OA20854A - Early management and prevention of sepsis and sepsis-like syndromes. - Google Patents

Abstract

The present invention relates to the early treatment, including pre-diagnosis treatment, of sepsis and acute inflammatory syndromes such as systemic inflammatory response syndrome (SIRS) by PLA2 and metalloprotease inhibitors to improve the performance of antibiotics and outcomes prior to and after confirmation of the diagnosis of sepsis and/or SIRS in a patient or subject. Additional embodiments include methods of treating sepsis, anthrax and severe acute respiratory syndrome coronavirus (SARS and SARS-CoV2) and related inflammatory syndromes and compositions, including pharmaceutical compositions and blood sample compositions. In further embodiments, the present invention is directed to embodiments which evidence that LY315920, LY333013 and related sPLA2 inhibitors are particularly effective COVID19/cytokine release syndrome therapeuticsprophylactics. In embodiments, the PLA2 inhibitor is varespladib (LY3 1 5920), methyl varespladib (LY3330I 3), AZD27 16-(R)-3-(5'-benzyl-2'carbamoyl-[1,1'-biphenyl]-3-yI)-2-methylpropanoic acid- as a racemic mixture or separately, as the "R" enantiomer) and LY433771 ((9[(phenyI)methyl]-5-carbamoylcarbazol-4-yl) oxyacetic acid), a pharmaceutical^ acceptable salt thereof or a mixture thereof. In embodiments, the metalloprotease inhibitor is Prinomastat, Batimastat, marimastat or vorinostat dosed alone or in combination with preferred sPLA2 inhibitors for the treatment of infection, inflammatory and wound conditions arising from various causes. Methods and compositions for achieving accelerated treatment of wounds and burns, anthrax metalloprotease toxin (lethal factor) driven complications, ARDS, neo-natal and pediatric acute respiratory distress syndrome (neonatal/pediatric ARDS), including meconium aspiration syndrome are also disclosed.

Description

EARLY MANAGEMENT AND PRÉVENTION OF SEPSIS AND SEPSIS-LIKE SYNDROMES

Related Applications

This application daims the benefit of priority of United States provisional application serial numbers 62/915.209, filed October 15, 2019; 62/990,020, filed Mardi 16, 2020; and 63/017,966, filed April 30, 2020, the entire contents of all three blood applications being incorporated by reference in their entirety herein.

Field of the Invention

The présent invention relates to the early treatment, including pre-diagnosis treatment, of sepsis and acute inflammatory syndromes such as systemic inflammatory response syndrome (SIRS) by PLA2 and métalloprotease înhibitors to împrove the performance of antibiotics and outcomes prior to and after confirmation of the diagnosîs of sepsis and/or SIRS in a patient or subject. Additional embodiments indude methods and compositions for treating wounds and lésions caused by toxins, trauma, or slow wound healing due to basement membrane or other damage. Further embodiments are directed to compositions, including pharmaceutical compositions and blood sample compositions. In further embodiments, the présent invention is directed to embodiments which evidence that LY315920, LY333013 and related sPLA2 înhibitors are particularly effective COVID-19/cytokine release syndrome therapeutics-prophylactics. In embodiments, the PLA2 inhîbitor is varespladib (LY3 1 5920), methyl varespladîb (LY3330I 3), AZD27 I 6-(R)-3-(5’-benzyl-2’-carbamoyl-[l,r-bîphenyl]-3-yl)-2methyl propan oie acid- as a racemic mixture or a stereoisomer thereof, often the “R” enantiomer of the racemic mixture, AZD Compound 4 (3-(5'-Benzyl-2'carbamoylbiphenyl-3-yl)propanoic acid) and LY43377I ((9-[(phenyl)methyl]-5carbamoylcarbazol-4-yl) oxyacetic acid), a pharmaceutically acceptable sait thereof or a mixture thereof- In embodiments, the métalloprotease inhîbitor is prinomastat, batimastat, marimastat or vorinostat dosed alone or in combination (prinomastat is often the métalloprotease of choice) with preferred sPLA2 înhibitors for the treatment of infection, inflammatory and wound conditions, including those wounds caused by bacterialiy, virally, venom-înduced, bums and traumatically arising from various causes at macro- and micro-scopic scales. Methods of accelerated treatment of wounds, acute kidney injury (AKI), anthrax léthal factor toxin associated complications, ARDS, néo-natal and pédiatrie acute respiratory distress syndrome (neo-natal/pediatrîc ARDS), including méconium aspiration syndrome are also disclosed with spécial attention to AZD2716-(R)-3-(5’-benzyl-2’-carbamoyl-[l,Γ-biphenyl]-3-yl)-2methylpropanoic acid- as a racemic mixture or a stereoisomer thereof, often the “R” enantiomer of the racemic mixture alone or in combination with a métalloprotease inhibitor (e.g. prinomastat) or as a composition for accelerating wound healing such as that arisîng from înjury or toxins affecting connective tissues associated with epithelia.

Background and OverView of the Invention

Préservation and/or accelerated healing of the basement layer membrane, epithelium, and endothélium is foundational for health in nearly every organ and tissue system of the body. These layers form the basic structure in nearly ail tissues and organs. Destruction of these layers or loss of function leads to pathogensis of immune function, pulmonary system, gastrointestinal system, rénal system, integumentary system, and circulatory system. Altogether, these experiments indicate that préservation of this critical structure through both the épithélial and endothélial layers results in significant improvement in cellular, systemic, and whole organism function that are unexpectedly and uniquely protected and treated by compositions and kits related to combinations of sPLA2 and métalloprotéase inhibitors individually and in combinations.

In pre-hospîtal emergency medical care as well as in early goal directed therapy (EGDT) for sepsîs, there has been a recent push to administer antibiotics earlier and earlîer to a patient or subject with severe bums and/or traumatic injury in order to ameliorate and/or prevent sepsîs and acute inflammatory syndromes. The principal problems with this approach is that is of debatable benefit and it is difficult to de-escalate, but while paramedics and first responders may be skilled at identifying the signs and/or symptoms associated with sepsîs and SIRS, their proximity to advanced diagnostic and care facilities limits accuracy and puts correct laboratory sampling (e.g. blood cultures) and correct antibiotic sélection at risk. An approach to mitigating the physiological disorder associated with sepsis/SlR.S, would be bénéficiai for initial stabilization, treatment and ultimate prognosis for these patients, particularly if such an approach could be admînîstered very early in the disease progression. Further, such an approach would hâve particularly useful benefit in remote and low-resource setiings as well as théâtres of war and in the treatment of mîlitary personnel who are often severely burned and/or înjured resulting in high risk of infection, sepsis and SIRS.

Injured military personnel are most often at risk of sepsis and/or SIRS pursuant to

Prolonged Field Care (PFC) which is the area of concem for the military dealing with holding a patient, often an injured patient, in a pre-hospital environment waiting for évacuation to a medical facility appropriate to further treatment of the injured patient. The military benefit of an effective pre-diagnostic option is substantîal.

Military medical Systems are arranged in five (5) échelons or rôles of care in the treatment of injured patients:

Rôle 1 - is directed to front line, typically self-aid/buddy care and TCCC (Tactical Combat Casualty Care). Small unit medics also operate at this level;

Rôle 2 - is directed to small aid station, advanced trauma management and emergency medical treatment including continuation of resuscitation started in Rôle 1; patient stabilization;

Rôle 3 - is directed to Military Treatment Facilitées (MTF’s) within the theater of operation (for example, Kandahar hospital in Afghanistan); staffed and equipped to provide care to ail categories of patients, to include resuscitation, initial wound surgery, 20 specialty surgery (general, orthopédie, urogénital, thoracic, ENT, neurosurgical) and postoperatîve treatment

Rôle 4 - Régional US or robust overseas hospital with many spécialists (for example Landstuhl hospital in Germany)

Rôle 5 - Large US based DoD or VA Hospital

Perhaps unsurprisingly, outcomes from trauma in milîtary/combat settings are heavily dépendent on transport time to higher ievels of care. Currently, for addressing the treatment of conventional troops, the military has become very efficient at transport back to Rôle 3 from Rôle 1, above, reducing times significantly. This has had the very positive resuit of împroving outcomes and reducing morbidity/mortality. But even in this best case scénario, sepsis leading to acute respiratory distress syndrome (ARDS) and other sequalae îs a high risk from trauma. Prolonged treatment with spécifie antibiotics is the norm.

However, United States Spécial Operations Command (USSOCOM) and other units of medics operate at the Rôle 1 échelon of care and operate in areas that are not well supported with transport and évacuation times taking up to 72 hours (or much longer) from time to call to pick up. This prolongs the amount of time that the medic needs to support a baltlefield trauma, substantially increasing the risk to the înjured patient.

One ofthe major issues that may occur îs infection leadîng to sepsis, particularly given the type of injuries that may be sustained. The military has searched for ways to treat for infection, but keep coming back to the need for diagnosis prier to treatment, or general antibiotic therapy that the medic has on hand along with wound cleansing and debridement. There are few or no diagnostics that can be supported by USSOCOM medics în the field and they carry only broad spectrum anti b soties, most often ineffective against several infections which cause sepsis.

The present invention îs directed to the addition of PLA2/MP inhibîtors to a therapeutic regimen that would support antibiotic administration to prevent or mitigate the effects toxins from infection and allow a higher level care and/or the front line medic to support the patient.

The înventors hâve, by surprise, enabied a key invention related to the treatment of inflammatory conditions for particular use in preventing and amelîorating inflammation associated with infection and attenuating the likelihood of sepsis in patients. There has been a big push in pre-hospital care to give antibiotics earlier and earlier to the point of ambulance personnel doing so in the field. This has raised many important treatment timing issues not just in the pre-hospital setting, but in the receiving facilitîes, as well. The present invention can be used to provide a pre-diagnostic treatment for amelîorating and/or reducing the likelihood of catastrophic treatment failure in a patient at risk for sepsis which meets a long-unmet need.

The present invention relates to the following concepts, among others as described herein. Early use of sPLA2 inhibîtors such as varespladib (LY315920), methylvarespladîb (LY333013) and (AZD2716-R. S, a racemic mixture of R. and S isomers or AZD Compound 4 from the same sériés) and carbazols such as LY433771, among others prevents and miti gates the ri se of sPLA2, stabilizing the patient sufficiently to improve and simplify pre-diagnosis care of the patients, promote wound healing, stabilize the patients and preserve the integrity of blood cultures, reduce blood culture contamination prior to anti biotics and improve the performance of anti biotics.

While much has been written on the need for early récognition and treatment of sepsis and related syndromes (e.g. SIRS), the focus has been on early treatment with antimicrobial agents, especially as it pertains to interventions available to pre-hospital and receiving emergency départaient personnel. Unresoived is the matter of EGDT is not only what antibiotic is best used initia lly by personnel in the advance of a diagnosis and conséquences related to:

1. Mîsuse/overuse and timing of fîuids and antibiotics; and

2. Disruption/adequacy of blood cuitures/samples drawn in the pre-hospital setting.

The current invention addresses the limitations of pre-hospital and pre-diagnosis fluids and antibiotics by safely mîtigating the effects of suspected or confirmed sepsis syndromes in the pre-hospital or early hospital setting and 1) eliminating the need to prématurély choose from a panel of available antibiotics while, additionally, mîtigating the conséquences of an incorrect or suboptimal antibiotic choice 2) preserving the întegrity of the blood for culture and testing at the receiving facility 3) préservation of vital signs allowing greater flexibility in critical areas such as fluid management.

Unmet Needs of the Présent Invention

1. Early antibiotics are a key to outcome in infection, acute inflammation and their sequelae and:

a. antibiotic choice is difficult in low resource environments and optimal prediagnosis antibiotic or antiviral choice may be difficult or impossible even in receiving clînic or hospital facilities

b. inappropriate/suboptimal antibiotic or antiviral choice may resuit in bad outcomes, overuse or corruption of blood cultures

c. small molécule anti-toxin therapy to assist in treatment of antibiotic résistant organisms to aid antibiotic treatment and preserve limited antibiotic availability

d. if pre-hospital personnel and preadmission patients had a single drug, drug mixture or drugs for co-administration to improve physiological parameters prior to rise in or to hait the progression of the infiammatory response to infection or biological agent then the protocol s for treatment could be simplified and made safer without complicating disease or treatment

e. prophylactic use, early use and pre-antibiotîc of sPLA2 and metalloprotease inhibitor agents could improve the performance of antibiotics, mitigate and reduce the incidence of inappropriate antibiotic use while stabilizing the patient and also hâve utility as bîodefense agents for civilians and personnel with occupation risk of exposure such as in warfare and hâve, by themselves, therapeutic efficacy (e.g. varespladîb/methylvarespladib/AZD2716 and related compounds for sPLA2 inhibition and/or for example, prînomastat, batimastat, marimastat, vorinostat for métalloprotéase inhibition)

f. Such agents could be stockpiled for bîodefense, be stocked in ambulances at low cost and in hospitals as part of the early response to suspected infections and SIRS such as those induced in the community by:

i. Pneumonîa, hemolytîc urémie syndrome, enterotoxîgenic E. coli, urinary tract infection, wound infection, infection derived toxins such as anthrax léthal factor, botulism and other types of bacteremia such as those induced by marine infections (e.g. Vibrio and mycobacterium) that spread rapidly and are often given inappropriate antibiotics such as cephalosporins.

ii. Viral infections prior to diagnosis such as ebola

g. Increase the safety and utility of connective tissue weakening antibiotics such as quinoloines (e.g. ciprofloxacin, levofloxacin)

h. Improve the performance of antibiotics, improve fluid management and other éléments of EGDT prior to the time sPLA2 is thought to peak in the progression of inflammatory conditions related to infection or systemic inflammatory response syndromes.

î. Improve wound management and healing from bums, trauma, toxin, venom, and acceptance and healing of skin grafts.

j. Reduce complications associated with infusion of CAR-T thérapies and other cell-based immunothérapies based on modîfied immune System cells.

k. Be combined with other PLA2 inhibitors (e.g. cPLA2, ÎPLA2, Lp-PLA2) and métalloprotease inhibitors to reduce unwanted inflammatory and tissue degradîng responses in a tailored or prophylactic fashion.

1. Be combined with antibiotics already possessing désirable properties such as intrinsic sPLA2 or métalloprotéase inhibition (e.g. doxycycline, Prînomastat, marimastat batimastat) enabling a narrower list of optimal antibiotic choices in the pre-hospital and early hospital setting for patients at risk of inflammatory syndromes due to infection, trauma or as a resuit of inflammation-inducing treatments for conditions such as blood cancers (e.g. CAR-T thérapies).

m. Métalloprotease inhîbitors may, unexpectedly hâve general application in ARDS and wound healing by themselves as single agents—especially surprising in ARDS and in combination with sPLA2 inhîbitors.

For treatment of ARDS, pulmonary surfactant, produced by Type 2 épithélial cells (T2C), is a key lung protectant and mechanical lubricant containing several classes of Hpids, including phospholipids, triglycérides, cholestérol, and fatty acids key to its function. It serves a cardinal part in lungs’ innate and adaptive immune response and plays a critical rôle in pulmonary function by the réduction of surface tension. Host defense properties of pulmonary surfactant include direct interaction of surfactant components with pathogens (viruses, bacteria) or their products (e.g. endotoxin, viral glycoproteins); stimulation of phagocytosis by surfactant components (as an opsonin or active ligand); influence of the chemotaxis of îmmune-competent cells; and régulation of cytokine release and reactive oxygen production by macrophages. The hydrophîlîc surfactant apoproteîns SP-A and SP-D hâve distinct functions in the innate immune response to mîcrobial challenge. In addition, the surfactant lîpids suppress a variety of immune cell functions, including activation, prolifération, and immune response of lymphocytes, granulocytes, and alveolar macrophages and can even promote bacterial lysis. Upon failure ofthis first lîne of defense by pulmonary surfactant, the innate immune system which relies on a sériés of germ-line encoded pattem récognition receptors (PRR) that are expressed on épithélial and innate immune ceiis, is activated. Toll-like receptors (TLR) constitutes one of four major classes of PRR, and these PRR are sensors for pathogen-derived, evolutionarily conserved molecular structures. Most TLRs are expressed at the cell surface where they interact with bacterial components and viral proteins. (47) In lung tissues, sPLA2 is a mediator of the normal inflammatory response through release of lipîd medîators and pulmonary mechanical function through its direct rôle in pulmonary surfactant turnover. Pulmonary sPLA2 is a critical mediator of lung function through its metabolism of surfactant and plays a key rôle in the homeostasis and recyclîng of surfactant proteins that résidé in these thin, but extraordinarily important fluid planes. Crîtically, elevated sPLA2 levels in pulmonary alveoli, results in surfactant catabolism and even greater release of factors, including TNF-a, TNF-β and IL-6. These cycles of inflammation and surfactant destruction act synergistically to the point at which the ïnnate immune response to insult becomes lethally maladaptïve.

Coronavirus disease 2019 (COV1D-19) is an infectious disease caused by severe acufe respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavîruses are prîmarily spread between people in close proximity to each other by coughing, sneezing, and talking. COVID-19 can affect the upper (sinuses, nose, and throat) and lower (trachea and lungs) respiratory tract. The lungs are the organ most affected by COVID-19 because the virus accesses host cells via the receptor angiotensin-converting enzyme 2 (ACE2), which is abundant in pulmonary alveolar épithélial type II cells (T2C) and is expressed heavily in alveolar capillary endothélial cells. Currently and urgently of interest, pandémie SARS-CoV-2 infections are resulting in an unusually high spike in incidence and mortality from acute respiratory di stress syndrome (ARDS). ARDS is a terri fying, lifethreatening and difficultto treat clinical complex of lung failure from direct or indirect physiological or physical insult. Patients with severe COVID-19 frequently hâve signs and symptoms of systemic inflammation, which may be mediated by Toll-like receptors and drîven by dysregulated feedback loops involving sPLA2, interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α). Systemic overexpression of sPLA2 may occur with infection that can resuit in dégradation of surfactant and cytokine overexpression, leading to the onset of ARDS. ARDS is associated with physical, physiological and infectious insults such as those caused by blunt trauma, barotrauma and, infections, including SARSCoV-2 infections. The most common clinical disorders associated with the development of ARDS are bacterial and viral pneumonia. When the lung is injured by infection, trauma, or inflammatory conditions, inflammatory pathways are activated. During the course of SARS-CoV-2 infection in the lower respiratory tract, damage to surfactant-producing alveolar épithélial T2C may exacerbate the overall severity of the résultant COVID-19. The inflammatory response and the innate immune barrier created by pulmonary surfactant, along with its rôle in innate and adaptive immune response can aid in pathogen clearance, however, excess inflammation can contribute to alveolar damage, loss of protective surfactant, increased endothélial and épithélial permeability, which resuits in pulmonary edema and reduced pulmonary compliance. At a molecular level, sPLA2 plays a key rôle in the homeostasis of surfactants and protein levels in the lung. During events that hâve increased pathophysiological effects, such as acute respiratory viral infection, elevated sPLA2 resuit în excess inflammation and enzymatic dégradation of surfactant phospholipids and associated proteins at a higher rate than it is formed. This contributes to ioss of protective surfactant layer followed by alveolar damage, increased endothélial and épithélial permeability, accumulation of débris, réduction of innate and adaptive pulmonary immune function, and widespread loss of functionîng lung tîssue, Ioss of lung tissue elasticity, and when severe, ARDS. Elevated sPLA2 levels or dysregulated phospholipase actîvîty may resuit in the development of MOF by directly injuring the lung, either by the cytotoxic effect of elevated sPLA2 on alveolar cells or by the ability of sPLA2 to dégradé surfactant. sPLA2 may also induce organ injury by producing a variety of pro-inflammatory molécules (eg, prostacyclin, thromboxane A2, leukotrienes. The resulting pulmonary edema and reduced pulmonary compliance further damages alveoli and surfactant producing cells required to protect these air sacs. Once edematous fluid accumulâtes in the interstitial and air spaces of the lungs, clinical outcomes include hypoxemia, impaired gas exchange, acid-base imbalances, reduced carbon dioxide excrétion, and ultimately, respiratory failure. Further cycles of inflammation and lung tissue loss resuit, such that there is a cascade of dysfunctional immune responses characteristic of ARDS. In addition to the disruption of the lung microvascular and surfactant barriers, increased tissue permeability can also lead to a new cycle of excessive inflammation such that there is a cascade of upregulated and dysfunctional immune responses. ARDS is further associated with coagulation abnormalities due to elevated cytokines and tissue factor expression. The rôle that sPLA2 plays in this syndrome is likeiy multifactorial as it can cause inflammation as well as damage surfactant, the substance that coats the alveolar epithelium, and prevents alveolar collapse by reducing surface tension.

Surfactant dysfunctîon in neonates and children may play a rôle as pulmonary surfactant typically recycled in a highly complex and regulated mechanism becomes dysregulated. Surfactant turnover is choreographed by Type II cells, macrophages and the alveolar 1 ining. Alveolar secreted apoprotein-rîch, active surfactant aggregates are converted into protein-poor, inactive forms by the cyclical changes in the alveolar surface and are ready for clearance by Type II cells and alveolar macrophages. SPs are re-secreted with surfactant by lamellar bodies, whereas endocytosed phospholipids are recycled and re-secreted by Type II cells. This process is slower in newboms (especially those born prématurély) than in adults or those with lung injury. Morbidîty and mortalîty in preterm and term neonates is due to defective surfactant metabolism secondary to accelerated breakdown of the surfactant complex by oxidatîon, proteolytic dégradation, and inhibition.

Respiratory Distress Syndrome (RDS) in preterm neonates is present shortly after birth with apnoea, cyanosis, grunting, inspiratory stridor, nasal flaring, poor feeding, and tachypnoea. It is one of the most common causes of morbidity and it occurs worldwide with a slight male prédominance Compared to a mature long, RDS is characterised by low volume of surfactant containing a lower percent of di-saturated phosphatidylcholîne species, phosphatidylglycerol, and surfactant proteins. Histopathological findings in lungs of RDS show alveolar atelectasis, alveolar and interstitîal edema and diffuse hyaline membranes in distorted small aîrways. The incidence, severity and mortality associated with RDS are significantly reduced by prénatal corticosteroids and postnatal surfactant replacement therapy, and surfactant therapy has become the standard of care in management of preterm infants with RDS.

Méconium Aspiration Syndrome (MAS) is an important cause of périnatal respiratory distress with increased morbidity and mortality, and it affects an estimated 25,000 neonates in the United States each year. Fêtai distress is indicated by méconium stainîng of the amniotic fluid or foetus. Physiologically, fêtai respiration is associated with movement of fluid from the aîrways out into the amniotic fluid. However, fêtai distress initiâtes aspiration of amniotic fluid and méconium into larger aîrways leading to gasping in utero. Méconium has been found to destroy the fibriiiary structure of surfactant and decreases its surface adsorption rate. An inflammatory response characterized by the presence of elevated cell count and pro-inflammatory cytokines IL-1 β, IL-6, and IL-8 has been found to be associated with MAS as early as in the first 6 hours hours of life. MAS induced acute long injury is characterized by airway obstruction, pneumonitis, pulmonary hypertension, ventilation/perfusion mîsmatch, acidosis and hypoxemia. In vitro studîes hâve demonstrated a méconium PLA2 mediated dose-dependent inhibition of surfactant activity through the compétitive displacement of surfactant from the alveolar film. PLA2 îs also known to damage the alveolar capillary membrane and induce intrapulmonary séquestration of neutrophils by free fatty acids and lyso-PC released by hydrolysîs of DPPC. Bolus or diluted exogenous surfactant replacement has been shown to reverse hypoxemia, reduce pneumothoraces, decrease duration of oxygen therapy and mechanical ventillation, reduce duration of hospital stay, and decrease requirement for extracorporeal membrane oxygénation (ECMO). However, comparison studîes, utilîsing various treatment regimens: standard, bronchoalveolar lavage (BAL) with diluted surfactant, or

H diluted surfactant BAL plus a single early dexaméthasone, did not demonstrate superiority of one form of therapy over another, and may be related to the heterogeneous nature of this form of lung injury. Another randomized trial, infants receiving surfactant lavage has significant improvements în oxygénation, decreases in mean airway pressure, and arterialalveolar oxygen tension gradients compared to bolus group. However, the study showed no significant différences in duration of nitric oxide, assisted ventilation, or hospitalization.

ARDS îs defined as a severe form of acute lung injury (ALI) and a syndrome of acute pulmonary inflammation, characterised by sudden onset, impaired gas exchange, decreased static compliance, and by a non-hydrostatic pulmonary edema. The pédiatrie ARDS incidence is higher in high income countries. Children are particularly vulnérable in the first year of life, and infection is the most common cause of ARDS. The group principally at risk are prématuré neonates with chronic lung disease who develop viral pneumonia, older children with immune deficiency syndromes, and those with chîldhood malignancîes. The hallmark of acute event is the injury to Type 1 alveolar cells and endothélium with increased permeability of the alveolar-capillary barrier leading to an influx of protein-rich edema-fluid into the alveoli, and decreased fluid clearance from the alveolar space. Host bacterial and chemotactic factors attract neutrophils into the airways which further damage the alveolar épithélial cells through expression of enzymes and cytokines. Injury to Type II épithélial cell leads to a decrease in surfactant production, with résultant alveolar collapse. Four clinical criterîa must be met to establish a clinical dîagnosis of ARDS: (i) acute disease onset, (ii) bilateral pulmonary infiltrâtes on chest radiograph, (iii) pulmonary capillary wedge pressure < 18 mmHg or absence of clinical evîdence of left atrial hypertension, and (iv), ratio between arterial oxygen partial pressure (PaO2) and the fraction of inspired oxygen (FiO2) < 200. (62) In contrast, patients that meet the first three criteria, but exhibit a PaO2/FiO2 ratio between 200 and 300, are defined as having ALI. The mortality from ARDS in the pédiatrie âge group, despite the introduction of novel treatments, sti 11 remains high. Efforts to treat with an SP-C surfactant were ineffective, and the use of caîfactant (Infasurf®) in younger children with ALI was effective in reducing ventilator days and increasing survival. The rôle of surfactant damage/loss in ARDS patients is recognized and surfactant replacement seems to improve clinical outcomes in pédiatrie primary, direct ARDS, as supported by some clinical trials. The incidence and mortality of néonatal ARDS are very similar to the pédiatrie one, according to preliminary data from the néonatal AR.DS network.

Bacillus anthracis, the etiological agent of anthrax, has been developed as a bioweapon by countries and terrorists largely because of a combination of the spore's durabîlity and the léthal toxemia ofthe végétative stage. This Gram-positive bacterium forms spores résistant to adverse environmental conditions and can survive for décades in pastures. If ingested or inhaled, even in small numbers, the spores germînate to establish explosive végétative growth and a resulting toxemia that is usually fatal to the host. The primary virulence factor is a secreted zinc-dependent metalloprotease toxin known as léthal factor (LF), which is léthal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Zinc métalloprotéases are classified into five distinct family groups based on the unique signature within the amino acid sequences around HEXXH motif: thermolysin, astaeîn, serratia, matrixin, and reprolysin métalloprotéases that include snake venom, anthrax and E. coii related enzymes. The latter four families hâve an extended zinc binding site, HEXXHXXGXXH, where the third histidine acts as the third zinc ligand instead of the more distant glutamic acid in thermolysin. Of note, the key zinc binding motif has the following: The zîne-binding motif, HEXXH (where H=his, E=glu, and x= any amino acid), found in many zinc métalloprotéases, including anthrax léthal factor (LF) and rattlesnake venoms.

H E X X H - General motif for zinc binding métalloprotéases

H E F G H - Conserved zinc binding site in anthrax léthal factor HE M GH- In zinc binding rattlesnake venom métalloprotéases Thus, we used rattlesnake venom as our mode! for anthrax LF. The only existing therapeutic intervention for naturally acquired or weaponized anthrax is antibiotic treatment that must be given early after infection and at a time when vtetims may expérience only mîld flu-iike symptoms. Delay of treatment, even by hours, substantîally reduces survival of infected patients. To date, physicians hâve antibiotic options to eliminate an anthrax infection, but they hâve no therapeutic options to combat the LFmediated toxemia and tissue destruction during an ongoing infection or the residual toxemia that persists even after the bacteria hâve been eliminated by antibiotics and previous attempts to invesligate hydroxamate MP inhibitors bave not yîelded satisfactory results despite favorable results in cell culture. Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. B. anthracis produces three proteins that make up anthrax léthal toxin (LT) and edema toxin (ET). The bipartite LT and ET, a combination of three proteins; protein protective antigen (PA), léthal factor (LF), and edema factor (EF), are important virulence factors for this bacterium. PA is a receptor-binding component common to both toxins that translocates LF (a protease) or EF (an adenylate cyclase) into cells. Immunization against PA is sufficîent for protection from infection. Group HA secreted phospholipase A2 (sPLA2I1A) is produced in particular by macrophages and possesses potent antibacterial açtivity especially against Gram-positive bacteria. The investigators previously showed, in vitro, that sPLA2-IIA kîlls both germînated B. anthracis spores and encapsulated bacilli. Studies hâve demonstrated that sPLA2-IIA might play a major rôle in host defense against anthrax. Conversely, this bacterium is able to disarm the host immune System, at least in part, through the inhibition of sPLA2-IIA expression by alveolar macrophages. Thus, the literature teaches away from the use of sPLA2 alone and in combination with MP inhibitors has not been contemplated in combination for the purpose of treating anthrax toxin effects. While these long peptide hydroxamates are highly potent LF inhibitors in vitro, their açtivity in inhibiting macrophage killing by LeTx is relatively weak, requirîng μΜ concentrations. There is evîdence to suggest that what effîcacy is observed in cultured cells may be at least partly attributable to weak inhibition of furîn by the polyArg sequence. Investigation has found that the hydroxamate group is susceptible to hydrolysis by prolonged incubation with LF, converting it to a weaker LF inhibîtor, potentially explaîning the low effîcacy in cells. Thus it is surprising that the inventors found that the métalloprotease inhibitor, prinomastat, was effective in treating, by itself or in combination with AZD2716, mice given doses of LPS/Oleic Acid (LPS/OA) suffîcient to induce severe, acute long injury and ARDS with the type of histological findings and endo/epithelial type findings common in directly induced ARDS such as with aspiration syndromes and Chemical irritants as well as diseases such as anthrax ail which hâve complex interplays between métalloprotease and sPLA2 normal and dysregulated processes.

It is envisaged that, depending on how and when administered, eîther an LF inhibitor (LFI) could block the proteolytic protection provided by LF in the macrophage and allow that cell to elimînate spores early in infection (which could be used prophylactically if intentional release of anthrax were suspected) or, more probably, an LFI would be used to block late stage effects of LF during an active infection and increase the probability of host survival. This latter aspect would unquestîonably be used in adjunct therapy with an antibiotic.

Importantly, the potential of sPLA2 inhibitors such as varespladib (LY315920), methyl-varespladib (L Y333013), AZD2716-(R)-3-(5’-benzyl-2’-carbamoyl-[ 1,1 ’-biphenyl]3-yl)-2-methylpropanoic acid- as a racemic mixture or stereoisomer thereof, preferably the “R” enantiomer of the racemic mixture, AZD Compound 4 (3-(5'-Benzyl-2'carbamoylbiphenyl-3-yl)propanoic acid) and LY433771 ((9-[(phenyl)methyl]-5carbamoylcarbazol-4-yl)oxyacetic acid) as therapeutic agents are now supported by the présent invention. Métalloprotéases are also supported for use in the présent invention. Metalloprotease inhibitors of importance include prinomastat, BB-94 (marimastat), BB-2516 (batimastat), vorinostat, cefixime, doxycycline, but these agents hâve not been recognized or realized in the treatment of these life threatening diseases, conditions or the timing of their use alone or in combinations that could prevent, mitigate and reverse conditions whose treatment could unexpectedly benefit from these compounds alone or in combination. Examples include ARDS type disease States and/or conditions ranging from néonatal to adult and for prévention and/or amelioration of lung and kidney damage from anthrax as well as for the accelerated healing of wounds and non-healing ulcers and residual toxidromes (e.g. from snakes) driven by maladaptive host responses or residual toxins.

Brief Summary of the Invention

The présent invention is directed to the use of an effective amount of at least one PLA2 înhibitor and/or at least one metalloprotease inhibitor alone or preferably in combination with at least one antibiotic to reduce the likelihood of an înjured patient or subject at risk of a life threatening inflammatory syndrome becomîng înfected such that the infection will produce one or more of sepsis, septic shock, an acute inflammatory syndrome such as systemic inflammatory response syndrome (SIRS) and/or acute respiratory dîstress syndrome (ARDS). These compositions and methods are also useful in the treatment of disease States and conditions which are associated with or hâve secondary effects of inflammatory syndrome such as anthrax (Bacillus ant.hracis) and coronavirus (especially including Severe Acute Respiratory Syndrome coronavirus (e.g. SARS or SARS-CoV2). In embodiments, the PLA2 inhibitor is varespladib (LY3 15920), methyl-varespladib (LY333013), AZD2716-(R)-3-(5’-benzyl-2’-carbamoyl·[l,l ’-biphenyl]-3-yl)-2methylpropanoic acid- as a racemic mixture or a stereoisomer thereof, preferably the “R” enantiomer of the racemic mixture, AZD Compound 4 (3-(5'-Benzyl-2'carbamoylbiphenyl-3-yl)propanoic acid) and LY433771 ((9-[(phenyl)methyl]-520854 carbamoylcarbazol-4-yl)oxyacetic acid), a pharmaceutically acceptable sait thereof or a mixture thereof. In embodiments, the metalloprotease inhibitor is prinomastat, BB-94 (marimastat), BB-2516 (batimastat), vorinostat. cefixime and doxycycline, a pharmaceutically acceptable sait thereof or a mixture thereof, among others.

In an embodiment, the présent invention is directed to a method of reducing the likelihood that an injured patient or subject at rîsk of a life-threatening infiammatory syndrome (especially including from an infection) will produce one or more of sepsis, septic shock, an acute infiammatory syndrome (whether iatrogénie or not) or acute respîratory distress syndrome, comprising administering to said patient or subject an effective amount of at least one PLA2 inhibitor and/or at least one metalloprotease inhibitor alone or in combination with at least one antibiotic. The présent invention is particularly useful for treating severe injuries, and infections which can resuit in infiammatory syndrome, including anthrax and SARS/SARS-CoV2 infections.

In an embodiment, the présent invention is directed to administering to a patient or subject who has been severely injured or burned and places that patient or subject at risk for one or more of sepsis, septic shock, slow or poor wound healing, including skin grafts, acute infiammatory syndrome, including infiammatory response syndrome (SIRS) and/or acute respîratory distress syndrome (ARDS), an effective amount of an antibiotic to treat infection in combination with an effective amount of at least one PLA2 inhibitor and/a metalloprotease inhibitor. In embodiments, the method comprises administering at least two antibiotics and at least one PLA2 inhibitor and/or at least one metalloprotease inhibitor. Pursuant to the présent invention, it has been discovered that the co-administration of effective amounts of at least one antibiotic and at least one PLA2 inhibitor and/or at least metalloprotease inhibitor will provide an unexpected inhibition, amelioration or avoidance of sepsis, septic shock, acute infiammatory syndrome, including infiammatory response syndrome (SIRS) and/or acute respîratory distress syndrome (ARDS) in the patient or subject at risk. In embodiments, the PLA2 inhibitor is varespladib (LY315920), methyl varespladîb (LY333013), AZD2716-(R)3-(5’-benzyl-2’-carbamoyl-[l,1 ’-biphenyl]-3-yl)-2-methylpropanoîc acid- (as a racemic mixture or a stéréo isomer thereof, preferably the “R” enantiomer of the racemic mixture), AZD Compound 4 (3-(5'-Benzyl-2'-carbamoylbiphenyl-3-yl)propanoic acid) and LY433771 ((9-[(phenyl)methyl]-5-carbamoylcarbazol-4-yl)oxyacetic acid), a pharmaceutically acceptable sait thereof or a mixture thereof. In embodiments, the metalloprotease inhibitor is prinomastat, BB-94 (marimastat), BB-25 i 6 (batimastat), vorinostat, cefixime and doxycycline, a pharmaceutically acceptable sait thereof or a mixture thereof, among others.

In an embodiment, the present invention is directed to treating a patient or subject with suspected sepsis, inciuding early sepsis or other inflammatory syndrome or comprising administering to said patient or subject in need, an effective amount of at least one antibiotic and an effective amount of at least one PLA2 inhibitor and/a metailoprotease inhîbitor. In embodiments, the method comprises administering at least two antibiotics and at least one PLA2 inhîbitor and/or at least one metailoprotease inhibitor. Pursuant to the present invention, it has been discovered that the co-administration of effective amounts of at least one antibiotic and at least one PLA2 inhibitor and/or at least metailoprotease inhibitor will safely provide an unexpected prévention, inhibition, amelioration or avoîdance of sepsis, septic shock. acute inflammatory syndrome, inciuding inflammatory response syndrome (SIRS) and/or acute respiratory distress syndrome (ARDS) in the patient or subject at risk. In embodiments, the PLA2 inhibitor is varespladib (LY315920), methyl varespladib (LY333013), AZD2716-(R)-3-(5’-benzyl-2’-carbamoyl-[l,l’-biphenyl]-3-yl)-2methylpropanoic acid- (as a racemic mixture or as a stéréo isomer thereof, preferably as the “R” enantiomer of the racemic mixture), AZD Compound 4 (3-(5'-Benzyl-2’carbamoylbiphenyl-3-yl)propanoic acid) and LY433771 ((9-[(phenyl)methyl]-5carbamoylcarbazol-4-yl)oxyacetîc acid), a pharmaceutically acceptable sait thereof or a mixture thereof. In embodiments, the metailoprotease inhibitor îs prinomastat, BB-94 (marimastat), BB-2516 (batimastat), vorinostat, cefixime and doxycycline, a pharmaceutically acceptable sait thereof or a mixture thereof, among others.

in an embodiment, the present invention is directed to treating a patient with an anthrax or severe acute respiratory syndrome coronavirus (SARS and SARS-CoV2) infection comprising administering to said patient or subject in need, an effective amount of at least one PLA2 inhîbitor and/a metailoprotease inhibitor, optionally in combination with at least one antibiotic or other therapeutic agent to reduce the likelihood of and/or amelîorate or inhibit the impact of any one or more of sepsis, septic shock, acute inflammatory syndrome, inciuding inflammatory response syndrome (SIRS) and/or acute respiratory distress syndrome (ARDS) in the patient or subject in need.

in embodiments, the present invention is also directed to a method for preserving a blood sample(s) taken from a patient or subject who has injuries or burns which place the patient or subject at risk for one or more of sepsis, septic shock, acute inflammatory syndrome, inciuding inflammatory response syndrome (SIRS) and/or acute respiratory distress syndrome (ARDS), the method comprising combining in said blood sample with an effective amount of at least one PLA2 inhibitor and/or at least one metalloprotease each alone, together or in combination with an effective amount of at least one antîbiotic. In embodiments, the PLA2 inhibitor îs varespladîb (LY315920), methyl varespladib (LY333013), AZD2716-(R)-3-(5’-benzyl-2’-carbamoyl-[l ,1 ’-biphenyl]-3-yl)-2- methylpropanoic acid- (as a racemic mixture or a stereoisomer thereof, preferably the “R” enantiomer of the racemic mixture), AZD Compound 4 (3-(5'-Benzyl-2'carbamoylbiphenyl-3-yl)propanoic acid) and LY433771 ((9-[(phenyl)methyl]-5carbamoylcarbazol-4-yl)oxyacetic acid), a pharmaceutically acceptable sait or mixture thereof. In embodiments, the metalloprotease inhibitor is prinomastat, BB-94 (marimastat), 10 BB-2516 (batimastat), vorinostat, ceflxime and doxycycline, a pharmaceutically acceptable sait or mixture thereof, among others.

In embodiments, the présent invention is directed to a pharmaceuticaï composition for ameliorating, inhibiting or reducing the likelihood of one or more of sepsis, septic shock, acute inflammatory syndrome, including inflammatory response syndrome (SIRS), hemolytic 15 urémie syndrome (HUS), and/or acute respiratory distress syndrome (ARDS) in a patient or subject in need, the composition comprising an effective amount of at least one antîbiotic in combination with at least one PLA2 inhibitor and/or at least one metalloprotease inhibitor. In embodiments, the PLA2 inhibitor is varespladib (LY315920), methyl varespladib (LY333O13), AZD2716-(R)-3-(5’-benzyl-2’-carbamoyl-[l,l’-biphenyl]-3-yl)-2- methylpropanoic acid- (as a racemic mixture or a stereoisomer thereof, preferably the “R” enantiomer of the racemic mixture), AZD Compound 4 (3-(5’-Benzyl-2'carbamoylbiphenyl-3-yl)propanoic acid) and LY433771 ((9-[(phenyl)methyl]-5carbamoylcarbazol-4-yl)oxyacetic acid), a pharmaceutically acceptable sait, a stereoisomer thereof or a mixture thereof. In embodiments, the metalloprotease inhibitor is prinomastat, 25 BB-94 (marimastat), BB-2516 (batimastat), vorinostat, cefixime and doxycycline, a pharmaceutically acceptable sait or mixture thereof, among others. In cases associated with anthrax, the antibiotic is ciprofloxacin, levofloxacin, moxifloxacin, penicillin g, doxycycline, chloramphenicol, ofloxacin and mixtures thereof.

In embodiments, the présent invention is also directed to a composition comprising a 30 blood sample(s) taken from a patient or subject who has injuries or bums which place the patient or subject at risk for one or more of sepsis, septic shock, acute inflammatory syndrome, including inflammatory response syndrome (SIRS) and/or acute respiratory distress syndrome (ARDS) in combination with an effective amount of at least one PLA2 inhibitor and/or at least one metalloprotease each alone, together or in combination with an effective amount of at least one antibiotic. In embodiments, the PLA2 inhibitor is varespladîb (LY315920), methyl varespladîb (LY333013), AZD2716-(R)-3-(5’-benzyl-2’-carbamoyl[l,l’-biphenyl]-3-yl)-2-methylpropanoic acid- (as a racemic mixture, or a stéréo isomer thereof, preferably as the “R” enantiomer of the racemic mixture), AZD Compound 4 (3(5'-Benzyl-2'-carbamoylbiphenyl-3-yl)propanoic acid) and LY433771 ((9[(phenyl)methyl]-5-carbamoylcarbazol-4-yl)oxyacetic acid), a pharmaceutically acceptable sait, stereoisomer or mixture thereof. In embodiments, the métalloprotéase inhibitor is prinomastat, BB-94 (marimastat), BB-2516 (batimastat), vorinostat, cefixime and doxycycline, a pharmaceutically acceptable sait or mixture thereof, among others.

The présent invention meets a long-felt need in the art, inasmuch as the methods and compositions may be used to treat patients or subjects with severe burns or injuries (without further diagnosîs) in orderto substantially ameliorate, inhibit or reduce the likelihood ofone or more of sepsîs, septic shock, acute inflammatory syndrome, including inflammatory response syndrome (SIRS) and/or acute respiratory distress syndrome (ARDS) in a patient or subject in need.

In additional embodiments, the présent invention also relates to the treatment of néonatal ARDS, including méconium aspiration syndrome (MAS) which is a life-threatening type of néonatal ARDS with high rates of mortality and no approved treatments. méconium aspiration syndrome (MAS) is known to damage surfactant. It is known that surfactant function is damaged and correlates with lung aération and as conséquence of these changes, surfactant nanostructure is also damaged. Néonatal ARDS has some shared traits with other forms of adult and pédiatrie ARDS and support the development of new surfactant protection and anti-inflammatory strategies such as combinations of surfactant (porcine and synthetic) or pretreatment of the patient with IV sPLA2 inhîbitors such as LY315920, AZD2716 (as a racemic mixture or stereoisomer thereof, preferably the “R” enantiomer of the racemic mixture) or mixtures thereof. Alternatively, în preferred embodiments, these drugs could be given intratracheally or via orogastric tube (LY333013, AZD2716 (as a racemic mixture or stereoisomer thereof, preferably the “R” enantiomer of the racemic mixture), see “Compound 7” in Giordanetto et al., Med. Chem. Lett. 2016, 7, 884-889, which is incorporated by reference hereîn. Off target effects may be mînimized by chiral séparation of LYS 15920 or LY333013. The unique features of this strategy include the combination of reduced cytokine production and préservation of surfactant. No other drug strategy can simultaneously produce these key clinical effects in MAS or néonatal ARDS.

In addition to néonatal and pédiatrie application of this strategy, including dosage, forms, carriers (eg surfactant carrying sPLA2 inhibitors), ail routes of delîvery, timing and weight based dosing can be applied to adult patients with ARDS from any cause and in combination with antiviral, antibiotic and anticoagulants such as heparin, low-molecular weight heparin or steroids. Unlike bîological response modifiers based on antibodies, synthetic small molécule inhibitors hâve superior tissue pénétration and prevent neutrophil stiffening.

In preferred embodiments, LY315920, AZD2716, as a stereoisomer or racemic mixture are combined. Alternatively, in preferred embodiments, these drugs could be given intratracheally or via orogastric tube (LY333013, AZD2716 (as a racemic mixture or stereoisomer thereof, preferably the “R” enantiomer of the racemic mixture), see “Compound 7” in Giordanetto et al, Med. Chem. Lett. 2016, 7, 884-889, provide this superior tissue pénétration and shorter half-life such that risk of réactivation of tuberculosis or hepatitis is lower risk than with long half-life antîbody thérapies putting patients and large populations at risk for pulmonary and systemic diseases of medium term and chronic immune suppression.

In preferred embodiments, LY315920, AZD2716 (as a racemic mixture or stereoisomer thereof) combined. Alternatively, in preferred embodiments, these drugs could be given intratracheally or via orogastric tube (LY333013, AZD2716 (as a racemic mixture or stereoisomer thereof, preferably as the “R” enantiomer of the racemic mixture), “Compound 7” in Giordanetto et al and hâve the unique feature of being a treatment for several diseases at once that affect poor populations, far forward military and expédition operations with purely military, scientific or combinations of military, scientific and civilian service in low and middle income countries.

The présent invention advances the State of the art with respect to at least six concerns:

1) Timing—especially use prior to dysregulated or exogenous sPLA2 and MP activîty in patients or subject for bénéficiai effect

2) Enhancing antibiotic effect

3) Improving flexibility of antibiotic choice/performance and fluid management 4) Prévention of CAR-T (and other cell-based therapy) inflammatory events

5) Prévention of cellular injury by endogenous and exogenous toxins such as anthrax léthal factor

6) Improvement in wound healing

The present invention îs described in further detail in the sections which are presented herein below.

Brief Description of the Figures

Figure 1, Table 1 Shows ARDS features and scoring system modeled to compare mouse lung injury based on American Thoracic Society (ATS) workshop report (Aeffner et al. Tox Path, 43:1074-1092, 2015). Exemplary si ides showing features of the ATS crîteria from Table 1 are presented as Figures 1 and 2.

Figure IA Shows the features of alveolar pathology as summarized in Table 1 in a mouse with ARDS induced by a combination of LPS/OA.

Figure IB Shows the features of normal alveoli and alveolar pathology as summarized in Table 1 in a mouse with ARDS induced by a combination of LPS/OA

Figure 2 Shows that întranasal AZD2716 protects and reduces alveolar damage from intranasally applied LPS/OA at 24 and 48 hours. Intranasal AZD2716 protects and reduces alveolar damage from intranasally applied LPS/OA at A) 24 (N=3 mice/group) and B) 48 hours (N=2 mice/group); minimum 25 High Power Fields (HPF) per analysis. *denotesp<0.05. Based on the ATS workshop scoring system (Table 1), Intranasal AZD2716 protected Black-6 mice from combîned LPS/OA-induced lung injury and ARDS. Treated animais were bright, aiert and responsive compared to léthargie, tachypneic Controls at 24 and 48 hours. Audio recordings of the mice at 24 hours clearly demonstrates the réduction in lung surface tension and increased lung compliance indicating préservation of lung surfactant (see below, Figure 3).

Figure 3 Shows the composite lung audiograms of mice demonstrating protection from ARDS by intranasally dosed AZD2716 demonstrate -dOdB différence between treated (quieter) and untreated (louder) mice. The fluid buildup in the mice also makes the lungs heavy and stiff, which decreases the lungs' ability to expand and causes crackling sounds when the alveoli suddenly expand with increased work of breathîng. This results from prévention of dégradation or restoration of surfactant via Type 11 épithélial cells responsible for production of surfactant. Measurement of the audiofiles showed roughly a 1 Odb différence between untreated and treated populations of mice exposed to ΓΝ LPS/OA mixtures: -38.5 db (treated, quieter) from maximum and -29.7db (control, louder) from maximum between control and treated animais.

Figure 4, Table 2 Shows that prinomastat was superior in potency to anthrax léthal factor inhîbitor. ICsos reported as micromolar (μΜ) concentrations and selected as test article for subséquent studies despite good to excellent performance of marimastat and batimastat in same assays as measured by potency. Prinomastat was more potent than other hydroxamate métalloprotease înhibitors, including anthrax léthal factor inhîbitor. Several venoms can cause ARDS and kidney damage with similarity in cellular changes seen in pulmonary and systemic anthrax. Zinc métalloprotéases are classified into five distinct family groups based on the unique signature within the amino acid sequences around HEXXH motif: thermolysin, astacin, serratia, matrixin, and reprolysin métalloprotéases. The latter four families hâve an extended zinc binding site, HEXXHXXGXXH, where the third histidîne acts as the third zinc ligand instead of the more distant glutamic acid in thermolysin. Further, we emphasize:

HEXXH- General motif for zinc binding métalloprotéases H E F G H - Conserved zinc binding site in anthrax léthal factor H E M G H- In zinc binding rattlesnake venom métalloprotéases Prinomastat was chosen for mouse lung-injury and ARDS study as well as in subséquent ECIS studies for this reason and to be combined with an sPLA2 inhîbitor. There is no known explanation for why Prinomastat is more potent than these other înhibitors for these embodiments and it has not been coniemplated as a therapeutic to combat toxin damage as seen caused by anthrax léthal factor, a toxic métalloprotease. Prinomastat has not been previously reported to be more potent than anthrax léthal factor inhîbitor, but demonstrably more potent as seen in Table 2. Sistrurus venom was used as surrogate for anthrax léthal factor inhîbitor because ofthe léthal factor inhibitor’s hîgh potency against Sistrurus venom métalloprotease activity (Table 2) which is similar to that of native anthrax léthal factor, which has also been termed “zincin” class métalloprotéases (Figures 6, 8).

Figure 5 Shows that both oral prinomastat and oral AZD2716 rescue and reduce damage from LPS/OA induced ARDS in mice at 48 hours. Unexpectedly, prinomastat treated mice were clinically the most bright, alert and responsive which has significant implications for the treatment ofboth ARDS and diseases such as anthrax. These results show the capacity of both AZD27I6 and Prinomastat to împrove clinical and histopathological findings when rescuing mice from LPS/OA înduced ARDS. Prinomastat treated mice were, surprisingly, clinically better than AZD2716 treated mice (Prinomastat > AZD2716 =Prinomastat + AZD2716 Controls) and indicates particular potentiai in the treatment of anthrax and other syncytia forming viral and bacterîal pathogens and their elaborated toxins (e.g. anthrax léthal factor, a metalloprotease). The combination of the two drugs significantly improved the outcomes in two key components of ARDS (intra-alveolar infiltrâtes and protein dépositions related to pulmonary edema) demonstrating the potentiai for these drugs alone and in combination to treat deadly conditions such as SARS-CoV-2 associated ARDS and SIRS as well as ARDS caused by anthrax bacterium, for example, that dépend on both métalloprotéases and sPLA2 to invade lung tîssues and cause widespread inflammation (SIRS). Sîgnificant prévention of pulmonary capillary leak, inflammation, hemorrhage implies préservation of both endo- and epithelial-cell layers. Figure 6 Shows that venom toxins produce cellular injury and detachment quantifiable by ECIS (Figures 7-12) that are comparable to that found in épithélial surfaces throughout the body including kîdney, lung and skin.

Figure 7 Shows that rescue of épithélial cell from a balanced metalloprotease/sPLA2 containing venom (Toxin: Whole A. contortrix laticinctus venom)

Figure 8 Shows the rescue of épithélial cell from a balanced métalloprotease/serine protease, but low sPLA2 content venom (Toxîn: Whole Sistrurus venom). Sistrurus venom is used as surrogate for anthrax léthal factor and prinomastat as comparator hydroxamate inhibitor.

Figure 9 Shows that AZD2716, like varespladib préserves cellular function and cell-cell junction integrity in the presence of high sPLA2 content venom toxins as measured by résistance (Toxin: C. scutulatus whole venom).

Figure 10 Shows that varespladib, like AZD2716 in previous figure 9 recovers and stabilizes épithélial cells across venom généra and geography (Toxins: C. scutulatus and Daboia rime///-Pakistan). Varespladib, like AZD2716 in previous figure 9 recovers and stabilizes épithélial cells across venom généra and geography. As a resuit varespladib is shown to préservé and restore kidney function or comparable structures in other organ Systems such as lung, skîn, or gastrointestinal tract from insuIts such as viral infection, organisms like anthrax as well as venom. Top Row: Raw data shown as function of résistance and then capacitance (left to right). Bottom Row: Cumulative data showing varespladîb recovers and stabilizes inter-cellular attachments (résistance) and cell viabîlîty (capacitance) left to right. C. scutulatus venom (North America), 4 doses, N=2 each; D. russelli (Pakistan variant) 2 dose concentrations, N=2 each) Varespladîb : 0.05mg/ml and 0.01 mg/mL no significant différence in response between the two doses the response was thus combined. Négative Controls: N=2 kîdney épithélial (vero) cells in media only.

Figure 11 Shows that low dose prinomastat + varespladîb prevents damage and accelerates wound healing in presence of rattlesnake venom containing simîlar enzymes responsible for chronic wounds and non-healing ulcers. Low dose prinomastat + varespladîb prevents damage and accelerates wound healing in presence of rattlesnake venom containing sîmilar enzymes responsible for chronic wounds and non-healing ulcers. Wounds produced by electrîcal current across ail wells. +Control cells (top line): Instability of healing demonstrated by large standard déviations and increasîng capacitance indicative of cell death and detachment from the basement membrane. Cells treated with lower doses of varespladîb and prinomastat than normal!y used with either one alone produced unexpected wound healing comparable to uninjured control cells (middle and bottom, respectively). B. In presence of rapidly toxic doses of Echis ocellatus venom, both prinomastat and a combination of low dose prinomastat plus varespladîb increase cellular junction tightness even în the ongoing presence of venom that will be useful for acute toxîcity and wound healing from Echis species and in general healing of cellular injury. In both studîes, drugs were applied following exposure to venom. Simîlar changes to épithélial cells are seen in ARDS and rénal and pulmonary effects of infections such as those caused by anthrax via metalloprotease and/or sPLA2 mediated pathways. Figure 12 Shows that low dose varespladîb + prinomastat accelerate rates of wound healing even in presence of toxins (Toxin: Sistrurus miliarius barbouri, whole venom acquired from the National Natural Toxins Research Group) even with addition of electrîcal wounding following exposure to venom. Cells treated with lower doses of varespladîb and prinomastat than normally used with either one alone produced unexpected synergistic wound healing raies. In presence of rapidly toxic doses of S. miliarius venom, both prinomastat and a combination of low dose prinomastat plus varespladîb increase cellular junction tightness even in the ongoing presence of venom that is a surrogate system for acute toxicity and wound healing. Simîlar changes to épithélial cells are seen in ARDS and rénal and pulmonary effects of infections such as those caused by anthrax via metalloprotease and/or sPLA2 mediated pathways. Rates of wound healing were unexpectedly fast as demonstrated by the combination of varespladib and Prinomastat that produced A) accelerated retum of cell-cell junctional tightness (résistance) as well as B) return of cellular viability. OU= Prinomastat. This has broad, unexpected significance for a multitude of wound healing therapeutics.

Detailed Description of the Invention

The following terms shall be used throughout the spécification to describe the présent invention. Where a term is not specifically defined herein, that term shall be understood to be used in a manner consistent with its use by those of ordinary skill in the art.

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 dictâtes otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. In instances where a substituent is a possibility in one or more Markush groups, it is understood that only those substituents which form stable bonds are to be used.

Unless defined otherwise, ail technicai and scientific terms used herein hâve the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or équivalent to those described herein can also be used în the practice or testing of the présent invention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended daims, the singular forms a, and and the include plural references unless the context clearly dictâtes otherwise.

Furthermore, the following terms shall hâve the définitions set out below.

The term “patient” or “subject” is used throughout the spécification within context to describe an animal, generally a mammal, especially including a domesticated animal and preferably a human, to whom a treatment, including prophyîactic treatment (prophylaxis) is provided. For treatment of those infections, conditions or disease States which are spécifie for a spécifie animal such as a human patient, the term patient refers to that spécifie animal. In most instances, the patient or subject is a human patient of either or both genders.

The term “compound” is used herein to describe any spécifie compound or bioactive agent disclosed herein, including any and ail stereoisomers (including diastereomers), individual optical isomers (enantiomers, including with respect to compound AZD2716) or racemic mixtures, pharmaceutically acceptable salts and prodrug forms. The term compound herein refers to stable compounds. Within its use in context, the term compound may refer to a single compound or a mixture of compounds as otherwise described herein.

The term “effective” is used herein, unless otherwise indicated, to describe an amount of a compound or component which, when used within the context of its use, produces or effects an intended resuit, whether that resuit relates to the prophylaxis and/or therapy of an infection and/or disease State or as otherwise described herein. The term effective subsumes ail other effective amount or effective concentration terms (including the term “therapeutically effective”) which are otherwise described or used in the présent application.

The term “pharmaceutically acceptable” as used herein means that the compound, composition, including a sali form, is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light ofthe severity of the disease and necessity of the treatment.

The terms “treat”, “treating”, and “treatment”, etc., as used herein within context, also refers to any action providing a benefit to a patient at risk for any of the disease States or conditions (which can be treated pursuant to the présent invention (e.g., ameliorate, inhibit, reduce the severity, cure, etc.). Treatment, as used herein, princîpally encompasses therapeutic treatment, but may also encompass both prophylactic and therapeutic treatment, depending on the context of the treatment. The term “prophylactic” when used in context, means to reduce the likelihood of an occurrence or in some cases, reduce the severity of an occurrence within the context of the treatment of a disease State or condition otherwise described herein.

The term “coadministration” shall mean that at least two compounds or compositions are administered to the patient at the same time, such that effective amounts or concentrations of each of the two or more compounds may be found in the patient at a given point in time. Although compounds according to the présent invention may be coadminîstered to a patient at the same time, the term embraces both administration of two or more agents at the same time or at different times, provided that effective concentrations of ail coadministered compounds or compositions are found in the subject at a given time. Compounds according to the présent invention may be administered with one or more additional bioactive agents to address spécifie disease conditions in a treated patient or subject.

The term “prévention” is used within context to mean “reducing the likelihood” of a condition or disease State from occurring as a conséquence of administration or concurrent administration of one or more compounds or compositions according to the présent invention, alone or in combination with another agent. Thus, the term prévention is used within the context of a qualitative measure and it is understood that the use of a compound according to the présent invention to reduce the likelihood of an occurrence of a condition or disease state as otherwise described herein will not be absoiute, but will reflect the ability of the compound to reduce the likelihood of the occurrence within a population of patients or subjects in need of such prévention.

The term “sepsis” is used to describe a clinical syndrome that complîcates infection. It is characterized by signs of inflammation (vasodilation, leukocyte accumulation, increased microvascular permeabîlity) occurring in tissues that are remote from the infection. Systemic inflammatory response syndrome (SIRS) is also a clinical syndrome that complîcates a noninfectious insult (e.g., acute pancreatîtis, pulmonary contusion). Théories about the onset and progression of sepsis and SIRS are focused on dysrégulation of the inflammatory response, including the possibility that a massive and uncontrolled release of proinflammatory mediators initiâtes a chain of events that lead to widespread tissue injury. This response can lead to multiple organ dysfunction syndrome (MODS), which îs the cause of the high mortality associated with these syndromes.

Sepsis is typically associated with a bacterial infection and is characterized by a whole-body inflammatory State (SIRS) and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune System to bacteria presence in the blood, urine, lungs, skin, or other tissues. Sepsis is also referred to as “blood poisoning” or septîcemia. Severe sepsis is the systemic inflammatory response, plus infection, plus the presence of at least one organ dysfunction. Septîcemia (also sometimes referred to as bacteremia) refers to the presence of pathogenic organisms in the bloodstream, leading to sepsis.

Sepsis is a lîfe-threatening condition in which the body is fighting a severe infection that has spread via the bloodstream. If a patient becomes septic, he or she will likely hâve low blood pressure leading to poor circulation and lack of blood perfusion of vital tissues and organs, resulting in shock. This condition of shock is often referred to as septic shock when an infection is the cause of shock to distinguish it from shock due to blood loss or from other causes. Sepsis and septic shock can develop either as a resuit of the body's own defense system or from toxic substances made by the infecting agent. Survival rates for sepsis dépend on the patient's underlying medical conditions, how quickly the diagnosis is made, the organîsm that causes the infection, and the patient's âge.

In the United States, sepsis is the second-leading cause of death in non-coronary ICU patients, and the tenth-most-common cause of death overall according to data from the Centers for Disease Control and Prévention (the first being heart disease). Sepsis is common and also more dangerous in elderly, immunocompromised, and crîtically ill patients. It occurs in 1-2% of ail hospitalizations and accounts foras much as 25% of intensive-care unit (ICU) bed utilization. It is a major cause of death in intensive-care units worldwide, with mortalîty rates that range from 20% for sepsis to 40% for severe sepsis to >60% for septic shock.

Septic shock is a medical emergency caused by decreased tissue perfusion and oxygen delivery as a resuit of severe infection and sepsis, though the microbe may be systemic or localized to a particular site. It can cause multiple organ dysfunction syndrome (formerly known as multiple organ faîlure) and death. Its most common vîctims are chîldren, immunocompromised individuals, and the elderly, as their immune Systems cannot deal with the infection as effectively as those of healthy adults. Frequently, patients suffering from septic shock are cared for in intensive care units. The mortalîty rate from septic shock is approximately 25%-50% (See United States Patent Application Document No. 20140162978). Many details of optimal EGDT remain unresolved and controversial. The current invention résolves or mitigates several key éléments of EGDT timing and protocol flexibility makîng a novel and critîcal advance to patient care for this long-unmet need of complex and crîtically ill and înjured patients.

Adéquate management of septic patients is often complicated by delay în adminîstering therapy after sepsis has been recognized. Every hour delay in the administration of appropriate antibiotic therapy there is associated with a significant rise in mortality.

“Sepsis” as used herein and within context includes ail of the aforementioned septic states, conditions and clinical symptoms, e.g. “sepsis” includes but is not limited to systemic inflammatory response syndrome (SIRS), septicemia and septic shock.

The term “early sepsis” is used to describe the initial stages of sepsis before a full septic State occurs. It is important to recognize the signs of early sepsis and immediately seek treatment if treatment has not yet been initiated because the infection can spread rapidly -- often in a matter of hours. Sepsis occurs when an infection in the body enters the bloodstream and spreads throughout the body; this can lead to septic shock, a potentially fatal condition. Some of the earliest signs of sepsis include a high fever, a feeling of fatigue, an increased heart rate, rapid breathîng or breathîng difficulty. Experts generally look for at least two symptoms to suspect and diagnose sepsis. A diagnosed infection îs also one of these symptoms.

If the original source of the infection is on the surface of the body, one of the best indicators of early sepsis is the presence of red streaks coming off the area and movîng up the înfected limb. Not ail infections are superficial however, which is why the other signs of early sepsis are also important to recognize.

Infections will often present with a fever that steadîly increases, but may not be évident without core température measurements. As the fever increases, muscle pain and weakness may become present, and some people may expérience pain in the joints as well. This fever may also cause chills, and some people notice that they become dîzzy and shaky, with a corresponding drop in blood pressure.

Accompanied by the chills and fever, the signs of early sepsis also often include a rapîd heartbeat and quick breathîng. People may fmd that they cannot slow the breathîng or the heart rate down no matter how much they try to relax and take deep breaths. These symptoms will also worsen as the infection progresses through the body. The signs of early sepsis typically only last for a short time; if they are not addressed with emergency treatment with antibiotics, other, more serions symptoms will quickly become apparent.

Some îndividuals will develop a rash on the skin in addition to the red streaks. This rash can show up anywhere on the body. In addition, urine output will generally decrease significantly, which is a symptom that the organs are slowîng function, which is extremely dangerous. Mental State may change as well; some people become confused and agîtated.

Rather than wait and see if these early symptoms of sepsis get better, it is important to receive emergency treatment as soon as possible, in this case at least one antibiotic în combination with at least one PLA2 inhibitor and/or metalloprotease inhibitor. This is another reason to be certain to care for any injuries or infections in the body, to clean cuts and scrapes well, and to take an entîre course of antibiotics when prescribed, to be sure ail infections are killed before they can spread.

The term “likelihood of sepsis” is used to describe a patient or subject who has extensive burns and/or a severe injury including an injury associated with an infection which likely will resuit in sepsis and includes any patient or subject with a several burn and/or injury which will likely resuit in an infection.

The term “severe burns” or “extensive burns” is used to describe a patient or subject who wili likely hâve an infection or will resuit in an infection which can cause sepsis or an acute infiammatory syndromes such as systemic infiammatory response syndrome (SIRS) or related infiammatory syndrome. The severity of a burn, along with the type of burn, is important to assess before planning a burn treatment regimen and providing treatment pursuant to the présent invention. Burn severity often indicates the recovery time needed and whether or not the burn patient will expérience permanent effects, such as scarring or an interférence or change in physiological function. More importantly, the severity of a burn may indicate that the patient or subject will become infected, thus substantially increasing the likelihood of sepsis or an acute infiammatory syndromes such as systemic infiammatory response syndrome (SIRS). Severe burns are typically classified by measuring the total body surface area (TBSA) of the burn injury. This system measuresthe percentage of bumed skin in comparison to the rest ofthe victim’s body. A burn injury of the same size will resuit in a higher TBSA for a child than for an adult, due to the child’s smaller body size. The American Burn Association has set forth guidelines for measuring TBSA and diagnosing severe burns.

Burn injuries are typically placed into three major categories. In adults, burns with a TBSA of 10 percent or less are classified as minor burns. In children, minor burns measure five percent TBSA or less. Moderate burns may cover roughly ten-to-twenty percent of adults. In children, moderate burns cover roughly five-to-10 percent. Minor and moderate burns often do not produce infection consistent with eventual sepsis and/or acute infiammatory syndrome, but the patient or subject must be monitored carefully and treated pursuant to the présent invention when there is evidence for risk of infection.

Major, or severe bums, measure more than 20 percent TBSA în adults and more than 10 percent in children. Major or severe burns in a patient or subject are a prima facie indication that the patient or subject should be treated pursuant to the présent invention.

Severe bums may be caused by a number of sources, including, but not limited to thermal, e.g. hot liquids or gases, open fiâmes and hot surfaces; Chemical, e.g. strong acids or bases, such as sulfurîc acid and bleach; electrical: high voltage exposure, electric arcs, and lîghting; radiation: ultraviolet light, micro waves, ionizing radiation such as from xrays or nuclear fallout. Severe bums may be accidentai or intentional (especially with children and/or elders).

The term “traumatîc injury” or “wound” is used to describe any injury of a severe or sufficient nature that has an identifiable risk of becoming infected or faîi to heal for other reasons. Traumatic injury may refer to physical injuries of sudden onset and severity which require immédiate medical attention. Initially small insults (e.g. a scratch or abrasion) as well as immédiate and severe insults (e.g. motor vehicle collision or blast injury) may cause systemic shock called “shock trauma” and may require resuscitation and interventions to save life and limb. Traumatic injuries are the resuit of a wide variety of blunt, penetrating and burn mechanisms. They include motor vehicle collisions, sports injuries, falls, natural disasters and a multitude of other physical injuries which can occur at home, on the Street, or while at work. Microscopie injuries may lead to serious injury or illness if not treated (e.g. tétanos)

With respect to major trauma, many accidents resulting in traumatic injury can be treated approprîately in hospital emergency departments. More severe and multiple traumatic injuries may be triaged by emergency responders as a Trauma Alert. A Level One Trauma Alert is a détermination based on a rapid physical assessment of the victim’s immédiate medical needs. Based on trauma alert criteria, first responders deliver the patient to the most appropriate hospital.

Trauma guidelines in the U.S. were first established in 1976, and an efficient sophisticated trauma network now serves us all wherever we live, work or travel. Hospitals are accredîted and designated as Level I, II, III or IV Trauma Centers based on the care they are able to provide, as well as the volumes they serve, urban and rural. The trauma System is designed to accommodate mass casualties and disaster situations. Level I Centers provide the highest level of care with optimal resources and capabilities, staff and specialties around-the-clock, and are continuously monitored to assure that they meet or exceed national standards. Trauma centers work closely with their respective EMS Systems so that care begins pre-hospital.

Typically, critically injured patients deemed a Trauma Alert are delivered to a resuscitation area which may look more like an operatîng room than a traditional emergency department. In this environment, a hîghly-skilled professional trauma team is ready to provide immédiate life-saving procedures in state-of-the-art trauma bays. Research shows that getting to the right place at the right time, commonly known as the “Golden Hour” or within the first 60 minutes after the occurrence of a major multi-system trauma, is critical. Adult and pédiatrie trauma surgeons, trauma staff and resources are ready and dedicated 24/7 to provide this unique level of response so that critically injured patients will bave the best possible chance of survival and the least residual disability from their injuries.

Following care in the trauma resuscitation area at a Level One facility, patients may proceed to surgery, an intensive care unît or the trauma nursing floor, with ail the resources and services of the hospital available in a true multi-disciplinary fashion. Patients brought to Level II-IV centers may remain at that hospital or be transferred to a higher level of care as appropriate.

Some common types of traumatic injuries include, but are not limited to traumatic brain injury, spinal cord injury, spinal fractures, amputations, facial trauma, acoustîc trauma, concussions, crush injuries, broken bone injury, broken jaw, skull fracture, cuts, puncture wounds, lacérations, collapsed tung, burns, myocardial contusions, electrîcal injuries, hypovolémie shock, subarachnoid hemorrhage and subdura] hematoma, among others.

The Injury Severity Score (ISS) is an established medical score to assess trauma severity. It correlates with mortality, morbidity and hospitalization time after trauma. It is used to define the term major trauma. The ISS classifies each injury in every body région according to its relative severity on a six point ordinal scale, e.g. minor, moderate, serious, severe, critical, Maximal in six body régions, e.g. head/neck, face, thorax, abdomen/pelvis, externai.

The term “systemic inflammatory response syndrome” or “SIRS” is used to describe an inflammatory syndrome which is caused by systemic response of the body due to severe inflammation or infection. This is characterized with high fever and rapid heartbeat and abnormal level of whîte blood cells în blood. The symptoms of SIRS vary widely depending on the triggering factor for the response and the victîm’s underlying prédispositions. Some of the common signs include high fever, chills and localized pain based on inflammation. Some of the criteria for diagnosing SIRS inciuding the following:

• Very high or very low level of white blood cells in blood. It may go up to 12,000 per liter or below 4,000 also.

• High fever and chills. The température may shoot up to 100.4 F or can go below 96 F also.

• Rapid heartbeat and • Fast respiratory rate.

SIRS can be caused by severe infection, ischemia or after effects of surgery. Infection can occur due to bacteria, virus and other microorganisms (fungi or parasites). It can be anything ranging from sepsis, cellulitis to diabetic foot infection. SIRS can develop due to non-infectious conditions like déhydration, burns, cirrhosis, autoimmune disorders, immune thérapies, acute ischemia, and myocardial infarctîon and due to hémorrhagie shock. In rare cases, SIRS can cause potentîal complications like hemolytic urémie syndrome (HUS), anémia, rénal failure, respiratory failure, gastritis and abnormal Ievels of electrolytes.

The method of treatment for SIRS is based on symptoms and health condition of the patient. Some patients may show signs of sepsis due to severe rate of infection without having any sign of SIRS. In case of myocardial infarctîon or respiratory failure emergency treatment may be given on ICU, îf avaiiable.

Blood pressure of the patient may be restored to normal by injecting vasopressor or similar drugs intravenously. If SIRS has developed due to surgical conditions like cholecystitîs or ruptured appendix suitable surgical measures are initîated. Antibîotics are gîven îf the root cause of infection is bacteria. Similarly antiviral médications are injected through vein if the doctor confirms the cause is viral infection. In the present invention, these agents are combined with PLA2 and/or metailoprotease inhibîtors in treating SIRS. Blood glucose level is monitored carefully and if required, insulîn therapy is given to stabîlize the level of blood glucose.

In the present invention, the administration of at least one antibiotic in combination with at least one PLA2 inhibitor and/or at least one metailoprotease inhibitor is used to reduce the likelihood that a patient suffering a severe injury or bum will contract an infection which will develop into sepsis, septic shock, acute inflammatory syndrome.

including systemic inflammatory response syndrome (SIRS) and/or acute respiratory distress syndrome (ARDS), as described herein.

Antibiotics which may be used in the présent invention include, for example, broad-spectrum antibiotics such as a broad spectrum β-lactam antibiotic or a broadspectrum carbapenem, or a mixture thereof, which can be used alone or combined with fluoroquinolones, macrolides, or aminoglycosides. In general, a combination of antibiotîcs may not be recommended for the treatment of sepsis but without shock and immunocompromised persons unless the combination is used to broaden the anti-bacterial activîty. The choîce of antibiotics is important in controlling infection, sepsis and ultimately determining the survival of the patient. It is often recommended that antibiotîcs are commenced within about an hour of making the diagnosis (e.g. within 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, 60 minutes, 75 minutes or 90 minutes), although the times can be slightly longer. In some embodiments, antibiotics and PLA2 inhîbîtors and/or métalloprotease inhibîtors are initîated îmmediately or as soon as the severity of a burn or injury is assessed in a patient or subject.

For severe sepsis and septic shock a broad spectrum antibiotic (often two such antibiotics) are administered intravenously or intravenously and orally to the patient or subject în combination with the PLA2 inhîbitor and/or métalloprotease at the First indication that the patient or subject has sepsis, including early sepsis. The antibiotîcs may include, for example a β-lactam antibiotic with broad coverage such as broad spectrum penicillin dérivatives (penams) amoxicillin and ampicillin, carboxylpenicillins (e.g.carbenicillîn and ticarcillin), cephalosporins (cephems) such as cefixime, doxycycline, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefdinir, ceftaroline fosnir ail of which are broad spectrum, monobactams (e.g. aztreonam, tigemonam, carumonam and nocardicin A) and carbapenems (e.g. doripenem, faropenem, imipenem, meropenem, ertapenem, panipenim, razupenem, tebipenem, thienamycin or cilastatin/imïpenem), a fluoroqunolîne (e.g. ciprofloxacin, levofloxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, perfloxacin, rufloxacin, balofloxacin, grepafloxacin, pazufloxacin, sparfloxacin, temafloxacin, garenoxacin, gatifloxacin, gemifloxacin, moxifloxacin, ciinafloxacin, sitafloxacin, prulifloxacin, besifloxacin, deiafloxacin and ozenoxacin, among others), a macrolide (e.g. (e.g., azithromycin, clarithromycin, erythromycin, fiaxomycin, telithromycin, carbomycin A, josamycin A, kitasamycin, midecamycin/midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin and roxithromycin, related ketolides inclouding telithromycin, cethromycin and solithromycin), each of which may be used alone or in combination.

In the case of anthrax, preferred antibiotics include ciprofloxacin, levofloxacin, moxifloxacin, penicillin G, doxycycline, chloramphenicol, ofloxacin and mixtures thereof.

“PLA2 Inhibitors” which term includes secretory Phospholipase A2 înhibitors and Phospholipase A2 (PLA2) inhibitors are inhibitors of lîpase enzymes that catalyze the hydrolysis of phospholipîds at the sn-2 position yielding a free fatty acid and a lysophospholipid. PLA2 contributes towards release and/or formation of at least three important lipid mediators from membrane-arachidonic acid, platelet activatîng factor and lysophosphatidic acid. The release of arachidonic acid from membrane phospholipîds by PLA is believed to be a key step in the control of eicosanoid production within the cell. PLA2 enzymes are usually grouped into cytosolic PLA2 (CPLA2), secretory PLA2 (sPLA2) and calcium independent PLA2 (1PLA2). Venom (e.g., snake venom) PLA2 are secreted (i.e., sPLA2s). Classification is based on molecular weight, calcium requirement, structural features, substrate specifïcity and functional rôle. See Ray, et al., “Phospholipase A2 in Airway Disease: Target for Drug Dîscovery,” Journal ofDrug Discovery and Therapeutics 1 (8) 2013,28-40.

Inhibitors of PLA2 hâve been identified in various sources and hâve been investigated as potential therapeutic agents for treatment of inflammatory diseases. See, Magrioti, Victoria, and George Kokotos. Phospholipase Α2 inhibitors as potential therapeutic agents for the treatment of inflammatory diseases. Expert opinion on therapeuticpatents 20.1 (2010): 1-18), and Dennis, Edward A., et al. Phospholipase A2 enzymes: physical structure, biological function, disease implication, Chemical inhibition, and therapeutic intervention. sPLA2 înhibitors which can be used in the invention include, but are not limited to, LY315920 and S5920 (varespladib), LY333O13 and S-3013, AZD2716-(R)-3-(5’-benzyl-2’carbamoyl-[l ,1 ’-bîphenyl]-3-yl)-2-methylpropanoic acid- as a racemic mixture or a stereoisomer thereof, often the “R” enantiomer of the racemic mixture, AZD Compound 4 (S-ÎS'-Benzyl-^'-carbamoylbiphenyl-S-yOpropanoic acid) and LY433771 ((9[(phenyl)methyl]-5-carbamoylcarbazol-4-yl)oxyacetîc acid), LY 311727, BMS 181162, YM 26567, Variabilin, SB 203347, S-2474 (methy I indoxam) and Indoxam. In some embodiments the PLA2 inhibitor(s) is varespladib and/or methy Ivarespladib.

Other PLA2 inhibitors, such as but not limited to other 1 H-indole-3-glyoxylamîdes, are also useful in treatment of envenoming. One of ordinary skill in the art guided by the présent disclosure will be able to identify PLA2 inhibitors and therapeutic combinations effective against a broad spectrum of venoms and/or tailored to a particular subset of venoms (e.g., particular species of snake, or venoms from particular types of invertebrates, for example).

Additional preferred sPLA2 inhibitors include those described in United States patent no. 5,654,326 (which is incorporated by reference in its entirety herein)represented by compounds according to the Chemical structure:

where X is O or S, preferably O;

Ri is Ct-Cîû alkyl, C7-C20 alkenyl, C7-C20 alkynyl, a carbocyclic radical (preferably a benzyl or ethylphenyl group) or a heterocyclic radical;

R2 is hydrogen, halo (F, Cl, Br, I), C1-C3 alkyl (preferably ethyl) or C3-C4 cycloalkyl;

R4 is H or an -O-(CH2)m-C(O)ORv group, where m is 1-3 (preferably 1) and Rv is FI or a

C1-C3 alkyl group, preferably CH3; and

R5, Rô and R? are H, or a pharmaceutically acceptable sait, solvaté or polymorph thereof.

Certain preferred sPLA2 inhibitor compounds (varespladib and methylvarespladib) for use în the présent invention are represented by the Chemical structure:

where Rv is H (varespladib) or methyl (methylvarespladib), or their pharmaceutically acceptable salts. The above compounds also may be used as prodrug forms Ci-C& alkyl esters, C2-C7 acyloxyalkyl esters, or C3-C9 alkyloxycarbonyloxyalkyl esters (each formed at R4), These and other related compounds for use in the présent invention are described in United States patent number 5,654,326 to Bach, et al., which is incorporated by reference in its entirety herein.

Additional PLA2 inhibitors include for example: Varespladib Mofetil, N-Acetyl Cysteine, LY329722 (sodium [3-aminooxyalyl-l-benzyl-2-ethyl-6-methyl-lH-indol-4yloxy]-acetic acid), ochnaflavone (a naturally occurring biflavonoid), BPPA (5-(4benzyloxyphenyl)-4S-(7-phenylhepatonoylamino) pentanoic acid, and pbromophenacylbromîde (p-BPB) and other benzophenone oximes derivatized with syndone. In certain embodiments, sPLA2 inhibitors for use in the current invention are selected from the group consisting of: {9-[(phenyl)methyl]-5-carbamoylcarbazol-4yljoxyacetic acid; 0-benzyl-6.f-dimethoxy-S-tetrahydrocarbazole-carboxylic acid hydrazide; 9-benzyl-5,7-dîmethoxy-l ,2,3,4-tetrahydrocarbazole-4-carboxamide; [9benzyl-4-carbamoyl-7-methoxy-l ,2,3,4-tetrahydrocarbazol-5-yl]oxyacetic acid; [9benzyl-4-carbamoyl-7-methoxycarbazol-5-yl]oxyacetic acid; methyl [9-benzyl-4carbamoyl-7-methoxycarbazol-5-yl]oxyacetic acid; 9-benzyl-7-methoxy-5cyanomelhyloxy-S-tetrahydrocarbazole-carboxamide; 9-benzyl-7-methoxy-5- (1 Htetrazol-5-yl-methyI)oxy)-l ,2,3,4-tetrahydrocarbazole-4-carboxamide; {9[(pheny l)methyl]-5-carbamoy 1-2-methyl-carbazol-4-yl)oxy acetic acid; {9-[(3fluorophenyl)methyl]-5-carbamoyl-2-methylcarbazol-4-yl}oxyacetic acid; {9-[(3methylphenyl)methyl]-5-carbamoyl-2-methylcarbazol-4-yl}oxyacetic acid; {9[(phenyl)methyl]-5-carbamoy 1-2-(4-tri fl uoromethylphenyl)-carbazol-4-yl)oxyacetic acid; 9-benzyl-5-(2-methanesulfonamido)ethyloxy-7-methoxy-l ,2,3,4-tetrahydrocarbazole-4carboxamide; 9-benzyl-4-(2-methanesulfonamido)ethyloxy-2-methoxycarbazole-5carboxamide; 9-benzyl-4-(2-trifluoromethanesulfonamido)ethyloxy-2-methoxycarbazole5-carboxamide; 9-benzyl-5-methanesulfonamidoylmethyioxy-7-methoxy-l , 2,3,4tetrahydrocarbazole-4-carboxamide; 9-benzyl-4-methanesulfonamidoylmethyloxycarbazoie-5-carboxamide; [5-carbamoyl-2-pentyl-9-(phenylmethyl)carbazol-4yl]oxyacetic acid; [5-carbamoyl-2-(l -methylethyl)-9-(phenylmethyl)carbazol-4yl]oxyacetic acid; [5-carbamoyl-9-(phenylmethyl)-2-[(tri(-l methylethylJsilyOoxymethyllcarbazol^A-ylloxyacetic acîd; [5-carbamoyl-2-phenyl-9(phenylmethyl)carbazol-4-yl]oxyacetic acid; [5-carbamoyl-2-(4-chlorophenyl)-9(phenylmethyl)carbazol-4-yl]oxy acetic acid; [5-carbamoyl-2-(2-furyl)-920854 (phenylmethyl)carbazol-4-yl]oxyacetic acid; [5-carbamoyl-9-(phenylmethyl)-2-[(tri(-l methylethylJsilyOoxymethyllcarbazoK-ylloxyacetic acid; {9-[(2-Fluorophenyl)methyl]-5carbamoylcarbazol-4-yl} oxy acetic acid; {9-[(2-trifluoromethylphenyl)methyl]-5carbamoylcarbazol-4-yl} oxy acetic acid; {9-[(2-benzyl phenyl )methyl]-5carbamoylcarbazol-4-yl}oxyacetic acid; {9-[(l -naphthyljmethyll-Ô-carbamoylcarbazol^Ayl}oxyacetic acid; [9-[(2-cyanophenyl)methyl]-5-carbamoy Icarbazol-4-y l}oxyacetic acid; {9-[(3-cyanopheny 1 )methyI]-5-carbamoyIcarbazo 1-4-y 1}oxy aceti c acid; {9-[(3,5dîmethylphenyl)nnethyl]-5-carbannoylcarbazol-4-yl}oxyacetic acid; {9-[(3iodophenyl)methy 1 ] -5 -carbannoy Icarbazol -4-y 1} oxyaceti c acid; {9-[(2Chlorophenyl)methyl]-5-carbannoylcarbazol-4-yl}oxyacetic acid; {9-[(2,3difluorophenyl)methyl]-5-carbannoylcarbazol-4-yl}oxyacetic acid; {9-[(2,6difluorophenyl)methyi]-5-carbannoylcarbazol-4-y]}oxyacetic acid; {9-[(2,6dichlorophenyl)methyI]-5-carbannoylcarbazol-4-yî}oxyacetîc acid; {9-[(2b iphenyî)methyl]-5-carbamoy lcarbazol-4-yl} oxy acetic acid; {9-[(2-Bi phenyl )methyî]-5carbamoyIcarbazo 1-4-y 1}oxyacetic acid methyl ester; [9-Benzyl-4-carbamoyl-l ,2,3,4tetrahy drocarbazo 1-5 -y 1 ]oxy acetic acid ; {9- [(2-Pyridy l)methy I] -5 -carbamoy Icarbazo 1 -4yl}oxyacetic acid; {9-[(3-Pyridyl)nnethyl]-5-carbannoylcarbazol-4-yl}oxyacetic acid; [9benzyl-4-carbamoyl-8-nnethyl-l ,2,3,4-tetrahydrocarbazol-5-yi]oxyacetîc acid; [9-benzyl5-carbamoyl-l -methyIcarbazo 1-4-yl]oxyacetic acid; [9-benzy l-4-carbamoyl-8-fluoro- 1 ,2,3,4-tetrahydrocarbazol-5-yl]oxyacetic acid; [ 9-benzy l-4-carbamoyl-8-chloro-l ,2,3,4tetrahydrocarbazo 1 -5-y I]oxyacetic acid; [5-carbamoy 1 -9-(pheny 1 n nethy l)-2- [ [(propen-3yl)oxy]methyl]carbazol-4-y 1]oxyacetic acid; [5-carbamoyl-9-(phenylmethyl)-2[(propyloxyjmethyllcarbazolylloxyacetic acid; 9-benzyl-7-methoxy-5((carboxamidonnethyloxy-tetrahy drocarbazo le-carboxannide; 9-benzyl-7- methoxy-Scyanonnethyloxy-carbazole-carboxannide; 9-benzy l-7-methoxy-5-((l H- tetrazol-5-ylmethyl)oxy)-carbazole-4-carboxannide; 9-benzy l-7-methoxy-5((carboxamidomethy l)oxy )-carbazo I e-4-carboxam ide; [9-Benzy 1 -4-carbamoy 1-1 ,2,3,4tetrahydrocarbazoie-5-y 1]oxyacetic acid; {9-[(phenyl)methyl]-5-carbannoyl-2-nnethylcarbazo 1-4-y 1}oxyacetic acid; {9-[(3-11 uorophenyl)methyl]-5-carbannoy 1-2nnethyIcarbazol- 4-y 1}oxyacetic acid; [9-[(3-methylphenyl)nnethyl]-5-carbannoyl-2nnethyIcarbazo 1-4- yIjoxyacetic acid; {9-[(phenyl)methyl]-5-carbamoy 1-2-(4trifluoromethylphenyl)-carbazol- 4-yl}oxyacetic acid; 9-benzy 1-5-(2methanesulfonamido)ethyloxy-7-methoxy-l ,2,3,4- tetrahydrocarbazole-4-carboxamide;

9-benzyl-4-(2-meÎhanesulfonamido)ethyloxy-2- methoxycarbazole-5-carboxamide; 9benzy 1 -4-(2-trifl uoromethanesul fonam i do)ethy loxy- 2-methoxycarbazole-5 -carboxam i de; 9-benzy l-5-methanesulfonamidoylmethyloxy-7- methoxy-1 ,2,3,4-tetrahydrocarbazole-4carboxamide; 9-benzyl-4- méthanes ulfo nam idoylmethy loxy-carbazole-5-carboxamide; [5carbamoyl-2-pentyl-9- (phenylmethyl)carbazol-4-yl]oxyacetic acid; [5-carbamoyl-2-(l methylethyl)-9- (pheny imethyljcarbazo 1-4-y 1] oxyacetic acid; [5-carbamoyl-9(phenylmethyl)-2-[(tri(-l - methylethylJsilyOoxymethyllcarbazolyloxyacetic acid; [5carbamoyl-2-phenyl-9- (phenylmethyl)carbazol-4-yl]oxyacetic acid; [5-carbamoy 1-2-(4chlorophenyl)-9- (phenylmethyl)carbazol-4-yl]oxyacetic acid; [5-carbamoyl-2-(2-furyl)-9(phenylmethyl)carbazol-4-y l]oxy acetic acid; [5-carbamoyl-9-(phenylmethyl)-2-[(tri(-l methylethylJsilyOoxymethyllcarbazol-yiloxyacetic acid; {9-[(3-fluorophenyl)methyl]-5carbamoy Icarbazo 1-4-yI}oxyacetic acid; {9-[(3-chlorophenyl)methyl]-5carbamoylcarbazol-4-yl} oxyacetic acid; {9-[(3-phenoxyphenyl)methyl]-5carbamoy 1 carbazol -4-y l}oxyaceticacid;{9-[(2-Fluorophenyl)methyl]-5carbamoylcarbazol-4-yl} oxyacetic acid; {9-[(2-trifluoromethylphenyl)methyl]-5carbamoylcarbazol-4-yl} oxyacetic acid; {9-[(2-benzylphenyl)methyl]-5carbamoylcarbazol-4-yl} oxyacetic acid; {9-[(3-trifluoromethylphenyl)methy!]-5carbamoylcarbazol-4-yl}oxyacetic acid; {9-[( 1 -naphthyijmethyll-Ô-carbamoylcarbazolyl}oxyacetic acid; {9-((2-cyanophenyl)methyI]-5-carbamoylcarbazol-4-yl}oxyacetic acid; {9-[(3-cyanophenyl)methyi]-5-carbamoy!carbazol-4-yl}oxyacetic acid; {9-((2methylphenyl)nnethyl]-5-carbannoylcarbazol-4-yl}oxyacetic acid; {9-((3methylphenyl)nnethyl]-5-carbannoylcarbazoi-4-yl {oxyacetic acid; {9-((3,5dimethylphenyl)nnethyl]-5-carbannoylcarbazol-4-yl {oxyacetic acid; {9-((3iodophenyl)methyi]-5-earbannoylcarbazol-4-yl{oxyacetîc acid; {9-((2Chlorophenyl)methy 1]-5-carbannoylcarbazol-4-y 1}oxyacetic acid; { 9 - ((2,3 dîfluoropheny 1 )methy 1 ]-5-carbannoyIcarbazo 1-4-y 1{oxyacetic acid; {9-[(2,6difluorophenyl)methyl]-5-carbannoy(carbazol-4-y 1 {oxyacetic acid; {9-((2,6dich loro pheny l)methyl]-5-carbannoy Icarbazo 1-4-y 1{ oxy ace tic acid; {9-((3trifluoromethoxyphenyl)nnethyl]-5-carbannoylcarbazol-4-yl}oxyacetic acid; {9-((2biphenyl)methy 1 ]-5-carbannoylcarbazol-4-y 1{oxyacetic acid; {9-[(2-BÎphenyl)methy 1 ]-5carbamoylcarbazol-4-yl{oxyacetic acid methyl ester; [9-Benzyl-4-carbamoyl-l ,2,3,4tetrahydrocarbazole-5-y 1]oxyacetic acid; {9-[(2-Pyridyl)methyl]-5-carbamoylcarbazol-4yl{oxyacetic acid; {9-[(3-Pyrîdyl)nnethyl]-5-carbannoylcarbazol-4-yl{oxyacetic acid; [9 benzyl-4-carbamoyl-8-nnethyl-l ,2,3,4-tetrahydrocarbazol-5-yl]oxyacetîc acid; [9-benzyl5-carbamoyl-l -methylcarbazol-4-yl]oxyacetîc acid; [0-benzyl^A-carbamoyl-ô-fluorol,2,3,4-tetrahydrocarbazol-5-yl]oxyacetîc acid; [0-benzyl-5-carbannoyl-i -fluorocarbazol4-yl]oxyacetic acid; [9-benzyl-4-carbamoyl-8-chloro-! ,2,3,4-tetrahydrocarbazol-5yl]oxyacetic acid; [9-benzyl-5-carbamoyl-l -chlorocarbazol-4-yl]oxyacetic acid; [9[(Cyclohexyl)methyl]-5-carbannoylcarbazol-4-yl]oxyacetic acid; [9[(Cyclopentyl)methyl]- 5-carbamoylcarbazol-4-yl]oxyacetic acid; [5-carbamoyl-9(phenylmethy 1)-2-(2- thienyl)carbazol-4-yl]oxy acetic acid; [5-carbamoyl-9(phenylnnethyl)-2-[[(propen-3- y[)oxy]methyl]carbazol-4-yl]oxyacetic acid; [5carbamoyl-9-(phenylnnethyl)-2- [(propyloxyjmethyllcarbazol-ylloxyacetic acid; 9-benzyI7-methoxy-5- ((carboxamidomethyloxy-tetrahydrocarbazole-carboxamide; 9-benzyl-7methoxy-ô-cyanomethyloxy-carbazole-carboxamide; 9-benzyl-7-methoxy-5-((l Htetrazol-5-yl-methyl)oxy)-carbazole-4-carboxamide; 9-benzyl-7-methoxy-5((carboxamidomethyl)oxy)-carbazole-4-carboxamide; [9-Benzyl-4-carbamoyl-l ,2,3,4tetrahydrocarbazole-5-yl]oxyacetic acid; (R,S)-(9-benzyl-4-carbamoyl-l-oxo-3-thia- 1 ,2,3,4-tetrahydrocarbazol-5-yl)oxyacetic acid; (R,S)-(9-benzyl-4-carbamoyl-3-thia- 1 ,2,3,4-tetrahy drocarbazo I -5-y l)oxyacetic acid; 2-(4-oxo-5 -carboxam i do-9 -benzy 1-9/7pyrido[3,4-ib]indolyl)acetic acid chloride; [N-benzyl-1 -carbamoyl-l-aza-1 ,2,3,4tetrahydrocarbazol-8-yl]oxy acetic acid; 4-methoxy-6-methoxycarbonyl-1O-phenylmethyl6,7,8,9-tetrahydropyrido[l ,2-a]indole; (4-carboxamido-9-phenyImethy 1-4,5dihydrothiopyrano[3,4-b]indol-5-yl)oxyacetic acid; 3,4-dihydro-4-carboxamidol-5inethoxy-9-phenylmethylpyrano[3,4-ib]indole; 2-[(2,9 bis-benzyl-4-carbamoyl-i ,2,3,4tetrahy dro-betacarbo 1 i n-5 -y i)oxy] acetic ac id; 2 -[4-oxo-5 -carboxam i do-9-(2methylbenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(3methylbenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(4methylbenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(4-tertbutylbenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9pentafluorobenzyl-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(2fluorobenzyl)-9/7-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(3fluorobenzy 1 ) -9/-/-pyrido [3,4- i b] indo 1 y I ] acetic ac i d ; 2-[4-oxo-5-carboxamido-9-(4fluorobenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(2,6difluorobenzyl)-9/-/-pyrido[3_i4-ib]indolyI]acetic acid; 2-[4-oxo-5-carboxamido-9-(3,4difluorobenzyl)-9/-/-pyrido(3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamîdo-9-(2,5 difluorobenzyl)-9/-/-pyrido[3,4-jb] indoly l]acetic acid; 2-[4-oxo-5-carboxamido-9-(3,5d i fl uorobenzy 1)-9/-/-pyπdo [3,4-ib] indo ly 1 ] acetic aci d ; 2- [4-oxo- 5 -carboxamido-9-(2_f4difluorobenzyl)-9/-/-pyrido[3,4-ib] indoly l]acetic acid; 2-[4-oxo-5-carboxamido-9-(2,3difluorobenzy 1)-9/-/-pyrido[3,4-îb] indoly l]acetic acid; 2-[4-oxo-5-carboxamido-9-[2(trifluoromethyl)benzyl]-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxam ido9- [2-(trifluoromethyl)benzyl]-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5carboxamido- 9-[3-(trifluoromethyl)benzyl]-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4oxo-5- carboxainido-9-[4-(trifluoromethyl)benzyl]-9/-/-pyrido[3₅4-ib] indoly l]acetic acid; 2-[4-oxo- 5-carboxamido-9-[3,5-bis(trifluoromethyl)benzyl]-9/-/-pyrido[3,4ib]indolyl]acetic acid; 2- [4-oxo-5-carboxamido-9-[2,4-bis(trifluoromethyl)benzyl]-9/-/py rido [3,4- i b] i ndo iy 1 ] acetic acid ; 2- [4-oxo-5 -carboxam îdo-9~(a-methy Inaphthy 1) -9/-/pyrido[3,4-Îb] indoly l]acetic acid; 2-[4-oxo-5-carboxamido-9-(b-methyInaphthy 1)-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4- oxo-5-carboxamido-9-(3_f5-dimethylbenzyl)-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo- 5-carboxamido-9-(2,4-dimethylbenzy 1)-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(2-phenyl benzy 1)-9/-/pyrido[3,4-ib]îndolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(3-phenylbenzyl)-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(4-phenylbenzy 1)-9/-/pyrido[3,4-îb]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(l -fluorenylmethy)-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(2-fluoro-3-methylbenzyl)9/-/-pyrido[3,4-ib]indolyi]acetic acid; 2-[4-oxo-5- carboxam ido-9-(3-benzoylbenzy 1)-9/-/py ri do [3,4-ib] indoly l]acetic acid; 2-[4-oxo-5- carboxamido-9-(2-phenoxy benzy I )-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(3-phenoxybenzyl)-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(4-phenoxybenzyl)-9/-/pyrido[3,4-îb]indolyl]acetic acid; 2-[4-oxo-5- carboxannîdo-9-[3-[2(fluorophenoxy)benzyl]]-9/-/-py^do[3,4-ibjindolyl]acetic acid; 2-[4- oxo-5-carboxamido9-[3-[4-(fluorophenoxy)benzyl]]-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5carboxalr)ido-9-[2-fIuoro-3-(trifluoronnethyl)benzyl]-9/-/-pyπdo[3,4- £>] indoly l]acetic acid; 2-[4-oxo-5-carboxamido-9-[2-fluoro-4-(trîfluoronnethyl)benzyl]-9/-/- pyrido[3,4ib]indoly 1]acetic acid; 2-[4-oxo-5-carboxamîdo-9-[2-fluoro-5- (trifluoromethyl)benzyl]9H-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9- [3-fluoro-5(trifluoromethyl)benzyl]-9/-/-pyrido[3_ï4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido9-[4-fluoro-2-(trifluoronnethyi)benzyl]-9/-/-pyrido[3,4-ib]indolyi]acetic acid; 2- [4-oxo-5carboxamido-9-[4-fli!oro-3-(trifluoronnethyl)benzyl]-9/-/-pyrido[3,4- ib]indolyl]acetic acid; 2- [4-oxo-5 -carboxam ido-9- [2-fluoro-6-(tri fl uoronnethy l)benzy l]-9/-/- pyrîdo [3,4ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(2,3,6-trifluorobenzy 1)-9/-/- pyrido[3,4ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(2_}3,5-trîfluorobenzy 1)-9/-/- pyrido[3,4ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(2,4.5-trifluorobenzy 1)-9/-/- pyrido[3,4ib]indolyl]acetic acid; 2-[4-oxo-5-carboxam îdo-9-(2,4,6-trifiuorobenzy 1)-9/-/- pyrîdo[3_f4ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamîdo-9-(2,3,4-trifluorobenzy 1)-9/-/- pyrîdo[3_;4ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-(3,4,5-trifluorobenzyl)-9/-/- pyrido[3,4rb]indolyl]acetic acid; 2-[4-oxo-5-carboxam îdo-9-[3-(tri fl uoronnethoxyl)benzy 1]- 9/-/pyridofS^-iblindolylJacetic acid; 2-[4-oxo-5-carboxamido-9-[4(trifluoronnethoxyl)benzyl]-9/-/-pyrido[3_î4-ib]indolyl]acetic acid; 2-[4-oxo-5carboxamido-9- [4-methoxy(tetraf1uoro)benzyl]-9/-/-pyrido[3,4-ib]indolyi]acetic acid; 2[4-oxo-5- carboxamido-9-(2-nnethoxybenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4oxo-5- carboxam ido-9-(3-nnethoxybenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4oxo-5- carboxamido-9-(4-nnethoxybenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4oxo-5- carboxannido-9-(4-ethylbeπzyl)-9/-/-ρyπdo[3_f4-ib]indolyl]acetic acid; 2-[4-oxo-5carboxamîdo-9-(4-isopropylbenzy 1)-9/-/-pyrîdo[3,4-îb]indolyl]acetic acid; 2-[4-oxo-5carboxamido-9-(3₅4,5-trinnethoxybenzyl)-9/-/-pyrido[3_s4-ib]indolyl]acetic acid; 2-[4-oxo5- carboxamido-9-(3,4-nnethylenedioxybenzyI)-9/-/-pyrido[3_!4-ib]indolyl]acetic acid; 2[4- oxo-5-carboxamido-9-(4-nnethoxy-3-nnethylbenzyl)-9/-/-pyndo[3,4-ib]indolyl]acetic acid; 2- [4-oxo-5-carboxamido-9-(3,5-dinnethoxybenzyl)-9/-/-pyTOdo[3,4-ib]indolyl]acetic acid; 2-[4- oxo-5-carboxamido-9-(2,5-dinnethoxybenzyl)-9/-/-pyπdo[3,4-ib]indolyl]acetic acid; 2-[4- oxo-5-carboxamido-9-(4-ethoxybenzyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(cyclohexylnnethyl)-9/-/-pyrido[3,4-ib]îndolyl]acetic acid; 2[4-oxo-5- carboxamido-9-(cyclopentylnnethyl)-9/-/-pyrido[3_}4-ib]indolyI]acetic acid; 2[4-ΟΧΟ-5- carboxamido-9-ethyl-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5carboxamido-9-(l - propyl)-9/-/-pyrido[3_i4-ib]indolyl]acetic acid; 2-[4-oxo-5carboxamido-9-(2-propy 1)-9/-/- pyrido[3,4-ib]indoiyl]acetic acid; 2-[4-oxo-5carboxamido-9-( 1 -butyl)-9H-pyrido[3,4-]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9(2-butyl)-9/-/-pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9-isobutyl-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-[2-(l -phenylethyl)]-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-[3-(l -phenylpropyl)]-9/-/pyrido[3_f4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-[4-(l -phenylbutyl)]-9/-/pyrido[3,4-ib]indolyl]acetic acid; 2-[4-oxo-5- carboxamido-9-(l -pentyl)-9/-/-pyrîdo[3,420854 ib]indolyl]acetic acid; 2-[4-oxo-5-carboxamido-9- (1 -hexyl)-9/-/-pyrido[3,4ib]indolyl]acetic acid; 4-[(9-benzyl-4-carbamoyl-l ,2,3,4- tetrahydrocarbazol-6yl)oxy]butyric acid; 3-[(9-benzyl-4-carbamoyl-l ,2,3,4- tetrahydrocarbazol-θyOoxylpropylphosphonic acid; 2-[(9-benzyl-4-carbamoyl-l ,2,3,4- tetrahydrocarbazol-6yl)oxy]methylbenzoic acid; 3-[(9-benzyl-4-carbamoyl-7-n-octyl- 1 ,2,3,4tetrahydrocarbazol-6-yl)oxy]propylphosphonic acid; 4-[(9-benzyl-4-carbamoyl-7- ethyl-1 ,2,3,4-tetrahydrocarbazol-6-yl)oxy]butyric acid; 3-[(9-benzyl-4-carbamoyl-7-ethyl- 1 ,2,3,4-tetrahydrocarbazol-6-yl)oxy]propyiphosphonic acid; 3-[(9-benzyl-4-carbamoyl-7ethyl-1 ,2,3,4-tetrahydrocarbazol-6-yl)oxy]propylphosphonic acid; (S)-(+)-4-[(9-benzyl-4carbamoyl-7-ethyl-l ,2,3,4-tetrahydrocarbazol-6-yl)oxy]butyric acid; 4-[9-benzyl-4carbamoyl-6-(2-cyanoethyl)-l ,2,3,4-tetrahydrocarbazoî-6-yl]oxybutyric acid; 4-[9benzyl- 4-carboxamido-7-(2-phenylethyl)-l ,2,3,4-tetrahydrocarbazol-6-yl]oxybutyric acid; 4-[9- benzyl-4-carboxamidocarbazol-6-yl]oxybutyric acid; methyl 2-[(9-benzyl-4carbamoyl- 1 ,2,3,4-tetrahydrocarbazol-6-yl)oxy]methylbenzoate; 4-[9-benzyl-4carbamoyl-7-(2- cyanoethyl)-! ,2,3,4-tetrahydrocarbazol-6-yl]oxybutyric acid; 9-benzyl7-methoxy-5- cyanomethyloxy-tetrahydrocarbazole-carboxamide; [9-benzyl-4-carbamoyl8- methyl-carbazole-5-yl]oxyacetic acid; and [O-benzyM-carbamoyl-carbazole-ôyl]oxyacetic acid, or pharmaceutically acceptable salts, solvatés, prodrug dérivatives, racemates, tautomers, or optical isomers thereof.

Direct and indirect PLA; inhibitors also include N,N-dimethylcarbamoylmethyl,44-guanidinobenzoyloxy-phenylacetate (Camostat, camostate) or ethyl-p[6guanidinohexanoyloxy]-benzoate methansulfonate (gabexate) and leukotriene synthesis inhibitor selected from the group consisting of methyl arachidony] fluorophosphonate (MAFP), pyrroxyphene, ONO-RS-082, l-[3-(4-octylphenoxy)-2-oxopropyl]îndole-5carboxylic acid, l-[3-(4-octylphenoxy)-2-oxopropyl]indole-6-carboxylic acid, arachidonyl tri fl uoro methyl ketone, D609, 4-{3-[5-chloro-2-(2-{([(3,4dichlorobenzyl)sulfonyl]amino}ethyl)-l-(diphenylmethyl)-l H-indol-3-yl]propyl}benzoic acid (WAY-196025), efipladib, 4-{2-[5-chloro-2-(2-{[(3,4dichlorobenzyl)sulfonyl]amino)-ethyl)-l-(diphenylmethyl)-l H-indol-3-yl]ethoxy)benzoic acid, Ecopladib, (E)~N-[(2S,4R)-4-[N-(biphenyl-2-ylmethyl)-N-2-methylpropylamino]-l[2-(2,- 4-difluorobenzoyl)benzoyl]pyrrolidin-2-yl]methy 1-3-(4-(2,4-dioxothiazolidî- n-5ylidenemethyl) phenyl]acrylamide (RSC-3388), berberine, glutamine, darapladib or a pharmaceutically acceptable sait thereof.

“Metalloprotease inhibitors” include, but are not limited to, prînomastat, BB-94 (marimastat), BB-2516 (batimastat), vorinostat, cefixime and doxycycline. Other metalloprotease inhbitors that may be used in the invention include but are not limited to, TAPI-2, TAPI-1, EGTA, EDTA, phosphoramadon, TAPI-0, Luteolin, alendronate, tanomastat, iiomastat, prinomastat, nafamostat, collagénase inhibitor I, Ro-32-3555, lactobionic acid, o-phenantroline, ecotin, 4-epi-chlortetracyclîne, teracycline, doxycycline or related antibiotic with additional, salutary antimicrobial effect, n-dansyl-dphenylalanine, 20[R]ginsenosideRh2, pro-leu-gly-hydroxymate, gm6001, actînonin, arp100, MMP9 inhibitor I, MMP2 inhibitor 1, SB-3CT, Thiorphan (DL), 4-epidemeclocychne, zinc méthacrylate, funalenone, and their analogs, dérivatives, pharmaceutically acceptable salts, enantiomers, diastereomers, solvatés and polymorphs and mixtures thereof.

Unexpectedly, use of at least one PLA2 inhibitor and/or a metalloprotease inhibitor as described herein in combination with one or more antibiotics as described herein has been found to be particularly effective to ameiiorate, inhîbit and/or reduce the likelihood of an înjured patient or subject at risk of becoming infected or who has become infected such that the infection will produce one or more of sepsis, septic shock, an acute inflammatory syndrome such as systemic inflammatory response syndrome (SIRS) and/or acute respiratory distress syndrome (ARDS).

The term “néo-natal ARDS” is used to describe a common clînical critical disease and îs one of the main causes of death and dîsabîlîty in neonates. The etiology and pathogenesis of néonatal ARDS are complîcated. It is an acute pulmonary inflammatory disease caused by the lack of pulmonary surfactant (PS) related to various pathological factors. It is often dîfficult to distinguish néonatal ARDS from other diseases. Prior to the present invention, there was no spécifie treatment method for this disease, although respiratory support, PS replacement, extracorporeal membrane oxygénation, nutrition support and liquid management are main treatment strategies. The term “méconium aspiration syndrome” or MAS is a life-threatening type of néonatal ARDS with high rates of mortality and no approved treatments. méconium aspiration syndrome (MAS) is known to damage surfactant. It is known that surfactant function is damaged and correlates with lung aération and as conséquence of these changes, surfactant nanostructure is also damaged. Méconium is the first feces, or stool, of the newbom. méconium aspiration syndrome occurs when a newborn breathes a mixture of méconium and amniotic fluid into the lungs around the time of delivery. méconium aspiration syndrome, a leading cause of severe illness and death in the newbom, occurs in about 5 percent to 10 percent of births. It typically occurs when the fétus is stressed during labor, especially when the infant is past its due date. Until the présent invention, there was no known effective therapeutîc method for méconium aspiration syndrome or MAS.

Pharmaceuticaï compositions comprising combinations of an effective amount of an antibiotic as disclosed herein, often according to the présent invention including one or additional PLA2 inhibitor and/or metalloprotease inhibitor as otherwise described herein, ail in effective amounts, in combination with a pharmaceutically effective amount of a carrier, additive or excipient, represent a further aspect of the présent invention. These may be used în combination with at least one additional, optional bioactive agent, especially agents which can be used to address additional symptoms of the patient or subject to be treated.

The compositions of the présent invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers and may also be administered in control led-release formulations. Pharmaceutically acceptable carriers that may be used în these pharmaceuticaï compositions include, but are not limited to, ion exchangers, alumina, aluminum stéarate, lecithin, sérum proteins, such as human sérum albumin, buffer substances such as phosphates, glycine, sorbîc acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloïdal silica, magnésium trisilicate, polyvinyl pyrrolidone, cellulosebased substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

The compositions of the présent invention may be administered orally, întratracheally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vagînally or via an implanted réservoir, among others. The term parentéral as used herein includes subcutaneous, întravenous, intramuscular, intra-articular, intra-synovial, intrasternal, întrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally (including via intubation through the mouth or nose into the stomach), intraperitoneally or întravenously.

Stérile injectable forms of the compositions of this invention may be aqueous, a stabilized liquid or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The stérile injectable préparation may also be a stérile injectable solution or suspension in a non-toxic parentéraliy-acceptable diluent or solvent, for example as a solution in 1, 3-butanedioL Among the acceptable vehîcles and solvents that may be employed are water, Ringer's solution and isotonie sodium chloride solution. In addition, stérile, ftxed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed inciuding synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride dérivatives are useful in the préparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especîally in theîr polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form inciuding, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnésium stéarate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingrédient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixîng the agent with a suitable non-irritating excipient which is solid at room température but liquid at rectal température and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also be administered topically, especîally to treat skin bacterîal infections or other dîseases which occur in or on the skin. Suitable topical formulations are readily prepared for each of these areas or organs. Topîcal application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-acceptable transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers or administered by mîcroneedle patch. Carriers for topical administration of the compounds of this invention include, but are not limited to, minerai oîl, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.

Altematively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, minerai oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonie, pH adjusted stérile saline, or, preferably, as solutions in isotonie, pH adjusted stérile saline, either with our without a preservative such as benzylalkonium chloride. Altematively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also be administered by nasal aérosol or inhalation. Such compositions are prepared according to techniques wellknown in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailabîlity, fluorocarbons, and/or other conventional soiubilizing or dîspersing agents.

The amount of compound in a pharmaceutical composition of the instant invention that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host and disease treated, the particular mode of administration. Preferably, the compositions should be formulated to contain between about 0.05 milligram to about 750 milligrams or more, more preferably about i milligram to about 600 milligrams, and even more preferably about 10 milligrams to about 500 milligrams of active ingrédient, alone or în combination with at least one additional compound which may be used to treat a pathogen, especially a bacterial (often a gram-negative bacterial) infection or a secondary effect or condition thereof.

Methods of treating patients or subjects in need for a particular disease State or condition as otherwise described herein, especially a pathogen, especially a bacterial infection, în particular, a gram-negative bacterial infection, comprise administration of an effective amount of a pharmaceutical composition comprising therapeutic amounts of one or more ofthe novei compounds described herein and optionally at least one additional bioactive (e.g. additional antibiotic) agent according to the present invention. The amount of active îngredient(s) used in the methods of treatment of the instant invention that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. For example, the compositions could be formulated so that a therapeutically effective dose of between about 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 mg/kg of patient/day or in some embodiments, greater than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 250 mg/kg of the novel compounds can be administered to a patient receiving these compositions.

It should also be understood that a spécifie dosage and treatment regimen for any particular patient will dépend upon a variety of factors, including the activîty of the spécifie compound employed, the âge, body weight, general health, sex, diet, time of administration, rate of excrétion, drug combination, and the judgment of the treating physician and the severîty of the particular dîsease or condition being treated.

A patient or subject (e.g. a human) suffering from a bacterial infection can be treated by administering to the patient (subject) an effective amount of a compound according to the présent invention including pharmaceutically acceptable salts, solvatés or polymorphs, thereof optionally in a phannaceutically acceptable carrier or diluent, either alone, or in combination with other known antibiotic or pharmaceutical agents, preferably agents which can assist in treating the bacterial infection or ameliorate the secondary effects and conditions associated with the infection. This treatment can also be administered in conjunction with other conventional thérapies known in the art.

The présent compounds, alone or in combination with other agents as described herein, can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid, cream, gel, or solid form, or by aérosol form.

The active compound is included in the phannaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desîred indication, without causing serious toxic effects in the patient treated. A preferred dose of the active compound for all of the herein-mentioned conditions is in the range from about 10 ng/kgto 300 mg/kg, preferably 0.1 to 100 mg/kg perday, more generally 0.5 to about 25 mg per kilogram body weight of the recipient/patient per day. A typical topical dosage will range from about 0.01-3% wt/wt in a suitable carrier.

The compound is conveniently administered in any suitable unit dosage form, including but not limited to one containing less than Img, 1 mg to 3000 mg, preferably 5 to

500 mg of active ingrédient per unit dosage form. An oral dosage of about 25-250 mg is often convenient.

The active ingrédient is preferably administered to achieve peak plasma concentrations of the active compound of about 0.00001-30 mM, preferably about 0.1-30 μΜ. This may be achieved, for example, by the intravenous injection of a solution or formulation of the active ingrédient, optionally in saline, or an aqueous medium or administered as a bolus of the active ingrédient. Oral administration is also appropriate to generate effective plasma concentrations of active agent.

The concentration of active compound in the drug composition will dépend on absorption, distribution, inactivation, and excrétion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, spécifie dosage regimens should be adjusted over time according to the individual need and the professional judgment ofthe person administering or supervîsîng the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to 1 imit the scope or practice of the claimed composition. The active ingrédient may be administered at once, or may be dîvided into a number of smaller doses to be administered at varying intervaîs of time.

Oral compositions will generally include an inert diluent or an edible carrier. They may be enciosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or îts prodrug dérivative can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible bînding agents, and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches, sachets and the like can contain any of the following ingrédients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnésium stéarate or Sterotes; a glidant such as colloïdal Silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring, When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents.

The active compound or pharmaceutically acceptable sait thereof can be administered as a component of an élixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplément the desired action, such as other anticancer agents, antibiotics, antîfungals, anti-inflammatorîes, or antiviral compounds. In certain preferred aspects of the invention, one or more chimeric antibody-recruiting compound according to the présent invention is co-administered with another anticancer agent and/or another bioactive agent, as otherwise described herein.

Solutions or suspensions used for parentéral, intradermal, subcutaneous, or topical application can include the foilowîng components: a stérile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycérine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acétates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental préparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid élimination from the body, such as a controlled and/or sustained release formulation, including implants and microencapsulated delivery Systems. Biodégradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrïdes, polyglycolic acid, collagen, poiyorthoesters, and polylactic acid. Methods for préparation of such formulations will be apparent to those ski lied in the art.

Liposomal suspensions or cholestosomes may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those ski lied in the art, for example, as described in U.S. Pat. No. 4,522,811 (which is încorporated herein by référencé in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholestérol) in an inorganic solvent that is then evaporated, leaving behind a thîn film of dried lipid on the surface of the container. An aqueous solution of the active compound are then introduced into the container. The container is then swîrled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby formîng the liposomal suspension.

EXAMPLES

Unbeknownst to the investigators, juvénile pigs in a herd shipped for research were not prophylaxed for Streptococcus suis (S. suis) a bacterium that is léthal to pigs and can also cause lethality in humans when exposed to sîck or colonized animais. One juvénile pig (Sus domesticus) became weak after becoming febrile 12 hours after major resuscitation and was euthanized. The pathologist noted an unusually high bacterial load immediately, which was visibiy apparent on microscopy. Tissues and cathéter tip were cultured, confirming S. suis infection. The médication itself was cultured and no bacterial contamination was detected. A second juvénile pig which was being treated with LY315920 alone and a single antibiotic dose given to the surviving members of the heard for experimental envenoming survived for >100 hours until euthanized as scheduled at the end of the study period with an non-curative dose of antibiotics. On autopsy it was noted that the pig had sîgnîficant evidence of S. suis infection, but the animal had only receîved antibiotics following realization that a first animal had died of septicemia rather than as a resuit of experimental envenoming by taipan venom.

Further Examples: Viruses and Bacteria Including COVID-19 and Anthrax

In severe cases of infection such as those caused by coronavîruses (e.g. SAKS, MERS, COVID-19, bacteria such as anthrax, fungi, or co- and secondary infections), the immune System may overreact and start attacking lung cells, neural tissues with accompanying or preceding endothélial damage ail resultîng în maladaptive élévations of MP and sPLA2 activity as well as other inflammatory species of phospholipase and cytokines. The lungs become obstructed with fluid and dying cells, making it difficult to breathe, neurological complications occur and a substantial percentage of infections can lead to ARDS and death (such as seen with severe coronavirus syndromes such as SARS, MERS and COV1D-19 or influenza with or wîthout secondary bacterial pneumonias).

Coronaviruses SARS-CoV and MERS-CoV hâve, for example, âge dépendent mortality that has been lînked to sPLA2 that was as high as 6% in the 25-44 âge group and 15% in the 45-64 year-old âge and up to more than 50% case fatality rates in older âge groups.¹ The inventors hâve unexpectedly realized that early administration of these drugs alone or in combination would abort and/or substantially inhibit the sPLA2 and métalloprotéase élévations that heralding severe outcomes resulting from acute pulmonary inflammation and cellular injury that can resuit in long-term pulmonary compromise in survîvors. When possible, sPLA2 and MP inhibitors should be administered orally before infection or resulting inflammatory, pulmonary, vascular, neurological and rénal sequelae become severe, The following examples are relevant to this analysis.

Example 1:

A Healthcare worker or fieid workers encounter a patients with an undefined respiratory illness or newly identified outbreak of a high fatality rate coronavirus or anthrax bacterium în the out of hospital or in hospital setting and suspect an infectious agent. Because of the high-risk occupational environment and risk of transmission from patient to Health care worker, an orally bîoav ai labié MP and/or PLA2 inhibitor is administered with or without an antiviral with or without a metalloprotease-suppressing antibiotic médication such as azithromycin (antibîotîc) for prophylaxis or to abrogate early symptoms or signs of infection heralding more life-threatening conséquences of virally-induced inflammatory responses. Therapy could be instîtuted prior to onset of severe signs or symptoms or prophylactically resulting in less severe conséquences of infection, avoiding intensive care and ventilatory support. This patient or Healthcare can be maintaîned on entîrely oral médications for the duration of disease or high risk occupational/high risk contact exposure.

Example 2:

A patient has a virally- or bacterially-mediated inflammatory response requiring intravenous médications and intensive care. The patient might or might not hâve ARDS, neurological sequelae but is sufficiently ill that intravenous infusion of médications are required. Intravenous infusions of an MP and/or PLA2 inhibitor decrease the acuity and dependence on intensive resources because of the suppression of maladaptive MP and/or PLA2-related inflammatory and cytotoxic responses. Once stabilized, this patient can be transitioned to an oral formulation of a MP and/or PLA2 inhibitor, reducing risk of relapse and long-term pulmonary, vascular or neurological damage.

Exainple 3 Systematic study of forty-one cases of documented inhalational anthrax from the Sverdlovsk épidémie of 1979 traced to release of aérosols of Bacillus anthracis were carried out. Respiratory function was compromised by médiastinal expansion, large pleural effusions, and hematogenous and rétrogradé lymphatic vessel spread of B. anthracis to the lung with conséquent pneumonia. These pathologie findings are consistent with previous experimental studies showing transport of inhaled spores to médiastinal lymph nodes, where germination and growth lead to local lésions and systemic spread, with resulting edema and cell death, owing to the effects of edema toxin and léthal toxin. An inhaled, intratracheally applied, IV or oral combination or single agent approach using MP or sPLA2 înhibitors is both prophylactic and therapeutic în human and non-human species. Vero (African green monkey) celi culture exposed to toxic métalloprotéases “zincins” and related toxin métalloprotéases and phospholipases demonstrates the efficacy of sPLA2 and métalloprotéase inhibition to protect cell junction integrîty.

Further Examples Methods

Mo use Studies: C57BL/6 mice weighing approximately 20grams were anesthetized using propylene gtycol/isoflurane with standard nose-cone procedure. Protection study: Controls (Figures IA, IB and Figure 2): 50pL mixture of 1:1:1 :l LPS (O55:B5):Oleic Acid (OA):PBS:Ethanol was instiiled intranasally (IN) under anesthésia and the mice recovered for observation. Treated animais received 50pL 1:1:1:1 LPS (O55:B5):Oleic Acid (OA):AZD2716:Ethanol by the same method. Final dose of AZD2716 administered was approximately 5mg/kg. Audio recordings of lung sounds were made using iPhone 10 and then transferred for amplitude analysis în terms of dB distance from peak (Figure 3). Rescue studies: (Figure 5) were conducted similarly but drugs were administered orally alone or in combination mixed in 8% gum Arabie at lOmg/kg (Prinomastat and/or AZD2716) at lOml/kg volume via recurved, stainless steel gavage needle five minutes after [N toxin instillation (75pL IN) under anesthésia as described, above.

Histology and Interprétation: After 24 or 48 hours, mice were euthanized under deep anesthésia using propylene glycol/isoflurane followed by cervical dislocation and rapid dissection of lungs and kidneys. The longs inflated with 10% neutral buffered formalin and the kîdney placed dîrectly into 10% neutral buffered formalin after nickîng the capsule. Tissues were sent to IDEXX Laboratories for mounting and staining with hematoxylin and eosin. Microscopie examination of stained lung sections was conducted and scores were assigned to each section of lung based upon ATS crîteria as described by Aeffner et al. Tox Path, 43:1074-1092, 2015. A minimum of 25 high power fields were examined by orienting the slide ID tag to the left of the left lung and altemating fields using the fine control outside the atelectatic tissues. Statistics: Scores were averaged and Students t-test applied (two tails, type 2, p<0.05 considered significant).

Enzymatic Assays: Viper, elapid and colubrid venom MP enzymatic activitîes were optimized Experiments to determine MP activity used validated substrate for MP (DQ Gelatin) and assay run per EnzChek gélatinase assay manufacturées instructions. These kits were stored as specified if stored at -20°C. PBS is the buffer in the case of MP assays, and absorbance is measured at 495nm. Substrate for MP îs DQ Gelatin and the assay is run per EnzChek gélatinase assay manufacturer’s instructions. Dose response curves were constructed by comparing absorbance with different doses of serially diluted inhibitors and compared to Controls to determine the IC50 for each venom-drug pair and for direct comparison of drugs for their effect on MP activity for each venom (Figure 4, Table 2). Cell culture and Electric Cell-Substrate Impédance Sensing (ECIS) studies: Figures 6-12: For cell culture and ECIS studies, veto épithélial cells (CCL-81) were cultured in Dulbecco’s Modîfied Eagle Medium (DMEM) supplemented with 5% EquaFETAL (EF) (Atlas Biologicals, Fort Collins, USA) (EF-DMEM) and 100 Units/mL of penîciilin and 100 pg/mL of streptomycin (Pen-Strep, see Maghalaes, et al., Insect Biochem. Mol. Biol., 2019, 111, 103169) and plated out on regular 8 or 96 well plates and ECIS plates (96 well) with or without wounding capability. In some studies venom and drug were pre-mixed to assess the ability of the drugs to protect cells from detachment. In others, venom was applied first, followed by drugs in order to assess the ability ofthe drugs to rescue cells from venom effects 5 to 15 minutes following exposure of vero cells to venom. Typically, samples were run in duplicate at varying concentrations of venom, drug, drug combinations with négative Controls (cells grown in media). Cellular wounding protocols were based on manufacturers pre-set recommendations for the spécifie plate models being used. Venoms and drugs were weîghed and diluted to l Omg/mL stocks in PBS (venoms) or 2.5 or Img/mL stocks in stérile water (drugs) before mixing with cell media at 2x the final concentrations to be used in the wells with final volumes of either 2mL or 250pL.

The ECIS-ΖΘ is an in vitro system that monitors real-time cellular behavîors and movements via gold film électrodes, cell membranes essentialiy act as insulators. Consequently current flows unrestrained in the absence of cells and constraîned once a cell monolayer is established. Changes in the current flow are measured as impédance (Z), which gives insight into two aspects of cellular behaviors and movements at different frequencies. At low frequencies (<10,000 Hz), the cell bodies force the current to flow basolaterally or through the intercellular space between the cell borders. Therefore, résistance (R) is measured at low frequencies and provides information on the barrier integrity. Conversely, the opposition created by the cell membrane is relatively small at high frequencies (>10,000 Hz), so current flows capacitively through the cell bodies.

Capacitance (C) îs a measure of the electrode coverage by the cells and is indicative of cell migration, as well as cell monolayer disruption following injuries. ECIS can also produce reproducible wounding models by mechanically disrupting a cell monolayer. The ECIS set up can be used to wound a cell monolayer using high current puise produced via the électrodes. The severity of injury is dépendent on the level of current and the duration of the application. The injured or dead cells then detach from the electrode surface which is measured as a rapid increase in the electrode capacitance and a réduction in the résistance. The system then returns to its normal operation and monitors the subséquent recovery as neighboring cells migrate to fl II the exposed electrode and re-establish a cell monolayer, see Gu, et a\.,Biosensors, (Basel), 2018, Oct. 11, 8(4), 90.

Methods and compositions for achieving accelerated treatment of wounds and bums, anthrax metalloprotease toxîn (léthal factor) drîven complications, ARDS, néo-natal and pédiatrie acute respiratory distress syndrome (neo-natal/pediatric ARDS), including méconium aspiration syndrome are described based on the expérimentais described herein above.

Thus, the expérimentais described above evidence the following observations, among others:

1. Oral prînomastat alone unexpectedly outperformed ail inhîbitors to treat LPS-Oleic Acid induced ARDS but there were additional benefits from combinations of sPLA2 and metalloprotease inhibition;

2. Locally (respiratory tract) applied of AZD2716 alone prevented and rescued young (~19-20g) mice from LPS-Oleic Acid induced ARDS, most notably from pulmonary edema. This has significant implications for treatment of néonatal and pédiatrie ARDS.

3. Low dose Prinomastat + varespladib potentiated both rescue of cultured tissue and accelerated wound healing of experîmentally înjured tissues in cell culture.

Références

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Claims (14)

1. Use of at least one PLA2 inhibitor and/or métalloprotease inhibitor in the manufacture of a médicament for use în a method of reducîng the likelihood that an injured patient or subject at risk of an inflammatory syndrome will produce one or more of 5 sepsis, septic shock, an acute inflammatory syndrome (whether iatrogénie or not) or acute respiratory distress syndrome, wherein the method comprises administering the médicament alone or in combination with at least one antibiotic to the patient or subject.

2. The use according to claim 1, wherein said médicament is combined with or administered with at least one antibiotic.

10

3. Use of at least one PLA2 inhibitor and/or métalloprotease inhibitor în the manufacture of a médicament for use in a method of treating an injured or burned patient or subject to improve wound healing of injured and/or burned tissue and/or to inhibit, ameliorate or reduce the likelihood of one or more of sepsis, septic shock, acute inflammatory syndrome, including inflammatory response syndrome (SIRS) and/or acute

15 respiratory distress syndrome (ARDS) in said patient or subject, wherein the method comprises administering the médicament in combination with at least one antibiotic to the patient or subject.

4. Use of at least one PLA2 inhibitor and/or métalloprotease inhibitor in the manufacture of a médicament for use in a method of treating a patient or subject with

20 sepsis, including early sepsis, wherein the method comprises administering the médicament in combination with at least one antibiotic to the patient or subject.

5. Use of at least one PLA2 inhibitor and/or at least one métalloprotéase inhibitor in the manufacture of a médicament for inhibiting or reducîng the likelihood of a COVID19/cytokine release syndrome in a patient at risk of same.

25

6. Use of at least one PLA2 inhibitor in the manufacture of a médicament for treating néo-natal acute respiratory distress syndrome (néo-natal ARDS), including méconium aspiration syndrome.

7. Use according to claim 6, wherein said néo-natal ARDS is méconium aspiration syndrome (MAS).

30

8. A pharmaceutical composition for use in a method of treating a patient at risk for or afflîcted with one or more of sepsis, septic shock, acute inflammatory syndrome, including inflammatory response syndrome (SIRS) and/or acute respiratory distress syndrome (ARDS), comprising at least one PLA2 inhibitor and/or at least one métalloprotease inhibitor, wherein the composition further comprises at least one antîbiotic or is administered în combination with at least one antîbiotic.

9. The use or the pharmaceuticaï composition according to any one of claims 1 -5 and 8, wherein said médicament or pharmaceuticaï composition comprises at least one PLA2 inhibitor.

10. The use or the pharmaceuticaï composition according to any one of claims 1 -5 and 8, wherein said médicament or pharmaceuticaï composition comprises at least one metalloprotease inhibitor.

11. The use or the pharmaceuticaï composition according to any one of claims 1 -5 and 8, wherein said médicament or pharmaceuticaï composition comprises at least one PLA2 inhibitor and at least one metalloprotease inhibitor.

12. The use or the pharmaceuticaï composition according to any one of claims 1-11, wherein said PLA2 inhibitor is varespladib (LY315920), methyl varespladib (LY333013). AZD2716- as a racemic mixture or enantiomer thereof, AZD Compound 4, LY433771 ((9-[(phenyl)methyl]-5-carbamoylcarbazol-4-yl)oxyacetic acid), a pharmaceutically acceptable sait thereof or a mixture thereof.

13. The use or the pharmaceuticaï composition according to any one of claims 1-5 and 8-12, wherein said metalloprotease inhibitor is prinomastat, BB-94 (marimastat), BB-2516 (batimastat), vorinostat, cefixime and doxycycline, a pharmaceutically acceptable sait thereof or a mixture thereof.

14. The use or the pharmaceuticaï composition according to any one of daims 1 -4 and 8-13, wherein said antibiotic is selected from the group consîsting of a penam, a carboxylpenicillin, a cephalosporin, a monobactam, a carbapenem, a fluoroqunoline, a macrolide or a mixture thereof.