WO2020007760A1

WO2020007760A1 - Tlr4 compounds or pharmaceutically acceptable salts thereof, corresponding pharmaceutical compositions or formulations, methods of preparation, treatment or uses

Info

Publication number: WO2020007760A1
Application number: PCT/EP2019/067513
Authority: WO
Inventors: Sabyasachi Bhattacharya; Gautam Dalwadi; Rajendra S SATHE; Rahul Parashar Shukla; Haoyan ZHOU
Original assignee: GlaxoSmithKline Intellectual Property Development Ltd
Current assignee: GlaxoSmithKline Intellectual Property Development Ltd
Priority date: 2018-07-03
Filing date: 2019-07-01
Publication date: 2020-01-09
Anticipated expiration: 2021-01-03

Abstract

The present invention relates to novel pharmaceutically acceptable salts of TLR4 agonist compounds and forms thereof, corresponding pharmaceutical compositions or formulations, processes or methods of compound preparation and uses or treatment methods for cancers, which includes associated cancer immunotherapies (i.e., also known as immuno-oncology).

Description

TLR4 COMPOUNDS OR PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF, CORRESPONDING PHARMACEUTICAL COMPOSITIONS OR FORMULATIONS,

METHODS OF PREPARATION, TREATMENT OR USES

FIELD OF THE INVENTION

BACKGROUND TO THE INVENTION

Toll-like receptors (TLRs) are a family of cell surface proteins primarily expressed on immune and epithelial cells that function as activators of innate immunity in response to microbial-related molecules known as Pathogen Associated Molecular Patterns (PAMPs). PAMPs include molecules, such as nucleic acids, flagellar proteins and lipopolysaccharide (LPS), the natural ligand for TLR4. TLR4 engagement results in the production of various inflammatory cytokines/chemokines, such as tumour necrosis factor alpha (TNFa), interleukin 6 (IL-6), granulocyte colony stimulating factor (GCSF), and type I interferons (IFNa, IFNP) and enhanced uptake, processing and presentation of antigens.

Accordingly, TLR4 agonists are being developed for a variety of therapies in which immune modulation is desirable, such as for vaccine adjuvants, treatments for allergy and asthma, chronic viral infections, and cancer. The TLR4 agonist may be administered in combination with other therapeutically active compounds for these treatments.

Activation of TLR4 forms an important bridge between innate and adaptive immunity by regulating the tumour microenvironment to reduce the immune suppression to anti-tumor immunity. TLR4 induces innate immunity via an intracellular signalling cascade in a MyD88 (an intracellular adaptor moleculej-dependent or -independent manner. Aminoalkyl glucosaminide phosphates (AGPs) are synthetic ligands of TLR4. Monophosphoryl lipid A (MPLA) is also a Toll-like receptor 4 agonist commonly used as a nontoxic, FDA approved adjuvant in vaccines.

Thus, there still exists a need for:

• development of novel pharmaceutically acceptable salts or forms of TLR4 agonist compounds, corresponding pharmaceutical compositions or formulations, where:

o such novel pharmaceutically acceptable salts or forms include, but are not limited to solvates, amorphous, semi-crystalline, mesophase semi-crystalline amorphous forms and the like;

• stable, aqueous solution for injection(s) of a TLR4 agonist with high biological activity and high physical stability for an extended period of time, as well as methods of treating cancer in a patient with same; • preparations of such pharmaceutical compositions or formulations via a dispensing vehicle; and

• development of such pharmaceutically acceptable salts or forms thereof of TLR4 agonist compounds to be used as immunological adjuvants in combination with immune system modulators for uses or treatment methods for cancers.

SUMMARY OF THE INVENTION

The present invention relates to a stable, aqueous solution for injection of a TLR4 active agent, pharmaceutically acceptable salts and forms thereof with high biological activity and high physical stability for an extended period of time.

In an aspect of the invention, there is provided a compound which is a 2-aminoethanol salt of a compound of Formula (III):

A specific pharmaceutically acceptable salt of the invention includes, but is not limited to (S)- 2-((R 3-(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R)-3-(decyloxy)tetradecanamido)-4- R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2- yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., the tris(2-aminoethanol) salt of the compound of Formula III and Compound 9 herein).

In another aspect of the invention, there is provided a 1 ml. unit dose pharmaceutical formulation comprising: a. about 0.0005 mg of a compound of Formula (III) or a 2-aminoethanol salt thereof, optionally the tris(2-aminoethanol) salt;

b. about 0.0005 ml. of dehydrated alcohol, optionally ethanol;

c. about 44.0 mg of dextrose monohydrate;

d. about 1 mg of P188;

e. sodium hydroxide and/or hydrochloric acid, if necessary, to adjust the pH of the formulation to about 7.0; and

f. water for injection to reach 1 ml_.

In a further aspect of the invention, there is provided a kit of parts comprising a compound or pharmaceutical formulation or composition as disclosed herein and instructions for use.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

The terms "a" and "an", as used herein, refer to "one or more" or "at least one" of the recited components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural, unless specifically stated otherwise.

An "acid", as used herein, is a substance that yields hydrogen ions in aqueous solution. A "pharmaceutically acceptable acid", as used herein, includes inorganic and organic acids which are nontoxic at the concentration and manner in which they are formulated.

The terms, "administering", and "administration", are used herein to mean any method, which, in sound medical practice, delivers the active agent or pharmaceutical composition thereof to a patient in such a manner as to provide the desired therapeutic effect.

The term "active agent" is defined for purposes of the present invention as any chemical compound or pharmaceutically acceptable salt thereof, pharmaceutical composition of the present invention, which can be delivered into an environment of use to obtain a desired result.

A "base", as used herein, is a substance that yields hydroxyl ions in aqueous solution. Pharmaceutically acceptable bases include inorganic and organic bases which are non-toxic at the concentration and manner in which they are formulated.

The term, "biological products", as used herein, means a category of products that are generally large, complex molecules. These products may be produced through biotechnology in living cells or systems, such as in microorganisms, plant cells, and animal cells. Examples of biological products include, for example, monoclonal antibodies, therapeutic proteins, and vaccines.

The term "buffering agent" refers to at least one component that, when added to an aqueous solution is able to protect the solution against variations in pH when adding acid or alkali, or upon dilution with a solvent. In addition to phosphate buffers, there can be used glycinate, carbonate, citrate buffers and the like, in which case, sodium, potassium or ammonium ions can serve as counterion. A "drug product" refers to the final formulation packaged in a container that may be reconstituted before use, such as with a lyophilized drug product; diluted further before use, such as with a liquid drug product; or utilized as is, such as with a solution drug product. Alternatively, the drug product can be the packaging as a final product of the drug in a liquid state.

A "drug substance", as used herein, refers to the starting material utilized in formulation of the final drug product.

The phrase an "effective amount" is synonymous with a "therapeutically effective amount" defined herein.

An "extended period of time" with respect to stability means about 12 to about 36 months' time period. Stability can include both physical and chemical stability. For example, the extended period of time may be a 24 months' shelf life kept at 2-8°C. Alternatively, the extended period of time can include at least a 24 months' shelf life under International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.

A "formulated bulk solution", as used herein, refers to the final formulation prior to filling of the container, such as the formulated solution, prior to filling the vials for lyophilization, or the formulated solution prior to filling a syringe for injection.

"Free" of a specified component refers to a composition where the specified component is absent.

The term "high biological stability", as used herein, refers to the AGP used in formulations of the present invention and that the AGP maintains at least 80%, 85%, 95%, or 100% of its biological activity upon storage at 2-8 °C for a period of about 12 to about 36 months, in a constituted or non- reconstituted state.

The term "high chemical stability" as used herein, refers to the AGP used in formulations of the present invention, and that the AGP maintains at least 80%, 85%, 95%, or more of its chemical integrity upon storage at 2- 8 °C for a period of about 12 to about 36 months, in a constituted or non- reconstituted state.

The term "high physical stability", as used herein, refers to the AGP used in formulations of the present invention, and that formulations of the present invention remain, clear, do not exhibit visible particular matter upon storage at 2 to about 8°C for a period of at least 21 months, 24 months, or up to 36 months' time. A Particulate Matter Test, as is well known in the art, is performed to confirm that particles are not increasing.

The term, "HLB", as used herein stands for "hydrophile/lipophile balance". HLB can be used to predict the surfactant properties of a molecule.

The term, "hypertonic", as used herein, describes a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. As used herein, the term, "iso-osmotic" refers to a solution that has the same concentration of water as to whatever solution to which it is being compared.

An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsmol/Kg H2O. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.

The term "lipopolysaccharide (LPS)" means purified and/or synthetic LPS. LPS is a constituent of the outer membrane of the wall of Gram-negative bacteria such as Neisseria spp. and Haemophilus spp. LPS comprises a lipid portion (lipid A) that is covalently bonded to a polysaccharide portion. Lipid A consists of two glucosamine units with attached acyl chains, for example, 3, 4, 5, 6, 7, 8 or 10 acyl chains. LPS is also known as lipoglycans and includes low-molecular weight forms of bacterial LPS referred to as lipooligosaccharide (LOS). For example, LPS can be obtained from Sigma (cat¹ L4516; E. coil 0127 :B8).

The term "mixture", as used herein, means a composition comprising at least two compounds. In one aspect or embodiment, a mixture is a mixture of at least two distinct compounds. In a further aspect or embodiment, when a compound is referred to as a "mixture", it means that it can comprise at least two "forms" of the compounds, such as, salts, solvates, or, where applicable, stereoisomers of the compound in any ratio. A person of skill in the art would understand that a compound in a mixture can also exist as a mixture of forms. For example, a compound may exist as a hydrate of a salt. All forms of the compounds disclosed herein are within the scope of the present disclosure.

As used herein, "patients" includes human patients, including adult, teens and children (e.g., paediatric patients). A paediatric patient, under FDA guidelines, can include individuals under the age of 21. A child for purposes herein is under the age of 12.

As used herein "a pH-adjusting agent", a "buffering agent" or "buffer" and grammatical variations thereof, means any component of a solution that can act to maintain the pH of a liquid composition or formulation.

The term, "pharmaceutically acceptable" as used herein, means approvable by a regulatory agency or listed in a Pharmacopeia, or other generally recognized guide for use in animals, and more particularly in humans.

The term "pharmaceutically acceptable salt thereof" as used herein, refers to salts that are safe and effective for use in the patient and possess the desired pharmaceutical activity. Such salts include salts formed when an acidic proton is replaced with a metal ion (e.g., alkali metal ion, alkaline earth metal ion, or aluminium ion).

As used herein, the term "polyol" refers to any sugar alcohol. Examples of polyols include, but are not limited to, mannitol, maltitol, sorbitol, xylitol, erythritol, and isomalt. Sugar alcohols may be formed under mild reducing conditions from their analogue sugars. A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized formulation in an aqueous carrier such that the TLR4 agonist, or pharmaceutically acceptable salt thereof, is dissolved in the reconstituted formulation. The reconstituted formulation is suitable for intravenous administration (IV) to a patient in need thereof.

As used herein, a "saccharide" means a tonicity agent that is pharmaceutically acceptable for use in injectables. A saccharide includes, but is not limited to, a disaccharide, monosaccharide or polysaccharide. The term, "sugar", can be used to refer to all saccharides. A disaccharide can be, for example, sucrose or trehalose, or a mixture thereof. A saccharide or a sugar can also serve as a stabilizing agent in the liquid compositions or formulations of the present invention.

"Substantially free" of a specified component refers to a composition with less than about 1% by weight of the specified component.

As used herein, a "surfactant", refers to any component that lowers the surface tension of the liquid composition. Surfactants can be anionic, ionic or non-ionic.

The term "tonicity" as used herein, refers to a measure of the effective osmotic pressure gradient, as defined by the water potential of two solutions separated by a semi-permeable membrane. In other words, tonicity is the relative concentration of solutes dissolved in solution, which determines the direction and extent of diffusion of such solutes.

A "tonicity agent", as used herein, is an agent that adjusts the tonicity of solutes in a solution. The combination of an aqueous solution or carrier, and a water-soluble organic solvent, sometimes referred to as a co-solvent, and at least one surfactant is often used in injectable formulations.

The phrase, "therapeutically effective amount", as used herein, refer to an amount of the active ingredient sufficient to have a therapeutic effect upon administration, e.g., that amount which will cause an improvement or change in the condition for which it is applied when applied to the affected area repeatedly over a period of time. Effective amounts will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the stage of advancement of the condition, the body surface area affected with the clinical condition, and the specific components of the composition. An effective amount of the active ingredient for treatment of a condition or disorder can be determined by standard clinical techniques. The exact amount required will vary from subject to subject depending on factors such as the patient's general health, the patient's age, etc. Appropriate amounts in any given instance will be readily apparent to those skilled in the art or capable of determination by routine experimentation. Thus, "an effective amount" is that amount which shows a response over and above the vehicle or negative controls.

The terms "treatment" or "treating" of a condition or disorder encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or the delay, prevention or inhibition of the progression thereof. Treatment need not mean that the condition or disorder is totally cured. Further, a treatment need not be effective in every member of a population. Other terms used herein are intended to be defined by their well well-known meanings in the art.

STATEMENT OF THE INVENTION

In general, aminoalkyl glucosaminide phosphates (AGPs) are synthetic ligands of Toll-like Receptor 4 (TLR4). AGPs are known to be useful as vaccine adjuvants and for stimulating cytokine production, activating macrophages, promoting innate immune response, and augmenting antibody production in immunized animals.

There are several variations of suitable AGP compounds that may be employed in the present invention. Compounds of Formula I and la are disclosed herein and may be made by a process as disclosed in US 7,960,522 and US 6,303,347, the disclosures of which are incorporated by reference in their entirety herein.

Suitable for use in the present invention are novel pharmaceutically acceptable salts of TLR4 agonist compounds or forms thereof generically encompassed by Formulas (I) or (II), respectively (i.e., including Formulas (la) and (Ila), respectively) and more specifically encompassed by Formulas (III) to (VI), respectively, as defined below.

Suitable pharmaceutically acceptable salts of compounds of Formula (I) used in the present invention are as set forth below:

(Formula I)

where:

m is 0, or an integer of 1 to 6;

n is 0, or an integer from 1 to 4;

X is O or S, preferably O;

Y is O or NH; Ri, R2, 3 are each independently selected from a C1-20 acyl or a C1-20 alkyl;

R₄ is H or Me;

Rs is selected from H, -OH, -C1-4 alkoxy, -P(0)(ORs)(OR9), -OP(0)(ORe)(OR9), -SO3R8, -OSO3R8,

-NR8R9, -SRs, -CN, -NO2, -CHO, -CO2R8, and -CONR8R9,

Rs and R9 are each independently selected from H and C1-4 alkyl; and

R6 and R7 are each independently selected from H or P(0)(OH)2.

In Formula I, the configuration of the 3' stereogenic centres to which the normal fatty acyl residues (that is, the secondary acyloxy or alkoxy residues, e.g., RiO, R2O, and R3O) are attached is in the R or S configuration.

Stereochemistry as applied to compounds of the present invention are defined by Cahn- Ingold-Prelog priority rules as conventionally known in the chemical arts. Configuration of aglycon stereogenic centres to which the R₄ and Rs are attached can be either R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the present invention.

The number of carbon atoms between heteroatom X and the aglycon nitrogen atom is determined by the variable "n", which can be 0 or an integer from 0 to 4. In one aspect or embodiment, n is 0 or an integer of 1 or 2.

Suitably, Ri, R2, R3 are each independently selected from a C 1-20 acyl or a C 1-20 alkyl. In one aspect or embodiment, the chain length of Ri, R2, and R3 can be from about 6 to about 16 carbons. In another aspect or embodiment, the chain length is from about 9 to about 14 carbons. In another aspect or embodiment, the chain lengths of Ri, R2 and R3 are 6, 10, 12 or 14. In yet another aspect or embodiment, the chain lengths of Ri, R2 and R3 are each 10. The chain lengths of Ri, R2, and R3 can be the same or different.

Formula I encompasses L/D-seryl, -threonyl, -cysteinyl ether and ester lipid AGPs, both agonists and antagonists and their homologs (wherein n=l-4), as well as various carboxylic acid bioisosteres (/.e., when Rs is an acidic group capable of salt formation; the phosphate can be either on 4- or 6- position of the glucosamine unit, preferably, is in the 4-position).

Suitable pharmaceutically acceptable salts of compounds of Formula (la) (i.e., a subgeneric AGP compound of Formula I, where n is 0, m is 0, Rs is CO2H, R₆ is P(0)(OH)2, and R7 is H) used in the present invention are as set forth below:

(Formula la)

where:

X is 0 or S; Y is 0 or NH;

each Ri, R2, and RB are independently selected from a C1-20 acyl or a C1-20 alkyl; and

R₄ is H or methyl.

Chain lengths of variables of Ri, R2, and R3 are defined as follows. In one aspect, the chain length of Ri, R2, and R3 can be from about 6 to about 16 carbons. In another aspect or embodiment, the chain length is from about 9 to about 14 carbons. In another aspect or embodiment, the chain lengths of Ri, R2 and R3 are 6, 10, 12 or 14. In yet another aspect or embodiment, the chain lengths of Ri, R2 and R3 are each 10. The chain lengths of Ri, R2, and R3 can be the same or different.

In Formula la, the configuration of the 3' stereogenic centres to which the normal fatty acyl residues (that is, the secondary acyloxy or alkoxy residues, e.g., RiO, R2O, and R3O) are attached is in the R or S configuration. In one aspect or embodiment, the configuration is R (as designated by Cahn-Ingold-Prelog priority rules). Configuration of aglycon stereogenic centres to which R₄ is attached can be either R or S.

All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the present invention.

Formula la encompasses L/D-seryl, -threonyl, -cysteinyl ether or ester lipid AGPs, both agonists and antagonists.

Suitable pharmaceutically acceptable salts of compounds of Formula (II) used in the present invention are as set forth below:

(Formula II)

where:

m is 0, or an integer of 1 to 6

n is 0, or an integer from 1 to 4;

X is 0 or S, preferably 0;

Y is 0 or NH;

Ri, R2, RB are each independently selected from a C1-20 acyl or a C1-20 alkyl;

R₄ is H or Me;

Rs is selected from H, -OH, -C i-₄ alkoxy, -P(0)(0Rs)(0R9), -0P(0)(0Rs)(0R9), -SO3R8, -OSO3R8, -NR8R9, -SRs, -CN, -NO2, -CHO, -CO2R8, and -CONR8R9,

Rs and R9 are each independently selected from H and C 1-4 alkyl; and

R6 and R7 are each independently selected from H or P(0)(0H)2.

In Formula II, the =0 adjacent to the Y moiety in Formula (I) is now a carbon with two hydrogen atoms.

Suitable pharmaceutically acceptable salts of compounds of Formula (Ila) (a subgeneric formula encompassed by Formula (II) used in the present invention are as set forth below:

(Formula Ila)

where: X is 0 or S; Y is 0 or NH; each of Ri, R2, and 3 are independently selected from a C1-20 acyl or a Ci-20 alkyl; and R₄ is H or methyl.

In one aspect or embodiment, the present invention relates to novel pharmaceutically acceptable salts or forms thereof of the following compounds of Formulas (III) to (VI) as shown below:

)

)

Pharmaceutically acceptable salts

Because of their potential use in medicine, the salts of the compounds of the present invention respectively, are preferably pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse J.Pharm.Sci. (1977) 66, pp 1-19.

When a compound of the invention is a base (contain a basic moiety), a desired salt form may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid or the like.

Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-1,6- dioates, benzoates, chlorobenzoates, methyl benzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, phenylacetates, phenylpropionates, phenylbutrates, citrates, lactates, g-hydroxybutyrates, glycollates, tartrates mandelates, and sulfonates, such as xylenesulfonates, methanesulfonates, propanesulfonates, naphthalene-l-sulfonates and naphthalene-2-sulfonates.

If an inventive basic compound is isolated as a salt, the corresponding free base form of that compound may be prepared by any suitable method known to the art, including treatment of the salt with an inorganic or organic base, suitably an inorganic or organic base having a higher pKa than the free base form of the compound.

When a compound of the invention is an acid (contains an acidic moiety), a desired salt may be prepared by any suitable method known to the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary), an alkali metal or alkaline earth metal hydroxide, or the like.

Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as ethylene diamine, dicyclohexylamine, ethanolamine, piperidine, morpholine, and piperazine, as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminium, and lithium.

Certain of the compounds of this invention may form salts with one or more equivalents of an acid (if the compound contains a basic moiety) or a base (if the compound contains an acidic moiety). The present invention includes within its scope all possible stoichiometric and non-stoichiometric salt forms. As such, compounds of this invention may form salts wherein there is one equivalent of counterion per equivalent of compound or more than one equivalent of counterion per equivalent of compound or less than one equivalent of counterion per equivalent of compound or may represent a mixture of species containing varying amounts of counterion per equivalent of compound. For example, compounds of this invention may form salts wherein there are two equivalents of counterion per equivalent of compound or three equivalents of counterion per equivalent of compound or may represent a mixture of species wherein some salt molecules contain one equivalent of counterion per equivalent of compound and others contain two equivalents of counterion per equivalent of compound and others contain three equivalents of counterion per equivalent of compound. Non-integer amounts of counterion per equivalent of compound are also included within the scope of this invention. Because the compounds of this invention may contain both acid and base moieties, pharmaceutically acceptable salts may be prepared by treating these compounds with an alkaline reagent or an acid reagent, respectively. Accordingly, this invention also provides for the conversion of one pharmaceutically acceptable salt of a compound of this invention, e.g., a hydrochloride salt, into another pharmaceutically acceptable salt of a compound of this invention, e.g., a sodium salt or a disodium salt.

Carboxylate functional groups of compounds of the present invention have coordinated mono or di-valent cations, where such cations may include, but are not limited to alkali metals, which may include, but are not limited to lithium (Li), sodium (Na), potassium, or mixtures thereof and the like.

Quaternary amine functional groups of compounds of the present invention, which are positively charged species, also may have coordinated anions, where such anions may include, but are not limited to halogens, which may include, but are not limited to chlorides, fluorides, bromides, iodides and the like.

Compounds or pharmaceutically acceptable salts of the present invention may also form a zwitterion(s) (formerly called a dipolar ion), which is a neutral molecule with a positive and a negative electrical charge (i.e., not dipoles) at different locations within that molecule. Zwitterions are sometimes also called inner salts.

In one aspect or embodiment, the present invention relates to a 2-aminoethanol salt of the TLR4 agonist compounds of the present invention as defined above. In another aspect or embodiment, the present invention relates to a tris(2-aminoethanol) salt of the TLR4 agonist compounds of the present invention as defined above.

Specifically, in another aspect or embodiment, a pharmaceutically acceptable salt of compounds or forms thereof for use in the present invention is selected from:

(S)-2-((R 3-(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R)-3- (decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., the tris(2-aminoethanol) salt of Formula III, and Compound 9 herein); and

(2S)-2-((R 3-(decanoyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R)-3- (decanoyloxy)tetradecanamido)-4-(((R)-3-(decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., the tris(2-aminoethanol)salt of Formula IV herein).

In another aspect or embodiment, the TLR4 agonist compound for use in the present invention is (S)-2-((R>3-(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R>3-((R)-3-

(decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt. In another aspect or embodiment, the TLR4 agonist compound for use in the present invention is (2S)-2-((R>3-(decanoyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R>3-((R)-3-

(decanoyloxy)tetradecanamido)-4-(((R)-3-(decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt.

Polymorphs

Polymorphism is a common phenomenon of crystalline materials. It describes the ability of a substance to exist as two or more crystalline phases that have different arrangements of the molecules in the solid state but are otherwise identical in terms of chemical content. It should be noted that 'arrangement' here includes not just the packing and orientation of molecules that may differ, but also their conformations - even if the packing is similar, if two crystals just differ in terms of the molecular conformations adopted then they are polymorphic.

When polymorphism exists as a result of a difference in crystal packing, it is called packing polymorphism. Polymorphism can also result from the existence of different conformers of the same molecule in conformational polymorphism.

In pseudopolymorphism the different crystal types are the result of hydration or solvation. This is more correctly referred to as solvomorphism as different solvates have different chemical formulae. An analogous phenomenon for amorphous materials is polyamorphism, when a substance can take on several different amorphous modifications.

Polymorphs have different stabilities and may spontaneously convert from a metastable form (unstable form) to the stable form at a particular temperature. They also exhibit different melting points, solubilities (which affect the dissolution rate of drug and consequently its bioavailability in the body), X-ray crystal and diffraction patterns.

Various conditions in the crystallisation process is the main reason responsible for the development of different polymorphic forms. These conditions include:

• Solvent effects (the packing of crystal may be different in polar and nonpolar solvents);

• Certain impurities inhibiting growth pattern and favour the growth of a metastable polymorphs;

• The level of supersaturation from which material is crystallised (in which generally the higher the concentration above the solubility, the more likelihood of metastable formation);

• Temperature at which crystallisation is carried out;

• Geometry of covalent bonds (differences leading to conformational polymorphism); and/or

• Change in stirring conditions.

The term polymorphism should not be confused with the notion of solvates, which are crystalline forms containing solvent molecules as well as the main compound(s) (nb. conventionally when the solvent is water, one refers to these as the hydrates). An unsolvated and a solvated crystalline form are distinct from each other in terms of chemical content and therefore are not polymorphs of each other. Nonetheless, of course, it is possible that solvates can exhibit polymorphism in their own right, by different arrangements of their constituent molecules in the crystal structures. In a crystallization screen developed primarily to explore polymorphism it is not uncommon to also observe a variety of solvates, so it is important to distinguish these from true polymorphism during any structural analysis.

Solvates and hydrates

For solvates of the compounds or pharmaceutically acceptable salts thereof of the present invention that are in crystalline form, the skilled artisan will appreciate that pharmaceutically- acceptable solvates may be formed wherein solvent molecules are incorporated into the crystalline lattice during crystallization.

Solvates may involve nonaqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethyl acetate, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent that is incorporated into the crystalline lattice are typically referred to as "hydrates."

Hydrates include stoichiometric hydrates as well as compositions or formulations containing variable amounts of water. The invention includes all such solvates of compounds or pharmaceutically acceptable salts of the present invention.

In one aspect or embodiment, novel pharmaceutically acceptable salt compounds or forms of the present invention exist in a solvate or hydrate form.

Forms

The present invention relates to novel pharmaceutically acceptable salts of TLR4 agonist compounds and forms thereof, corresponding pharmaceutical compositions or formulations, where such forms include, but are not limited to solvates, amorphous, semi-crystalline, mesophase semi crystalline amorphous forms and the like.

In another aspect or embodiment, the TLR4 agonist is a solvate, amorphous, semi-crystalline, mesophase semi-crystalline amorphous form and the like of (SJ^-ffRJ- - decyloxyJtetradecanamido)- 3-((Y2R,3R,4R,5S,6R>3-((R)-3-(decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)- 6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2- aminoethanol) salt or 2S 2-((R)-3-(decanoyloxy)tetradecanamido)-3- ( 2R,3R,4R,5S,6R)-3-((R)-3- (decanoyloxy)tetradecanamido)-4-(((R)-3-(decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt. In a further aspect or embodiment, the form is semi-crystalline or a mesophase.

Deuterated Compounds

The invention also includes various deuterated forms of the compounds of Formulas (I) to (VI), respectively, or a pharmaceutically acceptable salt thereof. Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. A person of ordinary skill in the art will know how to synthesize deuterated forms of the compounds or a pharmaceutically acceptable salt thereof of the present invention. For example, deuterated materials, such as alkyl groups may be prepared by conventional techniques.

Isotopes

The subject invention also includes isotopica I ly- la belled compounds which are identical to those compounds or a pharmaceutically acceptable salt thereof of the present invention, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.

Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as ³H, ⁿC, ^MC, ¹⁸F, i2³i or ¹²⁵I.

Compounds of the present invention and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention.

Isotopically labelled compounds or pharmaceutically acceptable salts of the present invention, for example those into which radioactive isotopes such as ³H or ¹⁴C have been incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e. ³H, and carbon-14, i.e. ^MC, isotopes are particularly preferred for their ease of preparation and detectability. ⁿC and ¹⁸F isotopes are particularly useful in PET (positron emission tomography).

Purity

Because the compounds or pharmaceutically acceptable salts of the present invention are intended for use in pharmaceutical compositions or formulations, it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85% pure, especially at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing forms with higher purity percentages used in the pharmaceutical compositions or formulations.

PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

In general, the present invention relates to pharmaceutical compositions or formulations comprising the compounds or pharmaceutically salts as described herein, which include compounds of Formulas (I) to (VI) or pharmaceutically acceptable salts thereof.

In accordance with the present invention pharmaceutical compositions or formulations of the present invention may also include, but are not limited to, suitable adjuvants, carriers, excipients, or stabilizers, etc. and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions, etc.

Typically, pharmaceutical compositions or formulations will contain compounds or pharmaceutically acceptable salts thereof or forms as described herein of the present invention, together with the adjuvants, carriers or excipients. For example, a pharmaceutical composition of the present invention may comprise, but is not limited to an effective amount of any of the compounds or pharmaceutically acceptable salts thereof, with any of the characteristics noted herein, in association with one or more non-toxic pharmaceutically acceptable carriers or diluents thereof, and if desired, other active ingredients.

Delivery systems suitable for use in accordance with the present invention, may include, but are not limited to materials as described generally in this section.

Solutions or suspensions of these active compounds for use in parental administrations can be prepared in water, suitably mixed with at least one surfactant.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil, etc. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, etc., are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms

In one aspect or embodiment, the present invention relates to pharmaceutical compositions or formulations, which comprise compounds of Formulas (I) to (VI) or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipient(s).

In one aspect or embodiment, the present invention relates to pharmaceutical compositions or formulations, which comprise a compound of Formula (III).

In one aspect or embodiment, the present invention relates to pharmaceutical compositions or formulations, which comprise a 2-aminoethanolsalt of a compound of Formula (III), in particular a tris(2-aminoethanol) salt.

In one aspect or embodiment, the present invention relates to a pharmaceutical formulation or composition, which comprises: a compound which is (S 2-((R 3-(decyloxy)tetradecanamido)-3- 2R,3R,4R,5S,6R 3-((R)-3-(decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6- (hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2- aminoethanol) salt and at least one pharmaceutically acceptable excipient.

In one aspect or embodiment, the present invention relates to a pharmaceutical formulation or composition, which comprises: a compound which is (2S)-2-((R)-3-(decanoyloxy)tetradecanamido)- 3 - 2R,3R,4R,5S, 6R)-3-((R)-3-(decanoyloxy )tetradecanamido)-4-(((R)-3- (decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2- yl)oxy)propanoic acid tris(2-aminoethanol) salt and at least one pharmaceutically acceptable excipient.

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes a pharmaceutically acceptable excipient that comprises water for injection. In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes a pharmaceutically acceptable excipient that comprises at least one surfactant. A surfactant is useful as a stabilizing agent in the instant compositions and formulations and may prevent absorption of the active ingredient onto the glass surfaces of the equipment used to make the formulation. In another aspect or embodiment, the surfactant is present in an effective amount in the composition from about 0.01% w/w to about 20% w/w by weight. In a further aspect or embodiment, the surfactant is present in an amount of 0.1 to about 1% of solution. One presentation of the amount of surfactant necessary in the formulation is a molar ratio of the drug: surfactant, such as from about 1: 1 to about 1: 10⁸. If the amount of drug is increased, then the amount of surfactant in the molar ratio, potentially, can be decreased to about 10⁶. For example, when the concentration of the active agent is at 7 ng/mL, a molar ratio of the surfactant to drug can be given, for example, the surfactant can be present in an amount as low as 3.5 x 10 ¹²M. Alternatively, the surfactant can be higher, such as at a concentration of 1.4 x 10 ⁴ M. In another example, when the active agent is present in a higher amount, such as at 210 ng/mL, the surfactant to drug ratio is 10⁶, e.g. the surfactant amount can be lowered accordingly. Surprisingly, it has been found that the surfactant, such as a polymeric ether, prevents loss of the AGP and allows systemic delivery of the AGP without loss of biological activity.

In another aspect or embodiment, the present invention relates to a pharmaceutical composition wherein the surfactant is poloxamer P188.

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, wherein a pharmaceutically acceptable excipient comprises at least one saccharide.

In one aspect or embodiment, the saccharide is a disaccharide which includes sucrose, lactulose, lactose, maltose, trehalose, raffinose, or cellobiose, and/or mixtures thereof. Other contemplated disaccharides include kojibiose, nigerose, isomaltose, bb-trehalose, ab-trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose, and xylobiose

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes a saccharide that is dextrose monohydrate.

In addition, the present invention is directed to a pharmaceutical composition or formulation for injection comprising a TLR4 agonist of the present invention, at least one non-ionic surfactant, a diluent, and optionally, at least one pharmaceutical excipient.

In one aspect or embodiment, the pharmaceutical composition or formulation is chemically and physically stable over an extended period of time, such as from 12 to 36 months.

In another aspect or embodiment, the pharmaceutical composition or formulation may, optionally, contain at least one pharmaceutical excipient chosen from: a pH -adjusting agent; a buffering agent; a co-solvent; a chelating agent; a tonicity agent; an antioxidant; and a preservative. In another aspect or embodiment, a liquid injectable pharmaceutical composition or formulation of the present invention comprises:

a) a TLR4 agonist;

b) at least one non-ionic surfactant chosen from: a polyol; a polyoxyethylene ester; a polyoxyethylene ether; a polymeric ether; a polyvinyl alcohol PEG diacetate; a nonylphenyl ether; a propylene glycol diacetate; a polyvinyl alcohol; a PEG castor oil derivative; a PEG ester; a sorbitan derivative; and an alkanolamide; in combination; and mixtures thereof;

c) a diluent;

d) optionally, at least one pharmaceutically acceptable excipient chosen from: a pH- adjusting agent; a buffering agent; a solvent; a co-solvent; a preservative; an antioxidant; a viscosity agent; a tonicity agent; a chelating agent; and a second pharmaceutically active agent.

In a further aspect or embodiment, the at least one buffer or pH-adjusting agent is chosen from: hydrochloric acid; and sodium hydroxide.

In a further aspect or embodiment, the at least one non-ionic surfactant is a polymeric ether. In another aspect or embodiment, the polymeric ether is a poloxamer. In yet another aspect or embodiment, the polymeric ether poloxamer is chosen from; poloxamer 124; poloxamer 181; poloxamer 182; poloxamer 184; poloxamer 188; poloxamer 237; poloxamer 331; poloxamer 338; and poloxamer 407 and mixtures thereof. In another aspect or embodiment, the poloxamer is chosen from: poloxamer 181 and poloxamer 407. In another aspect or embodiment, the poloxamer is poloxamer 188.

In one aspect or embodiment, the polymeric ether is present in the composition or formulation at a concentration of about 0.01 mg/mL to about 10 mg/mL

In one aspect or embodiment, the at least one non-ionic surfactant is a polyol that is a mono and di-fatty acid ester chosen from: PEG 300; PEG 400; and PEG 1750. In one aspect or embodiment, the at least one non-ionic surfactant is a PEG ester chosen from: PEG-4 dilaurate; PEG-150 distearate; PEG-12 glyceryl laurate; PEG-120 glyceryl stearate; PEG-6 isostearate; PEG-4 laurate; PEG-8 laurate; PEG-20 methyl glucose sesquistearate; PEG-5 oleate; PEG-6 oleate; PEG-10 oleate; PEG-25 propylene glycol stearate; PEG-2 stearate; PEG-6 stearate; PEG-6-32 stearate; PEG-8 stearate; PEG-9 stearate; PEG-20 stearate; PEG-40 stearate; PEG-45 stearate; PEG-50 stearate; and PEG-100 stearate; and mixtures thereof.

In further aspect or embodiment, the at least one non-ionic surfactant is a PEG ester chosen from: hydrogenated castor oil; PEG-7 hydrogenated castor oil, PEG-25 hydrogenated castor oil; PEG- 30 castor oil; PEG-33 castor oil; PEG-35 castor oil; PEG-36 castor oil; PEG-40 castor oil; PEG-40 hydrogenated castor oil; PEG-50 castor oil; PEG-54 hydrogenated castor oil; PEG-60 castor oil; and PEG-60 hydrogenated castor oil; and mixtures thereof. In another aspect or embodiment, the at least one non-ionic surfactant is a sorbitan derivative chosen from: polysorbate 20; polysorbate 40; polysorbate 60; polysorbate 65; polysorbate 80, sorbitan isostearate; sorbitan monolaurate; sorbitan monooleate; sorbitan monopa Imitate; sorbitan monostearate; sorbitan sesquioleate; sorbitan trioleate; and sorbitan tristearate; and mixtures thereof.

In another aspect or embodiment, the at least one non-ionic surfactant is a polyoxyethylene ester or ether is chosen from: macrogol 3350; macrogol 4000; macrogol 6000; macrogol 6 Glycerol Caprylocaprate; macrogol-15 hydroxystearate; Lauroyl polyoxyl-32 glycerides NF; and mixtures thereof.

Suitably, nonylpheyl ethers, also called nonoxinols, are ethoxylated nonyl phenols with a varying ethylene oxide chain length, such as nonoxynol-9, 10, and 11.

In an alternative aspect or embodiment, the at least one surfactant can be a mixture of bile salts and lecithins. Suitable lecithins include, but are not limited to, egg lecithin, soybean lecithin, or combinations thereof. Lecithins may also include various synthetic lipids or combinations of natural and synthetic lipids, such as phospholipids, e.g., Phospholipid 90H. Suitable bile salts include, but are not limited to, sodium cholate, sodium deoxycholate, taurine cholate, or combinations thereof.

In one aspect or embodiment, the at least one surfactant comprises at least one non-ionic surfactant. In another aspect or embodiment, the at least one surfactant comprises at least two non ionic surfactants, and the weighted average of the HLB values of the non-ionic surfactants is from about 10 to about 20.

In one aspect or embodiment, the pharmaceutically acceptable excipient is a tonicity agent. In another aspect or embodiment, the tonicity agent is chosen from: sterile saline solution; Ringer's solution; dextrose solution; dextrose monohydrate; and solidified high dextrose corn syrup. In another aspect or embodiment, the tonicity agent is dextrose monohydrate.

In another aspect or embodiment, the pH-adjusting agent is hydrochloric acid and/or sodium hydroxide.

In a further aspect or embodiment, the polymeric ether is a poloxamer. In another embodiment, the poloxamer is poloxamer 188.

In one aspect or embodiment the diluent is chosen from: USP water for injection; a pH- buffered solution; a sterile saline solution; Ringer's solution; a dextrose solution.

In another aspect or embodiment, the diluent is a dextrose solution chosen from: dextrose monohydrate; and a 5% w/w dextrose solution. In another aspect or embodiment, the diluent is a pH-buffered solution which is a phosphate-buffered saline.

In a further aspect or embodiment, the diluent is chosen from: USP water for injection; and sterile water for an injection chosen (SWFI); and bacteriostatic water for injection (BWFI).

In another aspect or embodiment, the pharmaceutical composition or formulation of the present invention comprises: (1) a lyophilized form of an AGP;

(2) at least one non-ionic surfactant; and

(3) optionally; at least one a pharmaceutically acceptable excipient chosen from: a buffer; and a pH-adjusting agent to maintain a pH in the range of 3.5 to 7.5; a preservative; an antioxidant; a tonicity agent; a viscosity agent; a co-solvent; a chelating agent; and a second pharmaceutically active agent.

In one aspect or embodiment, the pH is from about 6 to about 7.5. In another aspect or embodiment, the pH is from about 6.8 to 7.2.

In one aspect or embodiment, the co-solvent is a water miscible or water water-soluble organic solvent and includes, but is not limited, to: propylene glycol; ethanol; polyethylene glycol 300; polyetheylene glycol 400, glycerin; dimethylacetamide (DMA); and N-methyl-2-pyrrolidone (NMP). Other suitable solvents for use in the manufacturing process include other short chain alcohols, such as t-butyl alcohol, n-propanol, etc., and other organic reagents such as acetonitrile, DMSO, and ethyl acetate, ethyl lactate, and dimethyl isosorbide, etc.

A co-solvent can be added in the amount of about 1 to 50% w/w. In one aspect or embodiment, the co-solvent is in the range of about 1 to 20% w/w.

In another aspect or embodiment, the co-solvent is dextrose monohydrate. Upon re constitution, a suitable carrier or diluent is chosen from: buffered saline; a 5% w/w dextrose solution; and USP water for injection.

In one aspect or embodiment, tonicity or osmolarity agents may be added, which are any agents capable of rendering the formulations of the present invention iso-osmotic with human serum. Tonicity agents include, but are not limited to, sodium chloride, glycine, and sugar alcohols, such as mannitol, glucose and sorbitol, and mixtures thereof. Other suitable tonicity agents are saccharides and polyols, as further described herein.

In one aspect or embodiment, the invention relates to a composition (e.g., pharmaceutical composition) or formulation (e.g., a formulation for injection) comprising:

a 2-aminoethanol salt of a compound of Formula (III), i.e. Compound 9, (e.g., at a concentration of about 0.0005 mg/mL);

dehydrated alcohol (e.g., ethanol, e.g., at a concentration of about 0.0005 mg/mL);

dextrose monohydrate (e.g., at a concentration of about 44 mg/mL);

poloxamer PI 18 (e.g., at a concentration of about 1 mg/mL);

sodium hydroxide (e.g., as needed for pH adjustment);

hydrochloric acid (e.g., as needed for pH adjustment); and

water for injection (e.g., to bring total volume to 1 mL).

In some embodiments, the pH of the composition or formulation is 7.0 ± 0.2 (e.g., the pH range is 6.8 to 7.2). In some embodiments, the composition (e.g., pharmaceutical composition) or formulation is for the methods and uses described herein.

In some embodiments, the actual quantity of the 2-aminoethanol salt of a compound of Formula (III) (i.e. Compound 9) is adjusted to the equivalent to 0.0005 mg of the compound of Formula III as the free acid based on the weighing factor of input active ingredient.

In one aspect or embodiment, the extended period of time is a 24 months' shelf life kept at 2-8°C. In one aspect or embodiment, the extended period of time is 30 months' shelf life when frozen (e.g. -15°C to -25°C). In one aspect or embodiment, the product is protected from light. In one aspect or embodiment, the product includes a cold storage chain, e.g., 2 to 8 °C for a period of 12 months to 24 months or longer, such as up to 3 years' time, for improved product life.

In one aspect or embodiment, the TLR4 agonist is present in the composition from about 0.0001 mg/mL to about 10 mg/mL. In another aspect or embodiment, the TLR4 agonist is present in the composition from about 0.0001 mg/mL to about 0.001 mg/mL. In one aspect or embodiment, the TLR4 agonist is present in the composition from about 0.0005 mg/mL to O.OOlmg/mL.

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes about 1000 ng/mL of a 2-aminoethanol salt and/or solvate of a compound or pharmaceutically acceptable salt of any of the Formulas (I) to (VI).

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes about 1000 ng/mL of (S)-2-((R)-3- (decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R>3-((R)-3-(decyloxy)tetradecanamido)-4-(((R)-3- (decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2- yl)oxy)propanoic acid tris(2-aminoethanol) salt.

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes about 1000 ng/mL of (2S)-2-((R)-3-

(decanoyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R)-3-(decanoyloxy)tetradecanamido)-4- (((R)-3-(decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran- 2-yl)oxy)propanoic acid tris(2-aminoethanol) salt.

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes about 500 ng/mL of a 2-aminoethanol salt and/or solvate of a compound or pharmaceutically acceptable salt of any of the Formulas (I) to (VI).

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes about 500 ng/mL of (S)-2-((R)-3-

(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R)-3-(decyloxy)tetradecanamido)-4-(((R)-3- (decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2- yl)oxy)propanoic acid tris(2-aminoethanol) salt. In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation, which includes about 500 ng/mL of (2S)-2-((R)-3- (decanoyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R)-3-(decanoyloxy)tetradecanamido)-4- (((R)-3-(decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran- 2-yl)oxy)propanoic acid tris(2-aminoethanol) salt.

In another aspect or embodiment, the present invention provides a 1 ml. unit dose pharmaceutical formulation comprising:

a. about 0.0005 mg of a compound of Formula (III):

or a 2-aminoethanol salt thereof, optionally the tris(2-aminoethanol) salt;

b. about 0.0005 ml. of dehydrated alcohol, e.g. ethanol;

c. about 44.0 mg of dextrose monohydrate;

d. about 1 mg of P188;

f. water for injection to reach 1 ml_.

In addition, the present invention also provides for a process of preparing a stable, pharmaceutical composition or formulation comprising admixing:

a) an TLR4 agonist;

b) at least one non-ionic surfactant;

c) a diluent or carrier;

d) at least one buffer or pH-adjusting agent to maintain a pH in the range of 3.5 to 7.5; and e) optionally, a pharmaceutically acceptable excipient chosen from: an antimicrobial agent; a tonicity agent; a chelating agent; a co-solvent; or

f) a second pharmaceutically active agent, to form a solution.

The present invention also provides for a process of preparing a stable, pharmaceutical composition comprising admixing:

a) a TLR4 agonist;

b) at least one non-ionic surfactant;

c) USP Water for Injection;

d) at least one buffer or pH-adjusting agent to maintain a pH in the range of 3.5 to 7.5; and

e) optionally, a pharmaceutically acceptable excipient chosen from an antimicrobial agent; a tonicity agent; a chelating agent; a co-solvent; or

f) a second pharmaceutically active agent, to form a solution.

In one aspect or embodiment, the solution is prepared and maintained substantially free of dissolved oxygen and may further include a headspace of inert atmosphere above the solution at a value of less than about 4% by volume of oxygen.

In one aspect or embodiment, the AGP may be presented in the drug product as a lyophilized powder, to be constituted upon use with a suitable carrier, such as an aqueous carrier. An aqueous carrier is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Illustrative diluents for use herein include, but are not limited to, sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH-buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or a dextrose solution, such as dextrose monohydrate, or 5% w/w dextrose solution. Any of these diluents may further be used herein as a co-solvent in any of the compositions or formulations disclosed herein, if desired.

Concentrations, amounts, solubilities, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limit of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range, as if each numerical value and sub-range is explicitly recited. All numbers expressing quantities, percentages or proportions, and other numerical values used in the specification, are to be understood as being modified in all instances by the term "about".

Any concentration range, percentage range, or ratio range recited herein, is to be understood to include concentrations, percentages or ratios of any integer within that range and fractions thereof, such as one tenth and one hundredth of an integer, unless otherwise indicated. Unless otherwise indicated, all percentages are based on the percent by weight of the final composition prepared, and all totals equal 100% by weight.

Throughout the application, descriptions of various aspects or embodiments use "comprising" language. However, in some specific instances, an aspect or embodiment can alternatively be described using the language "consisting essentially of" or "consisting of".

Pharmaceutically Acceptable Excipients

The present pharmaceutical compositions or formulations may further comprise at least one additional pharmaceutically acceptable excipient. Exemplary additional acceptable excipients include, but are not limited to, a pH-adjusting agent, a chelating agent, a preservative, antioxidants, viscosity agents, a co-solvent, a colorant, and combinations or mixtures thereof.

In one aspect or embodiment, the additional pharmaceutically acceptable excipient is a preservative.

The optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. If the formulation is used as an injectable solution, sterility is required, whether by filtration sterilization or other suitable means. The AGP may be a powder prepared from a previously sterile filtered solution, e.g., the powder is prepared by any suitable technique, e.g., vacuum drying and/or freeze drying).

pH -adjusting agents

The present pharmaceutical compositions or formulations may further comprise a pH- adjusting agent.

Examples of buffering agents, include, but are not limited to, citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid- sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fuma rate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid- potassium lactate mixture, etc.) phosphate buffers (sodium phosphate monobasic/sodium phosphate dibasic) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, histidine or histidine HCI, and glycerine can also be used as buffering agents. In one aspect or embodiment, the pH-adjusting agent is an acid, an acid salt, or a mixture thereof. The acid may be chosen from: lactic acid; acetic acid; maleic acid; succinic acid; citric acid; benzoic acid; boric acid; sorbic acid; tartaric acid; edetic acid; phosphoric acid; nitric acid; sulphuric acid; and hydrochloric acid, and mixtures thereof.

In another aspect or embodiment, the pH-adjusting agent is a buffer. The buffer may be chosen from: citrate/citric acid; acetate/acetic acid; phosphate/ phosphoric acid; propionate/ propionic acid; lactate/ lactic acid; ammonium/ ammonia; and edetate/ edetic acid. In another aspect or embodiment, the pH-adjusting agent is a buffer which is citrate/citric acid.

In one aspect or embodiment, the pH-adjusting agent is present in the composition in an amount from about 0.01% to about 10% by weight, based on the total weight of the composition. The buffer systems suitable for the formulations of the present invention are those which maintain the pH of the aqueous solution formulation in the range of about 3.5 to about 7.5. In one aspect or embodiment, the pH is from about 4.5 to about 7.5. In another aspect or embodiment, the pH is from about 6.0 to about 7.5. In one aspect or embodiment, the buffer will maintain the pH of the aqueous solution at about 6. 5 - 7. 1, or at about 6.8. In another aspect or embodiment, the pH is from about 6.8 to about 7.2.

Normally, an equimolar amount of drug to buffer may be as high as 10 millimolar of drug.

In one aspect or embodiment of the present invention, the composition comprises a TLR4 agonist, such as an AGP, buffered using a zwitterionoic buffer. In another aspect or embodiment of the invention, the zwitterionic buffer is an aminoalkanesulfonic acid, or a suitable salt. Examples of amino alkane sulfonic buffers include, but are not limited, to HEPES, HEPPS/EPPS, MOPS, MOBS and PIPES. In one aspect or embodiment of the invention, the buffer is a pharmaceutically acceptable buffer, suitable for use in humans, such as in for use in a commercial injection product. In another aspect or embodiment of the invention, the buffer is HEPES.

Chelating Agents

The present pharmaceutical compositions or formulations may further comprise a chelating agent. In one aspect or embodiment, the chelating agent is a mixture of at least two chelating agents. As described herein, the compositions or formulations of the invention may comprise a mixture of a chelating agent and an antioxidant, where both excipients act to prevent or minimize oxidative degradation reactions in the composition.

Exemplary chelating agents include, but are not limited to, citric acid, glucuronic acid, sodium hexameta phosphate, zinc hexameta phosphate, ethylene diamine tetraacetic acid (EDTA), phosphonates, salts thereof, and mixtures thereof. Ethylene diamine tetraacetic acid is also known as edetic acid.

In one aspect or embodiment, the chelating agent is EDTA or a salt thereof, such as potassium, sodium or calcium salts of EDTA. In another aspect or embodiment, the EDTA or a salt thereof is disodium EDTA. In a further aspect or embodiment, the chelating agent is citric acid. In yet another aspect or embodiment, the compositions or formulations of the invention comprise a mixture of a chelating agent and an antioxidant which is a mixture of EDTA or a salt thereof and propyl gallate. In a further aspect or embodiment, the compositions or formulations of the invention comprise a mixture of a chelating agent and an antioxidant which is a mixture of EDTA or a salt thereof and BHT.

In one aspect or embodiment, the chelating agent is present in the composition in an amount from about 0.01% to about 1% by weight, based on the total weight of the composition. In another aspect or embodiment, the chelating agent is present in the composition in an amount of about 0.1% by weight, based on the total weight of the composition.

Preservatives

The present topical pharmaceutical emulsion compositions or formulations may further comprise a preservative. In one aspect or embodiment, the preservative is a mixture of at least two preservatives.

Examples of potential preservatives include, but are not limited to, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride.

Other types of preservatives include aromatic alcohols, such as phenol, butyl alcohol, benzyl alcohol, dichlorobenzyl alcohol, alkyl parabens, such as methyl, ethyl or propyl paraben, catechol, resorcinol, cyclohexanol, pentanol, phenoxyethanol, sorbic acid, benzoic acid, salts thereof, meta- Cresol, imidazolidinyl urea, and diazolidinyl urea, and mixtures thereof.

In one aspect or embodiment, the preservative is present in the composition in an amount from about 0.01% to about 2% by weight, based on the total weight of the composition. In another aspect or embodiment, the preservative is present in the composition in an amount of about 0.25% by weight, based on the total weight of the composition.

In another aspect or embodiment of the invention, there is no antimicrobial agent present, as the filtration method of sterility is sufficient to overcome the need for this agent.

METHODS OF TREATMENT AND USES

An aspect of the invention is a method of treating cancer in a human in need thereof comprising administering a therapeutically effective amount of a compound, pharmaceutically acceptable salt, pharmaceutical composition or formulation disclosed herein.

An aspect of the invention is the use of a compound, pharmaceutically acceptable salt, pharmaceutical composition or formulation disclosed herein in the treatment of cancer.

An aspect of the invention is the use of a compound or pharmaceutically acceptable salt disclosed here in the manufacture of a medicament for use in treating cancer. Another aspect or embodiment of the invention is a method of treating cancer in a human in need thereof comprising administering a therapeutically effective amount of a formulation for injection to the human comprising a TLR4 agonist, at least one polymeric ether surfactant, USP water for injection, and, optionally, at least one pharmaceutical excipient that is stable over an extended period of time.

Another aspect or embodiment of the invention is a compound for use in treating cancer in a human in need thereof comprising administering a therapeutically effective amount of a formulation for injection to the human comprising a TLR4 agonist, at least one polymeric ether surfactant, USP water for injection, and, optionally, at least one pharmaceutical excipient that is stable over an extended period of time.

In one aspect of the invention, the cancer to be treated is chosen from: melanoma; lung cancer; non-small cell lung cancer (NSCLC); kidney cancer; renal cell carcinoma; breast cancer; metastatic breast cancer; triple-negative breast cancer (TNBC); head and neck cancer; colon cancer; colorectal cancer (CRC); ovarian cancer; pancreatic cancer; liver cancer; hepatocellular carcinoma (HCC); prostate cancer; bladder cancer; gastric cancer; liquid tumors; solid tumors; hematopoietic tumors; leukemia; non-Hodgkins lymphoma (NHL); and chronic lymphocytic leukemia (CLL).

In another aspect or embodiment, the invention includes a formulation comprising a TLR4 agonist disclosed herein for use in the treatment of any of the above listed cancers.

In another aspect or embodiment, the invention includes the use of a formulation for treating cancer comprising a TLR4 agonist disclosed herein for the manufacture of a medicament to treat any of the above listed cancers.

Another aspect or embodiment of the invention is a use of a formulation of the present invention in the manufacture of a medicament for the treatment of cancer in a human in need thereof comprising administering a therapeutically effective amount of a formulation for injection to the human comprising a TLR4 agonist, at least one polymeric ether surfactant, USP water for injection, and, optionally, at least one pharmaceutical excipient that is stable over an extended period of time.

A compound of Formula (I) to (VI) or pharmaceutically acceptable salt thereof can be used as a monotherapy, or in combination with other active agents, whether biological products or small molecule compounds. In particular, (S)-2-((R>3-(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R> 3-((R)-3-(decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt can be used as monotherapy, or in combination with other active agents, whether biologicals or small molecule compounds. In one aspect or embodiment, the combination is with at least one antibody. In another aspect or embodiment, the combination is with an OX-40 antibody (e.g., an agonist OX-40 antibody). In a further aspect or embodiment, the combination is with an ICOS antibody (e.g. an agonist ICOS antibody), or with both an OX-40 antibody (e.g., an agonist OX-40 antibody) and an ICOS antibody (e.g. an agonist ICOS antibody). In an aspect or embodiment of the invention, the pharmaceutical composition or formulation disclosed herein is co-administered with at least one immunomodulatory agent.

In one aspect or embodiment, the immunomodulatory agent is chosen from: an ICOS antibody (e.g. an agonist ICOS antibody); an OX-40 antibody (e.g., an agonist OX-40 antibody); a PD-1 antibody (e.g. a PD-1 antagonist antibody); a PD-L1 antibody (e.g. a PD-L1 antagonist antibody); and a CTLA-4 antibody (e.g. a CTLA-4 antagonist antibody). In yet another aspect or embodiment, the formulation comprising the TLR4 agonist and the composition comprising the antigen antibody are delivered separately.

ROUTES OF ADMINISTRATION

In an aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation adapted for systemic administration.

In another aspect or embodiment, the present invention relates to a pharmaceutical composition or formulation which is suitable for intravenous injection.

In one aspect or embodiment, the pharmaceutical composition or formulation is administered via a route chosen from: intravenous; subcutaneous; intra-tumoural; and intradermal.

In an aspect or embodiment of the invention, the pharmaceutical composition or formulation is administered intravenously.

In an aspect or embodiment of the invention, the pharmaceutical composition or formulation is administered intra-tumouraly.

DOSAGES

It will be appreciated that the actual preferred dosages of the compounds being used in the compositions or formulations of this invention will vary according to the particular composition formulated, the mode of administration, the particular site of administration and the host being treated.

DEVICES AND KITS

Another aspect or embodiment of the present invention is an article of manufacture comprising a sealed vial that contains a sterile aqueous injectable formulation containing a TLR4 agonist of Formula (I) to (VI) as an active agent, or a pharmaceutically acceptable salt or solvate thereof, as an active agent.

Yet another aspect or embodiment of the present invention is an article of manufacture comprising a sealed vial that contains a sterile aqueous injectable formulation containing a TLR4 agonist chosen from:

• any one of compounds of Formula (I) to (VI) or a pharmaceutically acceptable salt or solvate thereof, as an active agent, packaged with printed instructions that indicate that the TLR4 agonist is useful for the treatment of cancer; • (S)-2-((R>3-(decyloxy)tetradecanannido)-3-(((2R,3R,4R,5S,6R>3-((R)-3- (decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt as an active agent, packaged with printed instructions that indicate that the TLR4 agonist is useful for the treatment of cancer; and

• (2S>2-((R>3-(decanoyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R>3-((R)-3- (decanoyloxy)tetradecanamido)-4-(((R)-3-(decanoyloxy)tetradecanoyl)oxy)-6- (hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2- aminoethanol) salt as an active agent,

packaged with printed instructions that indicate that the TLR4 agonist is useful for the treatment of cancer.

An aspect of the invention provides a kit of parts comprising: a compound or pharmaceutical formulation or composition as disclosed herein and instructions for use. In an embodiment, the compound or pharmaceutical formulation or composition is in the form of an injectable solution filled into a dispensing vehicle selected from the group consisting of: a sterile vial; a sterile multi-dose vial; and a syringe. In an embodiment, the kit further comprises at least one immunomodulatory agent selected from the group consisting of: an agonist ICOS antibody; an agonist OX-40 antibody; an antagonist PD-1 antibody; an antagonist PD-L1 antibody; and an antagonist CTLA-4 antibody.

SYNTHETIC SCHEME AND GENERAL METHODS OF PREPARATION

In general, the present invention also relates to processes for making compounds or a pharmaceutically acceptable salt thereof as described herein throughout the instant specification.

The compounds of the present invention as described herein, respectively, or corresponding pharmaceutically acceptable salts thereof, may be obtained by using synthetic procedures illustrated in the Scheme below or by drawing on the knowledge of a skilled organic chemist.

The synthesis provided in this Scheme is applicable for producing compounds of the invention having a variety of different Ri, R2, RB, R₄, and Rs groups employing appropriate precursors, which are suitably protected if needed, to achieve compatibility with the reactions outlined herein. Subsequent deprotection, where needed, affords compounds of the nature generally disclosed.

While the Scheme is shown with compounds for the synthesis of a salt of the compound of Formula (III), it is illustrative of processes that may be used to make the compounds of the present invention in general.

Intermediates (compounds used in the preparation of the compounds of the invention) may also be present as salts. Thus, in reference to intermediates, the phrase "compound(s) of formula (number)" means a compound having that structural formula or a pharmaceutically acceptable salt thereof. The compounds of the present invention as defined herein, or pharmaceutically acceptable salts thereof, are prepared using conventional organic syntheses.

A suitable synthetic route is depicted below in the following general reaction scheme.

The skilled artisan will appreciate that if a substituent described herein is not compatible with the synthetic methods described herein, the substituent may be protected with a suitable protecting group that is stable to the reaction conditions. The protecting group may be removed at a suitable point in the reaction sequence to provide a desired intermediate or target compound. Suitable protecting groups and the methods for protecting and de-protecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, Protecting Groups in Chemical Synthesis (3rd ed.), John Wiley & Sons, NY (1999). In some instances, a substituent may be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful as an intermediate compound or is a desired substituent in a target compound.

A description of the synthesis of (S)-2-((RJ-3-(decyloxy)tetradecanamido)-3-

(T(2R,3R,4R,5S,6R>3-((R)-3-(decyloxy)tetradecanamido)-4-(T(R>3-(decyloxy)tetradecanoyl)oxy)-6- (hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2- aminoethanol) salt is provided below and a flow diagram is provided in Scheme 1.

Scheme 1 Flow chart for the synthesis of Compound (9)

Abbreviations:

aq. Aqueous

Bn Benzyl

Pd/C Palladium on carbon

Ph Phenyl

T roc 2,2,2-T richlorethoxycarbonyl

Compound (1) was acylated with Compound (2) in the presence of dichloromethane (DCM), 4 dimethylaminopyridine (DMAP) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). After an aqueous work-up involving sodium bicarbonate, DCM and water, the crude product Compound (3) was isolated from the organic phase by concentration to dryness. Crude Compound (3) was subjected to silica gel chromatography, eluting with heptane/ethyl acetate (EtOAc). Product containing fractions were pooled and concentrated to yield the purified product, Compound (3).

Compound (3) was reacted with activated zinc powder in the presence of tetrahydrofuran (THF) and ammonium chloride at elevated temperature. The suspension was filtered and washed with THF to extract the product as a solution which was then concentrated to dryness and subjected to an aqueous work-up with DCM, sodium bicarbonate and water. The organic phase was concentrated to dryness to yield Compound (4).

Compound (2) was activated with A/^A/^A/'-Tetramethyl-CHbenzotriazol-l-yOuronium tetrafluoroborate (TBTU) in the presence of DCM and /y,/V-Diisopropylethylamine (DIPEA) before being treated with Compound (4). The solvent was exchanged to methanol before isolating the product Compound (5) by filtration, washing with methanol and drying under vacuum. Compound (5) was reacted with sodium cyanoborohydride in the presence of DCM and trifluoroacetic acid (TFA). After quenching the reaction with methanol, the DCM was replaced with additional methanol, before the resulting suspension was filtered and the solid product washed with methanol and dried under vacuum to obtain the product, Compound (6).

Compound (6) was reacted with 4,5-dicyanoimidazole (DCI) in the presence of DCM, dibenzyl /y,/V-diisopropylphosphoramidite and 30% aqueous hydrogen peroxide. The reaction was quenched with sodium bicarbonate solution. After a work-up involving DCM, water, heptane and methanol, the product containing organic phase was concentrated to dryness to obtain crude Compound (7). Crude Compound (7) was subjected to silica gel chromatography, eluting with heptane/ethyl acetate. Product containing fractions were collected and dried to obtain purified Compound (7).

Compound (7) was hydrogenated in the presence of palladium on activated carbon and THF. After removing the catalyst by filtration, the filtrate was concentrated to dryness to yield the product, Compound (8). Compound (8) was dissolved in methyl tert-butyl ether (MTBE) and treated with 2- aminoethanol and acetonitrile. The resulting suspension was filtered and the solid washed with MTBE and acetonitrile then dried in vacuo. The crude product thus obtained was subjected to reverse phase silica gel chromatography, eluting with DCM and methanol in the presence of 2-aminoethanol. Product containing fractions were collected and concentrated to obtain the product. After chromatography, the product was precipitated from methyl tert-butyl ether (MTBE), 2-aminoethanol and acetonitrile. After filtration, the product was washed with MTBE and acetonitrile then dried in a vacuum oven to deliver the final Compound (9), as a white to slightly coloured solid.

The structure of Compound (9) has been confirmed by a combination of proton ^H) and carbon (¹³C) NMR Spectroscopy, Mass Spectrometry (MS) and Infrared (IR) Spectroscopy. The mass spectrum of Compound (9) was obtained by negative ion electrospray ionisation and was concordant with the structure. The accurate mass of the deprotonated molecule of Compound (9) was measured at 1444.1162 Da. The calculated mass of this ion is 1444.1198 Da. The measurement accords with the elemental composition of this ion within an error of 5 ppm.

The purity of Compound (9) was confirmed by a combination of HPLC-uv, Ion Chromatography

(2-aminoethanol content), Gas Chromatography (residual solvent content) and Karl Fischer titration (water content).

While particular aspects or embodiments of the present invention are described, the skilled artisan will appreciate that various changes and modifications can be made without departing from the spirit and scope of the invention.

The invention includes the following items:

Item 1. A compound which is a 2-aminoethanol salt of a compound of Formula (III):

Item 2. The compound according to item 1, which is in an amorphous form.

Item 3. The compound according to item 1, which is in a semi-crystalline form.

Item 4. The compound according to item 1, which is in an mesophase semi-crystalline amorphous form.

Item 5. The compound according to item 1, which is in a solvate form.

Item 6. A compound which is (S 2-((R 3-(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R>3-(decyloxy)tetradecanamido)-4-(((R 3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt.

Item 7. The compound according to item 6 having a Diamond Attenuated Total Reflectance

(ATR) Infrared Spectrum pattern.

Item 8. The compound according to item 7 having a Diamond Attenuated Total Reflectance

(ATR) Infrared Spectrum pattern with characteristic peaks in wavenumbers (cm^-1) i.e., 3600-3000, 2955, 2919, 2851, 1741, 1717, 1646, 1636, 1552, 1466, 1101, 1073, 1026.

Item 9. A pharmaceutical formulation or composition comprising a compound of any of items

1-7 and at least one pharmaceutically acceptable excipient.

Item 10. The pharmaceutical formulation or composition of item 9 which is adapted for systemic administration.

Item 11. The pharmaceutical formulation or composition of any one of items 9 and 10, which is suitable for intravenous injection.

Item 12. The pharmaceutical formulation or composition of any one of items 9 to 11, wherein the pharmaceutically acceptable excipient is water for injection.

Item 13. The pharmaceutical formulation or composition of any one of items 9 to 11, wherein the pharmaceutically acceptable excipient comprises at least one surfactant.

Item 14. The pharmaceutical formulation or composition of item 13, wherein the at least one surfactant is poloxamer P188.

Item 15. The pharmaceutical formulation or composition of any one of items 9 to 11, wherein the pharmaceutically acceptable excipient comprises at least one saccharide.

Item 16. The pharmaceutical formulation or composition of item 15, wherein the saccharide is dextrose monohydrate.

Item 17. The pharmaceutical formulation or composition of any one of items 9 to 15 comprising about 1000 ng/mL of the 2-aminoethanol salt and/or solvate of the compound of item 1.

Item 18. A compound which is a 2-aminoethanol salt of a compound of Formula (IV):

Item 19. A compound which is 2S -2- R)-3-(decanoyloxy)tetradecanamido)-3- (((2R,3R,4R,5S,6R)-3-((R)-3-(decanoy\oxy)tetradecanam do)-4-(((R)-3- (decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2- yl)oxy)propanoic acid tris(2-aminoethanol) salt.

Item 20. A formulation for injection comprising:

(a) a TLR4 agonist;

(b) at least one non-ionic surfactant;

(c) a diluent; and,

(d) optionally at least one pharmaceutical excipient.

Item 21. The formulation for injection according to item 20, wherein the formulation is stable over an extended period of time from 12-36 months.

Item 22. The formulation for injection according to item 20, wherein the TLR4 agonist is selected from a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof:

(Formula I)

wherein:

m is 0, or an integer of 1 to 6; n is 0, or an integer from 1 to 4;

X is O or S, preferably O;

Y is O or NH;

Ri, R2, RB are each independently selected from a C1-20 acyl or a C1-20 alkyl;

R₄ is H or CH3;

R5 is selected from H, -OH, -C1-4 alkoxy, -P(0)(ORs)(OR9), -OP(0)(ORs)(OR9), -SO3R8, -OSO3R8, NR8R9, -SRs, -CN, -NO2, -CHO, -CO2R8, and -CONR8R9,

Re and R9 are each independently selected from H and C1-4 alkyl; and

R6 and R7 are each independently selected from H or P(0)(OH)2.

Item 23. The formulation for injection according to item 22, wherein the compound is present as the 2-aminoethanol salt and/or solvate.

Item 24. The formulation for injection according to item 20, wherein the compound is a 2- aminoethanol salt of:

Item 25. The formulation for injection according to item 20, wherein the TLR4 agonist is selected from:

(S)-2-((R)-3-(decy\oxy)tetra0ecanam 0o)-3-(((2R,3R,4R,5S,6R)-3-((R)-3- (decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., Compound 9 herein); and

(2S)-2-((R)-3-(decanoY\oxY)tetradecanam do)-3-(((2R,3R,4R,5S,6R)-3-((R)-3- (decanoyloxy)tetradecanamido)-4-(((R)-3-(decanoyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt.

Item 26. The formulation for injection according to item 25, wherein the TLR4 agonist is (S)-2-((R)-3-(0ec \oxy)tetra0ecanam\0o)-3-(((2R,3R,4R,5S,6R)-3-((R)-3- (decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., Compound 9 herein).

Item 27. The formulation for injection according to item 20, wherein the at least one non-ionic surfactant chosen is selected from: a polyol; a polyoxyethylene ester; a polyoxyethylene ether; a polymeric ether; a polyvinyl alcohol PEG diacetate; a nonyl phenyl ether; a propylene glycol diacetate; a polyvinyl alcohol; a PEG castor oil derivative; a PEG ester; a sorbitan derivative; and an alkanolamide; in combination; and mixtures thereof.

Item 28. The formulation for injection according to item 27, wherein the polymeric ether is a poloxamer chosen from: poloxamer 124; poloxamer 181; poloxamer 182; poloxamer 184; poloxamer 188; poloxamer 237; poloxamer 331; poloxamer 338; and poloxamer 407; and mixtures thereof. Item 29. The formulation for injection according to item 28, wherein the poloxamer is chosen from: poloxamer 181 and poloxamer 407.

Item 30. The formulation for injection according to item 29, wherein the poloxamer is poloxamerl81.

Item 31. The formulation for injection according to item 27, wherein the polyol is a mono and di-fatty acid ester chosen from: PEG 300; PEG 400; and PEG 1750.

Item 32. The formulation for injection according to item 27, wherein the PEG ester is chosen from: PEG-4 dilaurate; PEG-150 distearate; PEG-12 glyceryl laurate; PEG-120 glyceryl stearate; PEG- 6 isostearate; PEG-4 laurate; PEG-8 laurate; PEG-20 methyl glucose sesquistearate; PEG-5 oleate; PEG-6 oleate; PEG-10 oleate; PEG-25 propylene glycol stearate; PEG-2 stearate; PEG-6 stearate; PEG- 6-32 stearate; PEG-8 stearate; PEG-9 stearate; PEG-20 stearate; PEG-40 stearate; PEG-45 stearate; PEG-50 stearate; and PEG-100 stearate; and mixtures thereof.

Item 33. The formulation for injection according to item 27, wherein the PEG ester is chosen from: PEG-7 hydrogenated castor oil; PEG-25 hydrogenated castor oil; PEG-30 castor oil; PEG-33 castor oil; PEG-35 castor oil; PEG-36 castor oil; PEG-40 castor oil; PEG-40 hydrogenated castor oil; PEG-50 castor oil; PEG-54 hydrogenated castor oil; PEG-60 castor oil; and PEG-60 hydrogenated castor oil; and mixtures thereof.

Item 34. The formulation for injection according to item 27, wherein the sorbitan derivative is chosen from: polysorbate 20; polysorbate 40; polysorbate 60; polysorbate 65; polysorbate 80; sorbitan isostearate; sorbitan monolaurate; sorbitan monooleate; sorbitan monopa Imitate; sorbitan monostearate; sorbitan sesquioleate; sorbitan trioleate; and sorbitan tristearate; and mixtures thereof. Item 35. The formulation for injection according to item 27, wherein the polyoxyethylene ester or ether is chosen from: macrogol 3350; macrogol 4000; macrogol 6000; Macrogol 6 Glycerol Caprylocaprate; macrogol-15 hydroxysterate; Lauroyl polyoxyl-32 glycerides NF; and mixtures thereof.

Item 36. The formulation for injection according to item 20, wherein the diluent is chosen from: USP water for injection; a pH-buffered solution; a sterile saline solution; Ringer's solution; and a dextrose solution.

Item 37. The formulation for injection according to item 20, wherein the diluent is a dextrose solution chosen from: dextrose monohydrate; and a 5% w/w dextrose solution.

Item 38. The formulation for injection according to item 20, wherein the diluent is a pH-buffered solution that is a phosphate-buffered saline.

Item 39. The formulation for injection according to item 20, wherein the diluent is chosen from: USP water for injection; sterile water for injection (SWFI); and bacteriostatic water for injection (BWFI).

Item 40. The formulation for injection according to item 20, wherein the at least one pharmaceutical excipient is chosen from: a pH -adjusting agent; a co-solvent; a buffering agent; a chelating agent; a tonicity agent; an antioxidant; and a preservative.

Item 41. The formulation for injection according to item 20, wherein the formulation has at least 75% of the initial biological activity of the TL4 agonist.

Item 42. A formulation for injection comprising:

a. (S)-2-((R)-3-(deo/\oxY)tetradecanam do)-3-(((2R,3R,4R,5S,6R)-3-((R)-3- (decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., Compound 9 herein);

b. at least one non-ionic surfactant;

c. a diluent; and

d. optionally at least one pharmaceutical excipient.

Item 43. A formulation for injection comprising:

Item 44. The formulation for injection according to item 42, wherein the formulation is stable over a period of time from 12-36 months.

Item 45. The formulation for injection according to any of the preceding items, wherein the TLR4 agonist is present in the composition from about 1 ng to about 410 ng/mL.

Item 46. The formulation for injection according to item 45, wherein the TLR4 agonist is present in the composition from about 7 ng to about 210 ng/mL.

Item 47. The formulation for injection according to any one of items 9-17 and 20-46, wherein the polymeric ether is present in the composition at a concentration of about 0.01 mg/mL to about 10 mg/mL. Item 48. The formulation for injection according to any one of items 9-17 and 20-46, wherein the composition comprises a tonicity agent chosen from: sterile saline solution; Ringer's solution; dextrose solution; dextrose monohydrate; and solidified high dextrose corn syrup.

Item 49. The formulation for injection according to item 48, wherein the tonicity agent is dextrose monohydrate.

Item 50. The formulation for injection according to any one of items 9-17 and 20-46, wherein the pharmaceutical excipient is chosen from: a pH-adjusting agent and a buffering agent.

Item 51. The formulation for injection according to item 50, wherein the pH-adjusting agent or buffering agent maintains the pH of the solution in a range of about 6 to about 7.5.

Item 52. The formulation for injection according to any one of items 9-17 and 20-46, wherein the pharmaceutical excipient is a preservative.

Item 53. The formulation for injection according to any one of items 9-17 and 20-46, wherein the pharmaceutical excipient is a chelating agent.

Item 54. The formulation for injection according to item 42, wherein the diluent is USP water for injection, the at least one non-ionic surfactant is poloxamer 188, and the optional pharmaceutical excipient is a tonicity agent that is dextrose monohydrate.

Item 55. A formulation for injection comprising:

(a) a TLR4 agonist;

(b) mixed micelles;

(c) at least one bile salt;

(d) and at least one lecithin;

(e) a diluent or carrier; and

(f) optionally, at least one pharmaceutical excipient;

wherein:

the resulting formulation is stable over a period of time from 12-36 months.

Item 56. The formulation for injection according to item 55, wherein the TLR4 agonist is (S)-2-((R)-3-(decy\oxy)tetra0ecanam 0o)-3-(((2R,3R,4R,5S,6R)-3-((R)-3- (decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., Compound 9 herein).

Item 57. A formulation for injection comprising:

a TLR4 agonist (S)-2-((R)-3-(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R)-3-((R)-3- (decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt

wherein: the TLR4 agonist is dissolved along with:

i. at least one surfactant selected from pluronics or lecithin;

ii. bile salts;

iii. in a pharmaceutically acceptable solvent selected from ethanol,

DMA or DMSO; and

iv. is then added to a surfactant selected from lecithin, bile salt or PLURONIC F188.

Item 58. A method of treating a cancer chosen from: melanoma; lung cancer; non-small cell lung cancer (NSCLC); kidney cancer; renal cell carcinoma; breast cancer; metastatic breast cancer; triple-negative breast cancer (TNBC); head and neck cancer; colon cancer; colorectal cancer (CRC); ovarian cancer; pancreatic cancer; liver cancer; hepatocellular carcinoma (HCC); prostate cancer; bladder cancer; gastric cancer; liquid tumours; solid tumours; hematopoietic tumours; leukaemia; non-Hodgkins lymphoma (NHL); and chronic lymphocytic leukaemia (CLL) in a human in need thereof comprising administering to the human a therapeutically effective amount of a compound of any one of items 1-8, 18 and 19 or pharmaceutical compositions or formulations of any one of items 9-17 and 20-57.

Item 59. A use of a formulation according to any one of items 9-17 and 20-57 for the manufacture of a medicament to treat a cancer chosen from: melanoma; lung cancer; non-small cell lung cancer (NSCLC); kidney cancer; renal cell carcinoma; breast cancer; metastatic breast cancer; triple-negative breast cancer (TNBC); head and neck cancer; colon cancer; colorectal cancer (CRC); ovarian cancer; pancreatic cancer; liver cancer; hepatocellular carcinoma (HCC); prostate cancer; bladder cancer; gastric cancer; liquid tumours; solid tumours; hematopoietic tumours; leukaemia; non-Hodgkins lymphoma (NHL); and chronic lymphocytic leukaemia (CLL) in a human patient in need thereof.

Item 60. The method of treating cancer according to item 58, wherein the formulation is administered via a route chosen from: intravenous; subcutaneous; intra-tumoral; and intradermal. Item 61. The method of treating cancer according to items 58 or 60, wherein the formulation is co-administered with at least one immunomodulatory agent.

Item 62. The method of treating cancer according to item 61, wherein the immunomodulatory agent is chosen from: an ICOS antibody; an OX-40 antibody; a PD-1 antibody; a PD-L1 antibody; and a CTLA-4 antibody.

Item 63. A method for stimulating an immune response or for preventing or treating a disease associated with the presence of an antigen, the method comprising delivering to an individual in need thereof a formulation according to any one of items 9-17 and 21-57, and, separately, a pharmaceutical composition comprising an antigen. Item 64. The method for stimulating an immune response or for preventing or treating a disease according to item 63, wherein the formulation comprising the TLR agonist and the composition comprising the antigen are delivered separately.

Item 65. The method according to item 63, wherein the antigen is a J3-amyloid antigen comprising at least one of: AJ31- 6; AJ33-8; AJ3p(E)3-8; AJ311-16; and AJ3p(E)ll-16.

Item 66. The method for stimulating an immune response or for preventing or treating a disease according to item 63, wherein the TLR agonist is a TLR4 agonist chosen a compound of Formula (I) or (II) and a pharmaceutically acceptable salt thereof optionally, in combination with QS21 and liposome.

Item 67. The method for stimulating an immune response or for preventing or treating a disease according to item 63, wherein the formulation comprising the TLR agonist and the composition comprising the antigen are delivered at different times, and wherein the TLR agonist is delivered after the composition comprising antigen or the composition comprising the antigen is delivered before at least one delivery of the formulation comprising the TLR agonist.

Item 68. An article of manufacture comprising a sealed vial containing a sterile aqueous injectable formulation of a TLR4 agonist of Formula (la); or a pharmaceutically acceptable salt or solvate thereof.

Item 69. A preparation of a pharmaceutical formulation or composition according to any one of items 9-17 and 20-57 wherein the preparation is in the form of an injectable solution filled into a dispensing vehicle.

Item 70. The preparation of a formulation according to item 69, wherein the dispensing vehicle is chosen from: a sterile vial; a sterile multi-dose vial; and a syringe.

The following Examples illustrate the invention. These Examples are not intended to limit the scope of the present invention, but rather to provide guidance to the skilled artisan to prepare and use the compounds or pharmaceutically acceptable salts thereof, pharmaceutical compositions or formulations, and methods of the present invention.

EXAMPLES

Example 1: Preparative And Analytical Description Of Manufacturing Process Towards Compound (9)

Synthetic Preparation

The synthesis of Compound 9, which is defined by the chemical name:

(S>2-((R>3-(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R>3-((R)-3- (decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5- (phosphonooxy)tetrahydro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., the tris(2-aminoethanol)salt of Formula III herein)

is described in Scheme 1 above.

Analytical Data

Proton ( H) and Carbon (¹³C) Nuclear Magnetic Resonance Spectroscopy

The 500 MHz H NMR spectrum and the 126 MHz ¹³C NMR spectrum of (9) in pyridine-d5 (17 mg/mL at 60°C) were concordant with the proposed structure.

Infrared Spectroscopy

The interpretation of the Diamond Attenuated Total Reflectance (ATR) infrared spectrum of

(9) is given in Table 1. The absorption characteristics are concordant with the proposed structure.

Table 1: Interpretation of the Diamond ATR Infrared Spectrum of(9)

Example 2: Formulation

This example (2) and the following example (3) set forth alternate approaches or potential ways to make formulations of the TLR4 agonist of a Compound of Formula (I) or (II).

Compound of Formula (I) or (II): 0.005 mg

Poloxamer 188: 5 mg

Dextrose Monohydrate: 220 mg

USP water for Injection: qs to 5 mL

A pH-adjusting agent that is hydrochloric acid and/or sodium hydroxide (if needed).

Example 3: Formulation

Compound of Formula (I) or (II): 0.0005 mg

Poloxamer 188: 5 mg

Dextrose Monohydrate: 220 mg

USP water for Injection: qs to 5 mL

A pH-adjusting agent that is hydrochloric acid and/or sodium hydroxide (if needed)

The solutions of Example 2 and Example 3 contain the compound at 0.001 and at 0.0001 mg/mL, respectively, filled into a 5-mL vial with a 20 mm stopper and aluminium seal. Nominal overfill was used.

Example 4: Manufacture Of Bulk Solutions Of The Present Invention

A ) Preparation of a bulk dextrose solution:

A bulk dextrose solution is prepared, wherein the dextrose monohydrate is weighed and dissolved into a suitable amount of water for injection (WFI). For example, to make a 30 Kg solution, 30,000 g of WFI will be used with 1320 g of dextrose to prepare a stock solution.

B) Preparation of a bulk dextrose/surfactant solution:

To make a dextrose/surfactant buffer solution, one prepares the dextrose solution of Step a) above, and then takes 13,000 g thereof, and adds 13 g of at least one suitable surfactant to it, for instance, poloxamer 188, while sonicating if necessary, to dissolve the at least one surfactant accordingly.

C) Preparation of a bulk Dextrose/Surfactant/API solution To make a 1 mg/mL APG solution, add 13000 g of the dextrose/surfactant solution as prepared in step B) above, and with sonication, dissolve the API. To this sonicated solution is added a solution chosen from: 0.1N HCL solution; and a 0.1M NaOH solution to adjust the pH, as necessary.

If tonicity agents or other agents as described herein are necessary, they can be added accordingly at this step, such as sugar alcohols, or glycerol.

The resulting solution containing the active, dextrose monohydrate, and the at least one surfactant then undergo sterile filtration. The appropriate vials are depyrogenated and filled, as needed, with stoppering and crimping of the vials to product an injectable liquid solution.

The glass vials are washed and rinsed with USP water for injection and depyrogenated by dry heat prior to being filled. The stoppers will then be "ready to sterilize", and the overalls are sterilized by gamma irradiation.

To make a stock 1 mg/mL API solution:

First, prepare 500 mL of a 0.1 NHCL solution, 92 g of NaOH pellet for 500 mL of solution) and, similarly, prepare a 0.1 NHCL solution (90 g of 10% HCL for 500 mL of solution).

Prepare 30 L of dextrose monohydrate solution (1.32 kg of dextrose for 30 L of solution).

Prepare 13 L of dextrose monohydrate/poloxamer solution by adding 13 g of poloxamer 188 into 13 L of prepared dextrose monohydrate solution with stirring.

Transfer 100 g of dextrose solution into a 200 mL wide mouth clear glass flask. Wet the preweighed API by injecting through a syringe a suitable amount of vehicle and transfer the suspended API into a 400 mL flask. Rinse the API container several times with about 10 mL vehicle and stir gently to get a uniform suspension, approximately 15 minutes. Remove the stirrer bar/blade and rinse the container using vehicle. Sonicate the glass flask under chilled water for about 90 minutes. If any lumps/aggregated are observed after sonication, continue for to weigh 95% (about 950 g net weight) of final weight. 60 more minutes or until solution is uniform while maintaining sonicator water temperature < 15 °C. Transfer the contents of the sonicated flask to a 2 L glass vessel while stirring gently and rinse container with vehicle to extract all of the API suspension as needed. Continue stirring for about 30 minutes. Add dextrose/poloxamer solution. Check and adjust pH to about 6.9-7.1 with 0.1N HCI or 0.1 N NaOH as needed.

After interim analysis of the stock solution, final weight adjustments may be needed, and as such the final net weight of active may increase to account for these assays.

The stock solution of 1 mg/mL will undergo a bioburden reduction with inline filtration.

To make a 0.001 mg/mL API solution and a 0.001 mg/mL API solution from the bulk solution:

Using the 1 mg/mL stock solution added about 95% batch size of the vehicle (5.7L of prepared Dextrose Monohydrate/Surfactant solution) to a 15 L glass vessel while stirring and continue stirring at a moderate speed for about 5 minutes. Transfer the measured 1 mg/mL solution portion into a tared 15 L glass vessel and rinse 1 mg/mL solution holding contained approximately 3 times (each with about 50-80 ml. of vehicle). Sonicate the solution for about 60 minutes under chilled water. Check and adjust pH to about 6.9-7.1 with a solution chosen from: 0.1N HCL solution; and NaOH solution, as needed. Adjust the final weight by adding vehicle (dextrose monohydrate/surfactant solution) to make a 100% solution of active.

Example 5: Use of an Organic Co-Solvent

Purpose:

This example sets forth alternate approaches or potential ways to make formulations of the TLR4 agonist of Compound 9.

(1) Lyophilization Process

(2) Solvent diffusion oil/water process to generate mixed micelles to remove sonication.

Method Details:

(1) Lyophilization-based formulation

Compound 9, given its high log P = 80, remain challenging to formulate without the aid of a high energy process, e.g., sonication. The current formulation, which is being presented as a solution, is stable over at least 24 months at 5 °C. However, it is possible that such a formulation could be lyophilized and presented in more stable form at RT, with potentially 5 years' stability.

Lyophilization formulation could be achieved in following way:

(1) Dissolve API (Compound 9) in a lyophilisation-friendly solvent/co-solvent, e.g., acetonitrile/ tert-butyl alcohol (TBA), at 0.1 % to 50 % in water.

(2) Add at least one suitable surfactant, e.g, poloxamer 188, polysorbate, etc., anywhere from 7 ng to 100 mg/mL.

(3) Dissolve a suitable sugar in above admixture, e.g., Sucrose, Trehalose, mannitol, dextrose or lactose, anywhere from 1% to as high as 15 %.

(3) Sterile filter the solution (through a 0.2-micron filter), which could then be lyophilized under common conditions mentioned in steps below.

1. Freeze at 1 °C/min to -45 °C, at ambient pressure.

2. Annealing may be required, depending upon the type of sugars used (-45 to -15 °C to -45 °C) at 1 °C / min. at ambient pressure, with appropriate holding period at each temperature.

3. Then draw vacuum to < 100 mTorr, and ramp shelf temperature from -45 °C to -10 °C at 1 °C /min and hold it for sufficient time until primary drying is completed.

4. When primary drying is completed (Pirani gauge comes to chamber pressure or product temperature touches shelf temperature), start secondary drying at chamber pressure less than 100 mTorr, but at higher shelf temperature than primary drying, e.g., 10 °C, until sufficient moisture control is achieved, e.g., less than 1%.

This procedure should provide satisfactory cake with desired reconstitution time without change in chemical and biological properties of active ingredient.

(2) The current Compound 9 formulation process is based upon sonication, making it difficult to scale-up for manufacture. The following process can be adopted to improve formulation process, in a typical process of making mixed micelles, the active and surfactants (lipids, bile salt, or amphiphilic lipids/surfactants) can be dissolved in organic, water-miscible solvent (e.g., ethanol, dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), propylene glycol, polyethylene glycol 200 to 400 Da, glycofurol, etc.), and gradually discharged into water (aqueous phase) containing appropriate amount of amphiphilic surfactant/bile salts. This process can be called as oil/water (o/w) solvent diffusion method, where the solvent is miscible with aqueous phase. This process leaves the API entrapped in dissolved form within amphiphilic surfactant and become soluble / dispersed without aid of sonication like high energy processes.

Typical ratio of oil to water is about 0.1 %, or as high as 30 %. The oily or organic (solvent/cosolvent) can be removed using a rotary evaporator, often left within formulation if below their toxicity levels. The micellar solution (clear/opaque) can easily be filtered through a 0.2-micron filter as a sterile filtration, without any back-pressure issue, that is faced when sonicated formulation is filtered (Phase I manufacturing experience). The preliminary experiment proof of concept is described using lecithin and bile salt combinations, as discussed below in Example 6.

Example 6: Bile Salt Formulation

Purpose

The purpose of this experiment is to evaluate bile salts and/or lipids/surfactants as a mixed micellization system to solubilize insoluble/sparingly soluble drug, i.e., TLR4 agonist, (S)-2-((R)-3- (decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R)-3-(decyloxy)tetradecanamido)-4-(((R)-3- (decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2- yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e., Compound 9 herein).

Compound 9 is demonstrated for solubilization using mixed micelles approach as below.

Compound 9 is an insoluble molecule, currently requiring a sonication process to obtain meaningful solubility to make cGMP batch. Given that a sonication process is not feasible at cGMP scale going forward, solubilization without sonication is required to make process more feasible to scale up. These efforts are designed to evaluate the mixed micellization system, using solvent diffusion/evaporation (o/w) technique, as well as to micellize drug using various composition of bile salt/lipids- surfactant system. 702 mg of sodium cholate monohydrate was weighed and added to 30 ml. water for injection and dissolved to a clear solution using a magnetic stirrer. To this solution, 5.0 mg of Compound 9 was added, and followed by stirring with a magnetic stirring, a clear to translucent solution was obtained in less than 3.5 hours at 1500 rpm. No sonication was required to disperse/solubilize the drug at 0.166 mg/mL. The pH of this solution was found to be 8.0. The solution was sterile filtered through a 0.2- micron syringe filter 13 mm (using 3 ml. poly propylene syringe purchased from BENNETT DICKINSON COMPANY). No back pressure or blockage was noted for such a formulation containing bile salt micelles.

This solution was divided into 2 portions (15 ml. each). In one portion, (Formulation 1), 150 mg of egg phosphatidyl choline was added to form mixed micelles. The mixture was stirred at 1500 rpm for 3.5 h. A clear to translucent solution was obtained, with a pH of 8.0. The solution was sterile filtered through a 0.2-micron syringe filter 13 mm (using 3 ml. poly propylene syringe purchased from BENNETT DICKINSON COMPANY). No back pressure or blockage was noted for such a formulation containing bile salt and additional lecithin.

Both formulations above, when stored at 5 °C, maintained their clarity and remained physically stable.

It was possible to successfully repeat this process using optimal ratio of bile salts (various) and lipids. In the formulation art, one can produce combination of bile salts and various lipids to solubilize TLR-4 agonists, including Compound 9, under relatively mild conditions as compared to the conditions that required sonication.

The above experiment shows that sonication can be removed from the process, if additional excipients are considered. A solvent diffusion process using a water-miscible solvent could achieve the same results, in a case where Compound 9 is dissolved along with poloxamer P188 in a water miscible solvent, e.g., ethanol, glycofurol, N-methylpyrollidone (NMP), Dimethyl acetamide, PEG 200- 400, propylene, ethanol, etc., and added to aqueous phase containing a suitable amount of surfactants, such as Poloxamers, could generate micellar solution without aid of sonication which could be sterile filtered through 0.2 micron filter without back pressure issue.

In the formulation art, one could combine these processes/combinations to remove sonication from the currently adopted process.

Example 7: Formulation Containing fS)-2-ffR)-3-(decyloxy)tetradecanamido)-3-(7Y2R,3R,4R,5S,6R)-3- ((R)-3-(decyloxy)tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hvdroxymethyl)-5- (phosphonooxy)tetrahvdro-2H-pyran-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt (i.e.. Compound 9 herein)

In general, a formulation containing active pharmaceutical ingredient ("API") or drug product of the present invention is prepared by three (3) major steps: 1) Make vehicle solution. The vehicle is a solution, which contains all excipients of a formulation of the present invention, except the Active Pharmaceutical Ingredient ("API") and dehydrated alcohol as solubilizing agent.

2) Make Active Pharmaceutical Ingredient (i.e., "API") stock solution; and

3) Combine the two solutions to form the final formulation containing the API or drug product.

Procedure

To make the vehicle solution at 2 litre batch size, 1.6 Litre of water for injection, and 88 grams of dextrose monohydrate [0.44M] was added to a 3.5 litre beaker and mixed until complete dissolution. 2g of Kolliphor P188 [Kolliphor P188 = Poloxamer 188] was added subsequently to the solution as the stabilizer and also mixed until complete dissolution. The volume of the solution was brought up to 95% of the final volume before pH adjustment to 6.8-7.2 using 0.1N HCL and 0.1N NaOH. Then the volume was brought up to 100% of the batch size.

To make the API stock solution at 1 mg/mL concentration, 10 mg of API [6.9xlO ⁶M] was weighed and added to a 20 mL glass bottle followed by 10 mL of dehydrated alcohol, the resulting mixture was then vortexed until full dissolution.

Finally, to make the final formulation containing the API or drug product at concentration of 500 ng/mL at batch size of 200 mL, 200 mL of vehicle solution was then transferred to a 500 mL glass bottle followed by 0.1 mL of stock solution. Then the liquid was mixed until clear solution was achieved.

Formulation Table: Sterile Product

Example 8: Human Whole Blood Stimulation Assay

Peripheral whole blood samples were collected into anticoagulant-treated heparinized syringes from adult donors and cultured in flat-bottomed 96-well cell culture at 37°C, and 5% CO2. ( S)-2-((R>3-(decyloxy)tetradecanamido)-3-(((2R,3R,4R,5S,6R>3-((R)-3-(decyloxy ) tetradecanamido)-4-(((R)-3-(decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy) tetrahydro-2H-pyral-2-yl)oxy)propanoic acid tris(2-aminoethanol) salt was serially diluted in vehicle IV solution 0.9% Saline (No Dextrose) in a minimal volume of 2.5% of total volume of blood.

Samples were cultured immediately either with vehicle, (S)-2-((R )-3-(decyloxy) tetradecanamido)-3-(((2R,3R,4R,5S,6R 3-((R)-3-(decyloxy)tetradecanamido)-4-(((R)-3- (decyloxy)tetradecanoyl)oxy)-6-(hydroxymethyl)-5-(phosphonooxy)tetrahydro-2H-pyran-2- yl)oxy)propanoic acid tris(2-aminoethanol) salt (0.0625 to 20,000 ng/mL) or with a single concentration (20,000 ng/mL) of LPS (Sigma Chemical Co., St. Louis, MO) for the study of cytokine release.

Plasma supernatants were collected at 0 hours (baseline), and 0.5, 2, 6, and 24 hours post stimulation. Samples were then centrifuged for 10 minutes at 2500 rpm at room temperature (RT) and the plasma was collected and frozen at -80 °C until cytokine analysis.

Example 9: Human Cytokine Expression

Human Luminex (bead based liquid phase assay), cytokine plates were used to analyze plasma supernatants along with a human IFN beta ELISA kit.

The multiplex panel included IL-Ib, IL-1RA, IL-2, IL-6, IL-8, IL-10, IL-12p70, TNF-a, IP-10, RANTES, G-CSF, and MCP1 (EMD Millipore Corp, Billerica, MA).

The Luminex xMAP platform is a bead-based technology that allows capture antibodies to be coupled to colour coded beads or microspheres that contain different emission spectra.

A sandwich assay format is performed with the analyte added to the capture antibody bound beads, followed by the addition of a biotinylated antibody. The detection occurs by adding a streptavidin-conjugated flurochrome to the complex containing the sandwiched immunoassay. The fluorescent signal is detected using a Luminex xMAP which is a flow cytometry-based instrument. The microspheres are classified based on their emission spectra and the amount of analyte detected is directly proportional to the fluorescent signal.

Briefly, 25 mL of beads were incubated at 4 °C with 25 mL of undiluted or diluted (neat or 1: 100, respectively) plasma samples in the kit's assay buffer. Following overnight incubation, the liquid was removed by luminex magnet (EMD Millipore Corp, Billerica, MA). The beads were washed with wash buffer and then incubated for 1 hour with biotinylated detection antibody followed by incubation with streptavidin phycoerythrin (PE) for approximately 30 minutes. The beads were then washed with wash buffer and the plates were then read on the MAGPIX (EMD Millipore Corp, Billerica, MA) using xPonent software (EMD Millipore Corp, version 3.1). The detailed experimental kits procedures can be found in the manufacturer's protocol. An, enzyme linked immunosorbant assay (ELISA) with human IFNa capture antibodies spotted on the wells of a 96 well chromogenic microplate was also performed.

Experiments were performed using the manufacturer's recommended procedures for Milliplex and PBL commercial kits. (n=l). Data was generated using the LuminexTM MAGPIX reader and the SprectroMax 384 microplate reader.

Cytokines were induced between 1 and 2 hours with maximal cytokine levels observed at 24 hours (human) post stimulation, while TNF-a had a peak induction at 6 hours and returned to unstimulated levels by 24 hours. There was a potent IP-10 induction when stimulated with Compound 9. IL-10, G-CSF, and IL-Ib were also induced with similar kinetics.

Example 10: Formulation

An exemplary formulation of Compound 9 is:

The pH is 7.0 + 0.2 (range is pH 6.8 to 7.2).

Notes:

1. The actual quantity is adjusted to the amount of Compound 9 (the tris(2-aminoethanol) salt of the compound of Formula III) equivalent to 0.0005 mg of the compound of Formula III as the free acid based on the weighing factor of input active ingredient.

2. May be used to adjust pH. This formulation can be employed in the methods and uses provided herein. This formulation may be administered by injection, optionally intravenous injection.

Claims

1. A compound which is a 2-aminoethanol salt of a compound of Formula (III):

2. The compound according to claim 1, wherein the 2-aminoethanol salt is the tris(2- aminoethanol) salt.

3. The compound according to claim 1 or claim 2, which is in a semi-crystalline or a mesophase form.

4. The compound according to any of the preceding claims, which is in a solvate form.

5. The compound according to claim 2, having a Diamond Attenuated Total Reflectance (ATR) Infrared Spectrum pattern with characteristic peaks in wavenumbers (cm^-1) at: 3600-3000, 2955, 2919, 2851, 1741, 1717, 1646, 1636, 1552, 1466, 1101, 1073, and 1026.

6. A pharmaceutical formulation or composition comprising a compound of any one of claims 1- 5 and at least one pharmaceutically acceptable excipient.

7. The pharmaceutical formulation or composition according to claim 6, wherein the compound is present at 0.0005 mg/mL to 0.001 mg/mL.

8. The pharmaceutical formulation or composition according to claim 6 or claim 7, which is adapted for intravenous injection.

9. The pharmaceutical formulation or composition according to claim 8, comprising:

a. a diluent;

b. at least one non-ionic surfactant; and

c. about 500 ng/mL of the compound of any one of claims 1-6.

10. The pharmaceutical formulation according to any one of claims 6-9, wherein the formulation is stable for at least 12 months.

11. The pharmaceutical formulation or composition according to claim 9 or 10, wherein the at least one non-ionic surfactant is selected from the group consisting of: a polyol; a polyoxyethylene ester; a polyoxyethylene ether; a polymeric ether; a polyvinyl alcohol PEG diacetate; a nonyl phenyl ether; a propylene glycol diacetate; a polyvinyl alcohol; a PEG castor oil derivative; a PEG ester; a sorbitan derivative; an alkanolamide; and combinations thereof.

12. The pharmaceutical formulation or composition according to claim 11, wherein the polymeric ether is a poloxamer selected from the group consisting of: P124; P181; P182; P184; P188; P237; P331; P338; P407; and mixtures thereof.

13. The pharmaceutical formulation or composition according to any one of claims 9-12, wherein the diluent is selected from the group consisting of: USP water for injection; sterile water for injection (SWFI); and bacteriostatic water for injection (BWFI); a pH-buffered solution; a sterile saline solution; Ringer's solution; and a dextrose solution.

14. The pharmaceutical formulation or composition according to any one of claims 9-13, further comprising a pH-adjusting agent; a co-solvent; a buffering agent; a chelating agent; a tonicity agent; an antioxidant; and/or a preservative.

15. The pharmaceutical formulation or composition according to claim 14, wherein the tonicity agent is dextrose monohydrate.

16. A 1 ml. unit dose pharmaceutical formulation comprising:

a. about 0.0005 mg of a compound of Formula (III):

or a 2-aminoethanol salt thereof, optionally the tris(2-aminoethanol) salt;

b. about 0.0005 ml. of dehydrated alcohol, optionally ethanol;

c. about 44.0 mg of dextrose monohydrate;

d. about 1 mg of P188; e. sodium hydroxide and/or hydrochloric acid, if necessary, to adjust the pH of the formulation to about 7.0; and

f. water for injection to reach 1 ml_.

17. A method of treating a cancer in a human in need thereof, comprising administering to the human a therapeutically effective amount of a compound according to any one of claims 1-5 or a pharmaceutical formulation or composition according to any one of claims 6-16.

18. A compound according to any one of claims 1-5 or pharmaceutical formulation or composition of any one of claims 6-16, for use in treating cancer in a human.

19. Use of a compound according to any one of claims 1-5 in the manufacture of a medicament for use in treating cancer.

20. A method according to claim 17, a compound or pharmaceutical formulation or composition according to claim 18, or a use according to claim 19, wherein the cancer is selected from the group consisting of: melanoma; lung cancer; non-small cell lung cancer (NSCLC); kidney cancer; renal cell carcinoma; breast cancer; metastatic breast cancer; triple-negative breast cancer (TNBC); head and neck cancer; colon cancer; colorectal cancer (CRC); ovarian cancer; pancreatic cancer; liver cancer; hepatocellular carcinoma (HCC); prostate cancer; bladder cancer; gastric cancer; liquid tumors; solid tumors; hematopoietic tumors; leukemia; non- Hodgkins lymphoma (NHL); and chronic lymphocytic leukemia (CLL).

21. A method according to claim 17 or claim 20; a compound or pharmaceutical formulation or composition according to claim 18 or claim 20; or a use according to claim 19 or claim 20; wherein the compound, formulation or pharmaceutical composition is administered intravenously.

22. A method according to claim 17, claim 20 or claim 21; a compound or pharmaceutical formulation or composition according to claim 18, claim 20 or claim 21; or a use according to claim 19, claim 20 or claim 21; wherein the compound or pharmaceutical formulation or composition is administered with at least one immunomodulatory agent selected from the group consisting of: an agonist ICOS antibody; an agonist OX-40 antibody; an antagonist PD- 1 antibody; an antagonist PD-L1 antibody; and an antagonist CTLA-4 antibody.

23. A kit of parts, comprising:

a. a compound or pharmaceutical formulation or composition according to any one of claims 1-16; and

b. instructions for use.

24. A kit according to claim 23, wherein the compound or pharmaceutical formulation or composition is in the form of an injectable solution filled into a dispensing vehicle selected from the group consisting of: a sterile vial; a sterile multi-dose vial; and a syringe.

25. A kit according to claim 23 or claim 24, further comprising at least one immunomodulatory agent selected from the group consisting of: an agonist ICOS antibody; an agonist OX-40 antibody; an antagonist PD-1 antibody; an antagonist PD-L1 antibody; and an antagonist CTLA-4 antibody.