role of IS 217 in the treatment of sepsis.Description of inventionFollowing below are more detailed descriptions of various concepts related to, and embodiments of, inventive peptide, methods, use, medicament, compositions and formulations configured for treatment, amelioration, prevention and / or prophylaxis of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject in needthereof.“Treating” or “treatment” of a disease or condition includes:(1) preventing the disease or condition, i.e. causing the clinical symptoms of the disease or condition not to develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease or condition;(2) inhibiting the disease or condition, i.e., arresting or reducing the development of the disease or condition or clinical symptoms thereof; or(3) relieving the disease or condition, i.e., causing regression of the disease or condition or clinical symptoms thereof.A “therapeutically effective amount” means the amount of peptide or peptide variant thereof, formulation or combination of the present disclosure that, when administered to a patient for treating a disease or condition, is sufficient to effect such treatment for the disease or condition. The “therapeutically effective amount” will vary depending on the formulation, the symptom, the condition and severity of the disease, symptom or condition, and age, weight, etc., of the mammal to be treated.Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed by the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed by the invention, subject to any specifically excluded limit in a stated range. Where a stated range includes one or both of the endpoint limits, ranges excluding either or both of those included endpoints are also included in the scope of the invention.As used herein, “exemplary” means serving as an example or illustration and does not necessarily denote ideal or best.A “pharmaceutically acceptable additive or excipient” means an additive or an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non toxic and neither biologically nor otherwise undesirable, and includes an additive or an excipient that is acceptable for veterinary use as well as human pharmaceutical use. “A pharmaceutically acceptable additive / excipient” as used in the specification and claims includes both one and more than one such excipient. The pharmaceutically acceptable excipients are selected from the group consisting of suitable carriers, diluents, vehicles, disintegrants, swelling agents, antioxidants, buffering agents, bacteriostatic agents, emollients, emulsifiers, plasticizers, penetration enhancers, preservatives, cryoprotectants,neutralizers, fragrance additives, dispersants, surfactants, binders and lubricants.The present invention provides synthetic peptide LS217 or peptide variant thereof, and formulations, combinations and / or compositions thereof. The present invention relates to antimicrobial, immunomodulatory and anti-inflammatory effects of the synthetic peptide IS217 or peptide variant thereof, formulations, combinations and / or compositions thereof. The formulations, combinations and / or compositions of synthetic peptide IS217 or peptide valiant thereof demonstrate significant reduction in mortality, improved organ function, and protection against sepsis-induced damage. Accordingly, the present invention provides the synthetic peptide IS217 or peptide valiant thereof and the formulations, combinations and / or compositions thereof as a potential therapeutic for sepsis.The present invention provides a synthetic peptide IS217 of SEQ. ID NO 1 or peptide variant thereof for use in treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.The present invention provides antimicrobial, immunomodulatory and antiinflammatory pharmaceutical formulation for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis, said formulation comprising a peptide IS217 of SEQ. ID NO 1 or peptide variant thereof in an amount of from 0.01 pg / ml to 1000 pg / ml, preferably 0.5 pg / ml to 500 pg / ml and one or more suitable pharmaceutically acceptable excipients.The formulation or combination of the present invention is an injectable formulation, preferably for subcutaneous or intravenous route of administration.The formulation or combination of the present invention is administered to the subject one to three times per day, one to three times per week or one to three times per month.One or more symptoms of sepsis may include, but are not limited to, fever, low body temperature, chills, shivering, rapid breathing, difficulty in breathing, confusion and / or change in mental status, tachycardia, low blood pressure, skin rash, pain or discomfort, sweating and clammy skin.Conditions associated with sepsis include, but are not limited to, acute lung injury, septic shock, acute kidney injury, sepsis-induced cardiomyopathy, weakened immune system, sepsis-induced thrombocytopenia, hyperglycemia, hypoglycaemia, sepsis-induced coagulopathy, acute respiratory distress syndrome, sepsis-induced hypoperfusion.The peptide IS217 or peptide variant thereof, formulation or combination of present invention inhibits or reduces expression of one or more of inflammatory markers selectedfrom p38 kinases, IL-ip, IL-6, IL-12, TNF-a, IL-12P40, CCL2, VEGF, TLR4, IL-12p70, NO, TLR7, TLR8, TLR9 and GAPDH reduction in phosphorylation of ERK1 / 2.The peptide IS217 or peptide variant thereof, formulation or combination of present invention is capable of one or more of antimicrobial activity.The peptide IS217 or peptide variant thereof, formulation or combination of present invention completely or partially reduces release of at least one pro -inflammatory cytokine.The peptide IS217 or peptide variant thereof, formulation or combination of present invention downregulates phosphorylation of p38 mitogen activated protein kinase (MAPK).The peptide IS217 or peptide variant thereof, formulation or combination of present invention completely or partially increases release of at least one anti-inflammatory marker.The present invention provides an antimicrobial, immunomodulatory, antiinflammatory pharmaceutical combination for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis, said combination comprising a therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof in an amount from 0.01 pg / ml to 1000 pg / ml, preferably 0.5 pg / ml to 500 pg / ml and one or more additional active agent.The additional active agent in the combination of the present invention is selected from at least one antibiotic agent, at least one anti-inflammatory agent and at least one immunosuppressant agent, preferably wherein said additional active agent is selected from dexamethasone, lipopolysaccharide, ciproflaxin, penicillin, streptomycin or cefoxitin; and wherein said one or more additional active agent is in an amount of 0.01 pg / ml to 1000 pg / ml.The peptide of SEQ. ID NO 1 or peptide variant thereof can be used for manufacture of a medicament for the treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.The present invention provides a kit for treating or preventing sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, said kit comprising a peptide of SEQ. ID NO 1 or peptide variant thereof, and an additional therapeutically active agent, wherein the additional therapeutically active agent is an antibiotic agent, immunomodulatory agent or an anti-inflammatory agent.The present invention provides a product comprising a peptide of SEQ. ID NO 1 or peptide variant thereof, and an additional therapeutically active agent, as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention ofsepsis, one or more symptoms or conditions associated with sepsis in a subject, wherein the additional therapeutically active agent is antibiotic agent, immunomodulatory agent or an anti-inflammatory agent.The present invention provides a method comprising administering a therapeutic formulation comprising therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof to a subject, wherein said subject is suffering from sepsis, one or more symptoms or conditions of associated with sepsis, and wherein administering said therapeutic formulation reduces one or more symptoms of sepsis or conditions associated with sepsis.The present invention provides that therapeutic formulation is administered prophylactically or therapeutically to a subject at risk for sepsis or presenting symptoms of sepsis.The present invention provides that administering of therapeutic formulation to a subject reduces release of at least one pro-inflammatory cytokines; downregulates phosphorylation of p38 mitogen activated protein kinase (MAPK); and increases release of at least one anti-inflammatory marker in said subject.The present invention further provides a method of treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, comprising administering to said subject therapeutically effective amount of formulation of the present invention.The present invention additionally provides a method for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject comprising administering to the subject in need thereof a therapeutically effective amount of peptide of SEQ. ID NO I or peptide variant thereof of the present invention.The present invention still further provides that in a method for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis, a subject is administered from 0.1 pg / ml to 1000 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereof, preferably from 0.5 pg / ml to 500 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereof.The present invention additionally provides that in a method for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis, peptide of SEQ. ID NO 1 or peptide variant thereof is suitable as an injectable, preferably by subcutaneous or intravenous route of administration;and wherein the peptide of SEQ. ID NO 1 or peptide variant thereof is administered to the subject one to three times per day, one to three times per week or one to three times per month.1.1: Details of peptide IS217The simplified peptide sequence allows for rapid, cost-effective production and quicker clinical application. The synthetic peptide IS217, particularly IS 1217 (SEQ ID NO: 1- Phe-Ala-Lys-Lys-Phe-Ala-Lys-Lys-Phe-Lys of the present invention), a ten-amino- acid synthetic P-sheet peptide with broad-spectrum antibacterial and immunomodulatory properties, demonstrates great potential. One -Letter code and three-letter code for IS217 is FAKKFAKKFK and Phe-Ala-Lys-Lys-Phe-Ala-Lys-Lys-Phe-Lys, respectively. IS217 has molecular weight (Mr) of 1242.57 g / mol; isoelectric point of 11.2; net charge at pH 7.0: 5.0; average hydrophilicity: 0.6 and ratio of hydrophilic residues / total number of residues is 50%. The peptide IS217 of present invention has chemical and 3-D structure as provided in Figures 18 and 19.1.2 Materials and methods1.2.1 MaterialsDetails of reagents, cells, microbial cultures and antibodies, etc. used for conducting experimental studies in the present invention along with sources thereof are provided in Table 4 below. List of instruments and source thereof used for conducting experimental studies in the present invention is provided in Table 5 below.Table 4: List of reagents and antibodiesTable 5: List of instruments used in experimental studies of present invention1.2.2: Methods1.2.2.1: Confirmation of Mass and purity of synthetic test peptide:The test peptides (peptide code: IS 217) was synthesized by Issar Pharmaceuticals Pvt. Ltd., (Telangana, India). The peptide was synthesized manually in a stepwise manner at a 0.1 - 10 mmol scale on a using A-Fmoc (A-fluorenylmethyloxycarbonyl) solidphase peptide synthesis strategy (multichannel peptide synthesizer) and where the peptide of >10 -200 mmol were synthesized by automated peptide synthesizer. Peptide IS217 was purified by preparative reversed-phase HPLC (Agilent 1200) using with a C-18 coated, 10-micron bead column (Phenomenex Jupiter Cl 8, 10 «m, 300 A, 250 x 10 mm) using a gradient of 0.1% TFA in water (Mobile phase A) and 100% acetonitrile (ACN) (Mobile phase B)and characterized by RP-HPLC chromatography and MALDLTOF mass spectrometry at In - house. The molecular weights were confirmed by mass spectrometry HRMS -LCMS. The purity was about 93- 95% as determined by analytical HPLC. The peptide IS217 used in all biological assays was having higher than 90% purity.All commercially available chemicals and solvents of synthesis grade were used without further purification. The qualitative ninhydrin test was performed for each step to confirm completion of coupling. Purification was done by using reverse phase chromatography. The purity of the final compounds was examined by HPLC, (on Phenomenex C8 (150 * 4.6 mm, 5 pm, 100 A) double end-capped RP-HPLC column)) and was greater than 95%.1.2.2.2: In vitro anti-inflammatory activity of peptide IS 217 against LPS-induced inflammation in RAW 264.7 cellsThe methods for in vitro anti-inflammatory activity of peptide IS 217 against LPS- induced inflammation in RAW 264.7 cells, includes cytotoxicity and anti-inflammatory assays performed as depicted in Figure 6.1.2.2.2.1: Cell line cultureThe murine macrophage RAW264.7 cells were recovered from the stock and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% low- endotoxin heat-inactivated Fetal Bovine Serum (FBS), 2 pM glutamine, 100 U / ml penicillin, and 100 pg / ml streptomycin and cultured in a humidified atmosphere at 37°C under 5% CO2 until the cells were confluent around 70-80%. The growth medium wasfrequently replaced after 2-3 days. The cells were then washed and harvested using trypsin- EDTA. Cell culture has been done according to the methods described by Novilla et al. 2017; Rashad A. Al-Salahi et al., 2013 and Amit Kumar et al., 2021.1.2.2.2.2: Preparation of synthetic test peptide -IS217For in vitro experiments, the synthetic test peptidelS 217 was dissolved and diluted in 0.9% sterile normal saline (NS) for further concentrations used in the assay.1.2.2.2.3: Study designThe anti-inflammatory activities of IS217 were evaluated in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells. For establishment of in vitro model, lipopolysaccharide (LPS, Ipg / mL) was used to treat the cells for 18 h. RAW 264.7 cells were seeded at a density of 1 x 105cells / ml, either into 24 / 6-well plates for cytokines measurement by ELISA, western blot, and RT-PCR, or in 96-well plates for the cell viability assay. The experimental design is summarized in Figure 6. The microdilution assay representation and serial dilution of antimicrobial agents in bacterial culture are illustrated in Figure 7.1.2.2.2.3.1: Cytotoxicity test -MTT assayCell viability of synthetic peptide IS217 on growth of RAW264.7 cells was measured using the methyl thiazolyl tetrazolium (MTT) assay after 48 hours. Briefly, exponentiallygrowing macrophagesRAW264.7 cells were mechanically scraped, plated at a Q density of 7x10 cells in 100 pl were seeded into each well of a 96-well plate then incubated for 12 hours at 37°C under 5% CO2. The cells were then treated with test peptide: IS 217 (1.56, 3.12, 6.25, 12.5, 25, 50 and 100 pg / mL) or dexamethasone (500 ng / mL).The treated cells were incubated at 37 °C in the presence of 5% CO2 for 42 hours. After incubation, the morphology of cells was examined under a microscope. Following incubation, MTT was added to the cells (20 pL at 5 mg / mL MTT dissolved in lx PBS) and incubated for 4 hours in the presence of 5% CO2 at 37 °C. Formazan crystals were dissolved by the addition of 100 pL of 100% DMSO per well. Plates were then gently swirled for 5 minutes at room temperature to dissolve the precipitate. Absorbance was monitored at 575 nm using a microplate reader. Maximum cytotoxicity (100%) was determined by cells incubated with 1% Triton X-100; PBS was used as a negative control.Cells without treatment were considered as control. The average absorbance for each sample group was used to evaluate cell viability.Optical density (O.D.) was calculated as the difference between the absorbance at the reference wavelength and that at the test wavelength. The relative cell viability was determined by the amount of MTT converted to the insoluble formazan salt. The data were expressed as the mean percentage of viable cells when compared with untreated cells (control). The half maximal growth inhibitory concentration (IC50) value was calculated from the line equation of the dose-dependent curve of each compound. The results were compared with the cytotoxic activity of dexamethasone, a known anti-inflammatory drug. The concentrations of test peptide IS217 that showed significant (p < 0.001) cell viability was further selected for in vitro assays. Percentages of cell viability and LC50 of cells were calculated: Percent cell viability was calculated as (O.D. of drug-treated sample / control O.D.) xlOO. The data were expressed as percent cell viability compared with control (dimethyl sulfoxide / NS).The experiment was carried out in four samples of each concentration.The percentages of cell viability and LC50 of cells were calculated by using the formula below.% cell viability = Absorbance sample x 100Absorbance controlWhere Absorbance control is the absorbance of cells treated with DMSO 0.1% and Absorbance sample is the absorbance of cells treated with test sample.LC50 values will be determined from the dose-response curves. Data are to be expressed as a means of three independent experiments. Statistical analyses will be performed using Student’s z-test and one-way ANOVA.1.2.2.2.3.2: Anti-inflammatory test in LPS -stimulated RAW264.7 cellsThe methods of in vitro anti-inflammatory assay have been done according to the methods described by Laksmitawati et al., 2017; Rusmana et al., 2015; Sandhiutami et al., 2017 and Widowati et al., 2018].Cell Culture and LPS Stimulation:Cells were sub-cultured by scraping when plates reached 70-80% confluence with a 1:5 ratio in fresh medium. RAW 264.7 cells were plated at density of 1 x 105cells and allowed for attachment. The growth medium was then replaced with fresh medium withoutFBS and incubated for 6 hours then cells were pre-treated with different concentrations of synthetic peptide: IS 217 (3.12, 6.25, 12.5, 25, 50 and 100 pg / mL) or dexamethasone (500 ng / mL) as a positive control for 1 hour followed by stimulation of LPS (1 pg / mL) and then further incubated for 18 hours and were allowed to adhere. After 18 hours of incubation, plates were centrifuged for 6 minutes at 400x g and the supernatants and cell lysates were collected and kept frozen at - 80 °C until used for further analysis.At the end of the treatment period, cells were harvested in RNA isoplus reagent for subsequent cellular RNA extraction, for RTqPCR. The pro and anti -inflammatory cytokine production in supernatants was estimated by using ELISA, RTqPCR and western blotting in cell lysates. The experiment was carried out in triplicate. LPS and Dexamethasone were used as the controls.For analysis, samples of cells or culture supernatant were obtained after 18 hours of treatment.The following treatments were applied for the anti-inflammatory test:(1) The negative control: RAW 264.7 cells without being induced by lipopolysaccharide; no drug treatment.(2) The positive control: RAW264.7 cells that were induced by 10 pg / mL of lipopolysaccharide (LPS group).(3) The mixture of Synthetic peptide: IS 217(3.12, 6.25, 12.5, 25, 50 and 100 pg / ml), LPS (1 pg / mL) and RAW 264.7 cells; and(4) The standard positive control: The mixture of dexamethasone (500 pg / mL), LPS (Ipg / mL), and RAW 264.7 cells.Measurement of pro and anti -inflammatory cytokine markers;The levels of IL-6, TNF-a, IL-ip, and IL- 10 in the supernatants of RAW 264.7 cell cultures were determined using an ELISA kit, according to the manufacturer’s instructions (R&D Systems).Western blot analysis:As previously described, RAW264.7 cells were cultured and treated with LPS and synthetic peptide IS217. After 18 hours, cells were washed with PBS and lysed by incubating with RIPA lysis buffer cocktail (RIPA Lysis buffer, PMSF (ImM), Protease inhibitor (Ipg / mL), phosphatase inhibitor (ImM)) at -80°C for 30 minutes. Post incubation, cell lysate was collected by scraping cells and centrifuged at 12000 rpm for 15 minutes.The protein concentration was determined using the Bicinchoninic acid kit method with bovine serum albumin as the standard (provided in the kit), according to the manufacturer’s instructions. Protein bands were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane via semi-dry transfer blotting, Membranes were blocked with 5% BSA in PBST (1% Tween 20 in PBS) at 20-24 °C for 1 hour, then incubated with primary antibodies against IL- 10, TLR 4, VEGF, P38 p-p38,ERK and p- ERK, the dilutions are 1: 1000., at 4°C overnight. GAPDH was used as a protein-loading control. The membrane was washed 3 times (10 minutes each) with Tris-buffered saline / Tween 20 (TBST) and then treated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10000) for 1 hour. The membrane was washed again with TBST. The membrane -binding antibodies were visualized later with the ECL detection reagent and images were acquired in a Fusion SL Imaging System. Band density was analyzed by densitometry using Image J software.Reverse transcrip tion-polymerase chain reaction:Total RNA extraction, purification, and cDNA synthesis: Total cellular RNA was isolated using the RNA isoplus reagent (Takara Bioscience, India) following the manufacturer’s instructions. RNA was quantified spectrophotometrically by absorption measurements at 260 and 280 nm using the nanodrop system. cDNA synthesis steps: As per Tables 6 and 7, RNA and the reaction mixture were mixed in a tube and incubated in a thermal cycler for 5 minutes at 65 °C and then cooled immediately on ice. Further, in the same tube, the step 2 reaction mixture was added and further incubated in the thermal cycler as mentioned below.Table 6: cDNA synthesis procedure Step-1Table 7: cDNA synthesis procedure Step-2The ingredients mentioned in the Tables 6 and 7 were mixed gently to obtain a reaction mixture. The reaction mixture was incubated using the following conditions:30°C - 10 minutes (required when using Random 6 mers)42°C (50°C) - 30-60 minutesThe enzymein reaction mixture was inactivated by incubating at 95°C for 5 minutes and then cooled on ice. After cDNA synthesis, samples were stored at -80°C till used for RT-qPCR.RT-qPCR: Primers were designed using the Primer3 online tool. GAPDH was used as a normalizing control. The sequence of primers used is provided in below Table 12. Real-Time PCR was performed according to the manufacturer’s protocol using Takara biosystem's real-time PCR mix (Takara, Bio-India) with ROX as a passive reference dye using applied Biosystem’s step-one Real-Time PCR system. The following PCR program was used for all real-time PCR-based experiments: initial denaturation at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, extension at 60°C for 15 seconds.Real-time PCR was performed with steponeplus Real-Time PCR system (Thermo Scientific). PCR reactions were performed in 20-pL reactions with SYBR Green Realtime PCR Master Mix (Toybo, Osaka, Japan). The primer sequences used for PCR amplification are as depicted in the Table 8 below.Table 8: Primer Sequences used in the RT-qPCRGene expression analysis: For quantification of gene expression, CT values of each gene were normalized to GAPDH and calibrated to the appropriate control sample using the SYBR Green-based comparative CT method (2-AACt). Fold change was calculated using the following formula to determine relative gene expression between two samples:2-AAct = 2-AACt2A{-\Delta\Delta Ct}2-AACtwherein AACt is the difference in ACt values between two different samples or conditions, calculated by subtracting ACt of a control sample (often a calibrator or baseline sample) from ACt of experimental sample. AACt quantifies the fold change in gene expression between the experimental condition and the control condition. The base of logarithm used in the formula is taken as 2. In qPCR analysis, gene expression levels are often reported as 2 raised to the power of -AACt. Therefore, 2-AACt2A{-\Delta\Delta Ct}2-AACt calculates fold change in gene expression between two samples, with a result of 1 indicating no change, less than 1 indicating downregulation, and greater than 1 indicating upregulation of the gene of interest in the experimental sample compared to the control.1.2.2.2.3: Statistical analysisAll the experiments were performed in three replications, and data were expressed as the mean ± standard deviation (SD). Differences in mean values between groups were analyzed by a one-way analysis of variance followed by Dunnett's-test for comparisons between two independent groups. P<0.001, P<0.01& P<0.05 was considered to indicate a statistically significant difference and denoted as ****, *** and**, respectively. Graphs were plotted using Graph-pad prism-8.2 / 9.0.1.2.3: Stability of IS 217 in 0.9% NaCl at different conditions:Stability testing of the test peptide IS 217 was conducted by dissolving Img of IS 217 in ImL of 0.9% NaCl (Normal saline) freshly prepared and was kept under refrigerated (2-8°C) conditions and room temperature under light-resistant conditions. Stability testing of a 50% solution was determined that the solution was stable for at least 24hours. The exact mass of the pre-formulation IS 217 of initial and 24 hours assay kept at room temperature and refrigerated (2-8°C) conditions will be performed using HRMS as mentioned in Table 9.Table 9: Test sampling details1.2.4: Acute toxicity and ImmunogenicityWhile developing novel therapeutic proteins / peptides, preventing immunogenicity, and establishing the acute toxicity profile are important issues to consider and the animal models are used to study immunogenicity prediction and acute toxicity of therapeutic proteins. The acute toxicity studies were conducted in compliance with the guideline of Organization for Economic Cooperation and Development (OECD) and schedule Y Guidelines for acute oral toxicity and previous work [OECD401,402,423and425; SCHEDULEY, DCGI -CDSCO document] with some modifications.Details of synthetic test peptide (IS 217): The testpeptide IS 217 with a peptide content of 95% was supplied by Issar Pharmaceuticals Pvt.Ltd and was stored at -20°C until usage. During the study period, IS 217 was stored in a refrigerator (2-8°C) under light- resistant conditions. For the acute toxicity studies, the required amount of peptide IS 217 was weighed and dissolved and diluted in 0.9% sodium chloride as a vehicle. A 100% (wt / vol) solution was prepared for the acute toxicity study.Animals and maintenance: A total of 40 BALB / C and 40 C57BL / 6 male and female mice (6-8 weeks old at the beginning of the experiments) were used for toxicity and additional 15BALB / C male mice are used for immunogenicity assessments. Animals were weighing between 20 and 35 g aged 6-8 weeks, were obtained from Jeeva Life Sciences (Registered breeder: 1757 / PO / RcBiBt / S / 14 / CPCSEA), and the studies are carried and approved by Birla Institute of Technology and Science-Pillai (BITS-Hyderabad), IAEC Protocol number BITS-Hyd / IAEC / 2020 / 17), as specified by the Committee for Control And Supervision of Experiments on Animals (CCSEA) (Table 10). The animals were randomized and grouped and identified by marking by ear-punching after group assignment.The animals were acclimated to the laboratory conditions for 7 days prior to dosing, during which body weights and general conditions were observed. During the acclimation period, 4-5 mice were housed per cage, whereas during the study period, 2 animals were caged (acute study). The mice were housed in polypropylene cages and allowed free access to food and water, ad libitum and were maintained under specifically regulated environmental conditions (rearing conditions): 12-hour light / dark cycle, temperature 24°C + 2°C, humidity 55% + 10%, and ventilation 11 to 15 air exchanges perhour (all-fresh-air system). All the waste materials were disposed of in a safe and sanitary manner after performing the experiments.1.2.4.1: Experimental procedure of acute toxicity studyTable 10: Test systemOnce after acclimation period, a total of eighty mice for two routes of administration, fortymice (half male and half female) / route were randomized based on bodyweight divided into four groups (10 in each group / per route, in each group 5 per sex) per strain via two routes of administration (SC and IV); prior to dosing initiation (Table 11). On the day of dosing (designated as Day 0 of the study), the mice were 6 weeks old, and their body weights ranged from 20 to 25 g. The dosing volume was 0.1ml per mice and the actual volume of peptide IS 217 solution administered was calculated based on body weight measurements obtained on the day of administration and was administered SC and IV in increasing doses of the synthetic test peptide IS 217 0.6, 2.4, and 4.8 mg / kg; (administration once). The routes of administration were the same proposed for use in humans [Food and Drug Administration, 1988]. Prior to dosing the animals were deprived of food for 12 hours but had free access to drinking water. Food was provided 3 hours after dose administration and was available ad libitum thereafter.Table 11: Allocation of BALB / C and C57BL / 6 mice, for IVand SC administrationAfter a single dose administration, mortality and clinical signs associated with toxicity were observed and recorded daily for consecutive two weeks; body weight changes were measured before and after administration on the 14th day. The animals were observed frequently during the first 4- 6-hour period following dose administration and once daily, thereafter, for 7 days, during which animals were evaluated for mortality, clinical signs of toxicity, and behavior. Each animal was weighed on Day 0 (prior to dosing) and on day 7 during the observation period. At the end of the observation period, on day 14, after being weighed, animals were fasted for 12 hours (free access to water) and anesthetized with ketamine (80-100 mg / kgBW) and xylazine in a dose (5-10 mg / kg) intraperitoneally (for 20 g mice ketamine of 0.2ml and xylazine of 0.1ml) as in combination the anesthetized with 0.2 ml via, IP. Blood samples were collected from orbit into nonheparinized Eppendorf tubes for the determination of serum biochemical parameters. Then animals were euthanized with CO2 Inhalation / isoflurane, and a complete necropsy was performed. The main organs of the thoracic and abdominal cavities and the brain were examined macroscopically for gross abnormalities. The number of animals killed for each of the doses was noted and the LD50 calculated by the Up and Down method, which is one of the most used to reduce the number of animals used [P.A.Botham, 2004]. Some vital organs comprising brain, heart, lungs, liver, spleen, and kidney were harvested and weighed. Organs collected from animals were preserved in formalin solution (10%, pH7.4) for the further histopathologic examination [P.A. Botham. 2004].Relative organ weight was calculated according to the following formula:Relative organ weight (g) = organ weight / body weight x 100Functional Observation: The mortality, general health status, and toxic reaction symptoms of the experimental groups were documented. Before the first day of IS 217 administration and during the experiments, a detailed clinical observation was carried out and recorded the changes in skin, eyes and mucous membrane, respiratory system, nervous system, activity, and behaviors of the rats. The body weight was measured and recorded before the first day of administration.Biochemical analysis: Laboratory analysis of biochemical parameters was performed on serum samples. Serum aspartate transaminase (AST), alanine transaminase (ALT), urea, creatinine and BUN were performed in automated biochemical analyzer (AU2700 Beckman coulter chemistry analyzer (Beckman Coulter, Brea, CA, USA).1.2.4.2; In vivo ImmunogenicityIn acute toxicity, as a part of immunogenicity activity investigation, total 15 mice are randomized into three groups of each 5 / group which received 0.6 mg.kg, 1.2 mg.kg respectively as treatment groups and mice (n=5) received 10 pg LPS were used as a positive control. The blood was collected after 48 hours via retro orbital plexus in nonheparinized tubes using ketamine and xylene in saline solution anesthesia vialP injection and the sera was separated as mentioned above in the acute toxicity and stored at -80°C until for further cytokines analysis (TNF-a and IL-ip) [Julia Suhorutsenko et al., 2011]1.2.4.3: Statistical analysisFor the acute toxicity study, the body weight data measured on Days 1, 7 and 14 were subjected to statistical assessment using one-way analysis of variance was performed. All parameters measured were analyzed statistically, apart fromthe general condition of the mice and findings of the macroscopic and histopathological examinations. The results were expressed as mean +SEM of the groups and significance difference between groups was evaluated by using ANOVA. If ANOVA shows significant differences, post hoc analysis was performed with Dunnett’s test. The differences were considered statistically significant when p < 0.01 and denoted *** as data were analyzed using Graph Pad Prism 8.2 / 9.0 software.1.2.5. In vitro confirmatory studies for anti-microbial and anti-inflammatory activityI.2.5.I. In vitro evaluation of anti-microbial activityThe following experimental study was conducted to determine the spectrum of antimicrobial activity of synthetic test peptide IS 217:The anti-microbial activity was assessed by broth microdilution time-kill viability assays and inhibition of bio film assays.Microbial strains:The following were used as test bacteria: Staphylococcus aureus subsp. aureus (ATCC® 6538™), Pseudomonas aeruginosa (ATCC® 9027™), Escherichia coli (ATCC® 8739™) and Klebsiella pneumoniae subsp. Pneumoniae (ATCC® 700603™).Growth media:The bacteria cultures were maintained in cryotubes at -80°C in 15% glycerol. A singlecolony of bacteria were refreshed in Mueller Hinton broth and incubated for 12 hours at 37°Cand inoculated on Mueller Hinton agar plates for purity check. Overnight-cultured bacterialcells were transferred to MH medium and cultured to the exponential phase (optical density at 600 nm OD600 = 1.0). The culture was centrifuged and resuspended in sterile phosphate buffered saline (PBS) and adjusted to a final amount of 1X105CFU ml'1by using the equation CFU mF1> OD600 l-2.5x 108[Evelien Gerits et al., 2016]. The number of colony-forming units was determined, and the bacterial cultures were adjusted to 5 X 104cfu / mL. Plates were prepared under aseptic conditions.Peptide Details:The peptide used here (IS 217) was synthesized by Issar Pharmaceuticals Pvt.Ltd., (India) and the lyophilized peptide of lOmg vials was supplied and stored at -20°C until usage. For in vitro and in vivo experiments, IS 217 was dissolved in 0.9% normal saline while preparing stock and working solutions. The peptide purity used in biologic assays was higher than 90-95%.Determination of the minimal inhibitory concentration (MIC): Microdilution assayMinimum inhibitory concentration (MIC) of peptide and antibiotic was evaluated Q using the broth microdilution technique in BHI with an initial inoculum of l-2.5x 10 cells in non-treated polystyrene microtiter plates (Corning, USA) as described by [Wiegand et al., and Banfi et al., 2003]. The MICs were interpreted as the lowest concentration of peptide or antibiotic that completely inhibited the visible growth of bacteria after 24 hours of incubation at 37 °C.IS217 MICs against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027 and Klebsiella pneumoniae ATCC 700603 were determined using a standardized broth microdilution assay according to CLSI reference methods for bacteria [Clinical Laboratory Standards Institute 2006]. All thebacterial culturesgrowth, propagation, preservationwas done by following the ATCCbacterial culture guidebook and Stock solution (10 mg / ml)and Dilution series (0.195, 0.391, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 pg / ml) of the test compound IS 217 were prepared in 0.9% NaCl solution in micro-test tubes from where they were transferred to 96-well microtiter plates. Then, 100 pL of each dilution were transferred into a 96-well microplate in 3 x 8 columns. Bacterial suspension (100 pL) was inoculated in each well with 1-2.5 x 10 CFU / mL of all test organisms to obtain final concentrations of 2.5 x 10 CFU / mL and a final volume of 200 pL per well. The inoculum (positive control) and culture medium (negative control) were put into the first column of the microplate, and the ciproflaxin antibiotic control ranging from 0.5 and 1.0 pg / ml in the final column.Finally, the microplate was incubated with a sterile film cover for 24 hours at 37°C. Bacterial growth was detected by optical density using ELISA reader, Spectra Max M5 multi-detection reader and checked the OD600. The results were expressed in micrograms per milliliters. The MIC was defined as the lowest concentration of antibacterial agent that resulted in the complete inhibition of visible growth. The bacterial growth was indicated by the presence of turbidity meaning. Three independent experiments were performed, each with three biological replicates. Ciproflaxin was used as a standard antibacterial agent. Finally, microplate was incubated with a sterile film cover for 18-24 hours at 37°C Subsequently, 20 pL of 4% resazurin bacterial growth indicatorwas added to wells, which were then incubated for 30 minutes at 37 °C. The lowest concentration of essential oil that visually showed no growth was determined as MIC. The MIC was determined as the lowest tested concentration that leads to complete inhibition (100%) in comparison to the negativecontrol group.Antibacterial activity was expressed as the concentration of extract inhibiting bacterial growth by 50% (IC50)[MounyrBalouiri et al., 2016].Time-Kill assay:The survival of bacteria, time kill efficacy assay of IS 217 was further evaluated for activity against bacterial strain mentioned above according to CLSI reference method, with slightly modification. MICs were determined by broth microdilution assay as described above. The test compound IS 217 was incorporated into 4.9 ml Mueller Hinton Broth (MHB) at concentrations of 0.5 x MIC, 1 x MIC and 5 x MIC for each bacterial species. Test tubes of MHB without test compound IS 217 were used as vehicle controls. Overnight Q cultures of the bacterial strains at cell densities of approximately 1 x 10 CFU / ml were used to inoculate both test and control tubes. The standard tube dilution method was opted to evaluate the time-kill efficacy of bacteria. Bacterial inoculum (1 mL) was diluted by adding9 mL sterile saline solution and serially diluted up to 10 . On testing the bacterial dilution _0(10 ), bacterial colonies were observed in a discrete form which was easily countable. One mL of 10 bacterial cultures were then incubated with an equal amount of IS 217in a shaker at 37°C for 1, 2, 3, 4, 5, 12 and 24 hours.100 pL incubated suspension was transferred on the agar plates and spread through the spreader. Colony counts were performed after 24 hours incubation at 37°C. Plates with 10-300 colonies were used for these counts, and the kill rate was determined by plotting log 10 viable counts (CFU / ml) against time. Bactericidal activity was defined as a > 3 loglO decreases in CFU / ml of the initial microbial population, while bacteriostatic activity was defined was defined as a < 3 log 10 decrease in CFU / ml. The assay was performed in triplicate [Olufunmiso O Olajuyigbe et al., 2015 andSutthiwanThammawat et al., 2017].Efficacy of IS 217on Staphylococcus aureus biofilms:The efficacy of IS 217 to disrupt biofilms was conducted as follows, Briefly, the isolates of S. aureus (ATCC 6538) were grown overnight and diluted 1 : 100 in TSB + 1% Q glucose were inoculated with approximately 1 x 10 CFU / mL of S. aureus and incubated in 96-well plates at 37 °C for 48 hours. After removing media, wells were rinsed with PBS to remove planktonic bacteria before re-filling wells with fresh MHB daily. On the fourth day, peptide and antibiotic were added at desired concentrations to the matured biofilms and the plates are incubated at 37 °C for 48 hours. After incubation, the wells are washed, and biofdms are stained with 0.5% (w / v) crystal violet for 30 minutes. The dye was solubilized with ethanol (95%) and the optical density (OD) of biofilms was measured. Bacterial growth was detected by optical density. The results were expressed in micrograms per milliliters. The reaction mixture was read spectrophotometrically at 595nm. Inhibition mediated reduction of biofilm formation was calculated by the following formula. [Monroe D. 2007; 43. Hall-Stoodley et al., 2004 andStewart, P.S. et al., 2001]% of inhibition = OD in control -OD in treatment x 100OD in controlStatistical analysis:The experimental data were performed in triplicate and expressed as mean ± SEM. The proportions of cells surviving at different time points were calculated, as the mean and standard deviation. Proportions of surviving cells were compared using Student’s 2-tailed t- test, assuming unequal variance and differences were considered significant where P<0.005, P< 0.05 and denoted as *** &** and###&##when compared with standard drug and vehicle control groups respectively. The data were analyzed following Analysis of Variance (ANOVA) using graph pad prism 8.2 / 9.0.1.2.5.2: Invitro anti-inflammatory activity in mice macrophagesBALB / c mice (6-8 weeks old) of either sex was injected IP with 1 ml of 4% sterile thioglycollate broth in PBS. After 4 days, mice were sacrificed, and peritoneal macrophages were harvested as described below[Meurer SK , 2016] and this model is well known as ^Thioglycollate-induced peritonitis mode . The steps of method for collection of peritoneal macrophages are as illustrated in Figure 8.Preparation of peritoneal macrophages:After 4 days, mice werethen euthanized and the peritoneal macrophageswere harvested by lavaging the peritoneal cavity with 5-6 mL of harvest medium (EDTA 5mM + PBS). Isolated peritoneal exudate cells were washed twice with RPMI and centrifuged at 1000 rpm / 400xg for 840 minutes at 4 °C. Finally, the cell suspension was dispensed in complete RPMI- 1640 with 10% fetal bovine serum and allowed to adhere to the bottom of the 6 cm culture plate at 37 °C for 4 hours in 5% CCh- The plates were then washed with warm PBS to remove non-adherent cells. The attached cells were considered as macrophages with 90% purity [Meurer SK , 2016].Cell culture and treatments:Macrophages were seeded either in 96- well plates at a density of 0.2 X 106or in 24-well plates at a density of 0.8 X 106and were treated with different concentration (3.125, 6.25 and 12.5 pg / mL) of test peptide IS 217 for 1 hour and then activated with 1 mg / ml of bacterial LPS (Sigma- Aldrich) and were cultured Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 0.1 mg / ml streptomycin and maintained under sterile conditions of 37°C, 5% CO2, and 85% humidity. After 24 hours, culture supernatants were collected for estimation of cytokines by ELIZA and RTqPCR, whereas cells were harvested for western blotting. The procedure of ELISA, RTqPCR and other conditions are same as in the section 4.2.2.3[MinJee Kim et al., 2014].Western blot analysis:The cells were trypsinized, washed twice with phosphate buffered saline (PBS), and lysed with lysis buffer (modified RIPA buffer) at 4 °C. The pellet cellular debris was removed by centrifugation at 12500 rpm for 30 minutes and the supernatants were then either analyzed immediately or stored at -80 °C. Protein concentrations were measured by BCA protein assay kit (Pierce, Rockford, IL, USA). Lysates in sample buffer (2% SDS, 10%glycerol, 80mM Tris-base, 720mM DL-dithiothreitol, and 0.001% bromophenol blue) were denatured at 95 °C for 5 minutes. Equivalent amounts of protein (25 pg) from total cell lysates were subjected to SDS -polyacrylamide gel electrophoresis (PAGE) and the proteins were transferred onto polyvinylidene difluoride (PVDF) membrane. Nonspecific binding was blocked by soaking the membrane in Tris-buffered saline (TBS, 20mM Tris-base, and 300mM NaCl) containing 5% fat-free milk for 1 hour. The membrane was incubated with primary antibodies (antip38, and anti-ERK, atl: 5000 in TBS; anti-actin at 1: 7500 in TBS) overnight at 4 °C. The membrane was then incubated with a secondary antibody, a goat antirabbit IgG, or goat antimouselgG conjugated to horseradish peroxidase. The protein levels were determined by using enhanced chemiluminescence (ECL) plus western blotting detection reagents (Amersham Bioscience, Uppsala, Sweden) and the bands intensities were scanned. Densitometric analyses were conducted using the Quantity One software (Bio-Rad). Incubation with polyclonal mouse antihuman ?-actin antibody was performed for comparative control.Measurement of nitric oxide (NO):The nitrite concentration was measured in supernatants of cultured RAW 264.7 cells according to the griess reaction as an indicator of NO production by using NO colorimetric test [Min Jee Kim et al., 2014 andWidowati et al. 2018]. Aliquots (100 pl) of the culture supernatant were transferred in triplicates to a 96-well microplate, mixed with lOOpL of griess reagent, and incubated at room temperature for 10 minutes in the dark, followed by the measurement of absorbance at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The inhibition activity toward NO was calculated between the percentages (%) of NO concentration in each treatment compared to the positive control. Nitrite concentrations were calculated by referencing a standard curve plotted by known concentrations of sodium nitrite (NaNOo) [Kiemer and Vollmar, 2001].I.2.5.3. In vivo anti-inflammatory activity by using Carrageenan induced paw edema model:In vivo anti-inflammatory activity of test peptide IS 217 was evaluated based oninhibition of paw edema volume by using carrageenan-induced mouse hind paw edema [Sravani et al., 2014], which is highly reproducible model of inflammation as previously described by [Yonathan et al., 2006]. Carrageenanis is a sulfated polysaccharide promotes acute inflammation by activating proinflammatory cells. The inflammation was quantitated in terms of ml i.e., displacement of water by edema using a digital plethysmometer immediately before and after Carrageenan injection at different time points.In the present study, a total of 27 BALB / C female mice (6-8 weeks old weighing between 25 g at the beginning of the experiments) were used for carrageenan-induced mice paw edema model. Mice were obtained from Jeeva Life Sciences (1757 / PO / RcBiBt / S / 14 / CPCSEA), and the studies were carried and approved by Birla Institute of Technology And Science-Pilani (BITS-Hyderabad) IAEC Protocol number BITS-Hyd / IAEC / 2020 / 17) and the animals were maintained and acclimatized as mentioned above in the acute toxicity. Allocation of treatment groups in carrageenan-induced mice paw edema model is provided in Table 12 below.Once after the acclimatization of animals, they were fasted for 12 hours before the experiment; however, they were given access to water ad libitum. Mice were divided into five groups of six animals each (n=6) except the normal control group (n=3), weighed and numbered. In vivo anti-inflammatory activity of two dose levels of IS 217 {i.e., 0.6 and 1.2 mg / kg body weight,) suspended in 0.9% normal saline and a positive control group given dexamethasone (5 mg / kg body weight), while control group given with 0.9% normal saline (disease control) [Sharma Rakesh et al., 2011] (Table 12).Table 12: Allocation of treatment groups in carrageenan-induced mice paw edema modelThe treatments of IS 217 (0.1 ml) were administered intravenously or a similar volume of vehicle (10 ml / kg) 30 minutes prior to subplantar injection of 0.1 ml of 1% (w / v ^carrageenan) freshly prepared carrageenan in saline into the sub-plantar region of left hind paw. An equal volume of the vehicle (0.9% Normal Saline) was given to the control group. Initial paw sizes (basal volume) were measured by a volume displacement method using a digital plethysmometer [Kou et al., 2005].All mice were marked on the tail with an indelible pen for identification. The left paw is marked with ink at the level of lateral malleolus; basal paw volume is measured plethysmographically by volume displacement method using plethysmometerby immersing the paw till the level of lateral malleolus. The increase in volume and the percent change caused by the irritant were measured before, and 1, 2, 3, 4, 5 and 18 hoursafter injection of carrageenaninjection.The percentage inhibition of edema was calculated for each group with a vehicle-treated control group [Winter et al., 1962]. Paw volume was measured immediately prior to the injection of carrageenan and thereafter at an interval of 1 hourfor a period of 5 hours and then after 18 hours. Edema inhibitory activity, the increase in paw volume, i.e., inflammation expressed in percentage was calculated according to the following formula [Gupta AK et al., 2015]:Percentage inhibition = (Ct-Co) control - (Ct-Co) treated X 100(Ct-Co) controlWhere, Ct=mean paw volume for each group at time t, and Co=mean paw volume for each group before carrageenan injection.Statistical analysis:Values were expressed as the mean+SEM of at least three readings of experiments. Statistical analysis was performedusing a one-way analysis of variance (ANOVA). The multiple group comparisons between the mean values of two groups were performed using a Dunnett'stest. Column charts were generated using Prism 8.2 / 9.0 software (Graph pad Software, INC, La Jolla, CA), where P < 0.005, P< 0.05 and denoted as *** and** and ### and ## when compared with standard drug and vehicle control groups, respectively.1.2.6: Proof of concept in vivo animal studiesHow animals are currently used in sepsis research --translational and ethical issues:The central goal of animal models is to create reproducible systems of clinically relevant sepsis pathogenesis that mimic human disease and used for preliminary testing of potential therapeutic agents [Safiah Mai et al., 2012] Animal models are used for two main categories in the context of sepsis research:1) To identify involvement and changes in physiological, molecular and / or cellular pathways, and to understand the mechanism of sepsis syndrome progression.2) To study the efficacy / safety / phannacokinetics of therapeutic targets for proof of concept or regulatory studies.Requirements for animal models of sepsis to mimic human sepsis:An ideal animal model of sepsis would need to mimic the pace and severity of human sepsis (a severe and chronic syndrome), and its treatment in the ICU. Such success requires replication of the pathophysiology of sepsis, with particular emphasis on key patterns of inflammation and cardiovascular parameters: hemodynamic (warm shock followed by cold shock) and immunologic (proinflammatory stimulation, antiinflammatory counterregulation, i.e., immune depression) stages; mimic histology findings in key organs (lung, liver, spleen, kidney, etc.) that are frequently modest; and — perhaps counterintuitively for animal modelers — exhibit variability among animals. For these mechanistic models of inflammatory and immune pathways and enhancing the translational gap are in need. Furthermore, an ideal sepsis model would be low-cost and would not cause any distress to the animals involved [Marcin F. Osuchowski el al., 2018]. The perfect model of sepsis does not exist based on these characteristics. Thus, in order to mimic human sepsis for the purpose of therapeutic intervention, considering the points mentioned above, the screening has to be done in two or more distinct animal models [S. Manoj Kumar et aL2016;Wendy E. Walker ,2021 andCharles T Esmon et al., 2004], this helps in clinical trial prediction to be success.A full harm: benefit impact assessment is important while designing the animal model. Harm relates to welfare experience of animal whilst benefit relates to value of scientific data, considering (3Rs) principles [Sudhir Verma, 2016; Manasi Nandi, 2020 and Jean-Marc Cavaillon et al., 2020].To date, most used models based on initiating agent, include administration of an inflammatory trigger (e.g., endotoxin -LPS), amicrobial trigger (e.g., infusion or instillation of exogenous bacteria bacteria or peritonitis) or co-morbidity models (e.g., surgical‘immune barrier disruption models’: trauma plus infection). Considering regulatory guidelines, the present work involves the use of two Standard animal models of sepsis. The comparison of two rodent sepsis models is summarized in Table 13.Table 13: Comparison of two in vivo animal sepsis models used in the present invention [Sudhir Verma 2016]Justification and significance of C57BL / 6 and BALB / c mice in sepsis studies:The animal models of sepsis should be immunological and metabolic while designing the animal study, moreover in them vivo environment, local humoral and cellular factors probably alter the way that cells respond to pro-inflammatory stimuli. The key information gathered from the animal studies isefficacy and safety of the lead molecules (as determined by standard regulatory tests). As a part of preclinical research for sepsis, mice are most popularly used.At present, the most used inbred mouse strains in the laboratory are C57BL / 6 and BALB / c, as they can improve the reproducibility and reliability of study results and majorly used in immunology and antitumor activity studies involves inflammation and macrophage function.C57BL / 6 mice, in particular, are notable for their immunogenicity due to the initial strain development goal being their use in the study of cancer and immune responses, in addition due to their cell mediated immunity and NK cell activity are relatively higher in C57BL / 6 mice than in other mice, in respect to immunological characteristics and ,mortality rates, liver MPO activity, metallothione mRNA, leptin as well as IL- 10 levels are significantly higher in C57BL / 6J compared to other strains used for sepsis[Hyun Keun Song et al., 2017]. Whereas BALB / c mice tend to produce a stronger humoral response than C57BL / 6 mice; it is easier to induce Th2 immune response in BALB / c mice, which is very common in infectious diseases and allergic reactions.In the present study, the two gold standard models which mimic human sepsis are bacterial (E. colt) infection induced peritonitis model and cecal ligation and puncture (CLP) models are used in C57BL / 6 and BALB / cmice of aged 8 / 10-12 weeks (as illustrated in Figure 9). Figure 9(A) illustrates the injection of live bacteria E.Coli (8739™) (Intraperitoneally). Figure 9(B) illustrates the ceacal ligation and puncture (CLP) model by puncturing and ligating the cecum, faecescan reach the peritoneal cavity wasestablished. Hypothermia is one of the illness signs in both models, and it can be evaluated by measuring body temperature. The Figure 9 was adopted from BioRender.com and modified.Bacterial infection model: E. coli Induced peritonitis sepsis model:Because the rate of positive blood cultures is associated with increasing sepsis severity, (sepsis [17%], severe sepsis [25%], septic shock [69%]), it has been suggested that bacteremia plays an important role in the outcome of sepsis. Different aerobic bacterial species have been investigated to induce sepsis and septic shock. Escherichia coli is the most common one. Bacterial infection model involves the injection of live bacteria inside the body against which immune response is elicited. Animal inoculation with pure or mixed bacterial flora has long been used to explore septic processes [Wendy E. Walker et al., 2021]. There is a wide variability in the dose of and duration of infusion, as seen with endotoxicosis models. Low doses of E. coli administered over several hours in small animals have been associated with minimal early physiological changes, whereas higher doses have frequently resulted in a biphasic response, with an early rise and late fall in cardiovascular collapse and subsequent organ dysfunction and death.Cecal Ligation and Puncture (CLP) model: Polymicrobial sepsis:The CLP model is one of the most stringent clinically applicable models of sepsis, involve a localized infection, such as surgically induced polymicrobial sepsis, that gradually propagates a systemic immune response, compared to other models, CLP provides a better representation of the complexity of human sepsis and is the crucial pre-clinical test for any new treatment to human sepsis. CLP involves a combination of three insults:tissue trauma from laparotomy, necrosis from cecal ligation, and endogenous infection from microbial leaking. In this model, feces are leaking into the abdominal cavity, introducing peritonitis (Figure 9B) and further followed by translocation of bacteria into bloodstream which activates inflammatory response[Wichterman, Baue, and Chaudry, 1980],The advantage of CLP is that the pathogens are endogenous, simulating severe damage that leads to peritonitis in humans. Furthermore, the course of sepsis is strikingly comparable to that of human sepsis, with both hyper- and hypo-inflammatory responses [RittirschD, 2009 and Wendy E. Walker, 2021].Being one of the best representatives of human sepsis, it has been recognized as the gold standard for sepsis research [Dejager L, 2011].Peptide details:Peptide IS 217 (Batch No: IS 217-17-10) with 90% purity was supplied by Issar Pharmaceuticals Pvt.Ltd and was stored at -20°C until usage. During the study period, IS 217 was stored in a refrigerator (2-8°C) under light-resistant conditions. For in vivo animal studies, the solution was prepared freshly by dissolving the required amount of peptide IS 217 in 0.9% normal saline. 0.9% normal saline was used for dilution and delivery media. The appropriate concentrations in 0.1 ml volume were administered to mice by SC and IV injections. The standard drugs and other materials used for in vivo animal studies are mentioned in section 4.1.Animals and maintenance:8-10 weeks old BALB / c and C57BL / 6 mice weighing 25 -30g, were used in the study. The animals were randomized and grouped and identified by marking by earpunching after group assignment. BALB / c mice are used for IV route of administration, whereas C57BL / 6 mice are used for SC route of administration in two in vivo animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to dosing, during which time body weights and general conditions were observed. During the acclimation period, 5 mice were housed per cage, whereas during the study period, 3 miceper cage were limited. The mice were housed in polypropylene cages and allowed free access to food and water, ad libitum and were maintained under specifically regulated environmental conditions (rearing conditions): 12-hour light / dark cycle, temperature 24°C ± 2°C, humidity 55% + 10%, and ventilation 11 to 15 air exchanges per hour (all-fresh-air system).The number of mice were reduced using 3R principle (Reduction, Replacement and Refinement), the same control BALB / c mice strain were used for experiments involving BALB / C strain and the same applies to C57BL / 6 mice involved animal experiments.Ethics approval:The present study was approved by the Ethics Committee of Birla Institute of Technology and Science-Pilani, Hyderabad campus (BITS-Hyderabad) (IAEC approval numbers are mentioned in the below Table 14) and all procedures, care, and handling of the animals were performed according to the guide for the care and use of laboratory animals published by the CCSEA. The animals were obtained from Jeeva Life Sprovidegistered breeder: 1757 / PO / RcBiBt / S / 14 / CPCSEA). All the techniques / procedures have been refined to provide for maximum comfort / minimal stress to the animals. The male mice are used in PoC in vivo animal studies.Table 14: IAEC numbers approved for the in vivo animal studies1.2.6.1: CLP model: Induction of polymicrobial sepsisThe mice were subjected to the CLP in the sequence demonstrated in Figure 10.Polymicrobial sepsis was induced using the CLP method described by [Wendy E. Walker, 2021and Benjamimeta / . 2000], with minor modifications and the anesthetic used. Initially, the mice were derived of food for 12 hours before the CLP procedure and then anesthetized by intraperitoneal injection of ketamine (80-100 mg / kgBW) and xylazine in a dose (5-10 mg / kg) (for 20 g mice ketamine of 0.2 ml andxylazine of 0.1ml) according to Machado et al., 2012. Under sterile conditions a midline abdominal incision was made andthen, a laparotomy of 10 mm / 1 cm was performed, where the abdomen of mice was routinely disinfected and cut open in the middle to expose the abdominal cavity, and the cecum was mobilized, was then ligated below the cecal valve at the distal end of the cecum, 3 / 4 was ligated with a sterile No. 4 thread, and punctured (CLP) with a 20- gauge needle in 2 places at the center of the distal end of the occlusal cecum and squeezed gently to extrude a small amount of intestinal contents through the perforation site to induce sepsis. The cecum was located but not ligated or punctured in the control animals. The cecum was placed back into the peritoneal cavity, and the abdominal incision was then closed in two layers with absorbable ethilon suture 5.0, and the animals were resuscitated with 0.1-0.2 mL of normal saline by subcutaneous injection.The sham group of mice had the identical operation as the treated and disease control groups, which included opening the peritoneum and exposing the bowel but without ligation or needle perforation of the cecum. Betadine / tra adol was used to relieve the pain of mice after operation. During the surgery, the atropine solution was applied to the eyes to prevent dryness of the eyes. All the animals were returned to their cages, where they had free access to food and water. After 2hours of CLP, the treatment groups were administered with test peptide IS217 (0.6 and 1.2 mg / kg) and the standard drug through the subcutaneous and intravenous routes.To reduce variability between studies, the CLP method was always carried out by the same investigators [Zingarelli B el al., 2019; Libert C el al., 2019 and Hellman J el al., 2019],1.2.6.1.1: Pilot study: Standardizationof CLP model and test peptide dosesIn standardization model, the CLP procedure was done as described above. The treatment groups are divided into sham control and disease control group treated with test peptide IS 217 and one sham control group were considered as control and one disease control group was considered as positive control.The test peptide IS 217 at different doses were administered to treatment groups subcutaneously and the animals were observed for 24 hours for survival rate and physical activity. After 24 hours of infection, the mice are bled retro-orbitally and left to coagulate at room temperature for ~2 hours. Sera was then separated by centrifugation and the inflammatory biomarkers IL-10, TNF-a, IL-ip and IL-6 levels were determined by using ELISA kits. The standardization of this experiment was required to fix the doses of test peptide and sampling time points.1.2.6.2: Main CLP study: Experimental design and treatment groupsMice were subjected to CLP surgery and were discarded if they died during the procedure. In all the animal experiments, the test peptide IS 217 was administered in C57BL / 6 and BALB / c mice by SC and IV injections, respectively. According to the clinical dosage regimen, IS 217 was repeatedly administered at 2 hours after CLP surgery and recovery of animals from anesthesia. The mice were divided into the following treatment groups at random: BALB / c mice are used for IV route of administration, whereas C57BL / 6 mice are used for SC route of administration. In each group the half of the animals were killed after 4 and 18 hours, [Hubbard, W. J 2005 and Rittirsch D 2009]. In another half of the animals, IS 217 at two doses 0.6 and 1.2 mg / kg wereinjected SC andIV daily for 5 days following CLP. 0.9% Normal saline was administered in the sham group. The survival of mice was monitored for up to 18 hours and the other half of the animals are continued for the survival monitored up to 10 days (Table 15). Table 15: Allocating animals: Dosing paradigms*per group and the study parameters of the study -CLP model.The Figure 11 illustrates study design and treatment regimen in CLP model. The Figure 1 Iwas modified from Jon A Buras et al., 2005.Post-surgical procedures:To prevent post-surgical infection, the mice were placed in the cage supine position until the anesthetic effect wore off. To prevent hypothermia after surgery, the cage was warmed with a heater.Behavioral test:The behavioral assessments were carried out on the tenth day following CLP on all animals [Liu Y et al., 2017].Novel object recognition test (NORT):One of the most well-known tests for measuring recognition memory in rodents is the NORT. The apparatus is made up of a transparent Plexiglas box that measures 40 cmx 40 cmx 40 cm. Objects are around 4 cmx 4 cmx 4 cm in size. Objects, like building blocks, have simple shapes. Wooden, metal, and hard plastic objects are preferred, since they are resistant to biting damage [BharathiHattiangady et al.., 2014] .Figure 12 illustrates exposure of CLP post operated diseases control and IS 217 treated groups to NORT test for the evaluation of stress conditions and memory learning process.Procedure:Grouping and treatment were carried out in accordance with the procedure. To reduce stress, the mice were treated in a controlled environment. Mice were first moved for 10 minutes to a preparation area near the operative space. The mice are then placed in the apparatus for analysis for 10 minutes to acclimatize. This test also had several trials for each mouse, with a 60-minute break between each trial [Hattiangady and Shetty, 2012].As described above for the elevated plus maze test, the first two trials involved placing the mice in the middle of an empty open field box and letting them freely explore the box for 5 minutes (first trial, the habituation phase, Figure 12A), and then placing the mice in the middle of an open field box with two identical objects on opposite sides of the box and letting them freely explore the objects for 5 minutes (second trial, the training phase, Figure 12B).The third trial (the objection recognition memory testing phase) began 60 minutes after the second trial. The mice were given 5 minutes to explore the objects in the same open field box, which had one object from the second trial (the familiar object) and a new object in place of the second object from the second trial (the novel object, Figure 12C). A mouse is exploring an object when its nose is within 2cm of the object. The movement of mice in the third trial was continuously tracked and noted the time (sec)and the apparatus was cleaned with 70% alcohol and air-dried prior to the commencement of each trial for every mouse. The total amount of time spent exploring the new object, the known object, and both objects (z.e., the total object exploration time) were recorded. Furthermore, novel object discrimination index was calculated by using the formula, to calculate the object preference which is:Preference % = Time to explore the individual novel object / Total exploration time to objects X 100Then, within each group, the amount of time spent exploring the new object versus the familiar item was measured in percentages (%). The preference rates were calculated by dividing overall exploration time by item exploration time. This demonstrates the ability of each group of animals to investigate new item recognition under stressful conditions.The novel object discrimination index was also directly compared with control groups of mice. The mice in the control group were also tested on how well they could tell the difference between new and old objects. The speed and total distance moved during the third trial (test phase) were also recorded and compared between the two groups to see if depression (or lack of motivation) affected the object recognition memory test.The fact that the new object was looked at more than the known object reflects learning and (recognition) memory processes are used.Experiment 1 Survival study:In the survival study, survival rates were determined by setting the day 0 from 0 to 18 hours after surgery and continuing observation until day 10. In one set of animals / group, survival was monitored every 4 hours up to 18 hours and in another half subset of animalswas followed for 10 days for survival assessment every 12 hours after sepsis induction to verify mortality rates.The time of death was recorded as accurately as possible.Disease parameters: The following parameters are in common in both in vivo animal studies [Shrum B, 2014].Vital clinical symptoms evaluation:The physical activity of the mice was recorded independently by two independent observers who were blinded to treatment before sampling as previously described. In this study predetermined grading system was used with a scale of 1 (healthy) to 5 (agony). This scoring system is based on grading physical activity and food intake (Table 20) using spontaneous activity of mice, reactivity to external stimuli, and spontaneous food intake to differentiate between grades 1 and 5. Animals were closely observed for first 4- 18 hours for the development of symptoms, and where appropriate, time to death was recorded. Mice were observed at least every 6 hours and for next over a 5-day period for the development of symptoms, and where appropriate, time to death was recorded. The mice that were moribund and those with a body temperature less than 27°C were euthanized and counted as dead at each time point indicated. Body temperature was measured by an electronic thermometer with a mice rectal probe. Mice were manually restrained, the probe was lubricated with sterile glycerol, inserted for 5 seconds (or until the temperature reading stabilized), and the data were recorded. Malaise, immobility, and ruffled coat were noted in some animals. Humane endpoints were strictly observed (immobility, dyspnea and paralysis) so that no animal became distressed. The clinical symptoms, such as conjunctivitis, ruffling of fur coats, and activity on stimulation, were observed in all groups and graded as described earlier. A collective grade was assigned to each group depending on the severity of the symptom observed in most of the animals.The clinical score was calculated using a score system of 0-4 grade scored as follows: grade 0, normal; grade 1, slightly lethargic; grade 2, lethargic and hunched; grade 3, very lethargic, hunched, and shaky; and grade 4, dead. Body temperature was compared for physiological evaluation.Table 16: Scoring system for measuring mice physical activityExperiment 2: Acute hyper inflammation studyIn this study, pro and anti-inflammatory cytokines TNF-a, IL-6, IL- 10, IL- 12 and IL- 10 were measured in the serumand peritoneal lavage.Quantification of cytokine levels:The R&D Systems (Minneapolis, MN) IL- 1 , TNF -a, IL- 12 p70, IL-6 and IL- 10 and VEGF levels ELISA kits were used for the quantitative measurement of these cytokines either in mice lung (BALF supernatants), sera and peritoneal exudate cell (PEC) supernatants according to the manufacturer’s instructions. The results are expressed as picogram per millilitre (pg / mL) of samples analyzed.Experiment 3: Late immune paralysis studiesIn the immune paralysis study, along with cytokines estimation and the colonyforming units (CFUs) in the blood, PLF and organs were counted [Kieslichova E, et al., 2013].Sampling time points:The animals were observed for 18 hours and 10 days after the CLP surgery and the remaining animals are euthanized under humane conditions, the mice were anesthetized with ketamine (80-100mg / kgBW) and xylazine in a dose (5-10mg / kg) intraperitoneally for blood and peritoneal lavage fluid collection and euthanized with an overdose of (150 mg / kg ketamine hydrochloride and 120 mg / kg xylazine hydrochloride) / isoflurane for organ collection. For clinical observations, the time points and parameters are listed in the Table 17 below.Table 17: List of parameters screened for biochemical and clinical analysisExperimental outcomes: 1. Blood sampling:The whole blood and tissues were collected at 4 hour, 18 hour and on the last day of post CLP procedure in the surviving animals for hematologic and biochemical examination. The mice were anesthetized with ketamine (80-100 mg / kgBW) and xylazine in a dose (5- lOmg / kg) at a 2: 1 ratio via an intraperitoneal injection). Mice were bled retro-orbitally at specific timesin either 4% EDTAor 3.2% sodium citrate to collect whole blood and plasma, respectively and was left to coagulate at room temperature for ~2 hours and was centrifuged at 2,000 rpm for 10 minutes to separate the plasma and the serum was obtained by centrifugation at 1500 rpm for 10-15 minutes aliquoted and stored at - 20 °C until further use in ELISA.2. Assessment of clinical and biochemical parameters:Coagulation tests and Hematology: Blood in sodium citrate was immediately centrifuged for plasma collection to measure prothrombin time (PT), fibrinogen (FBG), and activated partial thromboplastin time (aPTT) by automated blood coagulation analyzer.From whole blood, WBC, and platelet (PLT) counts were determined by hematology analyzer.Liver and kidney function tests: The level of liver function indicators, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) and renal function indicator serum creatinine (SCr) were routinely measured using auto analyzersand hemagglutination analyzers.Differential leukocyte counts: For differential leukocyte count, blood was collected in EDTA tubes and analyzed on automatic cell analyzers, AD VIA 2120 (Hematology System).Serum procalcitonin (PCT), lactate and CRP levels: The serum PCT levelswere assessed using enzyme-linked immunosorbent assay (ELISA) Kit, according to the manufacturer’s instructions (R&D Systems).Peritoneal fluid collection:For some experiments, the peritoneal cavity of euthanized mice was washed with 5 ml sterile ice-cold PBS using an 18-gauge needle, and peritoneal lavage fluid was collected in sterile tubes and immediately placed on ice. Clear lavage was obtained by sequential centrifugation first at 2500 rpm to remove mouse peritoneal cells and 10-100pL was used for bacterial count and next at 5000 rpm to remove E. coli bacteria. Peritoneal cell free clear supernatant was used for cell count and ELISA and cell pellet was used for Western blotting and stored at-80°C until for further analysis (Figure 13). Figure 13 illustrates collection of peritoneal lavages from the mice [Meurer SK, 2016]. The goals of the study were (1) to measure bacterial growth and cell counts in peritoneal lavage fluid of anesthetized mice and (2) to investigate the direct influence of pro and anti-inflammatory cytokines (IL-ip, TNF -a, IL- 12 p70, IL-6 and IL- 10) in peritoneal lavage fluid [Stefan Wirtz et al., 2006].Mice bacterial clearance in PLF after treated with IS 217: CFU assayPeritoneal lavage fluid samples were used to count bacterial CFUs, the collection of peritoneal lavage fluid was as mentioned above.For bacterial clearance, serial dilution in peritoneal lavage fluid (1: 10, 1: 100, 1: 1000, 1: 10,000, and 1: 100,000). A lOOpL aliquot of each dilution was spread on a tryptic soy agar plate / Solid Luria-Bertani (LB) culture plates. All plates were incubated at 37°C for 48 hours. Colonies were counted and expressed as colony forming units CFU per milliliter for all samples.Isolation of leukocytes from the peritoneal cavity of mice:Cells present in the peritoneal cavity were harvested by introducing 3.0 ml of PBS containing 1 mM EDTA. Total cell numbers were estimated by counting cells with a haemocytometer. The peritoneal lavage fluid was centrifuged at 1,000 rpm for 10 minutes at room temperature, and the pellet was collected and resuspended in 200 pl PBS containing 0.6mM EDTA. Differential cell counts were determined by cytospin preparations. Following cytospin, the cells were stained with Wright-Giemsa stain and observed under 600x magnification under an oil immersion lens to differentiate between the different cell types.Preparation and processing of spleen tissue and cell harvesting:The spleen tissue was harvested from the all the mice in each experimental group and a part of the spleen tissue was immediately fixed in 10% neutral buffered formalin before further tissue processing and rest of the spleen tissue was homogenized and suspended in phosphate buffered solution (PBS) containing 10% Triton X-100. The cells obtained from individual spleens were suspended in RPMI 1640 containing antibiotics. Cells were placed in flat-bottomed wells of 96 well plates at cell density of 5xl05cells in a final volume of 150 pl and the spleen cells from uninfected mice were used as control. After 72 hours of incubation, the plates were centrifuged at 1200 rpm for 10 minutes and supernatants collected and stored at -80°C for cytokine analysis. Cytokine analyses in spleen tissue were determined in the supernatant from the spleen tissue by using ELISA using commercial assay kits (R&D systems). Briefly, the cytokine assay plate layout consisted of a standard series in duplicate, four blank wells and 20 pl duplicates of tissue supernatant samples and diluted to 50 pl with Bioscience mouse serum diluent. The quantification of the assay was done using a standard curve following manufacturer’s instructions and presented in pg / ml. [Iqbal J et al., 2016]Preparation of lung samples:The lung tissue of the mice was collected for histological analysis of lung edema and measurement of MPO activity. The lungs were collected on ice, weighed, and homogenized in ice-cold PBS. The resultant homogenates were centrifuged at 8000 xg for 10 minutes at 40°C and the supernatants were stored at - 80°C until analysis.Measurement of the lung wet / dry weight ratio:At the end of the experiment, the wet-to-dry weight ratio of the lung samples collected from the upper and middle lobes of the right lung in each mouse in all groups, the surface water of lung with filter paper was absorbed and was examined to assess lung tissue edema. All mice underwent whole left lung pneumonectomy on euthanizationday, and the lungs were excised, immediately recorded the wet weight. Subsequently, the samples were in aluminum foil and desiccated in an oven at 60- 80°C for 48 hours until a stable dry weight was achieved. The ratio of the wet weight to the dry weight was calculated by dividing the wet weight by the dry weight was calculated to evaluate the degree of pulmonary edema.In addition, lung index (lung index % = wet lung weight / body weight x 100) was calculated to further confirm the degree of pulmonary edema.BALF collection and analysis of total protein concentration and cell count of the BALF :BALF was prepared as previously described [Kuoet al., 2011]. BALF was obtained by washing the lungs flushed three times with 1.5 mL sterile saline via a tracheal cannula. BALF was centrifuged at 1500 rpm for 10 minutes at 4 °C, and the supernatant was stored at - 20°C for assessment of inflammatory cytokines and VEGF levels by ELIZA commercially available kits as mentioned above. The protein concentration was measured in the supernatant (BALF) with the BCA detection kit (Thermo Scientific, Rockford, IL, USA). The OD value was read at 540 nm according to the instruction provided by the kit.Proteins were expressed in milligram protein per milliliter BALF.The sediment cells were resuspended in saline, and the total number of cells was counted using a hemocytometer. The wright-giemsadye was used to find out the percentage of neutrophils.Measurement of MPO activity:To measure the myeloperoxidase (MPO) activity, lung tissue was homogenized in sterile PBS after the tissues are perfused with sterile PBS to eliminate myeloperoxidase activity in contaminating blood. After 4 hours and 18 hours of sepsis induction, mice wereanesthetized and the lung tissues were dissected, weighed, and homogenized in 0.5% HTAB buffer (hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer) to obtain a 10% homogenate. The homogenized tissues were stored at 80°C to lyse the cells. After thawing, the homogenates were centrifuged for 2-5 minutes at 10,000 g at 4°C. Supernatants were used to determine myeloperoxidase levels (U / mL) using the ELISA kits according to the manufacturer’s instruction. The supernatant samples were diluted in phosphate citrate buffer (pH 5.0) and samples absorbance was measured at 460 nm using a microplate reader. The amount of MPO in the lung was measured in units per mg of lung tissue.Western blot:Western blot was performed to detect the phosphorylation of MAPK. The protein expression of total ERK1 / 2, p38 and phosphor ERK1 / 2, p38 in lung tissue were assayed by Western blot. First, the lysate of lung tissues was prepared: 50 mg of lung tissue with 200 pl of cold RIPA lysis buffer, homogenized in ice bath, centrifuged at 4°C, 10000 rpm for 5 minutes, then 5x SDS loading buffer was added, boiled for 5 minutes at 95°C. Then, 20 pg of protein samples were taken to load at 10- 12% SDS / PAGE electrophoresis gel for running and transferred to polyvinylidene fluoride (PVDF) membranes at 110 V for 1.5 hours on ice bath. The membranes were than blocked with 5% milk at room temperature for~l hour. The primary antibody was diluted with 5% FBS solution and incubated the PVDF membrane under the diluted primary antibody at 4°C for overnight (~12 hours). The primary antibodies included p-ERK (1: 1000), ERK (1: 1000), and p -P38(l:2000), P38 (1:2000) (abclonal). Then, the membrane was washed with IxTBST solution for 10 minutes at room temperature andthe washing was repeated three times. The second antibody of anti-rabbit (1:10000) and anti-mouse (1:5000) were diluted with lx TBST solution and incubated the membrane at room temperature for 1 hour. The membrane was washed 3 times (for 10 minutes each) with Tris-buffered saline / Tween 20 (TBST) and then treated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10000) for 1 hour. The membrane was washed again with TBST. The membrane -binding antibodies were visualized later with the ECL detection reagent (ECL, Advansta) and images were acquired in a Fusion SL Imaging System (VILBER LOURMAT, France). Band density was analyzed by densitometry using Image J software.Histopathological evaluation: Macro- and microscopic assessment of the organs:The animals remained till the end of the study (at least n =3 for each group) and weresubjected to histopathological evaluation in in vzvoexperiments. Some vital organs comprising heart, brain, lung, liver, kidney, and spleen were harvested and weighed, immediately after blood sample collection. The macroscopic analysis of the collected organs considered the following parameters: size, weight, integrity, and presence or not of changes visually detectable.Relative organ weight was calculated by the following formula:Relative organ weight % = organ weight / body weight x 100For microscopic assessment, organs were fixed in formalin solution (10%, pH7.4) for the further histopathologic examination. The representative of one animal per group was presented.Hematoxylin-eosin (H&E) staining:The organs were collected, fixed with 10% formalin, embedded in paraffin, and sectioned at a 5pm-thickness were cut on a microtome and stained with hematoxylin and eosin. The sections were immersed in xylene I for 20 minutes, xylene II for 20 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, and 75% alcohol for 5 minutes to be dewaxed and rehydrated. After rinsing with tap water, the sections were stained with hematoxylin for 3-5 minutes, blued, dehydrated in increasing concentrations of alcohol (85% and 95%) for 5 minutes, and counterstained with eosin for 5 minutes. Then, the sections were cleared with absolute ethanol I for five minutes, absolute ethanol II for five minutes, absolute ethanol III for five minutes, and xylene I and xylene II for five minutes each. The sections were mounted with neutral gum and graphed with 100X / 200X lenses on a light microscope. At least 10 different fields were analyzed for each mouse. Pathologists, who didn't know about the experiment, observed and scored damage to the spleen, lungs, liver, kidneys, heart, and brain.Histopathologic observation:The total surface of the slides was examined and scored by pathologist who was unaware of the groups. Briefly, the criteria for scoring lung inflammation were as follows Murakami’s technique: Lung injury was assessed based on pulmonary edema as determined by alveolar wall thickening with vascular congestion and interstitial and alveolar leukocyte infiltration. Briefly, lung parenchyma was graded on a scale of 0-4 (0, absent and appearsnormal tissue; 1, light; 2, moderate; 3, strong; 4, intense) for congestion, edema, inflammation, and hemorrhage. A mean score for each of the parameters was then calculated. Lung injury scores were determined by assessing neutrophil infiltration, hemorrhage, necrosis, congestion, and edema as previously described. These criteria were scored as follows; 0 = normal, 1 < 25%, 2 = 25-50%, 3 = 50-75%, and 4 > 75%.To score liver injury, the following parameters were analyzed (16): interstitial inflammation, formation of thrombi, hepatocellular necrosis, and portal inflammation.1.2.6.2: Bacterial infection model: E. co / i lnduced Septic Peritonitis ModelThe bacterial infection model used in this study was E. co / z-induced septic peritonitis, which is Murine lethal infection by intraperitoneal injection of live bacteria E. coli. [Asma Ahmed et al., 2018].Preparation of bacterial suspension:Bacterial strain of E. coli ATCC 8739™ were maintained in laboratory of the Applciant and used for the mouse sepsis model. The strain was previously stored at -80°C in a Luria Bertani (LB) broth with 15-20% of glycerol. Single colonies from viable, growing Luria-Bertani (LB) agar plates were transferred to sterile liquid LB medium (containing 10 g tryptone, 10 g NaCl, and 5 g yeast extract per liter) and cultivated aerobically in 50-ml volumes at 37 °C in an orbital shaker for 24hours at 37°C. These cultures were transferred to 500 ml of fresh LB medium for another 12 hours. The optical density (OD) of the suspension was adjusted to 0.3 (exponential phase) using aQ spectrophotometer. Such OD corresponds to* 10 colony-forming units (CFU) / mL.When bacteria were in the log phase of growth, the suspension was harvested by centrifugation at 1500 xg for 5 minutes at 4 °C; the supernatant was discarded andwashed and resuspended three times in phosphate-buffered saline (PBS) at pH 7.4 and mixed by vortexing to achieve Q a concentration of approximately 1x10 colony-formation units (CFU) per milliliter. Finally, the bacterial suspensions were incubated in a water bath at 100 °C for 30 minutesto inactivate the cells. One hundred microliters of the suspension were serially diluted with sterile saline solution, plated on sheep blood agar plates, and incubated for 18 hours for determination of the bacterial concentration. The rest of the culture suspension was stored at 4°C until use. Just before the intraperitoneal injection, the bacterial suspension wasQ adjusted to a concentration of 1X10 colony forming units (CFU) per 0.1 mL with normalsaline solution / PBS. UTI chromogenic plate shown in Figure 14 illustrates confirmation of presence of E. coli strain used for sepsis studies in animal models.Infection of peritonitis in animals:This study was carried out within an animal containment BSL2 facility. The animals were infected with suspensions containing 2.5- 5X108CFU / mL E. coZZ8739™(200pL) inoculated intraperitoneally. The day of challenge was designated as day 1 of the experiment and considers it as 0 hr. [Wendy E. Walker, 2021 andKomal Dolasia et al., 2018],1.2.6.2.1: Pilot study- standardization of peritonitis in animalsLog phase E. coZZ8739™cells in 0.1 ml of sterile PBS and injected (IP) in age- and weight- matched male C57BL / 6 mice (8-10wk old) at a dose of 2.5 x 108CFU per mouse and 5.0 X Q10 CFU per mouse to induce septic peritonitis and survival rate and physical activity of the mice were observed. After 24 hours of infection, the mice were bled retro-orbitally and left to coagulate at room temperature for ~2 hours. Sera was then separated by centrifugation and the Inflammatory biomarkers IL- 10, TNF-a, IL-ip and IL-6 levels were determined by using ELISA kits. The standardization of this experiment was required to fix the dose of E. coli cells for infection and sampling time points.1.2.6.2.2: Main study: E. coZz induced septic peritonitis modelQMice received an intraperitoneal injection of E. coli ATCC 8739, 5.0 X 10 CFU per mouse in 200pL to induce septic peritonitis as mentioned above. After 2 hours of thc£. coli infection, mice (n =6) were treated subcutaneously and intravenously with two test doses of IS 217 (0.6mg / kg andl.2 mg / kg) in treatment groups. Control animals received 200 pL of normal saline (Table 18). The study design and treatment regimen in E. coZZ-induced septic peritonitis is as illustrated in Figure 15. The figure was adopted from Henrique G et al., 2020 and modified according to the present invention.Experimental design:In all the animal experiments, IS 217 was administered by SC and IVinjections, respectively. According to the clinical dosage regimen, IS 217 was repeatedly administered at 2 hours after the intravenous injection of heat-killed E. coli. BALB / c mice are used for IV route of administration, whereas C57BL / 6 mice are used for SCroute ofadministration.The survival of mice was monitored for up to 7days. In each group the half of the animals were killed after 18 hours. In other subset of the animals, IS 217 was injected SC and IV daily for 5 days following the infectionat two doses 0.6 and 1.2 mg / kg. The survival of mice was monitored for up to 18 hours and the other half of the animals are continued for the survival monitored up to 7 days.Table 18: Allocation of animals per group and dosing paradigms and the study parameters of the study -E. coli induced peritonitis modelExperiment 1: Survival studyIn the survival study, survival rates were determined over a period of 7 days with assessment every 12 hours, by setting the day 0 from 0 to 18 hours after surgery andcontinuing observation until day 7. In each group the half of the animals were killed after 18 hours, in another half subset of animals was followed for 7 days for survival assessment after sepsis induction to verify mortality rates. Two doses, 0.6 and 1.2 mg / kg, of IS 217 were injected SC and IV daily for 5 days following the E. coli infection.The animals were observed for 18 hours and 7 days after the E. coli infection and the remaining animals are euthanized under humane conditions, the mice were anesthetized (ketamine (80-100 mg / kgBW) and xylazine in a dose (5-10 mg / kg) intraperitoneally for blood and peritoneal lavage fluid collection and euthanized with an overdose of (150 mg / kg ketamine hydrochloride and 120 mg / kg xylazine hydrochloride) for organ collection. The disease parameterswere observedfollowing the procedures as samein CLP animal model mentioned above.Statistical analysis:Statistical analysis and data management were performed following recommendations on experimental design and analysis in pharmacology, and all experiments were blind and based on randomly assigned groups. Data were presented as the mean+SEM of n = 5 to 6 animals in each group.The survival of the groups was estimated using a Kaplan-Meier analysis. Comparisons were performed by the logrank test. The other experiments were analyzed using one-way ANOVA for comparison within groups under univariate conditions. For multigroup analysis, intergroup comparisons were performed via a Dunett’s t test or Bonferroni multiple comparison test.Data were analyzed by Graph pad Prism 8.2 / 9.0 (Graph pad Software, La Jolla, CA, USA). P<0.001, P,0.01 and p< 0.05was considered as statistically significant and denoted as***, **, *; and##and Compared to disease control and sham control groups respectively.2.0: Results and Discussion2.1: Confirmation of mass and purity of synthetic test peptide -IS 217Results:The synthetic peptide IS217 which were synthesized, and their mass and purity were confirmed by HRMS and HPLC details are as follows: The purity and the mass of the peptide were confirmed in -house and the details are mentioned in Table 19.Table 19: Details of the peptide used in the present inventionHRMS Results: The goal of the analysis was to confirm the peptide molecular weight. However, MS confirms that only m / z =2 belong to the peptide. The details of the instrument and mobile phase used are as mentioned in Table 20 and the obtained chromatograms of the peptide IS217 are shown in Figure 16. Table 20: Conditions used for HRMS analysisHPLC analysis results:The goal of the analysis was to confirm the purity of the peptide used in the present invention. The details of the instrument and mobile phase used are as mentioned in Table 21 and the obtained chromatograms of the peptide are shown in Figure 17? Physical characteristics of test peptide IS 217 are tabulated in Table 22 below.Table 21: Chromatographic conditions used for the peptide IS217Table 22: Physical characteristics of test peptide IS 217The 3D structure and chemical structure of test peptide IS217 are shown in Figures 18 and 19, respectively.Chemical properties of test peptide IS 217 are as follows: a) Sequence of Peptide— FAKKFAKKFK b) Molecular weight of the Peptide (MALDI) —1619.58; acetate salt c) No. of charged amino acids in sequence-5. d) No. of Hydrophobic and Hydrophilic amino acids — 5 hydrophobic; 5 hydrophilic e) PI / PK of the Peptide— pl is 11.4. f) No. of Acidic amino acids— 0 g) No. of Basic amino acids— 5 h) No. of Polar amino acids— 5 i) No of Non - Polar amino acids— 5 j) Helical wheel k) Amphipathicity of Peptide — 100%2.2: In vitro anti-inflammatory data of test peptide against LPS-induced inflammation in RAW 264.7 cellsResults:2.2.1: Effects of test peptide IS217 on the viability of RAW 264.7 macrophagesThe test peptide IS 217cytotoxic effects on RAW 264.7 mouse primary cells were determined by MTT assay. MTT assays were performed in RAW 264.7 cells treated with different concentrations of test peptide (1.56, 3.12, 6.25, 12.5, 25, 50 and 100 pg / mL) for 48 hours. Cell viability was measured by MTT assay based on the conversion of yellow tetrazolium salt to form a purple formazan product. The viability of the RAW 264.7 cellscan be seen on Table 23. The MTT assay revealed that test peptide’s concentrations (1.56, 3.12, 6.25, 12.5, 25, 50 and 100 pg / mL) exerted no significant cytotoxicity in the RAW264.7 macrophage cells, and safe to the cells with cell viability 80%. Hence, concentrations (3.12, -100 pg / mL) were applied for the next assays.Cell viability results revealed that treatment with peptide did not show above 80% viability at the tested concentrations.Table 23: Cell viability of the test peptide (IS217) in comparison with dexamethasone2.2.2: In vitro anti-inflammatory activity of test peptide (IS 217)2.2.2.1: For analysis of anti-inflammatory effects, the cells were pretreated with test peptide at different concentrations (3.12, 6.25, 12.5, 25, 50 and 100 pg / mL) before incubation with LPS for 18 hours. The morphology of macrophage RAW 264.7 cell visualized by optical microscopy at scale bar 60 pm (Figure 20) of peptide.2.2.2.2: Effect of early phase cytokines on the LPS -stimulated RAW 264.7 macrophages:2.2.2.2.1: Test peptidelS 217reduces the production of pro-inflammatory cytokinesCertain HDPs can modulate the innate immune response due to their ability to stimulate the induction of chemokines while suppressing potentially harmful pro-inflammatory cytokines. In the present study, the anti-inflammatory activities of test peptidelS 217 were evaluated on murine macrophage-like RAW 264.7 cells stimulated with 10 pg / mL LPS (Figures 21 and 22) and compared them with standard drug dexamethasone 500 pg / mL.The concentrations of TNF-a IL-6,IL-ipand IL- 10 in the culture supernatants of RAW 264.7 cells weremeasured by an ELISA kit. Treatment of RAW 264.7 cells with LPS alone resulted in insignificant increases in cytokine production as compared to the control group. RAW 264.7 cells stimulated with LPS alone produced low levels of IL- 10 while strongly inducing IL-6 and TNF-a levels. In the presence of test peptide IS 217, there was a significant increase in IL- 10 production, particularly in groups treated with test peptidecompared to groups exposed to standard drug dexamethasone (Figure23 b). The peptide caused a significant and almost complete reduction in the release of pro- inflammatory cytokines IL-ip, IL-6 and TNF-a in LPS -stimulated cells (Figures 26 and 27).The levels of TNF-a were significantly decreased as compared to the LPS group in all test groups (****&*** < 0.001 and p < 0.01, respectively) compared to IL-6, production TNF-a has been reduced to a greater extent. ELISA results revealed that treatment with LPS significantly increased the TNF-a, IL-ip and IL-6, whereasIL-10 levels are decreased. Treatment with IS217 reduced the levels of IL-6 but not in a dose-dependent manner. Further, treatment with IS217 significantly increased the anti-inflammatory marker IL- 10 levels. Similarly, treatment with IS217 significantly reduced the TNF-a level. However, these compounds failed to show dose-dependent inhibition of IL-ip. Dexamethasone also showed significant anti-inflammatory action by inhibiting the entire marker’s expression.2.2.2.2.2: Effects of test peptide IS217on LPS-Induced MAPK pathways activationThe mechanism by which test peptide IS217 inhibit LPS-inducedproduction of inflammatory cytokines was investigated by western blot which showed that IL- 10 levels were increased in IS217 treated cells. The effect of test peptide on the LPS-induced phosphorylation of ERK1 / 2 and p38 MAPK was examined in the cell lysate by western blottinganalysis using two different phospho-specific antibodies. The MAPK signaling pathway is known to be important for the expression of pro-inflammatory genesin LPS- treated RAW 264.7 cells. MAPKs act as specific targets for inflammatory responses. As shown in Figure 25, the phosphorylation level of the ERK1 / 2, and p38 MAPK increased dramatically after stimulation with LPS, whereas test peptide attenuated the LPS induced activation of p-ERKl / 2, M APKs in a dose dependent manner.However, the total protein expression levels of the unphosphorylated MAPKs were unaffected by LPS and test peptidetreatment. Results of this study confirmed that pretreatment with test peptide may block the LPS induced expression of pro inflammatory responses by inhibiting the MAPK signaling pathway and IS 217 significantly inhibited the phosphorylation of ERK and markedly prevented the LPS-induced increasing of p38 phosphorylation in a concentration-dependent manner (Figures23-25).The TLR- 2 and TLR-4 mediated signaling share the common target NFkB [Brach et al., 1993, 1992; and Carter et al., 1999]. Both TNF-a and IE-6 are dependent on NF-kB activation for full expression in macrophages [Collart et al., 1990 andLibermann and Baltimore, 1990]. Since EPS -stimulated NF-kB activation is dependent on p38 activation [Carter et al., 1999], the effect of test peptide on TLR -4 was examined.LPS stimulation significantly increased the levels of TLR-4 compared to control; treatment with IS217 (12.5 pg / mL) slightly reduced the TLR-4 levels. Further, VEGF levels did not increase in LPS control; however, VEGF levels were significantly reduced in IS217 treated samples (Figures 23-26).2.2.2.2.3: Effect of test peptide (IS 217) on mRNA expressions of cytokinesReal-time PCR analyses of the mRNA levels of IL-P, IL-6, CCL2 and TNF-a were performed to provide an estimate of the relative levels of expressions of these genes. In the present study, the mRNA expression levels of IL-P, IL-6, CCL2 and TNF-a were increased in the LPS treated groups compared to the control groups and, the mRNA expression levels of IL-P, IL-6, CCL2 and TNF-a were decreased in the test peptide treated groups compared to the LPS treated groups (Figures 26-28). IS217 reduced the expression of IL-1 p in a dose dependent manner as compared to LPS control.2.2.3: DiscussionIn vitro cell-based assays often play an important part in the preclinical screening of new lead molecules, and it is important to pay due care and attention to certain aspects of the design of cell-based assays. For example, the use of primary cell cultures might be desirable, rather than cultures of immortalized cell lines. LPS, a constituent of the outer membrane of gram-negative bacteria, apparently is one of the major toxins responsible for initiating this pathophysiological cascadeto release numerousproinflammatory mediators such as TNF-a, IL-6, IL-land IL-12 [Jiang T^etal., 2004]. In addition to cases caused directly by gram-negative microorganisms (accounting for 30-40%) in bacteremia involving gram-positive bacteria or in systemic inflammatory response syndrome, LPS may also play a pathogenic role due to bacterial translocation from the gut.Certain HDPs can modulate the innate immune response due to their ability to stimulate the induction of chemokines while suppressing potentially harmful pro- inflammatory cytokines. In the present study, the anti-inflammatory activity of peptide IS 217 was evaluated on murine macrophage-like RAW 264.7 cells stimulated with 10 ng.ml-1LPS (Figure 20) and compared them with the standard drug dexamethasone. It should be noted that this inhibitory effect was not due to cytotoxic activity of IS 217, because the cell viability was not affected by IS 217 treatments. Cell viability results revealed that treatment with peptide did not show above 80% viability at the tested concentrations. The results of TNF-a, IL-ip and IL-6 quantification which are shown in Figures 20 to 28 reveal that the test peptide IS217 had inhibition activity towards mentioned inflammatory mediatorsproduction in LPS induced RAW 264.7, but treatment with lower concentration which is 25 pg / ml resulted in better activity compared to dose of 100 pg / mL. TNF-a, IL-ip and IL-6 level in positive control are significantly higher compared to the level of negative control which proves that LPS can increase mentioned Inflammatory mediators production in RAW 264.7 cell. In this study, test peptide IS 217 could inhibit TNF-a, IL- ip and IL-6 production in RAW 264.7 cell lines which suggest that they have anti-inflammatory effect through down regulation of those pro-inflammatory cytokines. Our data revealed that the test peptide IS 217 inhibited TNF-a, IL-6 and IL-1 P, production in LPS -stimulated RAW 264.7 cells [Hong e lai., 2004] Treatment with IS217 reduced the levels of IL-6 but not in a dose-dependent manner. Similarly, treatment with test peptide IS217 significantly reduced the TNF-alpha levels. TNF-a is also a cytokine that plays a significant role in inflammation. This cytokine is produced chiefly by activated macrophages and play role during inflammatory response activating adhesion molecule inducer and nuclear factor kappa- light-chain-enhancer of activated cells (NF-KB) [An H el al., 2002]. TNF, being an endogenous pyrogenic, is able to induce fever, apoptotic cell death, cachexia, inhibit tumor genesis and viral replication, respond to sepsis via IL-ip and IL-6 producing cells. Deregulation of TNF production has been implicated in a variety of human diseases including Alzheimer's disease, cancer, major depression, psoriasis, and inflammatory bowel disease (IBD). With all effects caused, TNF-a inhibitory activity measurement is important in anti-inflammatory potential agent screening since this cytokine is an important mediator of inflammation. TNF-a may initiate an inflammatory cascade consisting of other inflammatory cytokines, chemokines, growth factors, endothelial adhesion factors and recruiting a variety of activated cells at the site of tissue damage. It is known that TNF-a can induce DNA damage, inhibit DNA repair, and act as a growth factor for tumor cells[McCoy SL et al., 2004]. Treatment of macrophages with LPS led to significant increase in the levels of both TNF-a and nitrites in the culture supernatants relative to control levels .IL-ip is important for the initiation and increase the inflammatory response to microbial infection during inflammation process due to its role to induces secretion of proinflammatory cytokines such as IL-6 and IL-8 [Liao JC et al., 2012]. IL-6 has a wide range effect on immune system cells resulting in the acute inflammation response. Increasing of IL-6 level was reported in rheumatoid arthritis, psoriasis, and encephalomyelitis individuals. Therefore, inhibition of IL-ip synthesis would indirectly be useful for autoimmune disease and inflammation treatment.RAW 264.7 cells stimulated with LPS alone produced low levels of IL- 10 while strongly inducing TNF-a IL-ip and 1-6 levels. In the presence of peptideIS217, there was a significant increase in IL- 10 , production, particularly in groups treated with IS 217 significant and almost complete reduction in the release of pro-inflammatory cytokines TNF-a IL-ip and IL-6 levels in LPS -stimulated cells (Figures 21-22). Dexamethasone also showed significant antiinflammatory action by inhibiting the entire marker’s expression [Eicher SD et al., 2004].Western-blot results showed that IL- 10 levels were increased in IS217 treated cells. LPS stimulation significantly increased the levels of TLR-4 compared to control, treatment with IS217 (12.5 pg / mL) slightly reduced the TLR-4 levels [Eicher SD et al., 2004; Schmalz Get al., 2011 andBhattacharyya Set al., 2007]. Further, VEGF levels were significantly reduced in IS217 treated samples. Treatment with LPS significantly increased the pERK levels, treatment with IS 217 showed significant reduction of pERK levels. The mechanisms of action underlying such a function were also investigated. MAPKs signaling pathways are involved in the LPS -induced pro-inflammatory mediators and cytokines expression, which play a critical role in the regulation of cell growth and differentiation as well as the control of cellular responses to cytokines and stresses [Chen HHet al., 2004]. Maximal MAPK expression is known to occur 20-30 minutes after LPS treatment in human and murine monocytes and macrophages. Inhibition of any of the three MAPK pathways (JNK, p38 MAPK, and ERK) is sufficient to block induction of TNF-a by LPS. The investigation results revealed that the test peptide (IS 217) obviously down-regulated LPS-induced phosphorylation of ERK1 / 2 in activated macrophage cells. This result suggests that p38 and ERK1 / 2 are involved in the inhibition by the test peptide (IS 217) in RAW 264.7 cells (Figures23-25) and participates in regulating the expression of cytokines and other mediators that are involved in the inflammatory response [RinaBarouchet al., 2001 andXia, Zet al., 1995]. Thus, inhibition of the production of these signaling pathwaysmay explain the potent activity of the test peptide (IS 217) as a suppressor of inflammatory cytokines. These results are supported by gene expression analysis. The results revealed that LPS stimulation significantly increased the expression of CCL2, TNF-a, IL-6 and IL-P compared to control (Figures26-28). Treatment with IS 217 showed reductions of CCL2 and IL-ipindose dependent manner. Treatment with IS217 showed significant reduction of TNF-a and IL-6 compared to LPS control. IS217 reduced the expression of IL- 1 pin dose dependent manner as compared to LPS control.2.2.3: SummaryThe test peptide (IS 217) showed significant anti-inflammatory activity against LPS induced inflammation in RAW 264.7 cells by inhibition of pro -inflammatory cytokines (CCL2, TNF-a, IL-6 and IL-P) and downregulated phosphorylation of p38 mitogen activated protein kinase (MAPK).2.3: Results of stability of IS 217 with the 0.9% NaCl used for pre formulationIn the present study, the preformulation of test peptide IS 217 was developed and determined maximum feasible concentration of developed formulation, which was used further through various routes in non-clinical efficacy studies. Four different aqueous based preformulations of test peptide IS 217 were prepared. All the preformulations were first subjected to stability study at intended use of temperature (RT) for 24 hours. During the period of 24 hours, preformulations showed no visual changes in the physical appearance from initial sample. Out of these four aqueous based preformulations, .0.9% NaCl solution was selected for IV and SC route and the aqueous vehicle for the co-solvent preformulation, based on its stability and suitability at physiological conditions. The maximum feasible concentration for IS 217 was greater than 50 mg / ml.2.3.1 Physical Description: White fluffy powder is not affected with solvents used. Stability of IS 217 in four different diluents was performed at 2-8 °C and room temperature (RT)-37°C.It was observed that after 24 hours the percentage of IS 217 remaining in different solutions was in the range of 81.23 to 112.98%.2.3.2 Results of confirmation of mass (m / z) of test peptide IS 217 in 0.9% NaCl solution after 24 hours:The mass(m / z)of test peptide IS 217 in 0.9% NaCl solution was confirmed by HRMS, at two different temperatures (2-8 °C and at room temperature) after 24 hours. The obtained spectra are shown in Figures 29-31.2.3.3: DiscussionIn the present study, it was proved that the test peptide IS 217 has good antiinflammatory activity from in vitro assays. Further to investigate the in vivo efficacy of test peptide IS 217 through four selected routes i.e., intravenous, intraperitoneal, intramuscular, and subcutaneous; preformulations of IS 217 were required.2.3.4: SummaryThis study summarized that 10% propylene glycol in 0.9% NaCl solution is suitable for IM and SC route and normal Saline (0.9% NaCl solution) is suitable for IV and IP route for the IS 217 peptide. As there were not much difference in the results of solvents of 10% propylene glycol, 0.9% NaCl solution and 0.9% NaCl solution; simple use of pre formulation was considered, and it was concluded to use 0.9% NaCl solution for both SC and IV routes in the animal studies.2.4: Acute toxicity studies and immunogenicity of IS 217 peptide2.4.1: Results of acute toxicity study in miceThe acute toxicity of test peptide IS 217 was then examined with single dose of SC and IV injections in C57 BL / 6 andBALB / C mice respectively. 0.9% of normal saline, single doses of IS 217 (0.6, 2.4, and 4.8 mg / kg) were administered subcutaneously and intravenously, respectively in C57 BL / 6 and BALB / C mice and were observed twice daily for clinical signs of toxicity and mortality and morbidity for 14 days of the study. As described in the method, blood and tissue samples were collected after 14 days. The results of acute safety investigation of IS 217 peptide showed that SCandIVinjection of IS 217 at 4.8 mg / kg are safe and does not cause mortality or organ damage.No deaths occurred throughout the 14-day observation period following single administration of IS 217 and higher dose of 4.8 mg / kg body weight (BW) appeared to be safe. Normal gain in body weight was observed in both the control and treatment groups.No macroscopic abnormalities were observed upon necropsy in any of the animals treated with IS 217. Based on these results, SC and IV administration of IS 217 was not associated with any toxic effects, and the LD50 was determined to be greater than the dose level tested in this study (z.e., 4.8 mg / kg BW). The summary of the results is provided in the following sections.2.4.3: Functional observational batteryIn any of the cases, no mice treated with IS 217 were found dead or dying by cage observation, and all mice were in a healthy mental state with actively free moments. The changes in the mice fur, eyes, and mucosa, along with their respiratory system, circulatory system, nervous system, as well as the behaviors were meticulously documented. All subjects showed no abnormalities after receiving a single subcutaneous and intravenous injection of IS 217 at a high dose of 4.8 mg / kg BW.2.4.3.1: MortalityDuring the period of 14 days, no death and noticeable clinical signs associated with toxicity were found in normal saline (NS) and all IS 217-treated groups in both routes of administration (IV and SC) (Table 24).Table 24: Summary of mortality of BALB / C-IV and C57BL / 6 -IV in IV and SC administration2.4.3.2: Bodyweight and food intakeA single SC and IV injection of IS 217 had no effect on food intake, body weight, and weight change in mice. On the 14th day, the mice’s body weight was also measured after an overnight fast. During the study period, the body weights of both male and female mice increase proportionately and found no significant differences in body weight or food intake among the experimental groups and the control group. The changes of body weight after 14 days of SC and IV injection of IS 217 are shown in Tables 25 and 26.Table 25: Effect of test peptide IS 217 (0.6, 2.4, and 4.8 mg / kg) on body weight inBALB / C mice, IV administrationTable 26: Effect of test peptide IS 217 (0.6, 2.4, and 4.8 mg / kg) on body weight inC57BL / 6 mice, SC administration2.4.1.2.1: Clinical biochemistry analysis:Biochemical examination on day 14 after single-dose administration revealed no abnormal parameters in male or female mice that received a single subcutaneous and intravenous injection of different doses of IS 217, reflecting the pathological changes of vital organs were determined, and results were shown in Tables 27 and 28 including hepatic and renal functions as well as other important parameters. In comparison to the control group, female mice had significantly lower levels of alanine aminotransferase (AST) in their blood. However, because this difference was only found in one sex and there was no evidence of a dose-effect relationship of IS 217, the reduction in AST level was not considered to be produced directly by IS217 treatment as this difference was observed in the control animals. Statistical analysis of these parameters indicated that there were no significant differences between NS and IS 217-treated groups. Other blood biochemical indicators in the blood were within the normal limits.Table 27: Effect of test peptide IS 217 (0.6, 2.4, and 4.8 mg / kg) on body weight in BALB / C mice, IV administrationTable 28: Effect of test peptide IS 217 (0.6, 2.4, and 4.8 mg / kg) on biochemical parameters in C57BL / 6 mice, SC administration2.4.1.3: Gross necropsy and relative organ weight of vital organs Gross necropsy showed that the SC and IV injection of different doses of IS 217(0.6, 2.4, and 4.8 mg / kg) showed no effect on the weight of the organs, the ratio of viscera to the body. The effects of IS 217 on relative weight of vital organs including brain, heart, lungs, liver, kidney, and spleen, were demonstrated in Tables 29 and 30. No significant differences in relative organ weight were noted between NS and IS 217 -treated groups. Internal (visceral) as well as external gross pathological examination did not reveal any test peptide IS 217 related abnormalities in the treated and control groups of both the sexes.Table 29: Effect of test peptide IS 217 (0.6, 2.4, and 4.8 mg / kg) on relative organ weight in BALB / C mice, IV administrationTable 30: Effect of test peptide IS 217 (0.6, 2.4, and 4.8 mg / kg) on relative organ weight in C57BL / 6 mice, SC administration2.4.1.4: DiscussionIn the present study the acute toxicity profile of test peptide IS 217 was established as it was necessary in identifying and characterizing the adverse effects associated with a test peptide molecule in laboratory animals (mainly rodents) is used for several purposes, which includes: clinical diagnosis, treatment and prognosis of acute human poisoning cases; and design (e.g., dose-setting, identification of potential target organs) of longer-term (e.g., 28-day) toxicity studies. Acute toxicity test is typically the first step to determine the LD50 and lethality estimated by the LD50 test has been a primary toxicological endpoint in acute toxicity tests, these studies are conducted following guidelines established by the Organization for Economic Cooperation and Development (OECD, 1992, 1996, 1998b, 2001). e.g., protocols that use new sequential dosing methods such as the Acute Toxic Class method (ATC, OECD TG 423; OECD, 2001) and the Up-and-Down Procedure (UDP, OECD TG 425; OECD, 2001).In present study, only a single-dose of SC and IV injections with an observation of 14-day interval was conducted to evaluate the safety of IS 217, and 4.8 mg / kg was taken as an upper limit dose. During the observation period, no death and noticeable clinical signs associated with toxicity were found in NS (Normal Saline) and IS 217-treated groups; there were also no significant changes in body weights (Tables 27 and 28), suggesting that the LD50 of IS 217 may be higher than 4.8mg / kg in mice [ICCVAM document, 2001;Directive 67 / 548 / EEC; Annex V. Revision. European Commission, 1997]. Then a complete necropsy and serum biochemical and histopathological examinations were performed to assess thetoxicity effects of IS 217 on inner organs [Xiaodan Yang,lJunjie Li ,et al., 2022]. During necropsy, no noticeable abnormalities were not noticed, and there were no significant differences in relative weights of vital organs including brain, heart, lungs, liver, kidney, and spleen between NS and IS 217 -treated groups (Tables 29 and 30). Another key profile to determine the in vivo injury degree of organs is serum biochemical parameters, such as ALT and AST, which are strongly associated to liver function, while CRE and BUN are important biomarkers of renal toxicity [Xiaodan Yang, et al., 2022 and Junjie Li, et al., 2022]. When compared with NC, no significant differences in AST, ALT, GLU, TG, BUN, and CRE were found in IS 217-treated groups (Tables 25 and 28) [Figure 90(a) to Figure 90(d), Figure 91(a), Figure 91(b)], indicating that SC and IV administration of IS 217 has no harm on absolute and relative organ weights of brain, heart, lungs, liver, kidney, spleen and testis / ovary and for future histopathological examination, in the brain, heart, lungs, liver, kidney, and spleen, the organs are fixed in 10% formalin solution and stored at specific storage conditions. Slight weight changes in several organs, such as the liver and kidney, were not considered treatment-relevant because neither gender showed such a similar appearance [Xiaodan Yang, 2022; Junjie Li, et al., 2022 and Katsuhiro Osajimaet al., 2009]. The results confirmed the test peptide IS 217 possesses high-safety property, even in the maximum dose of 4.8mg / kg.2.5.2: Results of immunogenicity of test peptide IS 217The results of ELISA assay of the separated serum samples also showed no evidence of enhanced IL-ip or TNF-a cytokine production after 48 hours of treatment with IS 217 peptide at doses of 0.01 and 0.06 pmol per animal (corresponding to 0.6 mg / kg and 1.2 mg / kg, respectively, as it is shown in Figure 32(a) and Figure 32(b) (Table 31). Indicated release of IL-ip and TNF-a cytokines was insignificant if compared to lipopolysaccharide (LPS). Possible toxic and immunogenic effects of IS 217 peptide were compared with the immunogenic activities of positive control (LPS).Table 31: IL- ip and TNF- a levels detection in vivo immunogenicity studyThe results expressed as mean +SEM, (n=6)2.5.2.1: DiscussionAlthough therapeutic protein agents and peptide used in clinical settings are generally considered nontoxic, anti-therapeutic protein antibodies can develop during the treatment and thus immunogenicity is a major disadvantage of these drugs. Almost all therapeutic proteins induce an antibody response and antibody formation may lead to a loss of efficacy, neutralization of the endogenous counterpart or cause infusion reactions, anaphylaxis, and anaphylactic reactions in response to immune response. To prevent these and thereby warrant better patient well-being and lower therapeutic costs, it is important to treat patients with therapeutic proteins that have no or minimal immunogenicity [Immunogenicity Assessment for therapeutic protein products: guidance for industry: by FDA, 2014],In the present study, the immunogenicity of the test peptide was established, as immunogenicity of therapeutic peptideis important for novel peptides as immunogenicity lowers patient well-being, which drastically increases therapeutic costs and considered as important issue to prevent immunogenicity while developing novel therapeutic proteins and applying them in the clinic. As peptides do not possess intrinsic toxicity due to harmful metabolites; they are broken down to amino acids, their use as drugs is under development for inflammatory diseases diagnosis and for many other applications in molecular biology. Thus, in vitro and in vivo assessments of therapeutic protein products including proliferation and cytokine release are recommended. Though, the animal models were used as predictive tools during drug development as considered low, they should be considered for selected immunogenicity studies [European Medicine Agency (EMA) Directive 67 / 548 / EEC, Annex V. Revision. European Commission, 1997)]. C57BL / 6 and BALB / c mice are inbred strains and being small and easy to real, little or no harm to laboratory personnel. The screening of diagnostic / immunological assay kits prefers mice over large animals at early drug developmental stages. The size, surface charge, surface hydrophilicity, surface morphology, and the amino acid sequence of the peptides also influence the immunogenicity of the peptide and the toxicity.The d-amino acids, particularly d-alanine (d-Ala) and d-glutamic acid (d-Glu), peptides and the peptides which are less than 15 amino acids are not immunogenic and lesstoxic [M. H. V. Van Regenmoret al., 2011]. In the current study, possible toxic and immunogenic activity of the test peptide IS 217 was investigated, which consist of alanine, lysine and phenylalanine amino acids and 10 amino acids sequence, trying to bring out the differences or possible enhanced influence of IS 217 peptide on the activation of the immune system. The results demonstrated the lack of viability reduction in RAW 246 cell line, as well as no indication of primary inflammatory effects after 48 hours of incubation with IS 217 peptide. Peptide showed no toxic influences on RAW 246 cell viability even at concentration of 100 pg / mL. Moreover, LPS exhibited strong cytotoxic effect, reducing cells viability up to 55%. The IS 217 peptide was not toxic at tested concentrations in vitro, and testing IS2 17 peptide in vivo was conducted [Vera Brinks et al., 2011; Julia Suhorutsenko et.al., 2011].Similar to the results from in vitro studies, the peptide did not induce an increase of the cytokine levels in blood serum (Figure 32), showing no influence on the host system and conclude that the peptides have no evidence of inflammatory effects in vivo. ELISA analyses of the separated serum samples also showed no evidence of enhanced IL-ip or TNF-a cytokine production after 48 hours of treatment with IS 217 peptide at doses 0.6 mg / kg and 1.2 mg / kg, respectively, as it is shown in Figure 32). Indicated release of IL-ip and TNF-a cytokines was insignificant if compared to lipopolysaccharide (LPS) [Rodrigo de Almeida Vauchera et al., 2011]. Additionally, no pathological deviations were observed in liver, kidney, lung, and spleen dissected from animals, which received 0.6 mg / kg, 2.4 mg / kg or 4.8 mg / kg of IS 217 peptide. Additonally, the biochemical parameters AST and ALT are in normal levels in treated groups compared to the normal control group.2.4.2: SummaryThe present study results showed that IS 217 peptide was nontoxic and non- immunogenic, suggesting promising perspectives for their future therapeutic applications without any risks of inflammation.2.5: In vitro anti-microbial study2.5.1: The anti-microbial activity was assessed in vitro by broth microdilution time -kill viability assays and inhibition of bio film assays.2.5.2: Results -In vitro anti-microbial study:Multi drug resistance is a global health problem, probably related to millions of deaths each year [WHO, Geneva; 2014], Nowadays, there are different “zn vitro ' methods to evaluate the antibacterial activity of novel test lead molecules; broth dilution method is the gold standard to determine the minimum inhibitory concentration (MIC). The antimicrobial activity was assessed by broth microdilution and time -kill viability assays against all tested pathogenic bacteria.The study results confirmed that the test peptide IS 217 did not exhibit cytotoxicity at the physiologically effective antibacterial concentrations.2.5.2.1: Minimum Inhibitory Concentration (MIC) assayIn the present study, MIC values were determined by the micro broth dilution method in Mueller Hinton broth procedure as described above. All MIC values calculated in the present study are listed in Table 32. IS 217 displays rapid killing activity against pathogenic bacteria. The study demonstrated that IS 217 exhibits antibacterial effects against different bacterial pathogens (MIC between 0.39 and 1.56 pg / ml). Results shown in the Table 32 are means obtained from triplicate experiments.Table 32: Minimum inhibitory concentration (MIC) of peptide IS 217 and Antibiotic (Ciprofloxacin) against S. aureus, P. aeruginosa, E.coli and K. Pneumoniae2.5.2.2: Time-killing assay / survival of microbial species: Time-kill kinetics of IS 217 against S. aureus, P.aeruginosa, E.coli and K.pneumoniaeThe time -kill assays have been widely used for in vitro investigations of new antimicrobial agents as these provide descriptive (qualitative) information on the pharmacodynamics of antimicrobial agents [Olajuyigbe and Afolayan, 2012] and can be classified as bacteriostatic or bactericidal, based on the characterization of the relationshipbetween agent concentration and activity over time. IS217 displays rapid killing activity against pathogenic bacteria. In the present study, time -kill assays were performed to analyze the killing rate of IS 217 and to compare it with that of conventional antibiotic“ Ciprofloxacin ' which is frequently used in clinical settings. MIC values for IS 217 and selected antibiotic (Ciprofloxacin) were determined and are listed in Table 33. The increase in viable count of bacteria in the control group shows these bacteria were actively growing from 1 to 24 h. Figures 33-36 show the killing curves of IS 217 and Ciprofloxacin for .S'. aureus, P. aeruginosa, E. Coli and K.pneumoniae [Boorn KL, et al., 2010; Pradhan, S., etal., 2020],Table 33: MIC concentration of test peptide IS 217 and standard antibiotic against test species2. 5.2.21: Time-kill kinetics of IS 217against .S'. aureusThe result obtained with the test compound IS 217 and S. aureus was shown in Figure 33. After 24 hours incubation with 0.5x MIC (0.78pg / mL) of IS 217, a 1 loglO CFU / mL reduction in viability of S. aureus occurred (Figure 33), indicating the IS 217 was bacteriostatic against this strain. At a concentration of lx MIC (1.56pg / mL), however, the IS 217 was bacteriostatic against S. aureus by 5 h. Also, increasing the concentration to 5xMIC (7.8pg / mL) resulted in rapid bacteriostatic activity against S. aureus by 1 hour(Figure 33 and Table 34) and the complete reduction of initial inoculum of S. aureuswas achieved by 2hours. These results indicate that anti-bacterial activity of IS 217 was both time- and concentration-dependent. In comparison, the killing activity of Ciprofloxacin at IxMIC was slowerand complete reduction of initial inoculum of S. aureus was achieved by 3 hours treatment. At 3 hours the IS 217 and ciprofloxacin display similar killing activities.Table 34: Time kill kinetics of IS 217 against S'. aureusResults shown in the table are Mean ± SEM obtained from triplicate experiments.2.5.2.2.2: Time Kill Kinetics of IS 217 against P. aeruginosaThe result obtained with the test compound IS 217 and P. aeruginosa was shown in Figure 34. After 24 h incubation with 0.5x MIC (0.39pg / ml) of IS 217, Complete reduction in viability of P. aeruginosa occurred (Figure 34), indicating the IS 217 was bacteriostatic against this strain. At a concentration of lx MIC (0.78pg / ml), however, the IS 217 was bacteriostatic against P. aeruginosa by 3h. Also, increasing the concentration to 5x MIC (3.9pg / ml) resulted in bacteriostatic activity against P. aeruginosa by Ih (Figure 34 and Table 35) and the complete reduction of Initial inoculum of P. aeruginosa was achieved by 2 h treatment with 5x MIC. These results indicate that anti-bacterial activity of IS 217 was both time- and concentration-dependent.In comparison, the killing activity of Ciprofloxacin at lx MIC was Similar to 5 x MIC of IS 217and Complete Reduction of Initial inoculum of P. aeruginosa was achieved by 2 h treatment. At 2h, the IS 217 and ciprofloxacin display similar killing activities.Table 35: Time kill kinetics of IS 217 against P. aeruginosaResults shown in the table are Mean ± SD obtained from triplicate experiments.2. 5.2.2.3: Time Kill Kinetics of IS 217 against E. coliThe result obtained with the test compound IS 217 and E.coli was shown in Figure 35. After 24 h incubation with 0.5x MIC (0.39pg / ml) of IS 217, a 1 log 10 CFU / ml reduction in viability of E. coli occurred (Figure 35), indicating the IS 217 was bacteriostatic against this strain. At a concentration of lx MIC (0.78 pg / ml), however, the IS 217 was bacteriostatic against E.coli by 6h. Also, increasing the concentration to 5x MIC (3.9 pg / ml) resulted in bacteriostatic activity against E. coli by 2h (Figure 35 and Table 36) and the complete reduction of Initial inoculum of E. coli as achieved by 3h treatment with 5x MIC. These results indicate that anti-bacterial activity of IS 217 was both time- and concentration-dependent. In comparison, the killing activity of Ciprofloxacin at lx MIC was Similar to 5 x MIC of IS 217 and Complete Reduction of Initial inoculum of E.coli was achieved by 3 h treatment. At 3h, the IS 217 and ciprofloxacin display similar killing activities. Table 36: Time kill kinetics of IS 217 against E. coliResults shown in the table are Mean ± SD obtained from triplicate experiments.2. 5.2.2.4: Time Kill Kinetics of IS 217 against K. pneumoniaeThe result obtained with the test compound IS 217 and K.pneumoniae was shown in 5 Figure 36. At a concentration of lx MIC (1.56 pg / ml), however, the IS 217 was bacteriostatic against K.pneumoniae by 2 h. Also, increasing the concentration to 5 x MIC (7.8pg / ml) resulted in rapid bacteriostatic activity against K. pneumoniae by 1 h (Figure 36 and Table 37). Complete Reduction of Initial inoculum of K.pneumoniae was achieved by 4 h treatment with 5x MIC. These results indicate that anti-bacterial activity of IS 217 was 10 both time- and concentration-dependent. In comparison, the killing activity of Ciprofloxacin at lx MIC was slower and Complete Reduction of Initial inoculum of K.pneumoniaevias achieved by 12 h treatment. At 12 h, the IS 217 and ciprofloxacin display similar killing activities.15 Table 37: Time kill kinetics of IS 217 against K. pneumoniaeResults shown in the table are Mean ± SD obtained from triplicate experiments.52. 5.3: Conclusion of time kill assaysTime-killing assay was performed treatment of organisms with test peptide IS 217 for 24 h and IS 217 has showed good antimicrobial activity at the concentration of lx MIC (1.56pg / ml) against S. aureus, lx MIC (0.78pg / ml) against P. aeruginosa, and at 2h,5x 10 MIC of IS 217 showed the completed reduction of initial inoculum. 5x MIC of IS 217 and lx MIC of ciprofloxacin display similar killing activities, whereas IS 217 has showed good antimicrobial activity at the concentration of lx MIC (0.39pg / ml) against E.coli and at 3h,5x MIC of IS 217 and lx MIC of ciprofloxacin display similar killing activities. IS 217 has showed good antimicrobial activity at the concentration of lx MIC (1.56 pg / ml) against15 K.pneumoniaeanA at 12 h,5x MIC of IS 217 and lx MIC of ciprofloxacin display similar killing activities. Qualitative analysis of bacteria survival / time -killing assay, at different time intervals.2. 5.4: Effect of IS 217 on 48 h old biofilms of S.aureus20 Since IS 217 presented a lower MIC and comparable to ciprofloxacin andin the present study, IS 217 was investigated whether the peptide IS 217 was able to inhibit the viability of S. aureus biofilms. A significant reduction in CFU / mL was observed after 48 h exposure of a biofilm to IS 217 (Figure 37).25 Table 38: MIC concentration of test peptide IS 217 and the standard antibiotic against S.aureusBiofilm formation is one of the major virulence factors of S. aureus [Mah, T. F.et aZ.,2001; Lowy FD 1998]. The polysaccharide matrix of biofilm shields microscopic organisms from host immune defenses and obstructs the capacity of anti-microbial to target profound situated microorganisms living inside the biofilm. Moreover, biofilms go about as an irresistible specialty with continued arrival of microbes inside the host, which prompts unending contamination, backslides, hazardous circulation system diseases, and treatment failure. Given the genuine difficulties related with staphylococcal biofilms and their job in advancing repeating diseases in the host, we next moved to survey whether our peptide is equipped for disturbing full-grown biofdms (formed after 48 hours) of S. aureusto compare it with that of conventional antibiotic “Ciprofloxacin” which is frequently used in clinical settings. MIC values for IS 217 and selected antibiotic (Ciprofloxacin) were determined.The results in Table 50 clearly indicate the enhanced anti-biofilm effect of IS 217 with 0.5 x MIC,1 x MIC and 5 x MIC concentrations recorded 65, 43.7 and 25.7% of biofilm formation. The result obtained with the test compound IS 217 and S. aureus was shown in Figure 37. The adherent biofilm stained by crystal violet, then the dye was extracted with ethanol, measured at 595 nm absorbance, and presented as percentage of biofdm reduction compared to untreated wells “control” and standard drug.Although some conventional antibiotics might be capable of disrupting 24 hour- mature bacterial biofilm, most antibiotics are not effective against 48 hour-mature biofilms due to the dormant state of growth of the bacterial cells present within the mature -biofilms. To examine whether the potential therapeutic application of IS 217 is having the ability to disrupt 48 hours-mature staphylococcal biofilm was tested. As expected, it was observed in Figure 37, the IS217 (at 1 x MIC) significantly disrupted the 48 hours of matured biofilms of S. aureus, reducing biofilm mass by 50%. Ciprofloxacin at 1 X MIC were able to decrease only 40% of the biomass (p < 0.05).Table 39: Effect of IS 217 on 48 h old biofilms of S.aureusResults shown in the table 39 above are mean values of percentage obtained from triplicate experiments. 5.5: DiscussionIt is well known that the incidence of pathogen resistance to antibiotics is increasing day by day and is the most serious issue in today's world [Infectious Diseases Society of America, 2004; Peters NK et al. 2008]. Bacterial pathogens (ethological agents of human illnesses) are a severe hazard to human health and the antibiotic abuse and misuse generate major environmental concerns, as well as the evolution and rapid spread of antibiotic resistance bacteria [Gums JG 2002 ; Jorgensen and Ferraro, 2009], resulting in an increasing number of deaths each year [Nathan and Cars, 2014]. Most of the current antimicrobials have natural origin deriving from microbes, plants or animals [Bdrdy, 2005] like Host defense peptides (HDPs) and / or antimicrobial peptides (AMPs) [Steinstraesser et , 2009]. Antimicrobial peptides are classified based on their mode of action, which can include interfering with cell wall synthesis, protein, DNA, or RNA synthesis, as well as inhibiting various metabolic pathways or the cell cycle [Hancock and Sahl, 2006; Hale and Hancock, 2007; Hilpert et al., 2010; Mardti and Kondorosi, 2014]. Due to some toxicity profile, the synthetic HDPs are the focused area of research to combat the resistance to the various pathogens.In the present study, the IS 217, one of the synthetic peptide known as Host Defense Peptide (HDP / AMP) was investigated for its anti-bacterial profile by estimating MIC and time kill survival rates. Our study results showed that IS 217 was significant inhibitory properties against .S'. aureus fATCC® 6538™) 1.56(pg / ml), P. aeruginosa (ATCC® 9027™) 0.78(pg / ml), E. coll fATCC® 8739™) 0.39(pg / ml) and K. pneumoniae A CC®700603™)1.56(pg / ml). Based on the MIC results, the inventor of the present invention continued to investigate the time kill survival to determine how long all bacteria are necessary for IS 217 to eliminate above mentioned pathogens. Unlike an MBC / MIC assay, this assay enables the measurement of the compound's rate of cidal action [Aiyegoro, Afolayan, &Okoh, 2009]. The curves were determined to assess the correlation between MIC and bactericidal activity of IS 217 at concentrations ranging from 0.5 -fold MIC to 5- fold MIC [Mohamed F. et al., 2016].The compound was rapidly bactericidal at 1 x MIC for all four pathogens after 1 h incubation. Meanwhile S. aureus and P. aeruginosa were completely eliminate after 2h incubation, whereas E.coli after 3h incubation and K.pneumoniae after 4h incubation at concentration of 5 x MIC. The 5x MIC concentration of IS 217 are comparable with lx MIC of ciprofloxacin and display similar killing activities. The results of the time -kill assay are presented in Figure 33 to 36 and the data showed that the response of the bacteria to the tested compound IS 217 varied among the strains, concentration, and time dependent. The differences in susceptibility may be due to the differences in cell wall composition and / or genetic content of their bacteria. The antibacterial activity is most likely due to the adsorption of compounds causing membrane disruption, subsequent leakage of cellular contents and cell death.Based on literature study, Peptide IS 217 is designed antimicrobial host defense peptide (HDP). In general, the anti-microbial activity of AMP’s might be as follows, where the AMPs must interact with membranes as part of their direct antibacterial mechanism (or mechanisms) of action, leading to membrane perturbation, disruption of membrane- associated physiological events such as cell wall biosynthesis or cell division, and / or translocation across the membrane to interact with cytoplasmic targets and destroy the cell by changing membrane conductance and altering intracellular function and alterations in membrane structure results in the reorientation of peptide molecules in the membrane culminating in eventual pore formation and lysis of the target microbe.The concentrations of the peptides also play an important role which promotes the cell lysis, and capability of channel formation [Jaynes, J. M. Drug News & Perspectives 3: 69

[1990] ; and Reed, W. A. et al. Molecular Reproduction and Developments 1:061992]. Thus, at a certain concentrations, these peptides stimulate or create channels that can be advantageous to the normal mammalian cell in a benign environment where it is not necessary to exclude toxic chemical compounds.The test compound at a concentration equal to 5 x MIC was rapidly bactericidal, achieving complete elimination of both test bacterial strains within 3h. All the time -kill data obtained with the test compound IS 217 showed its antibacterial activity to be time - and concentration-dependent. In follow-up studies, the test peptide will be examined to see if the in vitro time-kill statistics are predictive of in vivo efficacy.After confirming that IS 217 possessed excellent antimicrobial activity against Staphylococcus aureus, the invetor of the present invention next assessed the efficacy of IS 217 on 48h old biofilms of S. aureus. Biofilms serve as an infectious niche, allowing bacteria to be released continuously inside the host, resulting in chronic infection, relapses, life-threatening bloodstream infections, and treatment failure. S. aureus is the key virulence factors in biofilm development. Furthermore, biofilms given the serious challenges associated with staphylococcal biofilms and their role in promoting recurring infections in the host, we next moved to assess whether our peptide IS 217 can disrupt mature biofilms (formed after 48 hours) of S. aureus to compare it with that of conventional antibiotic “Ciprofloxacin” which is frequently used in clinical settings [Archer, N. K. et a / .,2011;Nickel, J.C. et al., 1994;Hojo, K. et al. 2009;Hojo, K. et al., 2009]. In the present study, anti-biofilm effect of IS 217 against Staphylococcus aureus has been studied adopting biofilm inhibition spectrophotometric assay. All the concentrations tested inhibited biofilm in a dose dependent manner and the result was shown in Figure 37. Although some standard antibiotics may be capable of disrupting 24 hour-mature bacterial biofilms, most antibiotics are ineffective against 48 hour-mature biofilms due to the dormant state of growth of the bacterial cells present within mature-biofilms. As appeared in Figure 37, IS217 (at 1 x MIC) significantly disrupted the 48 h matured biofilms of S. aureus, reducing biofilm mass by 50%. Ciprofloxacin at 1 X MIC were able to decrease only 40% of the biomass [iberio SA et al., 2011; SoeryaDewiMarliyana et al., 2017]In summary, it was shown that IS 217displays rapid bactericidal activity along with the efficacy of IS 217 on 48h old biofilms of S. aureus. Taking all these factors into account, it was believed that IS 217 has the potential to serve as a backbone molecule for the development of new anti-infective therapies, even as coatings for implants. Further investigations such as physicochemical studies, toxicity tests, animal efficacy and pharmacokinetic / pharmacodynamic studies will be needed to fully reveal the therapeutic potential of this class of molecules. .6: SummaryIS 217 has showed good antimicrobial activity, almost like standard antibiotic Ciprofloxacin. Furthermore, IS 217 disrupted the mature biofilms of 48 h old of S. aureus at a more significant rate than antibiotics of choice. Moreover, the anti-biofilm effect of IS 217 would suggest the possible utilization of synthetic peptides as effective anti-bacterial agents against pathogenic bacteria.Collectively, the qualities of IS 217 presented here have the potential to be used for different clinical applications including chronic infections, dental biofilms, since the pathogenesis of the cariogenic process is related to the formation of a biofilm.2.6: Confirmatory studies for anti-inflammatory activity (In vitro and In vivo)2.6.1: Effect of IS 217 on viability of BALB / C mice peritoneal macrophagesThe viability and cytotoxicity of different concentrations of IS 217 to peritoneal macrophages were examined by MTT assay.The peritoneal macrophages were incubated with IS 217 in different concentrations ranging from (1.56, 3.12, 6.25, 12.5, 25, 50 and 100 pg / mL) and cell viability was measured by an MTT assay 18 h later. It was found that IS 217from 1.56 to lOOpg / mL had no cytotoxic effects on murine peritoneal macrophages. These results confirmed that the effects of IS 217 on murine peritoneal macrophages were not due to a reduction in cell viability.Therefore, subsequent assays were carried out at concentrations less than lOOpg / mL.2.6.1.1: Effect of IS 217 on the levels of cytokines in LPS -stimulated peritoneal macrophages: Immunomodulatory activityIS 217 inhibits LPS -induced macrophage activation and the inflammatory response. Macrophages are known to produce all kinds of inflammatory factorsto be involved in the progression of sepsis. When stimulated with LPS, the number of mouse peritoneal macrophages increases, and an excessiveimmune response is triggered. Cell viability assay was used to evaluate the cytotoxicity of IS 217 onperitoneal macrophages; the result indicated that the concentrations less than 100 pg / mL had no obvious cytotoxicity. In the subsequent experiments, concentrations of IS 217 did not exceedlOO pg / mL. To further confirm the anti-inflammatory effects of IS 217 on macrophages, the peritoneal macrophages from mice were collected to measure the production of IL-6, IL-ip, IL-12P70 and TNF-a and the same inhibitory effects were observed (Figure 38) and IL- 10 levels (Figure 39).TNF-a and IL-6 are known to be a pro-inflammatory mediator in inflammatory diseases. In an attempt to determine whether IS 217 regulates TNF-a and IL-6 production, peritoneal macrophages at a range of 0.8 -1 X 106were treated with IS 217 (3.12, 6.25 &12.5 pg / mL) in the presence or absence of LPS stimulation. As shown in Figure 39, LPS challenge markedly increased the levels of pro-inflammatory cytokines IL-6, IL-ip, IL- 12P40 and TNF-a in peritoneal macrophages as compared with those in the normal control group (P < 0.001), Following pre-treatment with IS217, obviously reduced LPS-induced production of IL-6,IL-ip,IL-12P40 and TNF-a levels were significantly decreased in a dose-dependent manner. This confirmed the anti-inflammatory effect of IS 27 on secretion of IL-10 Levels (Figure 38) also along with the pro inflammatory cytokines, IL-6, IL-ip, IL-12P40 and TNF-a levels in LPS-stimulated murine peritoneal macrophages.2.6.1.2: Effect of IS 217 on the levels of NO in LPS-stimulated peritoneal macrophagesIn murine macrophage RAW 264.7 cells, LPS stimulation alone has been demonstrated to induce iNOS transcription and its protein synthesis, with a corresponding increase in NO production [Xieet al., 1994; Henkel et al., 1993]. The Griess reaction, a spectrophotometric determination for nitrite, was carried out to quantify the nitrite levels in the conditioned medium of RAW 264.7 cells treatedwith LPS. Figure 40 shows the inhibitory activity by test peptide IS 217 towards NO production by LPS -activated macrophages.2.61.3: Effect of IS 217 on mRNA expressions of cytokinesThe mRNA levels of IL- 12, IL-P, IL-6, and tumor necrosis factor (TNF)-a were performed to provide an estimate of the relative levels of expressions of these genes. In the present study, the mRNA expression levels of IL- 10, IL- 12, IL-P, IL-6, and TNF-a were increased in the LPS treated groups compared to the control groups, and the mRNA expression levels of IL- 12, IL-P, IL-6, and TNF-a were decreased in the IS 217 treated groups compared to the LPS treated groups (Figure 41, Figure 42).2.6.1.4: In vitro effects of test peptide IS 217 on MAPK PhosphorylationAs NF-KB pathway is closely related to theexpression of iNOS and proinflammatory cytokines, modulationof test peptide IS 217 was examined. Figure43shows western blot results of LPS-induced RAW264.7 cellsunder treatment with different test peptide IS217 concentrations. To further investigatewhether inhibition of inflammatorymediators by test peptide IS 217 was modulated by the MAPK pathway, the effects of test peptide IS 217 on LPS-induced phosphorylation of ERK1 / 2, and p38 were examined. The test peptide IS 217 significantly reduced phosphorylation of ERK1 / 2, and p38 in a dose dependent fashion (Figures 43 to 45).2.6.2: Effect of IS 217 on paw volume and percentage inhibition in carrageenan- induced paw edemaIn the present study, sub plantar injection of carrageenan caused an increase in paw size in mice due to edema, thus indicating acute inflammationof paw. Figure 46(b) shows the thickness of paw volume in different groups of mice following treatment with test doses of IS 217 and Dexamethasone. The paw edema of mice increased progressively and reached its maximum after 3 h of carrageenan injection (Figure 46(a)). The study revealed that, the percent reduction in paw edema on 5th hour recorded highest in IS 217 (90.2) at 1.2 mg / kg b.wt, as shown in Table 44. The anti-inflammatory effect of 1.2mg / kg of IS 217 was comparable to that of the standard (dexamethasone) (Table 40). The highest antiinflammatory activities for both IS 217 88.9%) and dexamethasone (88.9%) were observed after 24 h of carrageenan injection. Pre-treatment with IS 217 (0.6 mg / kg and 1.2 mg / kg) and DEX (5 mg / kg) significantly decreased the amount of edema 3 h after injection when compared with control groups (P<0.05, P<0.05, P<0.01, respectively). After 18 h, IS 217(1.2mg / kg) reduced swelling by 88.9% (P<0.01), almost curing the edema completely Figure 47 (a) and Figure 47 (b).Photographs of the paw of various groups of mice i.e., normal control or carrageenan induced paw edema mice treated with the placebo or IS 217or Dexamethasone have been shown in Figure 47. These images clearly showed significant reduction in paw thickness in IS 217orDexamethasone treated groups as compared to control group (Figure 47).Table 40: Percentage Inhibition (%) of paw edema at different time intervals2.6.3: DiscussionThe purpose of this study was to investigate the effect of IS 217 on acute inflammation model both in in vivo and in vitro and to explore the possible mechanism. It was found that carrageenan-induced mice paw edema was significantly attenuated by IS217 pretreatment. In addition, IS217 markedly reduced TNF-a and IL-6 production in LPS- stimulated mouse peritoneal macrophages. These findings suggest that IS 217 possesses significant anti-inflammatory activities in vivo and in vitro.Furthermore, the production of TNF-a in the peritoneal macrophages was markedly inhibited by IS217. Lastly, IS217 produced a significant inhibition of pro -inflammatory cytokines protein expression in LPS -stimulated mouse peritoneal macrophages. Taken together, these results demonstrate thatIS217 was an effective inhibitor of inflammation.Edema is the typical feature of inflammation not only in systemic inflammation, but also in local inflammation [XiaofengNiu et al., 2012]. Carrageenan induced paw edema model, a classical animal model of acute inflammation, has beenbroadly used for the evaluation of anti-inflammatory compounds. In present study, the results of carrageenan- induced paw edema assay showed that sub-plantar injection of carrageenan in mice displayed an obvious increase in paw volume, and the paw edema attained its maximum 3 h after carrageenan stimulation, while the paw swelling was effectively inhibited by pretreatment with IS217at all time -points. These results reflect that IS 217 exhibits its antiinflammatory effects on carrageenan-induced rat paw edema based on suppression of local edema [Inmaculada Posadas et al.,2Q \. The acute inflammatory response induced by carrageenan injection involves two phases that consist of the sequential release of several mediators. The early phase is observed during the first hour of exposure and is related to the release of histamine, serotonin, bradykinin, and to a lesser extent, prostaglandins. The delayed phase (after 1 h) is attributed to polymorphonuclear (PMN) leucocyte infiltrationand the continuation of prostaglandin generation and pro -inflammatory cytokines, such as TNF-a and interleukin- ip (IL-1), are also involved in the delayed phase of carrageenan- induced inflammation [Sravani Edula et.al,2014]. LPS, the major constituent of the Gramnegative bacterial cell wall, induces the expression of various inflammatory cytokines such as TNF-a and IL-6 when administered to cells [XiaofengNiu et al., 2012]. Macrophages are extraordinarily versatile cells, playing a key role in the host's defense against bacterial infection by nature of their phagocytic, cytotoxic and intracellular killing capacities. In response to LPS, the peritoneal macrophages easily secrete different inflammatory cytokines including TNF-a and IL-6, which play a critical role in the process of macrophage activation and are related to acute and chronic inflammation. Previous studies have demonstrated that over production of TNF-a and IL-6 by macrophages contributes to chronic hepatitis, rheumatoid arthritis, pulmonary fibrosis and skin inflammation. In present study, the effect of IS 217 on LPS-induced TNF-a and IL-6 production was evaluated in peritoneal macrophages by ELISA assay and Western blotting. ELISA results showed that the levels of pro-inflammatory cytokines, like TNF-a and IL-6, were elevated in LPS -challenged peritoneal macrophages. In contrast, the administration of IS217 significantly reduced the supernatant levels of TNF-a and IL-6. In summary, these results suggest that the anti-inflammatory effect of IS217 in LPS-stimulated peritoneal macrophages may relate to its inhibition on TNF-a and IL-6 production [Stephen B. et al., 2010]. The test peptide IS 217 demonstrated inhibitory activities against NO production, which may have therapeutic potential for the treatment of inflammation accompanying overproduction of NO [Min Jee Kim et al., 2014]. Real time PCR analyses of the mRNA levels of IL- 12, IL-P, IL-6, and TNF-a were performed to provide an estimate of the relative levels of expressions of these genes. In the present study, the mRNA expression levels of IL- 12, IL-P, IL-6, and TNF-a were increased in the LPS treated groups compared to the control groups. And, the mRNA expression levels of IL- 12, IL-P, IL-6, and TNF-a were decreased in the IS 217 treated groups compared to the LPS treated groups. The MAPKs are a family of serine / threonine kinases thatare involved in a variety of cellular processes. The MAPK molecules, ERK, and p38are activated in responseto certain extracellular stimuli such as LPS or carrageenanchallenge. These kinases have different downstream targetsand mediate diverse cellular responses, including regulationof apoptosis, proliferation, and inflammation [XiaofengNiu, et al., 2015]. The present studyshows that treatment by test peptide IS 217 significantly inhibited LPS induced ERK1 / 2, and p38 phosphorylation in LPS stimulated macrophages (Figure 44), which maycontribute to the inhibitory effect of test peptide IS 217 on the production of proinflammatory mediators in LPS -induced macrophages.Taken together, it is possible that IS217 reduced LPS-induced pro inflammatory cytokines through the inhibition of MAPK pathway and increases the IL- 10 levels. However, the exact mechanism(s) of the anti-inflammatory effects of IS217 observed in this study are needed to establish its mechanism(s) of action.2.6.4: SummaryThis section of the study summarized the LPS -activated mouse macrophage cell model and carrageenan induced mice paw edema model are an economic approach for basic screening anti-inflammatory activity for treatment or mitigation of inflammation. The present study demonstrated that IS 217 was shown to inhibit not only TNF-a and IL-6 production, but also the activation of MAPK pathway in LPS -challenged peritoneal macrophages, which confirms the in vitro results on RAW 246.7 cells. Furthermore, IS 217 has a protective effect on carrageenan in induced mice paw edema. Carrageenan-induced inflammation model is a significant predictive test for anti-inflammatory agents acting by the mediators of acute inflammation. The results of this study suggest that IS 217 has antiinflammatory and immunomodulatory activity, and thus, may be of value in the treatment of diseases of immunopathological origin characterized by macrophage hyperactivation. These findings suggest that IS217 may be a new therapeutic agent for the prevention of inflammation.2.7: In vivo Proof of Concept (PoC) studies by using Cecal Ligated and Puncture (CLP) model and E.coli induced peritonitis model in mice2.7.1: Pilot study of CLPAfter 24 h of observation, the surviving animals are euthanized in a humane manner and collected blood for cytokines estimation. The survival rate of disease control is > 50% when compared to treated groups and sham control, whereas the treated groups with IS 217 -0.6 mg / kg &1.2 mg / kg survival rate was > 60 & 80%, respectively (Figure 48, Figure 49 and Table 41).Table 41: Percent survival rate of all the groups of CLP for 5 daysAfter the observation that treatment with test peptide IS 217 activates phagocytes at the site of infection and considering that the evolution of sepsis occurs in parallel to systemic inflammation, we chose to investigate some cytokines that are related to the progression of sepsis and the activation of phagocytes. The test peptide IS 217 was observed to inhibit the production of the inflammatory cytokines IL-6, IL-ipandTNF-a (Figure 49 and Figure 50). The anti-inflammatory cytokine IL- 10 was also decreased by IS 217 treatment (Figure 50). Early in the infection, IL- 12 is produced and induces production from NK and T cells of IFN-gamma, which contributes to phagocytic cell activation and inflammation and so we investigated the IL- 12 levels and the IS 217 treatment showed the inhibition of IL- 12 levels along with other pro inflammatory cytokines. [Figure 49(d)]. It was observed that IS 217 negatively regulates inflammatory cytokines and subsequently improved survival during sepsis. But it remains unclear whether improved survival is due to inhibition of TNF-a, IL-ip, and IL-6 together. Keeping in mind that TNF-a and IL-ip are induced within hours [ArashKarimi el al., 2019; Cai J, Cui X et al., 2021], mice were treated with IS 217 within 2h after the CLP surgery. In sham control groups, which are treated with IS 217, it was observed that there was no effect on the pro inflammatory cytokines and this also proved that the test peptide IS 217 was non immunogenic and showed similar response with previously reported immunogenicity results. As there was no effect of test peptide on inflammatory cytokines in sham control groups, for the main experiment, we excluded the sham control+ treatment groups.IS 217 administrations protect animal from death by CLP-induced sepsis and inhibits the systemic inflammatory cytokines.6.7.2: Main CLP studyThe aim of the present study was to investigate anti-inflammatory and immunomodulatory properties of IS 217 doses (0.6 &1.2 mg / kg) on the control of the systemic inflammatory response, the activation of phagocytes and the control of bacterial growth in a sepsis experimental model.10-12 male C57BL / 6 &BALB / c mice were submitted to the sepsis model by cecal ligation and perforation (CLP group) or laparotomy only (sham group). After 2h post CLP surgery, the animals received IS 217 (0.6 & 1.2 mg / kg), Intravenously (IV) in BALB / c mice &subcutaneously (SC) in C57BL / 6 mice. The animals received saline in sham control. Blood was isolated for cytokine analysis and other biochemical markers at 18 h and 10 days after CLP. A subset of animals was followed for 10 days for survival assessment, and then behavioral tests were performed. The administration of IS 217 restored the elevation of IL- ip, TNF-a, IL-6, and IL-10 cytokine levels in the sera even after 10 days of post CLP.After 4h and 18h after the CLP, the lungs, spleen and blood were collected to measure the serum cytokines and the animals were killed for the evaluation of cytokines estimation, colony-forming units (CFUs). The results showed that only the test peptide IS 217 treatment inhibited bacterial growth in the peritoneum and inflammatory cellular influx, especially influx of macrophages and neutrophils. However, test peptide IS 217 treatments decreased the pro -inflammatory cytokines in the serum, indicating a systemic anti-inflammatory effect of both.In the present study, goals of the inventor were (1) to investigate the direct influence of proinflammatory cytokines (IL-ip, IL-6, and TNF-a) in abdominal lavage fluid, lungs, spleen, and serum and (2) to measure bacterial growth in abdominal lavage fluid of anesthetized mice.2.7.2.1: Activity and vital parametersThe activity of mice in the sham, IS 217 treated groups was significantly higher (each P < 0.05 and demoted as ***, Table46) compared to the sepsis-control group 18 hours after CLP. The general activity and body temperature results at 18 h after CLP surgery are shown in Table 42. Both the CLP animals exhibited tachypnea and hypothermia. The test peptide IS 217 treated animals’ body temperatures are in range of normal values. [Asma Ahmed, et al., 2018]Table 42: Activity Index and body temperature after 18 h of sepsis2.7.2.2: Results of Behavioral testResults of Novel object recognition test (NORT)In NORT, naive mice displayedaclearpropensityfor exploring the novelobjectincomparisontothefamiliarobject,as these mice spent80%oftheirobjectexplorationtimeinthetestphase withthenovelobject (Table 43). On the other hand, diseased control showednopreferenceforthenovelobject, as their totalobjectexplorationtimewasmoreorlessproportionatelysplitbetweenthenovelobject(57%) and the familiar object (Table 43). indicating the effect on memory impairment due to CLP surgery and infection. The mice treated with IS 217 at 0.6& 1.2 mg / kg, showed normal and increased tendency to recognize the new object (69.61&75.97 %, respectively) and which is evident by increase in tendency torecognize the new object in a dose dependent manner in comparison to control group. Theseobservationsimplythatany underlying depressionlikelydidnotinterferewiththenovelobject recognition memorytestingin IS treated groups.Thus, exposure to IS 217 treated groups has the normal tendency of novel objectrecognition memory function.Table 43: Effect of IS 217 (0.6 &1.2 mg / kg) injected IV, on percentage preference for new object recognition at 10thdayEffect of IS 217 (0.6 &1.2 mg / kg) injected IV (n = 5-6) on time spent in open arms, in comparison with diazepam and disease control groups in the elevated plus maze.Data are expressed as means +SEM. p<0.05 was considered as significant and denoted as *** in comparison with disease control group.DiscussionThe sepsis survivors present acute and long-term cognitive impairment and the pathophysiology of neurological dysfunction in sepsis involves microglial activation leading to memory impairment in sepsis-surviving rats and performed two behavioral tests performed at 10 days after sepsis [LucineiaGainski D et. al., 2020]. Wu and colleaguesrevealed a reduction for long-term memory in the freezing time of animals submitted to the fear conditioning test at 7 days after sepsis and treatment with peptide SS- 31, and the same pattern was observed in another study that tested animals at 2 weeks after inducing sepsis and treating with MCC950 or a caspase-1 inhibitor. In 2018, Zarbato and colleagues exhibited the protective effect of dimethylfumarate on the short-term object recognition memory of rats submitted to the CLP model [Wu J, 2014]. In contrast, Della Giustina and colleagues demonstrated that fish oil-treated rats had positive effects only on long-term memory [Danielski LG, Della Giustina A et al., 2017].As already described in the literature, we made an attempt to evaluate the memory impairment and activity of the mice undergone CLP and we performed two behavioral tests: elevated plus maze test and Novel object recognition test. The study results showed the IS 217 at 0.6 & 1.2 mg / kg doses produced a clear anxiolytic effect, reducing avoidance of the open arms without changing the locomotor activity of the rats in the closed arms and this was compared to control animals. It was assessed the behavioral elements derived from ethological analysis to assess EPM anxiety in rodents. Cruz and colleagues, in a study of factor analysis of mice behavior in the EPM, identified four distinct factors with loadings greater than 0.4: Factor 1 (anxiety); Factor 2 (activity); Factor 3 (decision making); Factor 4 (displacement). In addition, they found that their measure of risk assessment co-loaded in three factors (1, 3 and 4) that measure different aspects of anxiety (avoidance of danger,decision making, approach-avoid conflict). Assessment of the ethological measures, in this study, revealed that IS 217 treatment and diazepam produced an anxiolytic profile of action in some behavioural parameters. Both treatments reduced rearing and peeping out (decision making-related behaviour) and increased end-arm activity (exploratory activity-related behavior), indicating an enhanced tendency to explore actively the potentially dangerous areas of the EPM [Perry VHet al., 2014] and may represent an attempt to avoid threatening situations associated with the height clues in the apparatus. On the other hand, diazepam showed a clear anxiolytic effect, reducing avoidance of the open arms without changing the locomotor activity of the mice in the closed arms.These results provide further support for the anxiolytic potential of test peptide IS 217, and point the inventor towards the necessity of extending this study through a series of tests involving different stressful stimuli (e.g. novelty, and openness), increasing the number of tests, the range of stressful stimuli and the behavioral tasks involved would certainly contribute to gaining a broad understanding about the underlying mechanisms of the emotional behavior of rodents. [LucineiaGainskiDanielski et al. ,2020] so, the inventor performed the Novel object recognition test.To determine whether IS 217 treated groups exhibit memory impairments once after post CLP surgery, these mice were examined for object recognition memory function using another stress-free test. For this, an NORT was employed comprising exploration of two identical objects in the acquisition phase and comparison of the exploration of a familiar and a novel object an hour later in the test phase [Bussey et al., 2000; Langston et al., 2010]. Naive mice spent more time exploring the novel object than the familiar object, which confirmed their ability for object recognition memory. However, disease control animals showed no preference for the novel object as they spent nearly equivalent amounts of time with the novel and familiar objects, implying that these CLP operated animals have object recognition memory dysfunction, where IS 217 treated groups, particularly IS 217 - 1.2 mg.kg dose demonstrated normal object recognition memory function when minimal inter-trial interval (5 min) was maintained between the acquisition and test phases. This suggests that IS 217 treated groupsmice display deficits for long-term object recognition memory function.SummaryThis test demonstrated the IS 217 treated groups display deficits for long-term object recognition memory function and having normal activity after the post CLP surgery as comparable to control groups.2.7.2.3: Experiment 1 Survival studyIS 217 treatment enhanced survival of mice after induction of sepsisThe Kaplan-Meier curve for survival analysis of mice subjected to polymicrobial sepsis and treated with test peptide IS 217 (0.6 &1.2mgZkg) showed lower mortality after sepsis when compared with non-treated septic mice (Figure 51 and Figure 52). Peritonitis induced by CLP with a 21 -gauge needle results in 100% lethality at day 8 [YonaKalechman, Uzi Gafter, et al., 2002] CLP was performed as described in the methods section and animals were Intravenously (IV) & subcutaneously (SC) injected with various doses of IS 217 or vehicle at 2h after CLP procedure and then mice were observed for 18 h (Figure 51(a) , 52 (a) and subset of animals are continued for observation for 10 days [Figure 52(b) and Figure 53 (b)] [Tables 44 and 45]. No significant difference was observed when IS 217 was injected at the time of injury (time zero). Notably, treatment with IS 217 18 h decreased survival, compared with NS -treated mice [Konstantin Tsoyi, et al., 2009; Kim, Y. K., 2017].Table 44: Percent survival rate of all the groups of post CLP surgery for 18 h & 10 days with IV administration of IS 217Table 45: Percent survival rate of all the groups of post CLP surgery for 18 h & 10 days with SC administration of IS 217Mice treated Intravenously with IS 217 2 h after CLP showed improved survival rates (62.5%) in 0.6 mg / kg and (83.3%) in 1.2 mg / kg, after 18h compared with CLP mice (28.5%; P, 0.001) or sham control (0%; P, 0.001) [Figure 51a ,b]. whereas the 18h survival rates of mice subcutaneously treated with IS 217 2 h after CLP showed were 30% lower compared with that of mice treated intravenously, showed survival rates (33.3%) in 0.6 mg / kg and (50%) in 1.2 mg / kg, after 18h compared with CLP mice (37.5%) or sham control (0%; [Figure 52 a, b]. Simultaneously, mice treated Intravenously with IS 217 2h after CLP showed improved 10-day survival rates (55.5%) in 0.6 mg / kg and (100%) in 1.2 mg / kg, (Figure 51b) compared with CLP mice (22.3%;) or sham control (0%;) (Figure 51b). Whereas, the 10-day survival rates of mice subcutaneously treated with IS 217 2 h after CLP showed survival rates (55.5%) in 0.6 mg / kg and (83.3%) in 1.2 mg / kg, [Figure 52(b)] compared with CLP mice (22.2%) or sham control (0%). In intravenous administration, the first sign of the death started from 2h and continued for 8 days in disease control and whereas in treatment groups it was observed first 4hrs and last death observed at 3rdday Analysis of the survival rates showed that IS 217 at 1.2 mg / kg, (IV& SC) protected mice from death caused by CLP-induced sepsis. Compared with the CLP control group, the IS 217 (0.6 &1.2mg / kg) treated group showed a better disease score. Thus, it appears that IS 217 could protect mice from sepsis-induced death only if injected after induction of sepsis.2.7.2.4: Biochemical markers, Serum lactate and DICCLP-induced organ injury is ameliorated in IS 217-treated mice sepsis frequently causes multiple organ failure, a condition that leads to death. To understand the mechanism whereby IS217-treated mice were resistant to CLP, experiments were conducted to assess the organ damage induced by CLP and its response to IS 217. For this purpose, biochemical markers of liver damage (AST, ALT) were measured and renal failure (creatinine, BUN) with IS 217 IV & SC administration at 18h following CLP along with other parameters are mentioned in Tables 46 and 47 and Figure 53 and Figure 54. shows that control CLP mice demonstrated biochemical evidence of more severe multiple organ damage than IS 217-treated mice, as reflected by higher ALT, AST, creatinine, and BUN concentrations (all p <0.05). The levels in IS 217- treated mice were comparable with those in mice not subjected to CLP, suggesting that organ injury was avoided in IS 217-treated mice [Asma Ahmed, et al., 2018; Ajaz Ah made / al., 2020].Table 46: Effects of IS 217 (0.6 & 1.2 mg / kg -IV) administrations on serum liver &kidney function tests in cecal CLP-induced sepsis in mice. IS 217 shows protective effect on liver injury in CLP miceTreatment with IS 217 ameliorates organ injury induced by CLP. IS 217-0.6 & 1.2 mg / kg, SC was injected into mice 2 h after CLP. At 18 h after CLP, mice were killed, and the amounts of AST, ALT, BUN, and creatinine, in sera were measured. Table 47: Effects of IS 217 (0.6 & 1.2 mg / kg -SC) administrations on serum liver function tests in CLP-induced sepsis in miceHowever, to have potential as a tool for treatment of sepsis, it is important to study if IS 217 can reduce inflammation and DIC when administered therapeutically. Next, it was examined whether IS 217 could also inhibit DIC when injected therapeutically. BALB / c mice were administered IS 217 2h post CLP surgery and 18 h later, blood was collected and coagulation parameters for DIC were examined. Administration of IS 217 (IV) therapeutically helped to reduce clotting time as evidenced by decreased PT and aPTT ■(Table 48) values in IS 217 treated group as compared with PBS-treated septic mice. Thus, IS 217 can prevent inflammation and DIC in septic mice, even when administered therapeutically.Table 48: Effects of IS 217 (0.6 & 1.2 mg / kg -IV) administrations on Coagulation tests in CLP-induced sepsis in miceThe serum lactate levels are higher in CLP group, whereas sham control and treatment groups with IS 217 IV administrations at 18h following CLP along with other parameters are mentioned in (Table 49 and Figure 55) [Chen H, Zhao C,2019].Table 49: Effects of IS 217 (0.6 & 1.2 mg / kg -IV) administrations on serum lactate in cecal CLP-induced sepsis in mice2.7.2.5: Experiment 2: Acute hyper inflammation study: Cytokine estimation in SerumTo investigate the underlying potential mechanism of the protective effect of IS 217, the levels of the representative proinflammatory cytokines TNF-a, IL-ip and IL-6 in the peritoneal cavity, lungs and spleen of severe CLP mice were measured. In blood sera the levels of the representative cytokines TNF-a, IL-ip, IL-6, IL12 and IL- 10 were measured by ELISA.Both intravenous and subcutaneous administration of IS 217 markedly decreased the TNF-a, IL-ip and IL-61evels in peritoneal fluid and serum 18 h after CLP surgery compared with CLP mice. More importantly, the concentrations of both TNF-a, IL- ip and IL-6 were lower after the treatment IS 217 than after intravenous administration, whereas IL-10 levels increased slightly only at high dose of IS 217(1.2 mg / kg). Pro- inflammatory macrophages with an Ml phenotype play an important role in mediating inflammation. The same trend was observed in samples collected after 10 days with treatment of IS 217 daily for 5 days. [Burgelman, M et.al, 2021]Effect of IS 217 (0.6 & 1.2 mg / kg -IV& SC) administrations on proinflammatory cytokines levels after CLP surgeryOrgan injury observed in sepsis is due to the explosive release of cytokines into the serum / plasma. We therefore sought to determine the serum levels of cytokines following CLP and their response to IS 217 treatments. The classic cytokines produced in the initial period of an inflammatory insult are TNF-a, IL-ip IL-6 and IL- 12, which are followed by overproduction of IL- 10. TNF- a and IL-1 p levels were undetectable in the serum at 1 h post-CLP. Maximal levels of these cytokines were found at 6 and 12 h. The inflammatory response was not persistent, and it gradually declined, until at 24-48 h after CLP it was only minimal and so, in the present study, the inventor collected the samples at 4h and 18 h. [Wendy E. Walker, 2021]. The results are as follows:TNF-a, IL-ip IL-6 and IL- 12 were increased in the sepsis-control (CLP)group and significantly decreased in the IS 217 -1.2 mg / kg compared to the sepsis-control group (P < 0.001) and the trend of TNF-a levels at 4h, 18 h and 10 days in treatment groups (Figure 56) intravenously.TNF-a, IL-ip IL-6and IL- 12 were increased in the sepsis-control (CLP)group and significantly decreased in the IS 217 -1.2 mg / kg compared to the sepsis-control group (P < 0.001)and the trend of TNF-a levels at 18 h and 10 days in treatment groups (Figure 57) subcutaneously.Treatment with IS 217 -0.6 mg / kg did not reduce IL-6 release as compared to IS 217-1.2 mg / kg and the trend of IL-6 levels at 4h, 18 h and 10 days in treatment groups (Figure 58) intravenously.Treatment with IS 217 -0.6 mg / kg did not reduce IL-6 release as compared to IS 217-1.2 mg / kg and the trend of IL-6 levels at 18 h and 10 days in treatment groups (Figure 59) subcutaneously.The trend of IL-ip levels at 4h, 18 h and 10 days in treatment groups [Figure 60(a)] intravenously and the trend of IL-ip levels at 18 h and 10 days in treatment groups [Figure 60(b)] subcutaneously.Likewise, the IL-12 levels were significantly reduced in the IS 217 -1.2 mg / kg (P < 0.001), but not much in the IS 217 -0.6 mg / kg groups compared to the sepsis- control group and the trend of IL-121evels at 4h, 18 h and 10 days in treatment groups (Figure 61) intravenously and the trend of IL- 12 levels at 18 h and 10 days in treatment groups (Figure 62) subcutaneously.IL- 10 levels were significantly decreased in the IS 217 treated groups compared to the sepsis- control group (Figures 63 and 64). Quantitation of IL- 10 levels revealed a gradual increase in serum content of this anti-inflammatory cytokine that peaked at 18 hfollowing CLP. IS217 injected at 2 h after CLP promptly and significantly, inhibited IL- 10 levels at both 18 h and 4 h (Figure 63). In 10 day samples, IL- 10 levels in the IS 217 treated group were negligible and the same trend was observed in groups.The trend of IL- 10 levels at 4h, 18 h and 10 days in treatment groups (Figure 63) intravenously and the trend of IL- 10 levels at 18 h and 10 days in treatment groups (Figure 64) subcutaneously.VEGF concentrations, with peak levels occurring at 18 h. In contrast, circulating levels of IL-6 and TNF-a were maximal at the earliest time point measured (6 h). In a cecal ligation puncture (CLP) model of sepsis, peak levels of VEGF occurred at 18 h, respectively (Figure 65 a). The trend of VEGF levels at 4h, 18 h and 10 days in treatment groups intravenously and the trend of VEGF levels at 18 h in treatment groups of subcutaneous was shown (Figure 65b) and VEGF levels was detected at 10 th day samples of SC administration.2.7.2.6 Experiment 3: Late immune paralysis studiesEffect of IS 217 on the inflammatory cytokine concentration in the BALF of sepsis miceSince the inflammation in sepsis induced acute lung injury (ALI) disease, here, in the present study, we estimated the levels of pro-inflammatory factors of TNF-a and IL-6, and VEGF levels and the anti-inflammatory factors of IL- 10 by ELIS . . Measurements of the inflammatory responsevery relevant [Eun Jung Park et al., 2014].To analyze the effect of IS 217 on sepsis-induced inflammatory cytokine production, wedetermined the concentrations of TNF-a, IL-6, IL- 10, and VEGF in the BALF of mice in the different treatmentgroups of CLP by intravenously after 4h and 18 h of post -CLP and after 18 h of post -CLP in subcutaneous treated groups using an ELISA assay.The results showed that the levels of TNF-a, IL-6 and VEGF in CLP group were significantly increased, while the levels of IL- 10 were dramatically decreased in CLP group, compared with the control group. After IS 217 treatment (0.6 and 1.2 mg / kg), the levels of TNF-a and IL-6 in BALF were significantly reduced, and the levels of IL- 10 in BALF wereincreased compared with the CLP group. The result exhibited that IS 217 peptide treatment can decrease the levels of VEGF, TNF-a and II., -6, increase the levels of IL- 10 in BALF, which suggested that IS 217 has an anti-inflammatory effect in ALI mice.As shown in figures, sepsisinduction significantly increased the pro inflammatory cytokines concentration of TNF-a (Figure 66), IL-6 (Figure 67), VEGF(Figure 68) and IL-10 (Figure 69), [Kengo Tomita et al., 2020] (Figures 66 to 69) in the BALF of sepsis- induced mice when comparedto the sham group micevza IV administration of IS 217 at 0.6 &.2 mg / kg. The test peptide IS 217 treatmentsinhibited the elevation of these pro- inflammatory cytokines, as observed in the IS 217- treated sepsis mice. These results suggest that IS 217 could inhibit sepsis-inducedinflammatory responses in ALI mice.As shown in figures, sepsisinduction significantly increased the pro inflammatory cytokines concentration of TNF-a, IL-6 and IL- 10 (Figure 70), [Kengo Tomita et al., 2020] in the BALF of sepsis-induced mice when comparedto the sham group mice via SC administration of IS 217 at 0.6 &.2 mg / kg. The test peptide IS 217 treatmentsinhibited the elevation of these pro-inflammatory cytokines, as observed in the IS 217- treated sepsis mice.Changes in the Lung Wet / dry Ratio, of lungs after the treatment with IS 217The lungs were collected, and the wet / dry weights were measured to determine lung oedema, which can partly indicate the effects of intravenous treatment of IS 217 treatments. The wet / dry ratio of the lungs in the IS 217 administration mice was significantly lower than that in the CLP group or isotype-treated animals (P,0.01, respectively) by intravenously and by subcutaneously (Table 50 and Figure 71a, b).Table 50: Effects of IS 217 (0.6 & 1.2 mg / kg -IV& SC) treatments on Lung w / d ratio changesEvaluation of MPO levels in LungsTissue damage in ALI is related to pulmonary MPOactivity, and neutrophil extravasation is one of the majorhistological markers of inflammatory and immunologicalresponses in injured lung tissue [Abraham 2003; Zhou et al., 2012]. Furthermore, pulmonary MPO activity is also areliable marker of pulmonary neutrophilinfiltration [McCabe et al. 2001]. Therefore, we determined the MPOactivity in the lung tissue homogenates (in BAL fluid) and the number ofinfiltrated neutrophils in the BALF of mice in the differenttreatment groups at 24 h after sepsis induction. As shown in (Figure 72), sepsis induction significantly increased the pulmonary MPO activity in the sepsis- induced CLP group, when compared to the non-sepsis-induced and non-treated sham group mice. Furthermore, pulmonary MPO activitywas dramatically lower in the IS 217 peptide - treated mice than inthe sepsis group mice (Table 51). These results were furthersupported by the data of neutrophil infiltrates in BALF.MPO level is an indirect means of determining the recruitment of neutrophils to the infected organs [Yung- Yang Liul, 2008]. As can be seen in Figure 72, MPO levels in the lung, 18 h after CLP, were elevated in CLP mice as compared with IS 217-treated mice. This increase was significant in lungs (p<0.001; p<0.01). The level of MPO in lungs in control CLP mice was 10-fold increased as compared with normal untreated mice. Whereas MPO levels in the lungs of IS 217-treated mice were only slightly elevated as compared with untreated mice (7-4-fold) in intravenous treatment (Figure 72a). IS 217 subcutaneous treatments also decreased the level of MPO in the lungs (2 folds), when compared to CLP mice (Figure 72b).Table 51: Effects of IS 217 (0.6 & 1.2 mg / kg -IV& SC) treatments on MPO levelsEffect of IS217 (0.6 & 1.2 mg / kg) via IV treatment on mitogen-activated protein kinases signaling pathway in CLP induced sepsis miceIn addition, to pro inflammatory cytokines estimation, we continue detection of the mi togen- activated protein kinases (MAPKs) pathway (Figure 73). The levels ofphosphorylation ERK1 / 2, and phosphorylation p38 in lung tissue of sepsis mice were detected. The results showed that the levels ofthe phosphorylation ERK 1 / 2, and phosphorylation p38 in lung tissue weremarkedly increased in the CLP group, compared with the control group (Figure 82, F<0.01 ). IS 217 -treated group (0.6 and 1.2 mg / kg) showed a reducing expression of phosphorylation of ERK1 / 2, and phosphorylation of p38, compared with the CLP group, especially in the IS 217 -1.2 mg / k -IV route group (Figure 82, ZJ<().01andZJ<0.05). These results suggested that IS 217 alleviated the Sepsis induced acute lung injury (ALI) mice relatedwith the MAPKs signaling pathway. [Eun Jung Park et al., 2014]SummaryIn conclusion, IS 217 peptide showed better activity on InvitroRaw 246.7 cells and mice macrophages stimulated with LPS and induced production of TNF-a and IL-6. From theperspective mechanisms, IS217 dose-dependently inhibited LPS-induced ERK phosphorylation in macrophages.In mice model with ALI, pretreatment with IS 217 significantly attenuated LPS- induced pulmonary edema, pathological changes, inflammatory cytokines in serumand BALF, inflammatory cell infiltration, inflammatorycytokines and p38, ERK phosphorylation.This presents the possibility that IS 217 mightserve as potential agents for the treatment of ALI. Althoughthe anti-inflammatory mechanism and underlyingtargets are still unknown, the beneficial effects of this peptide on LPS-induced inflammation make IS 217 peptide oneof important leads in the continuing drug developmentand research.Cytokines estimation in spleenCytokines production was expressed as pg / mL supernatant of spleen and the difference in relative organ weight of spleen are shown (Table 52 and Figure 75) and the macroscopic representation of spleens of all groups are as shown in Figure 74.Table 52: Effects of IS 217 (0.6 & 1.2 mg / kg -IV& SC) treatments on spleen relative organ weight.5In the spleen of sham controls (371.1+26.0 pg / mL) at 4 h post CLP, TNF-a levels were significantly higher than in IS 217-0.6 & 1.2 mg / kg treated mice (272.0+13.0, 249.1+15.7, respectively). However, TNF-a levels of CLP group are higher than IS 217 treated groups at 18h and 10 days post CLP and not high as at 4h (Figure 76).A significant difference was found in IL- ip expression between disease control and IS 217 treated group mice spleen at 4h, 18h and 10 days post CLP. IL-ip production in CLP mice at 4h, 18 h (402.1+21.3 and 310+13.5pg / mL) was less (208.7+4.2pg / mL) at 10 days (Figure 77). Post CLP Mice in treatment groups showed a significant increase in IL-ip levels in comparison to sham control at 4h, 18h and 10 days post CLP, whereas at 18 h, the levels are decreased in comparison to 4h.The IL-6 expression in controls was not expressed and the levels of IL-6 expression was high in disease control CLP mice when compared to IS 217 treated groups and in treatment groups IS 217 -1.2 mg / kg was showed better results when compared to IS 217 - 0.6 mg / kg, when treated intravenously (Figure 78).Cytokine estimation in abdominal peritoneal lavage fluid (PLF)Preclinical and clinical studies demonstrate that intra-abdominal injury / ischemia, especially with subsequent infection, induces an excessive inflammatory / protein mediator production and uncontrolled inflammation in the peritoneum mostly via the lymphatic pathway) into the systemic circulation may precipitate the deleterious effects of sepsis and multiple organ dysfunction [Stefan Wirtz et al., 2006;Yoon Ju Choi, et al., 2011]. In the present study, the cytokines, bacterial count and total no of cells are measured in PLF. The amount of secreted cytokines levels in the abdominal lavage fluid was expressed as pg protein / mL.Mice in the present study showed a significant increase in TNF-a, (Figure 79), IL- ip (Figure 81) and IL-6 expression (Figure 80) in CLP mice than treated and sham groups and IS 217 treatment groups at 4h, 18h and 10 days post CLP. The IL-6 levels of treatmentgroups showed a similar trend with sham control group when treated intravenously. The IL- 6 expression showed no significant difference between controls and the other groups at any time (Figure 80), whereas the expression of TNF-a, and IL-6 levels are increased in CLP group when compared to the IS 217 SC treatment at 18 h (Figure 82), but not as observed in intravenous treatment.Recently, it has been suggested that septic patients demonstrate a state in which monocytic TNF secretion capacity is severely depressed, while the capacity to produce IL- 10 is at least temporarily preserved [Donghong Yan et al., 2002]. This predominantly antiinflammatory state, in contrast to the assumption of persistent hyperinflammation, was associated with a high risk of death from persistent infection and multiple organ failure. Figure 82shows that peritoneal macrophages obtained from CLP induced mice gradually lose the ability to secrete TNF-a and IL- ip. This loss of function is significantly reflected at 24-48 h following CLP. At 18 h, peritoneal macrophages completely lose their ability to secrete those proinflammatory cytokines. This capacity is significantly restored (p <0.01) in IS 217-treated mice at all time points following is 217 injections. Similar results were obtained when TNF-a and IL-ip in supernatants from adherent splenocytes were quantitated (Figure 82). In contrast to the loss of macrophage ability to secrete TNF-a and IL-ip, the capacity of PEC cells to secrete IL- 10 at 24-48 h was preserved. Nevertheless, PEC cells from IS 217-treated mice secreted significantly decreased amounts of IL- 10 at 18h post-CLP (Figure 90), when IS 217 was treated subcutaneously (p<0.001)Measurement of procalcitonin in peritoneal lavage fluidProcalcitonin levels are directly reflected in the severity of the sepsis. The higher levels of PCT levels are observed in CLP mice at 18 h post CLP and whereas the treatment groups also showed higher PCT levels when compared to sham control, and considered as significant in comparison to disease control CLP mice and the PCT levels of IS 217 IV treatment was mentioned in (Table 57 and Figure 83a) and whereas the PCT levels of IS 217 SC treatment was mentioned in (Table 53 and Figure 83 b).Table 53: Effects of IS 217 (0.6 & 1.2 mg / kg -IV& SC) treatments on procalcitonin levels in peritoneal lavage fluid after 18h of CLP surgeryMeasurement of peritoneal bacterial load and leukocytes in PLFInduced by CLP, considering the role of macrophages and neutrophils in the control of bacterial growth, CLP was observed to induce an evident influx of inflammatory cells, with the majority being neutrophils. IL- 10 has been shown recently to impair bacterial clearance from the peritoneal cavity and to facilitate dissemination of bacteria to distant organs . The significant decrease in serum IL- 10 levels in CLP-induced mice treated with IS 217, as well as the increased survival of these mice led us to evaluate the role of IS 217 in the bacterial clearance of mice subjected to CLP. [Donghong Yan et al., 2002]To this end, in the present study the inventor quantified the total and differential cell count in the peritoneal cavity andthe bacterial load in the peritoneal fluids of IS 217 and PBS -treated mice were examined.IS 217 treatments decreased this influx of total cells. The pattern of response in relation to macrophages and neutrophils was the same as that observed in the total cell count, although not significant (Figure 84) in intravenous administration.No bacteria were detected in abdominal lavage fluid of mice after a 48-h culture. However, in the CLP model, there were animals with negative cultures or very small numbers of bacteria, and individual differences were large. The peritoneal fluids at 18 h after CLP mice contained a significant number of bacteria in the peritoneum (Table 58). At this time point, the bacterial load in the peritoneum of IS 217-treated mice was significantly decreased ( p 0.01). At 18 h, IS 217-treated mice who shivered and had bristled hair, clinical symptoms of active sepsis, had increased peritoneal bacterial load (>300 ). These amounted to 40%. In the remaining mice, the bacterial load recovered from the peritoneal bacterial load of IS 217-treated mice was significantly lower than that in control PBS- treated mice.Treatment with IS 217 decreases the number of CFUs in the peritoneal cavity fluid at 18 h after 24 h culture. The IV treatment was mentioned in the table and whereas SC treatment was mentioned in the Table 54.Table 54: Bacterial CFU counts from peritoneal lavage from mice treated with IS 217 (0.6 & 1.2 mg / kg -IV) of CLP induced sepsisat 18 h of sham and CLP induced miceData are expressed as mean ± SEM. Mice (n =5- 6 / group) and estimated at 18 h post-CLP challenge via IV route.P< 0.001, P<0.01& p< 0.05 was considered as statistically significant and denoted as ***, **&* and###’##&#compared to disease control and sham control groups, respectively.2.7.2.7. Histopathological changes of lung and liver tissues: IS 217treatement ameliorated organ injury in CLP miceOrgan damage is a leading cause of death in patients with sepsis. Thus, we investigated whether the organ protection afforded by IS 217inCLP mice. No significant changes were observed in the body weight of the animals and in relation to the organs weight, besides, no macro- or microscopic alteration was detected in the brain, heart, lungs, liver, kidney, and spleen.All the tissues from different experimental groups were harvested after 18 h, considering the early phase of immunosuppression and most of the animals of the CLP group are not survived while the other treated groups (IS 217 -0.6 mg / kg and IS 217 -1.2 mg / kg groups) lived longer, to assess lung and liver damage, histological examinations. The presence of congestion in the groups analyzed was due to the euthanasia procedure. Necrosis was not observed in any analyzed organs.Briefly, lung and liver tissues werefixed in buffered 10% formaldehyde and then embeddedin paraffin. The embedded tissue samples were sectioned (5 pm) and stained withhaematoxylin and eosin to examinegeneral histological features. These investigations showed thatCLP-induced sepsis in mice caused hepatic inflammatorycellular infiltration, hepatic steatosis, and hepatic fibroplasiain the portal tract.A semi-quantitative scoringsystem was used. For liver tissue evaluation, hepatocytedegeneration and portal / lobular inflammation were scored (each 0-3), lung injury scores were determined by assessing neutrophil infiltration, hemorrhage, necrosis, congestion and edemas previously described. The score ofeach tissue sample represented the mean score of ten differentfields. The stained tissue sections were evaluated undera light microscope (Eclipse E200-LED; Nikon, Kawasaki, Japan) at x200 magnification.The general architectures of the lung and liver in the sham and normal control groups were of normal histological structure (Figure85). There was also no statistically significantdifference between both the sham and Normal control groups (P<0.01). However, lung tissue in the CLP-controlshowed histopathological changes inthe alveolar walls (Figure 85). Also, interstitialedema, infiltration of polymorphnuclear leukocytes andmonocytes, hemorrhage, vascular congestion, and cellular hyperplasia were observed, and the tissue damage was more prominent in the disease control group. Inflammatory cell types weregenerally neutrophils and macrophages. In the lungs, congestion and neutrophil infiltration were observed in both groups (Table 55). Neutrophil infiltration into the alveolar space was not observed in any case, and no traces of pneumonia were noted in any of the lung samples (Figure 93). The morphologic study showed that the lungs of CEP mice were damaged. Severe oedema, wider interalveolar septa, severe alveolar haemorrhage, and extensive inflammatory cell infiltration was observed. But the lungs and livers ofthe IS 217 -0.6 mg / kg group and IS 217 -1.2 mg / kg group had normal histologicalstructure, when compared with the CLP group (Figure 85). Mild lung oedema, haemorrhage, and inflammatory cell infiltration were seen in the IS 217 -0.6 mg / kgtreatment group. Furthermore, it was evident that IS 217 (1.2mg / kg) reduced these injuries by reducing edema and macrophage infiltration. Histological evaluation of lung tissue revealed that IS 217 reduced macrophage infiltration and alleviated lung tissue damage (Figure 85).Table 55: Histopathology evaluation of lungs and livers from all the groups of 18h post -CLP surgeryThe results are expressed as mean ± SEM of the scores:0-absent,l-mild,2- moderate, 3 -intense of n=3-4 mice / group.According to total histologyscore, IS 217 -1.2 mg / kg group groups had significantly reducedscores of lungsand liver injury induced by sepsis. The administration of IS 217 -0.6 mg / kg had mild lung injury (Figure 85) induced bysepsis. In IS 217 -0.6 & 1.2 mg / kg groups mice, the septic effects statically decreased when compared to CLP-sepsis group (P<0.01). So, there was no statistically significant differencein lung histological structures between sham group and especially IS 217 -0.6 & 1.2 mg / kg treated animals (P<0.01).Hence, our study demonstrated the ability of IS 217 toreduce inflammatory cytokines such as IL-ip, IL-6 andTNF-a and ameliorate the negative alteration in the tissuelevels of MPO and histopathological changes in lung sections under these conditions. In addition, reduced macrophage infiltration into the liver by IS 217 wasinhibited concentration-dependently. Furthermore, the results in Table 55 show that CLP increased the levels of ALT and AST and that IS 217 concentration-dependently and significantly reduced liver damage marker levels. A few neutrophil infiltrates were found in the portal veins, central veins of the liver and renal small vessels in the IS 217 -1.2 mg / kg treatment group (Figure 85).IS 217 treatments inhibiting TNF-a, IL-6 & IL-ip and organ damage in vivo in septic mice. BALB / c mice were given either0.6 mg / 1.2 mg / kg of IS Tlvia IV injection after 2h of post CLP surgery. The uninfected control group received an equivalent volume of normal saline alone. TNF-a, IL-6 & IL-ip levels in BALF were measured by ELISA at 4h,18h and lOd post infection. At 24 h post infection, mice were bled retro-orbitally, and sera were separated to measure ALT levels. Also, after 18 h mice were sacrificed and observed for the histopathological changes indicated lung and liver tissue in CLP mice. Furthermore, itwas evident that IS 217 (1.2 mg / kg) reduced these injuries by reducing edema and macrophage infiltration (Figure 93) and showed that minimized CLP-induced lung &liver damage. In addition, IS 217 (0.6 mg / kg) reduces the injuries to moderate extent. Furthermore, the results in Table 51 show that CLP increased the levels of ALT and AST and that IS 217 peptide concentration-dependently and significantly reduced liver damage marker levels.2.7,2.7: DiscussionCLP model induced polymicrobial infection (blood cultures positive for Escherichia coli, Streptococcus bovis, Proteus mirabilis, Enterococcus, and Bacteroides fragilis) and bacteremia (peritoneal cavity fluid positive for the above microbes as well as Streptococcus viridians and Clostridium sporogenes) and a 70% mortality rate [Konstantin Tsoyi.c / al., 2009]. Mildly ill mice sacrificed 10 hours following CLP demonstrated the early hyperdynamic phase of sepsis (increased blood flow to organs, hyperinsulinemia, and hyperglycemia) [ Burgelman, M et al., 2021 ; Hotchkiss, R. S., et al., 2013]. Thus, in the present study we collected PLF, lungs and sera after 4h of CLP surgery to know the effect of IS 217 treatments on early hyperdynamic phase of sepsis. While mice sacrificed 16-24 hours post-operative represented a hypodynamic late septic state (decreased blood flow to organs, hypoinsulinemia, hypoglycemia, and high serum lactate levels). Thus, in the present study we collected PLF, lungs and sera after 18h of CLP surgery to know the effect of IS 217 treatments on late hypodynamic phase of sepsis. Multiple aspects of the CLP procedure address the complex of the clinical course of sepsis. CLP induces polymicrobialinfection of the peritoneum with a localized infectious focus, release of bacteria and endotoxic molecular components of pathogens (pathogen-associated molecular patterns or PAMPs) into normally sterile areas in the host, and subsequent translocation of enteric bacteria into the bloodstream, modelling the stages of intraabdominal clinical sepsis [Fourrier, F., C. et al., 1992].Thus in the present study the peritoneal lavage fluid was examined for cytokines levels and for bacterial CFU, to know the efficacy of IS 217 treatment.The pro-inflammatory response is also characterized by significant increases in cytokines TNFa and IL-6 and remain elevated over an 8-hour period [Schulte, W., J. Bernhagen et al., 2013; Matsukawa, A., 2003; KYUNG-JUN JANG et al., 2016 &Gil, M., 2016]. Several studies have demonstrated the importance of an early pro -inflammatory- response in the progression of sepsis [Schuerholz et al. 2013; Singleton K, et al., 2003; Silva, O. N., 2016]. To screen the host response to CLP, as part of this the following estimations are done in PLF, lungs, spleen and sera and the results are mentioned as in the results section. [Stefan Wirtz et al., 2006; Yung-Yang Liul 2008].As only a fraction of patients with severe sepsis and septic shock displays signs of DIC, this level of effectiveness is not surprising [Vincent, J. L. et al., 2005; Levi, M. 2010 &1999]. In the present study A strategy of controlling bacterialmultiplicationalong with inhibition of excessive proinflammatory cytokines andDIC by use of peptide IS 217 treatment might be more effective incontrolling human sepsis. Recent studies demonstrated that systemic levels of both the proinflammatory cytokines and IL- 10 are correlated directly with severity of illness [YonaKalechman, Uzi Gafter, et al., 2002] However, during septic peritonitis induced by cecal ligation and puncture (CLP), 3 neutralizing IL- 10 was associated with an increased mortality [Grace Y. 1999] In this study, we show that in mice subjected to cecal ligation and puncture (CLP), treatment with IS 217 2h afterCLP significantly increased survival of septic mice. This was associated with a significant decrease in serum IL- 10 and in IL- 10 secretion by peritoneal macrophages 24-48 h after CLP. At that time, the ability of these cells to secrete TNF-a, IL-6, IL- 12 and IL-ip was restored in IS 217-treated mice for 5 days and this reflects in survival rate which observed for 10 days and also in tissue damage. Recent data of Song et al. suggest that at later time points after the onset of infection, IL- 10 may have a net detrimental effect on host antimicrobial clearance mechanisms. In a cecal ligation model, when administered 2 h after CLP, this treatment was protective. This data indicates the critical importance of timing of manipulations that affect IL- 10 activity in sepsis.Moreover, IS 217 treatment ameliorated bacterial clearance in the peritoneum and blood and decreased severe multiple organ damage, as indicated by clinical chemistry. Furthermore, myeloperoxidase levels in the lung of IS 217-treated mice, an indirect means of determining the recruitment of neutrophils, were significantly decreased. We suggest that non immunogenic peptide IS 217, with the capacity to inhibit IL- 10 and stimulate macrophage functions, may have clinical potential in the treatment of sepsis, provided they are administered during the phase of sepsis characterized by immune suppression. Sepsis survivors present acute and long-term cognitive impairment and the pathophysiology of neurological dysfunction in sepsis involves microglial activation. Thus, in the present study, we investigated two memory tests: elevated plus maze and novel object recognition test to elevate the memory and learning activity of post CLP surgery mice. IS 217 treated groups display deficits for long-term object recognition memory function and having normal activity after the post CLP surgery as comparable to control groups.Acute lung injury (ALI) is a common complication of sepsis. Sepsis-induced ALI is thought to be polymorphonuclear neutrophil dependent, which results in a cytokine / chemokine storm in the lungs that leads to intense ALI and acute respiratory distress syndrome [Yibin Zeng et al., 2022; Yali Zhang et al., 2015]. Sepsis starts as a process of system inflammation mediated by pro -inflammatory cytokines / chemokines including TNF-a, IL-ip, IL-6, and IL- 12 as well as anti-inflammatory cytokines, e.g. IL- 10 [Stefan Wirtz et al., 2006;Jinbao Li et al., 2012]. These proinflammatory cytokines result in recruitment and activation of neutrophils, NK cells, and monocytes / macrophages which produce deleterious reactive oxygen species and lysosomal enzymes [Anasuya Patel, et al., 2020]. Studies have shown thatendotoxin can activate the MAPK signaling pathway, regulate cytokines TNF-a and IL-6, recruit and activate whiteblood cells through the production and activation of inflammatory mediators. These cascades of inflammation inducedby activation of inflammatory signaling pathways promote the occurrence and development of ALI [Eun Jung Park et al., 2014], Whilein our study, we found that treatment with IS 217 can reduce the protein levels of phosphor ERK1 / 2, and phosphor p38in lung tissue, which implied that the effect of IS 217 involved in the signaling pathway of MAPK.In the present study, the inventor explored the effects and the potential mechanisms of IS 217in sepsis induced ALI mice in vivo. The resultof the study showed that IS 217 can significantly reduce the expression of phosphor ERK1 / 2, and phosphor p38 in lung tissue. In addition, IS 217 can also dramatically decreasethe cytokine levels of the pro-inflammation (TNF-a and IL-6), along with VEGF levels and increase the cytokine levels of anti-inflammation (IL- 10). Furthermore, IS 217 also alleviates the histopathological injury of lung in ALI mice. The currentresults exhibited that the effect of IS 217 on ALI may be associated with the signaling pathway of MAPK. In conclusion, IS 217 showed a significant protective effect on ALI mice, it can reduce the levels of inflammationcytokine, alleviate the lung histopathology, reduce the white blood cells, mainly related with the influence on the signaling pathway of MAPKs. agent against lethal sepsis.Thus, this study provide evidence that IS 217 peptide treatment mayhave a protective effect on sepsis-induced ALI by reducinginflammatory responses in the lung tissue, ourresults suggestthat IS 217 may be a potent protective agent forpulmonary injuryand particularly in covid pneumonia.2.7.2.8: SummaryHence, the present study provides evidence for the anti-bacterial and antiinflammatory effects of IS 217 in CLP-inducedsepsis. The present findings indicate that this protectioncould be attributed to: IS 217-induced inhibition ofMAPK- mediated inflammatory responses, thus attenuatinglung injury.These findings reveal administration of IS 217 peptide treatment decreases bacterial growth likely by activation of phagocytes and, in parallel, attenuates inflammatory cytokine release and tissue damage in mice subjected to cecal ligation and puncture (CLP) and protects these mice from the lethality of sepsis and shed light on the development of Host defense peptides-targeted therapeutics for sepsis.2.7.3: In vivo Proof of Concept (PoC) studies by using E. coli induced peritonitis model in mice:2.7.3: Results of pilot study -Standardization of E. coli induced peritonitisThe present study was carried out to assess E. coli infection induced peritonitis. Following Escherichia coli isolation, identification and counting, the lethal dose (LD-50) was determined before infection. The E. coli bacterial strain (8739™) used in this study, as expected for nonpathogenic bacteria, mice can clear a large number without mortality and compared tohumans, rats and mice are considerably less sensitiveto the pro -inflammatory and lethal effects of E.coli infection.Furthermore, in acute E. coli infection, theinflammatoryresponse is exclusively deleterious; therefore, most effective antiinflammatory agents tend to improveorgan function and / or survival. QHowever, 1.5x 10 per mouse routinely yields 10-20% mortality, so we standardized the model with 2.5x 10 & 5x 10 per mouse, indicating that this is a sufficient dosage to identify decreased resistance to sepsis, which would cause higher mortality. This model is expected to be representative of sepsis in humans that begins with loss of gastrointestinal barrier function (caused, e.g., by trauma, appendicitis, diminished liver function, or other conditions). In human peritonitis, a single species of bacteria often predominates, and in approximately half of cases, E. coli is the species isolated in blood cultures [De Waele et al., 2008]. Thus, administration of a single strain of indigenous E. coli in our model allows more controlled conditions than cecal ligation and puncture but yields peritonitis and sepsis similar to that observed in humans [Asma Ahmed et al., 2018]. QIn the present study, the E. coli induced infection was standardized with2.5x 10 & Q5x 10 CFU in eighteen mice of 8-10 weeks old and after 24h of infection, the cytokines were estimated in the serausing ELIZA kits (R&D biosystems), to evaluate the degree of infection with 2.5x 10 & 5x 10 CFU and standardized the infection dose (Figure 86).2.7.4: Main E. coli induced infection model studySynthetic peptide IS 217 provided broad-spectrum protection against lethal infections caused by E. coli in mice.Due to the potent and very promising antimicrobial properties of IS 217, the inventor decided to test its immunomodulatory activity. In the present study, an aggressive bacterial infection mouse model of bacterial infection was establishedwith E. coli ATCC 8739™bacteria via intraperitoneal (IP) injection, and 2 hours later treated SC & IV with 0.6 & 1.2 mg / kg of IS 217 peptide suspended in sterile saline. The in vivo protective activity of IS217 was also evaluated by using an invasive infection in the E. coli model. All treatments were performed daily for 5 days. After 18 h of infection, half of animals from each group are anaesthetized for blood and euthanized for organ collection and other subset will continue to be observed for survival rate for 7 days. Mice treated with IS 217 appear clinically healthier after induction of septic shock, when compared to infected animals.BALB / c mice were given either normal saline or0.6 mg / kg & 1.2 mg / kg of IS 217IV after 2h of infection with 5.0 X108E. coli CFU. 18h later parameters listed in Table60were observed (Table 60). Each group consisted of 5-6 mice and photographic representation of mice is shown in the Figure. 95. This also confirms the development of E.Q coli infection with 5.0 X 10 E. coli CFU and the mice were graded according to the severity of the symptom: normal, mild, marked, and severe. The same observations were observed in SC treatment of IS 217, but not up to the mark as IV treatment. Mice treated with IS 217 appear clinically healthier after induction of septic shock.Table 56: Activity index of mice after 18hr of induction of septic shock2.7.4.1: Body temperatureMice were observed at least every 6 h after the challenge, and mice that were moribund and those with a body temperature less than 33°C were euthanized and counted as dead at each time point indicated. In this model of sepsis (as in some cases of sepsis in humans), profound hypothermia is observed. However, in mice that will ultimately survive, hypothermia is less severe. It should be noted that severe illness typically proceeded to death very quickly, such that most deaths occurred between the 6-h observation points, and only a few cases of moribund mice or mice with very low body temperatures were removed from the study (Table 57).Table 57: Body temperatures of mice treated with IS 217at 18 h after induction of septic shock.2.7.4.2: Experiment 1: Survival studyIS 217 treatment enhanced Survival of mice after induction of sepsisThe Kaplan-Meier curve for survival analysis of mice subjected to polymicrobial sepsis and treated with test peptide IS 217 (0.6 &1.2mg / kg) showed lower mortality after sepsis when compared with non-treated septic mice (Figures 88 and 89). Peritonitis induced byE. coli infection results in 100% lethality at day 8 (Figures 88 and 89) Induction of infection was described in the methods section and animals were Intravenously (IV) &subcutaneously (SC) injected with various doses of IS 217 or vehicle at 2h after post infection and then mice were observed for 18 h (Table 58 and Table 59 and Figure 88a, 88b) and subset of animals are continued for observation for 7 days (Table 62, and Figure 89). No significant difference was observed when IS 217 was injected at the time of injury (time zero). Notably, treatment with IS 217, 18 h decreased survival, compared with NS- treated mice.Table 58: Percent survival rate of all the groups of post E. coli infection for 18 h &7 days with IV administration of IS 217As shown in Figures 88a and 88b, both IS 217 0.6 and 1.2 mg / kg of IV treatment prolonged the survival of infected mice in the treatments (IV). After 7 days of infection, 37.5% survivalwas observed for animals infected with E. coli ATCC 8739™(table 69a, b&figure 88 a,b) and treated with 0.6 and 1.2 mg / kg of IS 217 was 53.5 % and 68.71 % respectively. E. co / zinfectedmice treated with IS 217 at a dose of 1.2mg / kgshowed a survival rate of 50 % after 18h of Infection (Figure 88a,). In this case, the lethality rate in the PBS control group was 100%. All groups treated immediately after infection with IS 217 (0.6 and 1.2 mg / kg) showed a better outcome than disease control groups (P< 0.05). The survival rates in mice were 71 % in bothgroups treated with IS 217 (0.6 and 1.2 mg / kg). The IS 217-IV treatment shows similar results to the standard drug Cefoxitin (Figure 88c).Table 59: Percent survival rate of all the groups of post E. co / infection for 18 h with IV administration of IS 217, compared with standard marketed drug (Cefoxitin)Table 60: Percent survival rate of all the groups of post E. co / zinfection for 18 h &7 days with SC administration of IS 217As shown in Figure 89(a, b) both IS 217 0.6 and 1.2 mg / kg of SC treatment prolonged the survival of infected mice in the treatments (SC). After 7 days of infection, 37.5% survivalwas observed for animals infected with E. coli ATCC 8739™ (Table 70 and Figure 89) and treated with 0.6 and 1.2 mg / kg of IS 217 was 71.4 % and 85.71 % respectively (Figure 89). E. co / zinfectedmice treated with IS 217 at a dose of 1.2mg / kgshowed a survival rate of 60 % after 18h of Infection (Figure 89a). In this case, the lethality rate in the PBS control group was 100% (Figure 89b). All groups treated immediately after infection with IS 217 (0.6 and 1.2 mg / kg) showed a better outcome than disease control groups (P< 0.05). The survival rates in mice were 40, and 60 % in the groups treated with IS 217 (0.6 and 1.2 mg / kg, respectively).2.7.4.3: Biochemical markers, CRP levels and DICTo understand the mechanism whereby IS217-treated mice were resistant to E. coli infection, experiments were conducted to assess the organ damage induced by the infection and its response to IS 217. For this purpose, the inventor measured biochemical markers of liver damage (AST, ALT), and renal failure (creatinine, BUN) with IS 217 IV & SC administration at 18h following infection along with other parameters are mentioned in Table 61, Table 62, Figures 90 and 91. shows that infected mice demonstrated biochemical evidence of more severe multiple organ damage than IS 217- treated mice, as reflected by higher ALT, AST, creatinine(Figure 90a, b), and BUN (Figure 91a, b) concentrations. The levels in IS 217- treated mice were comparable with normal control mice, suggesting that organ injury was avoided in IS 217-treated mice.Table 61: Effects of IS 217 (0.6 & 1.2 mg / kg -IV) administrations on serum liver and kidney function tests in E. coli- induced sepsis in miceTable 62: Effects of IS 217 (0.6 & 1.2 mg / kg -SC) administrations on serum liver and kidney function tests in E. coli- induced sepsis in miceEffect of IS 217 treatments on serum CRP levelsCytokines are the key molecules that can inhibit or propagate inflammation by activating or deactivating the genes involved in cellular response. The pro inflammatory cytokines like TNF-a, IL-ip, and IL-6 can stimulate the liver to produce C-reactive proteins (CRP) which are increased several folds during acute inflammation. In the present study, due to infection, the CRP levels are increased in disease control animals when compared to treated groups with IS 217 -0.6 mg / kg & 1.2 mg / kg by both routes (IV & SC) Table 63 and Figure 92.Table 63: Effects of IS 217 (0.6 & 1.2 mg / kg -IV & SC) administrations on serum CRP levels in E. coli- induced sepsis in miceHowever, to have potential as a tool for treatment of sepsis, it is important to study if IS 217 can reduce inflammation and DIC when administered therapeutically. Next, it was examined whether IS 217 could also inhibit DIC when injected therapeutically. BALB / c mice were administered IS 217 2h post infection with 5 x 108E. coli ATCC 8739 CFU and 18 h later, blood was collected and coagulation parameters for DIC were examined. Administration of IS 217 (IV) therapeutically helped to reduce clotting time as evidenced by decreased PT and aPTT (Table 68) values in IS 217 treated group as compared with PBS-treated septic mice. Thus, IS 217 can prevent inflammation and DIC in septic mice, even when administered therapeutically.Table 64: Effects of IS 217 (0.6 & 1.2 mg / kg -IV) administrations on Coagulation testsat 18 h in E. coli- induced sepsis in mice2.7.4.3 Experiment 2: Acute hyper inflammation study: Cytokine estimation in serum IS 217 prevents E. co / i-induced septic death and inhibits TNF-a, IL-ip, IL-12 and IL- 6 release in mice serumIn the present study, the IS 217 two doses are assessed using an E. coli induced peritonitis sepsis mice model. Although serum levels of TNF-a and IL-ip were remarkably induced in mice challenged by an intraperitoneal injection of E.oli strain. The treatment with a single dose of IS 217 (0.6 or 1.2mg / kg) by IV at 18 h, 7 days and by SC at 18 h of post infection resulted in a significant protection from lethal septic shock (Figure 93-96). Proinflammatory cytokines (IE-6, IE-12, IL-ip and TNF-a), anti-inflammatory cytokines (IL- 10) were detected in serum.Most mice that received IS 217 treatment doses (0.6 or 1.2mg / kg) for 5 days were alive at 7days after the injection; these findings indicate that IS 217 can protect mice from peritonitis. Furthermore, while the E. coli vca cd mice showed a dramatic increase in their serum levels of TNF-a, IL-6, IL- 12, and IL-ip, treatment with IS 217 significantly attenuated the serum level of TNF-a and the serum level of IL- 10 at 18 h. These results show that acute administration of IS 217 significantly increases the survival rate of mice at risk of E. co / z-induced septic death and that these effects are exerted via an antiinflammatory effect on cytokine production.Similar data were obtained with cytokines analysis7days after bacterial injection showed that mice infected by E. coli and treated with IS 217 by IV route, compared with the disease control group (E.co...

Claims

I claim:

1. A peptide of SEQ. ID NO 1 or peptide variant thereof for use in treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.

2. An antimicrobial, immunomodulatory and anti-inflammatory pharmaceutical formulation for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis, said formulation comprising a peptide of SEQ. ID NO 1 or peptide variant thereof in an amount of from 0.01 pg / ml to 1000 pg / ml, preferably 0.5 pg / ml to 500 pg / ml and one or more suitable pharmaceutically acceptable excipients.

3. The formulation of claim 2, wherein said one or more suitable pharmaceutically acceptable excipients are selected from the group consisting of suitable carriers, diluents, vehicles, disintegrants, swelling agents, antioxidants, buffering agents, bacteriostatic agents, emollients, emulsifiers, plasticizers, penetration enhancers, preservatives, cryoprotectants, neutralizers, fragrance additives, dispersants, surfactants, binders and lubricants.

4. The formulation of claim 2, wherein said peptide variant is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the SEQ. ID NO 1.

5. The formulation of any of claims 2 to 4, wherein said formulation is an injectable formulation, preferably for subcutaneous or intravenous route of administration, and optionally wherein said one or more symptoms of sepsis are fever, low body temperature, chills, shivering, rapid breathing, difficulty in breathing, confusion and / or change in mental status, tachycardia, low blood pressure, skin rash, pain or discomfort, sweating, clammy skin, and optionally wherein said conditions associated with sepsis are acute lung injury, septic shock, acute kidney injury, sepsis-induced cardiomyopathy, weakenedimmune system, sepsis-induced thrombocytopenia, hyperglycemia, hypoglycaemia, sepsis-induced coagulopathy, acute respiratory distress syndrome and sepsis- induced hypoperfusion.

6. A formulation for treatment, prevention, prophylaxis and / or amelioration of inflammation in a subject in need thereof, comprising a therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof, wherein said formulation:(ij inhibits or reduces expression of one or more of inflammatory markers selected from p38 kinases, IL-lp, IL-6, IL-12, TNF-a, 1L-12P40, CCL2, VEGF, TLR4, !L-12p70, NO, TLR7, TLR8, TLR9 and GAPDH reduction in phosphorylation of ERK1 / 2;(ii) is capable of one or more of antimicrobial activity;(hi) completely or partially reduces release of at least one pro-inflammatory cytokine;(iv) downregulates phosphorylation of p38 mitogen activated protein kinase (MAPK); and / or(v) completely or partially increases release of at least one anti-inflammatory marker.

7. An antimicrobial, immunomodulatory, anti-inflammatory pharmaceutical combination for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis, said combination comprising a therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof of claim 1 in an amount from 0.01 pg / ml to 1000 pg / ml, preferably 0.5 gg / ml to 500 gg / ml and one or more additional active agent, and wherein said additional active agent is selected from one or more of antibiotic agent, one or more of anti-inflammatory agent and one or more of immunosuppressant agent, preferably wherein said other active agent is selected from dexamethasone, lipopolysaccharide, ciproflaxin, penicillin, streptomycin or cefoxitin; and wherein said one or more additional active agent is in an amount of 0.01 pg / ml to 1000 pg / ml.

8. Use of a peptide of SEQ. ID NO 1 or peptide variant thereof of claim 1 in a method for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, wherein the method comprises administering to the subject in need thereof therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof.

9. The use of claim 8, wherein the subject is administered from 0.1 pg / ml to 1000 pg / ml, preferably from 0.5 pg / ml to 500 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereof.

10. The use of claim 8 or 9, wherein one or more symptoms of sepsis are fever, low body temperature, chills, shivering, rapid breathing, difficulty in breathing, confusion and / or change in mental status, tachycardia, low blood pressure, skin rash, pain or discomfort, sweating and clammy skin; and wherein conditions associated with sepsis are acute lung injury, septic shock, acute kidney injury, sepsis-induced cardiomyopathy, weakened immune system, sepsis-induced thrombocytopenia, hyperglycemia, hypoglycaemia, sepsis-induced coagulopathy, acute respiratory distress syndrome and sepsis-induced hypoperfusion.

11. The use of any of claims 8 to 10, wherein said peptide of SEQ. ID NO 1 or peptide variant thereof is suitable as an injectable, preferably by subcutaneous or intravenous route of administration, and wherein the peptide of SEQ. ID NO 1 or peptide variant thereof is administered to the subject one to three times per day, one to three times per week or one to three times per month.

12. Use of a peptide of SEQ. ID NO 1 or peptide variant thereof of claim 1 for manufacture of a medicament for the treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis.

13. Use of formulation of any of claims 2 to 6 for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associatedwith sepsis in a subject, comprising administering to said subject therapeutically effective amount of said formulation.

14. A kit for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, said kit comprising a peptide of claim 1, and one or more of additional therapeutically active agent, wherein the additional therapeutically active agent is at least one antibiotic agent, at least one immunomodulatory agent and at least one anti-inflammatory agent.

15. A product comprising a peptide of claim 1, and one or more of additional therapeutically active agent, as a combined preparation for separate, simultaneous or sequential use in the treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, wherein the additional therapeutically active agent is at least one antibiotic agent, at least one immunomodulatory agent and at least one anti-inflammatory agent.

16. A method comprising administering a therapeutic formulation comprising therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof to a subject, wherein said subject is suffering from sepsis, one or more symptoms or conditions associated with sepsis, and wherein administering said therapeutic formulation reduces one or more symptoms of sepsis or conditions associated with sepsis; and wherein said administering reduces or inhibits release of at least one pro-inflammatory cytokine; reduces or inhibits expression of at least one inflammatory marker; downregulates phosphorylation of p38 mitogen activated protein kinase (MAPKj; exhibits antimicrobial effect; and / or increases release of at least one anti-inflammatory marker in said subject.

17. A method for treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject comprising administering to the subject in need thereof a therapeutically effective amount of peptide of SEQ. ID NO 1 or peptide variant thereof of claim 1.

18. The method of claim 17, wherein the subject is administered from 0.1 pg / ml to 1000 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereof.

19. The method of claim 17, wherein the subject is administered from 0.5 pg / ml to 500 pg / ml of peptide of SEQ. ID NO 1 or peptide variant thereof.

20. The method of claim 17, wherein one or more symptoms of sepsis are fever, low body temperature, chills, shivering, rapid breathing, difficulty in breathing, confusion and / or change in mental status, tachycardia, low blood pressure, skin rash, pain or discomfort, sweating and clammy skin; and wherein conditions associated with sepsis are acute lung injury, septic shock, acute kidney injury, sepsis-induced cardiomyopathy, weakened immune system, sepsis-induced thrombocytopenia, hyperglycemia, hypoglycaemia, sepsis-induced coagulopathy, acute respiratory distress syndrome and sepsis-induced hypoperfusion.

21. The method of claim 17, wherein said peptide of SEQ. ID NO 1 or peptide variant thereof is suitable as an injectable, preferably by subcutaneous or intravenous route of administration.

22. The method of claim 17, wherein the peptide of SEQ. ID NO 1 or peptide variant thereof is administered to the subject one to three times per day, one to three times per week or one to three times per month.

23. A method of treatment, prevention, prophylaxis and / or amelioration of sepsis, one or more symptoms and / or conditions associated with sepsis in a subject, comprising administering to said subject therapeutically effective amount of formulation of claim 2.

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