INFECTIOUS AND INFLAMMATORY DISORDERS

PRIORITY CLAIM

This application claims priority to United States Provisional

Application Serial No. 61/386,345 filed September 24, 2010 and to United States Provisional Application Serial No. 61/387,335 filed September 28, 2010, the contents of both of which are hereby incorporated in their entiries herein. GRANT INFORMATION

Federal funding was not used in the development of the subject matter of the invention.

1. INTRODUCTION

The present invention relates to methods of treating infectious, inflammatory and post-traumatic disorders by administering various compounds newly discovered to have TLR4 inhibitory activity.

2. BACKGROUND OF THE INVENTION

The innate immune receptor Toll-like receptor 4 ("TLR4") has been recognized to be the receptor on hematopoietic and non-hematopoietic cells for endotoxin (lipopolysaccharide, "LPS") as well as a variety of endogenous molecules that are released within the body during inflammatory or infectious disorders.

Strategies to discover molecules that are capable of neutralizing the ability of TLR4 to signal are likely to show promise as novel anti-infective and/or antiinflammatory agents.

3. SUMMARY OF THE INVENTION

The present invention relates to methods of treating infectious, inflammatory and post-traumatic disorders by administering various compounds newly discovered to have TLR4 inhibitory activity. Compounds that may be used according to the invention are set forth in TABLE 1, below. In addition to methods of treatment, the present invention further provides for pharmaceutical compositions comprising said compounds, together with a suitable pharmaceutical carrier. Because

NY02:723955.1 1 TLR4 is the most upstream receptor in the pro- inflammatory LPS signaling cascade, treatments of the invention, which inhibit or antagonize TLR4 action, may avoid the pitfalls associated with other cytokine inhibitors that act further down the pathway and accordingly play a less specific (and perhaps non-critical) role.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 A-B. NFkB luciferase reporter mice treated with either (A) LPS (positive control, left) or saline (negative control; right) or (B) Bay- 11-7082 + LPS (left) or compound 1 + LPS (right), imaged using the IVIS Lumina 3D Optical system.

FIGURE 2 A-B. NFkB luciferase reporter mice treated with either (A) LPS (positive control, left) or saline (negative control, right) or (B) compound 3 + LPS (left) or compound 2 + LPS (right), imaged using the IVIS Lumina 3D Optical system.

FIGURE 3 A-C. Intestines from NFkB luciferase reporter mice of

FIGURES 1 A-B and 2A-B, in particular (A) mice treated with LPS (left) or saline (right) or (B) Bay-11-7082 + LPS (left) or compound 1 + LPS (right) or (C) compound 2 + LPS or compound 3 + LPS (left), imaged using the IVIS Lumina 3D Optical system.

FIGURE 4A-B. NFkB luciferase reporter mice treated with either (A) saline (left) or LPS (right) or (B) compound 4 + LPS, imaged using the IVIS Lumina 3D Optical system.

FIGURE 5. NFkB luciferase reporter mice treated with either saline (left), compound 5 + LPS (center) or compound 6 + LPS (right) imaged using the IVIS Lumina 3D Optical system.

FIGURE 6 A-B. Intestines from NFkB luciferase reporter mice of FIGURE 4A-B, in particular mice treated with (A) saline or (B) LPS (left) or compound 4 + LPS (right), imaged using the IVIS Lumina 3D Optical system.

FIGURE 7 A-B. Intestines from NFkB luciferase reporter mice of FIGURE 5, in particular mice treated with (A) saline or (B) compound 5 + LPS (left) or compound 6 + LPS (right), imaged using the IVIS Lumina 3D Optical system.

FIGURE 8A-B. Summary of results of FIGURES 1-7, above.

NY02 :723955.1 2 FIGURE 9. ELISA results showing IL6 production. (*) denotes the endotoxicity score, where 0= healthy, 1= mild, 2=moderate, 3=¾evere, and 4-lethaL Highlighting denotes particular "hits" of interest.

FIGURE 10A-B. NFKB luciferase reporter mice treated with either (A) LPS (in the animal on the right with an animal treated with saline control on the left) or (B) LPS _. plus compound (with the animal on the right treated with C34 + LPS and the animal on the left treated with C 16 + LP S) .

FIGURE 11. i OS mRNA levels in breast-fed (BF) controls and a CFW- EC model induced by formula feeding (FF).

FIGURE 12. TNFa mRNA levels in human NEC tissue explants treated with LPS as compared to LPS + C34.

FIGURE 13. iNOS mRNA levels in human NEC tissue explants treated with LPS as compared to LPS + C34.

FIGURE 14. TLR4 mRNA levels in breast-fed untreated or C34- treated controls as compared to formula-fed (CFW-NEC) rats that were untreated or treated with C34.

FIGURE 15A-B. (A) AST levels in control animals as compared with hemorrhage-shock (H/S) model animals, and H/S animals treated with C34. ALT levels in control animals as compared with hemorrhage-shock (H/S) model animals, and H/S animals treated with C34.

5. DETAILED DESCRIPTION OP THE INVENTION For purposes of clarity a d not by way of limitation, the detailed description of the invention is divided into the following subsections:

(i) TLR4 inhibitory compounds;

(ii) pharmaceutical compositions;

(iii) disorders that may be treated; and

(iv) methods of treatment.

5.1 TLR4 INHIBITORY COMPOUNDS

Compounds that may be used to inhibit TLR4 (also referred to herein as "T4lCs") according to the invention are set forth, below, in TABLE 1.

NY02:723955.1 3 In specific, non-limiting embodiments of the invention, compounds that maybe used to inhibit TLR ("T4Ics") include compounds 1, 3, 4, 5, 6, 8, 16, 21, 22, 27, 28, 29, 30, 45, and 47.

In one specific, non-limiting embodiment of the invention, the T4IC compound is compound 3, which is 4-0-(3-0- {2-(acetylamino)-2-deoxy-4-0-(6- deoxyhexopyranosyl)-3-0-[2-0-(6-deoxyhexopyranosyl)

hexopyranosyl]hexopyranosyl} hexopyranosyl)hexopyranose, having the structure:

NY02:723955.1 4 In another specific, non-limiting embodiment of the invention, the T4IC compound is compound 4, which is 3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2JJ-pyran-2-yl dihydrogen phosphate, sodium salt, having the structure:

In another specific, non-limiting embodiment of the invention, the T4IC compound is compound 8, cyclohexanamine compound with 1,6-di-O- phosphono-beta-D-glycero-hexopyranose (4:1) hydrate, having the structure:

In another specific, non-limiting embodiment of the invention, the

T4IC compound is compound 16, 2-(acetylamino)-2-deoxy-D-galactopyranose hydrate, having the structure:

NY02:723955.1 5 In another specific, non-limiting embodiment of the invention, the T4IC compound is compound 27, 2-(acetylamino)-2-deoxy-4-0- hexopyranosylhexopyranose, having the structure:

In another specific, non-limiting embodiment of the invention, the T4IC compound is compound 34, isopropyl 3,4,6-tri-0-acetyl-2-(acetylamino)-2- deoxyhexopyranoside, having the structure:

NY02:723955.1 6 In related non-limiting embodiments, the present invention provides for derivatives of isopropyl 3,4,6-tri-0-acetyl-2-(acetyIamino)-2- deoxyhexopyranoside. In one set of non-limiting embodiments, said derivatives may have FORMULA I, as follows:

where R is selected from R and -O-R , where R may be a substituted or

unsubstituted alkane or alkene, where the substituent if present may be methyl or ethyl, where the alkane or alkene portion optionally comprises a branched or cyclic component, and may have between 1 and 12 or between 1 and 6 carbon atoms.

In further non-limiting embodiments, said derivatives may have FORMULA II, as follows:

where R is selected from R ¹ and - -R ¹ , where R ¹ may be a substituted or

unsubstituted alkane or alkene, where the substituent if present may be methyl or ethyl, where the alkane or alkene portion optionally comprises a branched or cyclic component, and may have between 1 and 12 or between 1 and 6 carbon atoms.

In specific, non-limiting embodiments, a derivative of isopropyl 3,4,6- tri-0-acetyl-2-(acetylamino)-2-deoxyhexopyranoside may be selected from the following group of compounds:

NY02.723955.1 7

NY02:723955.t 8 In particular non-limiting embodiments, isopropyl 3,4,6-tri-0-acetyl-2- (acetylamino)-2-deoxyhexopyranoside may be prepared by the following method:

1.5g scale 656mg, 25%

700mg scale 350mg, 29%

141.3mg, 88%, 200mg scale

238.2mg, 94%, 300mg scale 80.3mg, 49%

Scheme 1. Synthesis of Isopropyl 3,4,6-tri-0-acet l-2-(acetylamino)-2-deoxyhexopyranoside

A working example containing further details of the synthesis is set forth below as section 9, Example 4, and is incorporated by reference herein.

The present invention provides for pharmaceutical compositions comprising therapeutically effective amounts of any of the foregoing compounds, for example but not limited to together with a pharmaceutical carrier such as water or other physiologic solvent. A therapeutically effective amount inhibits TLR4.

A compound is considered to inhibit TLR4 (that is, be a T4IC) if it inhibits one or preferably more sign or symptom of inflammation, such as, for example, activation of NFkB, increased expression/levels of interleukin 6 ("iL-6"), elevated erythrocyte sedimentation rate, elevated C reactive protein, fever, tachypnea, lethargy, swelling, redness, and/or pain. The ability for a compound and/or a particular concentration of a compound to inhibit TLR4 may be determined using an assay for TLR4 activity which may assess one of the abovelisted signs or symptoms. For example, TLR4 inhibition may be assayed using a method that measures the affect of a compound on NFkB activity, for example, but not limited to, the NFkB luciferase reporter mouse model, stimulated with a TLR4 ligand such as LPS,

NY02:723955.1 9 described in the example below or HEK-Blue-4 cells (InvivoGen). As other non- limiting examples of systems for testing compounds to determine TLR4 inhibitory activity, CWT mice may be treated with LPS and a test compound and monitored for signs and symptoms of inflammation, and/or C3H/WT cells (InvivoGen) may be treated with LPS and a test compound and tested for NFkB activation, IL6 production, or other markers of the inflammatory process.

5.2 PHARMACEUTICAL COMPOSITIONS

The present invention provides for pharmaceutical compositions comprising a T4IC, as described above, in a suitable pharmaceutical carrier. . The amount of T4IC present in the composition may be calculated to provide, when administered to a subject in need of such treatment, an effective amount of T4IC.

In non-limiting embodiments, the T4IC may be comprised in a coated particle, micelle, liposome, or similar structure.

A pharmaceutical composition may be a liquid, comprising a T4IC in a liquid pharmaceutical carrier comprising, for example, water (an aqueous carrier) or saline. Said liquid composition may optionally further contain one or more of a buffer or a preservative.

Alternatively, a pharmaceutical composition may be a solid, for example in the form of a tablet, capsule, sachet or suppository, comprising a dose of T4IC that provides an effective amount of T4IC to a subject in need of such treatment when administered according to a dosing regimen. Said solid pharmaceutical composition may further comprise one or more excipients, for example, but not limited to, lactose, sucrose, mannitol, erythritol, carboxymethylcellulose,

microcrystalline cellulose, hydroxypropylmethylcellulose, starch,

polyvinylpyrrolidone, etc

A pharmaceutical composition according to the invention may, in non- limiting embodiments, comprise an additional agent that may have antimicrobial and or antiinflammatory activity, for example, but not limited to, an antibiotic agent, a steroid, or a non-steroidal antiinflammatory agent. In additional non-limiting embodiments of the invention, a pharmaceutical composition may comprise an analgesic agent. In additional non-limiting embodiments of the invention, a pharmaceutical composition may comprise an agent that improves cardiac function and/or reduces cardiac stress, for example, but not limited to, an angiotensin

NY02:723955.) 10 converting enzyme inhibitor, a beta blocker, nitroglycerin or a related nitrate compound, digoxin or a related compound, or a calcium channel blocker.

5.3 DISORDERS THAT MAY BE TREATED

The present invention may be used to treat any disease/disorder

("disorder") involving TLR4 activation, including, but not limited to, infectious diseases and inflammatory disorders such as sepsis, necrotizing enterocolitis, autoimmune diseases, Crohn's disease, celiac disease, ulcerative colitis, rheumatoid arthritis, cardiovascular disease including myocardial infarction, epilepsy, gram negative bacterial infections, aspergillosis, periodontal disease, Alzheimer's disease, cigarette smoke mediated lung inflammation, viral hepatitis (including hepatitis C virus hepatitis), alcoholic hepatitis, insulin resistance in adipocytes, and others. See, for example, United States Patent Application Publication No. US2008-0311112-A1 , published December 18, 2008. The present invention may also be used, in non- limiting embodiments, to treat post traumatic conditions, including ischemic injury and traumatic injury to the heart, liver, lung, kidney, intestine, brain, eye and pancreas.

5.4 METHODS OF TREATMENT

In a non-limiting embodiment, the present invention provides for a method of treating an infectious or inflammatory disorder comprising administering, to a subject in need of such treatment, an effective amount of a T4IC that reduces one or more sign or symptom of inflammation in the subject.

In another non-limiting embodiment, the present invention provides for a method of treating an intestinal inflammatory disorder in a subject comprising administering, to a subject in need of such treatment, an effective amount of a T4IC that reduces intestinal inflammation in the subject.

In another non-limiting embodiment, the present invention provides for a method of treating a cardiovascular disease in a subject comprising administering, to a subject in need of such treatment, an effective amount of a T4IC that reduces myocardial ischemia in the subject. In various non-limiting embodiments, the subject may be suffering from cardiac angina, may have suffered or is suffering a myocardial infarction, and/or may be at risk for suffering a myocardial infarction.

NY02:723955.1 11 In another non- limiting embodiment, the present invention provides for a method of treating an inflammatory pulmonary disease in a subject comprising administering, to a subject in need of such treatment, an effective amount of a T4IC that reduces pulmonary airway inflammation in the subject,

In another non-limiting embodiment, the present invention provides for a method of treating a traumatic injury in a subject comprising administering, to a subject in need of such treatment, an effective amount of a T4IC that reduces TLR4- induced post-traumatic injury. In non-limiting embodiments, the traumatic injury is to an organ selected from the group consisting of the heart, the liver, the lung, the kidney, the intestine, the brain, the eye and the pancreas.

A subject may be a human or a non-human subject.

A T4IC may be administered by any standard route including but not limited to oral, intraperitoneal (i.p), intravenous (i.v.), subcutaneous (s.c),

intradermal, intramuscular (i.m.) intraarticular, intrathecal, intraarterial, intravaginal, rectal, nasal, pulmonary, etc..

An effective dose may be determined using methods known in the art (including but not limited to TLR4 activity assays described herein). In specific, non- limiting embodiments of the invention, an effective dose may be between about 0.01 and 50 micromoles of T4IC per kilogram weight of the subject, or between about 0.1 and 20 micromoles of T4IC per kilogram weight of the subject. In specific, non- limiting embodiments, the dose of T4IC may be between about 0.001 and 100 milligrams per kilogram weight of the subject, or between about 0.01 and 10 milligrams per kilogram weight of the subject, or between about 0.1 and 10 milligrams per kilogram weight of the subject, or between about 0.5 and 5 milligrams per kilogram weight of the subject.

6. EXAMPLE 1

6.1 MATERIALS AND METHODS

In silico similarity screen. In order to identify inhibitors of TLR4, the structure of a known TLR4 inhibitor, E5564, was utilized. E5564 is a second- generation synthetic analogue of the lipid A component of endotoxin

(lipopolysaccharide [LPS]). Based on the published structure of E5564, an in silico similarity screen was conducted using the on-line iResearch library of ChemNavigator (San Diego, CA) accessed at www.chemnavigator.com. This compound library was

NY02.-723955.1 12 chosen to maximize structural diversity while including drug-like structures, and also to include multiple Food and Drug Administration-approved drugs, known bioactive compounds, metabolites, natural products and related compounds.

Compound preparation and delivery. All 65 identified compounds were received in solid form and stored at 4°C until use. Stock solutions of each compound were made by dissolving the appropriate amount of each compound in dimethyl sulfoxide (DMSO) to yield a concentration of lOmM. Immediately prior to injection into experimental animals, the compounds were diluted to an experimental concentration of lOOuM in phosphate-buffered saline (PBS). Total concentration of DMSO in the final diluted drug was 1%. Compounds were closely examined to insure that no precipitate formed prior to injection and were stored on ice until injection. All compounds were given via intraperitoneal (i.p.) injection in a total volume of 200ul using a lec syringe and 27 gauge needle. In all experiments listed below, the compounds were delivered to the experimental mice 30 minutes prior to injection with lipopolysaccharide (LPS) to induce endotoxemia. Control animals not receiving compound received 1% DMSO dissolved in PBS, Following drug injection, the mice were observed closely in their cages for signs of immediate toxicity or adverse reaction to the drug, including but not limited to piloerection, tachypnea, bleeding, abnormal or aggressive behavior, signs of altered mental status, and level of activity.

Induction of endotoxemia. All mice were housed and cared for at the Rangos Research Center, Children's Hospital of Pittsburgh (Pittsburgh, PA). All experiments were approved by the Children's Hospital of Pittsburgh Animal Care Committee and by the Institutional Review Board of the University of Pittsburgh. Swiss Webster (CFW) and NFkB-luciferase reporter mice were obtained from The Jackson Laboratory. Throughout the course of all experiments, mice were housed 4 per cage with access to food, water, and standard bedding. Endotoxemia was induced in all experiments by i.p . injection of LPS (Escherichia coli 0111 :B4 purified by gel filtration chromatography, >99% pure, Sigma- Aldrich) at a dose of 2mg/kg for 6 hours. At the end of each experiment, all animals were euthanized by C02 hypoxia and cervical dislocation.

Two separate in vivo experimental designs were utilized to assess the effect of pretreatment with each individual compound in a model of endotoxemia.

NY02.-723955.1 13 The first series of experiments involved the use of the NFkB reporter mice, while the second utilized CFW.

NFkB-luciferase reporter mice assay. NFkB-luciferase reporter mice, in which the NFkB is downstream of the luciferase gene, were subjected to endotoxemia with or without pretreatment with experimental compounds. The experimental compounds treated by this assay were compounds 1-6, with compounds 1-3 tested first and compounds 4-6 tested later. For both experiments, the mice were 6 weeks old on the date of the experiment Controls for each included 1% DMSO alone, LPS alone (2mg/kg, 6hrs), and pretreatment with the known NFkB inhibitor Bay-11-7082 (20mg/kg, 30 minute pretreatment via i.p. injection, Cayman Chemical). Following 6 hours of endotoxemia, each mouse was given an i.p. injection of luciferin (160 ug/kg, Invitrogen), then after 10 minutes a whole animal image was obtained using the WIS Lumina 3D Optical in vivo imaging system (Caliper Life Sciences, Hopkinton, MA) under 1.5% isofluorane anesthesia. After whole body imaging, an additional injection of luciferin is given and animals were euthanized, organs were harvested, and the extent of NFkB activation within various tissues was further analyzed by an additional luminescence image of the individual organs.

CFW endotoxemia assay. In order to effectively screen the entire compound list, the remaining compounds (7-60) were screened in a model of endotoxemia using CFW mice. Three separate experiments were performed: First, Compound 7 along with a repeat of compound 4 (n=3 mice per compound, mice age 5 weeks) were tested. Controls were saline injection alone (n=l), LPS 2mg/kg 6hrs (n=2). Second, compounds 3, 4, 6, 8-32 were tested (n=l mouse each, 3 week old mice). Controls were saline injection alone (n-1), LPS 2mg kg 6hrs (n=2), LPS 1 Omg kg 6hrs (n=2), Bay 1 1 pretreatment 20mg/kg for 30 minutes prior to LPS

2mg/kg 6hrs. Third, compounds 8, 16, 33-60 were tested (n=l mouse each, 3 week old mice). Controls were saline injection alone (n=l), LPS 2mg/kg 6hrs (n=2), Bay 11 pretreatment 20mg/kg for 30 minutes prior to LPS 2mg/kg 6hrs. Following 6 hours of endotoxemia, mice from the second and third experiments were observed within their cages and videotaped to document behavior and phenotypic differences to assess for an inhibitory effect of compound pretreatment on the effects of LPS. Mice were identified by a number previously placed using permanent marker on their tails. The degree of piloerection, tachypnea, location in the cage (center of cage versus

NY02:723955.1 14 comers), degree of activity, and behavior relative to other animals was observed and documented via video for each mouse.

Enzyme linked immunosorbent assay (ELISA). Prior to being euthanized, mice from the NFkB luciferase and all three CFW endotoxemia experiments were anesthetized (1.5% isofluorane) and a retro-orbital sinus puncture was performed to obtain a blood sample. Serum was obtained via centrifugation and an ELISA was performed to assess for IL-6 expression using a pre-made kit (R&D Biosystems). Results are reported relative to a standard curve for each experiment as pg/ml.

6.2 RESULTS

With a view to identifying TLR4 inhibitors amongst previously known compounds (that were not hitherto known to have TLR4 inhibitory activity), a library of compounds was screened for those bearing structural similarity to the known TLR4 inhibitor, E5564. A total of 124,413,264 samples within the library were screened, and a total of 100 structures were identified with a similarity greater than 70%.

Excluding the known TLR4 agonist, LPS, which was identified in the search, the remaining 99 compounds, were then researched for commercial availability. A total of 65 commercially available compounds were identified and obtained in amounts ranging from 1 -lOOmg from ChemNavigator. These 65 compounds are shown in TABLE 1. Compounds 9, 10, 11, 13, 14, 17, 21, 33 and 35-39 are available from InterBio Screen Ltd., compounds 3, 5, 6, 8, 23, 30 and 49-58 are available from Carbosynth Ltd., compounds 12, 22, 24 and 34 are available from Enamine, compound 59 is available from Synthon Lab Ltd., compounds 1, 4, 7, 15, 16, 18, 19, 32, 40, 41 and 60 are available from Sigma- Aldrich, compounds 42 and 43 are available from Bosche Scientific LLC, compounds 2, 20, 25, 26, 27, 44, 45, 47 and 48 are available from Toronto Research Chemicals, compound 65 is available from PBMR Labs, Ukraine, compounds 31 and 46 are available from Maybridge Ltd., compound 28 is available from Labotest and compounds 29 and 61-64 are available from CehDiv Inc.

TABLE 1

NY02:723955.1 15 /][1 ,3]dioxin

-8-yloxy)propanoic add

dodecyl 3,4,6-tri-0-acety[-2-(acetylamino)-2-deoxy-beta-D- glucopyranoside 2010752 butyl 2-(acetylami no)-2-deoxy-3,4-di -O-met hyl-beta-D- glucopyranoside 2010750 isopropyl 3,4,6-tri-0-acetyl-2-(aoetylamino)-2-deoxyhexopyranoside 2010760 cydohexyl 3,4,6-tri-0-aoety!-2-(acetylamino)-2-deoxy-alpha-D- gluoopyranoside 2010748 hexyl 3,4,6-tri-0-acetyl-2-(acetylamino)-2-deoxy-beta-D- glucopyranog ^' de 2010747

N-[(2R3R4R5¾6R)-2-[(1'S2'R,6 ^, 8 ^, R9'S)-dispiro[q clohexane-1 ,4'- [3,5,7,10,12]

pentaoxatricyclo[7.3.0.0 ^A {2,6}]dodecane-i r,1"-i-yclohexane3-8 ^l - ylmethoxy]-4,5- dihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide 2010754

(2f¾334F?5^6/^-5-acetamido-2-(acetoxymethyl)

^-(((Sa^S^Sa^SaSSb^^JJ-tetramethyltetrahydro

-3a/+bis[1,3]dioxolo[4,5-0:4\5'-clpyran-5-yl)methoxy)tetr ahydro-2l-l- pyran-3,4-diyl-diaGetate 2010753

W-{(2f?3F?4^536F¾- ,5 dihydroxy-6-(hydrox nnethyl)

-2-(((3a^5^5a$8a$8b^-2,2,7J-tetramethyltetrahydro-3a

H-bist1 ,3]dioxolo[4,5-J :4',5'-c]pyran-5-yt)methoxy)tetrahydro-2l-t- pyran-3-yl) acetamide 2010759 propyl 3,4,6-tri-0-aGetyl-2-(acetylamino)-2-deoxyhexopyrano9de 2010761

1 , 3,4,6-t etra-0-acetyl-2-deoxy-2-(palmitoylamino)hexopyranose 2010762

6-0-[2-(acetylamino)-2-deoxy-beta-[ glucopyranosyl]-3-0-isopentyl- 1 ,2-0-(1-methylethylidene)

-al pha-D-xy io-hexof uran ose 2010757

6-0-[2-(ai_etylamino)-2-deoxy-beta-D-glu »pyrario¾fl]-1 ,2-0-(1 - methylethy!idene)-3-0-propy!- alpha-D-xyio-hexofuranose 2010758

1 ,2- (1-methylethylidene)-3- propyl^- [3,4,6-tri-0-acetyl-2-

(acety lami n o)-2-deoxy-bet a-

D-g!ucopy ranosy I ] -al ph a-D-xy I o-h exof u ranose 2010755

1,2-CK1-methylethylidene)-3-0-pentyf-6-0-[3,4,6-tri-(- -acetyl-2- (acety I ami no)-2-deoxy-beta- D-glucopyranosyl]-alpha-D-xylo-hexofuranose 2010756 odyl 2-(acetylamino)-2-deoxy-beta-D-glycero-hexopyranoside 2010751 sec-butyl 2-(acetylamino)-2-deoxyhexopyranoside 2010763

(2S4$5 6 ¾-5-acetamido-2-((2f¾3$4§5R

,6¾-3,5-dihydroxy-2-(hydroxymethyl)-6-((2^334f¾5^-

4,5,6-trihydroxy-2-(hydroxymethyl)tetrahydro-2/+pyran-3-y !oxy) 2010745 sodium (2S3$4/¾5R6f¾-3-((2Si3^5S

6/¾-3-ac¾tamido-5-riydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran- 2-yloxy)-4,5,

6-trihydroxytetrahydro-2 +pyran-2-carboxylate 2010739

2-(acety!amino)-4-0^2-(ao3tylamino)^0-[2'(acety!amino)-2- deoxy- beta-D-glucopyranosyt]

-2-deoxy-beta-[ glucopyrano¾il}-2-deoxy-D-gluoopyranose 2010744

3-aoetamido-4,5-d!hydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran- 2-yl dihydrogen

phosphate, sodium salt 2010742 sulfuricadd compound with (2R)^-amino-N-{(1R,253£i4R5g-5- amino-2-[(3-amino-3-deoxy- 2010737 al p ha-D-gl ucopy ranosy I )oxy]-4-[ (6-ami no-6-deoxy-al pha- D- gl ucopy ranosy I )oxy]

-3-hydroxycydohexyl}-2-hydroxybutanamide (1 : 1)

{4 ¾^-((2¾-2-((2^-2-((3^4f?5g6f¾-3-acetamido- 2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyrari-4- y lox ) propan amido)propanamid o)

-5-amino-5-oxopentanoicadd 2010746

Uridine S'-diphospho-N-acetylglucosamine sodium salt 2010741

Uridine 5'-diphospho-N-acetylgalactosamine disodium salt 2010743

2-(acetylamino)-3-0^4-0-[2-(aoetyiamino)-2-deoxy-3-0-alph a-D- xylo-hexopyranuronosyl-beta

-D-ribo-hexopyranosyl]-beta-D-xylo-hexopyranuronosyl}-2-deox y-D- gluoopyranose 2010792

2-(acetyl ami no)-2-d eoxy-3-0-(6,8-d ideoxy-beta-L-gl ycero- octopyranosyl-7-ulose)-4-0-sulfo-

L-eryihro-hexopyranose 2010793

2-(aoety I ami no)-2-d eoxy-4-O-hexopy ranosy I hexopy ranose 2010777

N-{(13253F¾-1-[(beta-L-glyoero-hexopyranosyloxy)methyl]- 2,3- dihydrox heptadecyl}hexacosanamide 2010778 dimethyl 5-(acetylamino)-3,5-dideoxy-D-erythro-non-2- ulopyranosidonate 2010764 methyl 2-(acetylamino)-2-deoxy-3-0-hexopyranosylhexopyranoside 2010776

8-{[2-(acetylamino)-4-0-[2-(acetylamino)-2-deoxyhexopyran osyl]-2- deoxy-6-0-(6-deoxy hexopy ranosy I)

hexopyranosyl]oxy}octyi acetate 2010781 octyl 2-(acetylamino)-2-deoxyhexopyranoside 2010775

2-(acetylami no)-2-deoxy-4-0-(6-deoxyhexopyranosyl )-3-0- hexopy ranosy i hexopy ranose 2010774

2-(acetylamino)-2-deoxy-alpha-D-!yxo-hexopyranose 2010772

2-(acetylamino)-2-deoxy-D-glucopyranose 2010773 ally! 34,6-tri-0-acetyi-2-(acetylamino)-2-deoxy-beta-D-lyxo- hexopyranoside 2010780

N-[(152 3E)-1-[(beta-L-ribo-hexopyranosylox| )methyl]-2-hydroxy-3- heptadeceny I }octadecanami de 2010779 sodium ((3¾6i¾-5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2- yl)methyl

phosphate 2010787

2-((2^5¾-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)tetr ahydro- 2H-pyran-

4-yloxy)propanoic acid 2010782 aliyl 2-(acety!amino)-2-deoxy-beta-D-glycero-hexopyranoside 2010765

1 , 3,4,6-tet ra-O-acety l-2-(acety I ami no)-2-deoxy-bet a-D-glycero- hexopyranose 2010766

2-(acetylamino)-2-deoxy-beta-D-glycero-hexopyranose 2010769

4-0-[2-(acetylamino)-2 ieoxyhexopyranosyl]-1,5-anhydro-2- deoxyhexitol 2010791 ethyl 2-(acetylamino)-2-deoxy-beta-Oglycero-hexopyranoside 2010785 ethyl 3,4,6-t ri-0-acetyl-2-(acetylami no)-2-deoxy-beta-D-glycero- hexopyranoa ^' de 2010784

5-acetamido-6-((1R2R>)-3-(3-(3-acetamido-5-hydroxy-6- (hyd roxy met hyl)-4-(3,4, 5- trihydroxy-6-(hydroxymethyi)tetrahydro-2H>-pyran-2- yioxy)tetrahydro-2H-pyran-2- 2010770

Next, 60 of the 65 compounds were tested to determine whether they exhibit TLR4 inhibitory activity in a murine LPS-induced model for endotoxemia . The first series of experiments involved the use of the NFkB luciferase reporter mice (testing compounds 1-6), while the second utilized CFW mice (testing compounds 3- 65). In all cases, each mouse received 20 nanomoles of test compound, or approximately I micromole per kilogram weight.

The results of the studies in NFkB luciferase reporter mice are shown in FIGURES 1-8; FIGURES 1-7 are photographs generated by the IVIS Lumina 3D Optical in vivo imaging system, and FIGURE 8A-B are bar graphs summarizing the results. Bay- 11-7082 was used as a positive control for TLR4 inhibitory activity. These results indicate that, at the concentration tested, with the exception of compound 2, the other putative TLR inhibitors 1 and 3-6 all inhibited LPS-induced

NY02:723955.1 18 NFkB activation to some extent, with compound 4 having the greatest inhibitory activity in this assay.

The results of the study of compounds 3-65 in the CFW endotoxemia model are presented in TABLE 2 below. Less activity, tachypnea, and piloerection are considered symptomatic of endotoxemia, so a decrease in these is consistent with TLR inhibition by the administered compound. Note that compunds listed as negatives may still be T4Ics at other concentrations or conditions.

TABLE 2.

NY02:723955.1 19 25 More piloerection, less mobile than other in the cage

31 More tachypneic, more piloerection

32 More tachypneic, more piloerection

37 More tachypneic, less active

38 Less active

40 Less active, huddled in corner

41 Less active, huddled in corner

62* Nearly static, little movement, rigors

* Indicates most profound phenotypic differences from each group (positive or negative).

The results of the ELISA study showing IL6 production are presented in FIGURE 9.

7. EXAMPLE 2

Experiments were performed to further explore the activity of certain "positive" TLR4 inhibitors described in TABLES 1 and 2.

In a first set of experiments, NFKB luciferase reporter mice, as described above, in which NFKB is downstream of the luciferase gene, were treated with either (i) saline (FIGURE 10A, animal on the left); (ii) LPS alone (2 mg/kg; FIGURE 10A, animal on the right)); (iii) LPS plus C16 ((lmg/kg, intraperitoneal (i.p.) injection); FIGURE 10B, animal on the left) or (iv) LPS plus C34 ((l mg/kg, i.p. injection); FIGURE 10Β, animal on the right). As the amount of luciferase reflects NFKB signalling, the imaging studies indicate that both compounds CI 6 and C34 reduced NFKB signalling.

Inducible nitric oxide synthase (iNOS) is associated with necrotizing enterocolitis (NEC) in human infants. In a second set of experiments, the CFW-NEC mouse model system for NEC was used to evaluate the effect of various compounds from TABLES 1 and 2 above on iNOS mRNA levels. A model system for NEC was induced in newborn mouse pups by formula feeding, where control animals were breast-fed (Sodhi et al., 2010, Gastroenterol. 138(11:185-196: Richardson et al., 2010, Gastroenterol. 139£3}:904-917). As shown in FIGURE 11, the level of iNOS was substantially increased in the formula-fed (FF) group. This increase was much less when FF pups were also administered C27 (lmg kg, i.p., once daily for the first four days of the five day model) and the increase was almost eliminated by C34 (lmg/kg, i.p., once daily for the first four days of the five day model). Note that RPLO stands for 50S ribosomal subunit protein LI 5, an acknowledged housekeeping gene, which

NY02:723955. ! 20 means that it is a constitutive gene expressed at relatively constant levels by all cells and is thus used as a reference for comparing other genes which may vary under experimental conditions. In this same model system, levels of TLR4 mRNA levels were also evaluated. The level of TLR4 mRNA seen in breast-fed animals, including breast-fed animals treated with C34, was substantially increased in the FF animals. In contrast, the level of TLR4 was not increased in FF animals treated with C34

(Img/kg, i.p. once daily for the first four days of the five day model) and indeed mRNA levels were 30 percent below normal (see FIGURE 14).

The effect of C34 was then tested on explants of human NEC tissue. These explants were obtained from human infants suffering from NEC and were prepared from NEC-affected portions of the intestine. When explants were treated with LPS (25ug/ml, 37°C, 3 hours) the level of TNFa mRNA (FIGURE 12) and iNOS mRNA (FIGURE 13) increased precipitously. These increases were substantially reduced by treatment with C34 (lOuM, tissue was pretreated for 30 minutes prior to LPS being added; C34 was not removed and remained in solution during incubation with LPS; FIGURES 12 and 13).

All the foregoing data supports the use of compounds C34, C16 and C27 as TLR4 inhibitors, as inhibitors of inflammation, and as inhibitors and agents for treatment of NEC.

8. EXAMPLE 3

An experiment was performed to test the effect of TLR4 inhibitors and C34 in particular on the tissue damage associated with hemmorhagic shock. A murine model was used in which, under sterile conditions and anesthesia induced using i.p. sodium pentobarbital (20mg/kg), a left groin exploration was performed, and the left femoral artery was cannulated with tapered polyethylene (PE)-10 tubing and connected to a blood pressure transducer for continuous mean arterial pressure (MAP) monitoring for the duration of the experiment (6 h) as described in Sodhi, et al., 2011 , Am. J. Physiol. Gastrointest. Liver Physiol. 300f5):G862-G873. To induce hemorrhagic shock, blood was withdrawn to allow the mean arterial pressure to drop to 25 mmHg over 5 minutes, and the blood pressure was maintained at this level for 150 min. The mice were then resuscitated over 0 min with lactated Ringer's solution. Sham-operated mice underwent anesthesia and femoral cannulation only. In this model system, the extent of liver damage is reflected in increased serum levels of

NY02:723955. I 21 aspartate aminotransferase (AST) and alanine transaminase (ALT). As shown in FIGURE 15A-B, administration of C34 (lmg/kg, i.p. divided in two doses with one given 30 minutes prior to H/S and the second given immediately prior to

resuscitation) inhibited the increases in AST (FIGURE 15A) and ALT (FIGURE 15B) observed in H/S animals receiving phosphate buffered saline (PBS), supporting the effectiveness of compound C34 in reducing the extent of tissue damage/injury associated with trauma and/or shock.

9. EXAMPLE 4

Synthesis of Isopropyl 3,4,6-tri-0-acetyl-2-(acetylamino)-2- deoxyhexopyranoside (C34)

2-Acetamido-2-deoxy-l,3 ₅ 4,6-tetra-0-acetyI-C-D-glucopyranose. A dry, 1 OOmL round-bottom flask was equipped with a Teflon coated stir bar, septum and an ice-salt bath. The flask was put under an argon atmosphere and acetic anhydride (8.40 g, 7.76 mL, 82.27 mmol) which was stored in the freezer at 5 °C was added via syringe. The acetic anhydride was cooled for 15 min at 0 °C. After 15 min, the septum was replaced with a funnel and N-acetyl-D-glucosamine (0.692 g, 3.13 mmol) and montmorillonite K-10 (2.4 g) were added sequentially and slowly over 15 min. The stopper was then replaced the ice bath removed and the reaction was stirred for 24 hours. The reaction mixture was then filtered through a medium porosity sintered glass funnel precoated with a pad of celite moistened by methyl acetate. The flask and filtered solids were rinsed with methyl acetate (100 mL) and the combined filtrate was concentrated under rotary evaporation (40 °C). The resulting orange residue was dislodged with a spatula and twice recrystallized from hot methanol (2mL) over 24 h in an explosion proof freezer. The solution was decanted by pipette and the crystals were rinsed with ice-cold diethyl ether (3 χ 2 mL) to afford 2- Acetamido-2-deoxy-l,3,4,6-tetra-0-acetyl-B-D-glucopyranose as a white crystal solid (350.2 mg, 29%). 1H-NMR (400 MHz, CDC1 ₃ ): δ 5.69 (d, J = 8.8 Hz, 1H), 5.41 (d, J = 9.6 Hz, 1H), 5.17-5.09 (m, 2H), 4.33-4.25 (m, 2H), 4.13 (dd, J = 2.4, 12.6 Hz, 1H), 3.78 (ddd, J = 2.4, 4.4, 9.6 Hz, 1H), 2.12 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H). Note: Commercially available through Alfa Aesar (Cat. No. = L09020). See Knapp et al., 2009, Organic Synthesis 84:68-76.

NY02:723955. I 22 2,3-Dihydrooxazole-3,4,6-tri-0-acetyl-fi-D-glucopyranoside. 2-

Acetamido-2-deoxy-l ,3,4,6-tetra-0-acetyl-B-D-glucopyranose (299 mg, 0.768 mmol) was dissolved in dichloro ethane (21.3 mL, 0.036 M) in a 100 mL round bottom flask and then trimethylsilyl trifluoromethanesulfonate (TMSOTf, 0.149 mL, 0.806 mmol, 1.05 eq) was added. The mixture was stirred at 50 °C for 55 min after which TLC (100% EtOAc) indicated full conversion. The mixture was then removed from the heat and triethylamine (0.327 mL, 2.30 mmol, 3 eq) was added. The mixture was then stirred at room temperature for 10 min and then passed through a short plug of silica which was washed carefully with dichloromethane (25 mL) and EtOAc (15 mL). The solvent was removed under reduced pressure and the crude orange oil was purified by flash chromatography with 100% EtOAc (the column was based washed with 1% triethylamine in EtOAc prior to use) giving 2,3-Dihydrooxazole-3,4,6-tri-0- acetyl-B-D-glucopyranoside (238.2 mg, 94%) as a colorless oil. ¹ H-NMR (500 MHz, CDC1 ₃ ): δ 5.96 (d, J = 7.5 Hz, 1H), 5.25 (app. t, J = 2.5 Hz, 1H), 4.92 (dq, J = 1.5, 9.3 Hz, 1H), 4.17-4.11 (m, 3H) ₃ 3.61-3.56 (m, 1H), 2.10 (s, 3H), 2.09 (s, 3H), 2.08 (d, J = 2 Hz, 3H), 2.07 (s, 3H). ¹³ C-NMR (100 MHz, CDC1 ₃ ): δ. 170.6, 169.5, 169.2, 166.7, 99.4, 70.4, 68.4, 67.5, 65.0, 63.3, 20.9, 20.8, 20.7, 14.0. See Norberg et al., 2011, Analytical Chem. 83: 1000-1007. Isopropyl 3,4,6-tri-0-acetyI-2-(acetylamino)-2- deoxyhexopyranoside. 2,3-Dihydrooxazole-3,4,6-tri-0-acetyl- β-D-glucopyranoside (0.140 g, 0.424 mmol) and anhydrous CuCl ₂ (57 mg, 0.424 mmol, 1 eq) were coevaporated with toluene (Note: some material was lost due to the mixture bumping on the rotovap and this is reflected in the yield). Anhydrous chloroform (0.80 mL, 0.53 M) and anhydrous 2-propanol (0.13 mL, 1.72 mmol, 4.05 eq) were added to the sugar and CuCl ₂ in a 5 mL conical sealed vessel under argon atmosphere and the reaction mixture was heated at 62 °C for 2.25 hr. After cooling to room temperature, the mixture was diluted with acetone (15 mL) and saturated aqueous sodium bicarbonate (7 mL) and the precipitated copper carbonate salts were removed by filtration through a short plug of celite which was washed with acetone (20 mL). The filtrate was removed and the residue coevaporated with toluene to remove residual water. The remainder was shaken with chloroform and weakly acidic ion-exchange resin (Amberlite IRC-86, ca 1.5 g) in order to remove remaining sugar-oxazoline starting material. Filtration, evaporation and flash chromatography (hexane/efhyl

NY02:723955. 1 23 acetate, 1 :3, Rf = 0.24) gave isopropyl 3,4,6-tri-0-acetyl-2-(acetylamino)-2- deoxyhexopyranoside (80.3 mg, 49%) as a white solid. Ή-NMR (400 MHz, CDC1 ₃ ): δ 5.57 (d, J = 8.0 Hz, 1H), 5.40 (dd, J = 9.6, 10.4 Hz, 1H), 5.02, (app. t, J = 9.6 Hz, 1H), 4.83 (d, J = 8.0 Hz, 1H), 4.24 (dd, J = 4.8, 12.2 Hz, 1H), 4.1 1 (dd, 2.4, 12.0 Hz, 1 H), 3.92 (sept, J - 6Hz, 1H), 3.73-3.61 (m, 2H), 2.07 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.93 (s, 3H), 1.22 (d, J = 6.4 Hz, 3H), 1.13 (d, J = 6.0 Hz, 3H). ¹³ C-NMR (100 MHz, CDC1 ₃ ): δ 170.7, 170.7, 170.2, 169.5, 99.18, 72.6, 72.2, 71.6, 69.0, 62.4, 55.6, 23.3, 23.3, 22.0, 20.7, 20.7, 20.6. HRMS (+ESI-TOF) calcd for Ci ₇ H ₂₇ N0 ₉ Na

[M+Na]: 412.1584. Found: 412.1555. See Wittmann et al., 2002, Eur. J. Org. Chem. 8: 1363-1367.

Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.

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