WO2012047656A1 - Lipid a analog compositions - Google Patents

Abstract

Provided are compositions compirsing a Lipd A analog, e.g., a compound of formula (I): or a pharmaceutically acceptable salt thereof, and an anti-sequestration component, such as a component of LDL. Also provided are methods for treating disorders, e.g., sepsis, using such compositions.

Description

LIPID A ANALOG COMPOSITIONS

Related Applications

This application is related and claims priority to U.S. Provisional Application Serial Number 61/386,736, filed September 27, 2010. The entire contents of this application are hereby incorporated by this reference.

Background

Lipid A analogs potentially have a number of clinical applications. For example, eritoran, also called E5564 and/or [a-D-glucopyranose, 3-0-decyl-2-deoxy-6-0-[2- deoxy-3-0-[(3R)-3-methoxydecyl]-6-0-methyl-2-[(l lZ)-l-oxo-l l-octadecenyl)amino]- 4-0-phosphono- -D-glucopyranosyl]-2-[(l,3-dioxotetradecyl)amino]-l-(dihydrogen phosphate), is an antagonist of LPS, possibly due to its ability to compete with LPS at the cell-surface receptor complex containing TLR4 and MD-2. As such, eritoran can be used in the treatment of endotoxemia, sepsis and various other indications. See, e.g. , U.S. Patent No. 5,750,664.

Although eritoran typically has a fairly long pharmacokinetic half-life, it experiences a dose dependent loss of activity, generally over an 8-hour period. See, e.g. , Wong et al. J. Clin. Pharmacol., 43:735-742 (2003). It has been suggested that this loss of activity may be attributable to the ability of eritoran to associate with and/or bind to lipoproteins, predominantly high-density lipoprotein (HDL) which, in purified form, has been demonstrated to inactivate eritoran. For example, studies have shown that about 60% of eritoran rapidly associates with HDL upon administration to a subject and generally does not re-distribute among lipoprotein classes once associated, thus leading to time-dependent inactivation of the eritoran. See, e.g. , Wasan et al. Antimicrob.

Agents Chemother., 47:2796-2803 (2003) and Rossignol et al. Antimicrob. Agents Chemother., 48:3233-3240 (2004).

Summary of the Invention

It would be useful to reduce the interaction of Lipid A analogs, such as eritoran, by generating a composition which would maintain its activity, e.g. , which would not be inactivated by HDL. The present invention is based, at least in part, on the discovery of compositions which can allow for an extended pharmacodynamic half life of a Lipid A analog. In some embodiments, the present teachings provide compositions which include a compound of formula (A) and an anti-sequestration component, wherein the compound of formula

or a pharmaceutically acceptable salt thereof

where R¹ is selected from:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R is selected from:

where A is straight or branched C7 to C12 alkyl; and each B and D, independently, is straight or branched C4 to C9 alkyl;

R⁴ is selected from:

straight or branched C8 to C12 alkyl, and

where U is straight or branched C2 to C4 alkyl; V is straight or branched

C5 to C9 alkyl; and W is hydrogen or -CH₃;

R_A is R⁵-0-CH₂-, where R⁵ is hydrogen or straight or branched CI to C5 alkyl; R⁶ is hydroxy; and A¹ and A² are each independently

O O P OH

OH embodiments,

R¹ is:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R³ is:

where A is straight or branched C7 to C12 alkyl; and B is straight or branched C4 to C9 alkyl;

R⁴ is:

where U is straight or branched C2 to C4 alkyl; V is straight or branched C5 to C9 alkyl; and W is hydrogen or -CH₃;

R_A is R⁵-0-CH₂-, where R⁵ is straight or branched CI to C5 alkyl;

R » 6 i ·s hydroxy; and

A¹ and A² are each independently

O

-o- -P OH

OH In some embodiments, the present teachings provide compositions which include a compound of formula (I) and an anti-sequestration component, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the present teachings provide methods for extending the pharmacodynamic half-life of a compound of formula (A). The method generally includes contacting the compound of formula (A) with an anti-sequestration component, wherein the compound of formula (A) is:

or a pharmaceutically acceptable salt thereof

where R¹ is selected from:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R is selected from:

where A is straight or branched C7 to C12 alkyl; and each B and D, independently, is straight or branched C4 to C9 alkyl;

R⁴ is selected from:

straight or branched C8 to C12 alkyl, and

where U is straight or branched C2 to C4 alkyl; V is straight or branched

C5 to C9 alkyl; and W is hydrogen or -CH₃;

RA is R⁵-0-CH₂-, where R⁵ is hydrogen or straight or branched CI to C5 alkyl; R⁶ is hydroxy; and

A¹ and A² are each independently

O O P OH

OH such that the pharmacodynamic half-life of the compound of formula (A) is extended relative to the pharmacodynamic half-life of free compound of formula (A).

embodiments,

R¹ is:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R³ is:

where A is straight or branched C7 to C12 alkyl; and B is straight or branched C4 to C9 alkyl;

R⁴ is:

where U is straight or branched C2 to C4 alkyl; V is straight or branched

C5 to C9 alkyl; and W is hydrogen or -CH₃;

R_A is R⁵-0-CH₂-, where R⁵ is straight or branched CI to C5 alkyl;

R⁶ is hydroxy; and

A¹ and A² are each independently

O O P OH

OH

In some embodiments, the present teachings provide methods for extending the pharmacodynamic half-life of a compound of formula (I). The method generally includes contacting the compound of formula (I) with an anti-sequestration component, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof; such that the pharmacodynamic half-life of the compound of formula (I) is extended relative to the pharmacodynamic half-life of free compound of formula (I).

In some embodiments, the anti-sequestration component is an anti-sequestration lipoprotein component. In some embodiments, the anti-sequestration component is a component of HDL, LDL, IDL or VLDL. In some embodiments, the anti-sequestration component is a component of LDL or VLDL. In some embodiments, the anti- sequestration component is at least one component selected from an apolipoprotein, a triglyceride, a cholesterol, a cholesterol ester, a phospholipids, a lipoprotein fragment and mixtures thereof. In some embodiments, the anti- sequestration component is at least one component selected from apolipoprotein C2, apolipoprotein C3 and mixtures thereof.

In some embodiments, the anti-sequestration component is a lipid bound apolipoprotein or a lipid bound apolipoprotein fragment. In some embodiments, the anti-sequestration component is selected from lipid bound apolipoprotein A2, lipid bound apolipoprotein CI and mixtures thereof.

In some embodiments, the lipid bound apolipoprotein is an apolipoprotein bound to or associated with reconstituted HDL. In some embodiments, the lipid bound apolipoprotein is selected from rHDL-A2, rHDL-Cl, rHDL-SAA and mixtures thereof. In some embodiments, the lipid bound apolipoprotein is selected from rHDL-A2, rHDL- Cl, and mixtures thereof.

In some embodiments, the ratio of anti-sequestration component to compound of formula (A) is between about 1:10 and about 1000:1 by weight.

In some embodiments, the present teachings provide methods for treating sepsis in a subject in need thereof. The method generally includes administering a composition as described herein to the subject, such that sepsis is treated.

The composition can be administered in a single bolus or in a bolus followed by one or more maintenance doses. Additionally or alternatively, the composition can be administered in an intermittent intravenous infusion or in a continuous intravenous infusion.

In some embodiments, the composition is administered no more than once about every 12 hours. In some embodiments, the composition is administered no more than once about every 18 hours. In some embodiments, the composition is administered no more than once about every 24 hours. In some embodiments, the composition is administered no more than once about every 48 hours.

In some embodiments, the compound of formula (A) in the composition is administered in a dosage of about 0.1 mg/kg to about 10 mg/kg per day. In some embodiments, TNF-a release in the subject is inhibited by at least about 80% for at least about 24 hours. In some embodiments, TNF-a release in the subject is inhibited by at least about 90% for at least about 72 hours. Description of the Drawings

Figure 1 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with apolipoprotein A2 at low (A), intermediate (B), and high (C) concentrations. *Denotes a statistically significant difference from LPS/Eri (p<0.05).

Figure 2 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with apolipoprotein CI at low (A), intermediate (B), and high (C) concentrations. *Denotes a statistically significant difference from LPS/Eri (p<0.05).

Figure 3 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with apolipoprotein C2 at low (A), intermediate (B), and high (C) concentrations.

Figure 4 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with apolipoprotein C3 at low (A), intermediate (B), and high (C) concentrations.

Figure 5 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with apolipoprotein E at low (A), intermediate (B), and high (C)

concentrations. *Denotes a statistically significant difference from LPS/Eri (p<0.05).

Figure 6 depicts TNF-a release from human whole blood after stimulation with lOng/mL LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri", final concentration ΙΟηΜ) pre-incubated with increasing concentrations of high-density lipoprotein (HDL). HDL concentrations are expressed in mg/mL by protein and cytokine release in % of LPS control. *Denotes statistically significant differences between groups to the LPS/Eri group as assessed by Dunnett's test.

Figure 7 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with rHDL-Al. *Denotes a statistically significant difference from LPS/Eri (p<0.05). Figure 8 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with rHDL-SAA. *Denotes a statistically significant difference from LPS/Eri (p<0.05).

Figure 9 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with rHDL-Cl .

Figure 10 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with rHDL-A2.

Figure 11 depicts TNF-a release from human whole blood after stimulation with LPS in the presence of an exemplary compound described herein (eritoran, denoted "Eri") pre- incubated with "normal" reconstituted HDL (NrHDL). The NrHDL model is composed of all native HDL apolipoproteins (Al , A2, CI , C2, C3, and E) in the molar ratio they would be found associated with HDL in a healthy person.

Detailed Description of the Invention

The present invention is based, at least in part, on the finding that the compounds taught herein, e.g. , compounds of formula (A) can be used in combination with one or more anti- sequestration components, such as various components of HDL, LDL, IDL or VLDL. Without wishing to be bound by any particular theory, it is believed that the compounds of formula (A) can bind to or otherwise associate with the anti- sequestration components (e.g. , the component of HDL, LDL, IDL or VLDL), such that the resultant composition is able to exhibit an extended pharmacodynamic half life. It is believed that such extension of pharmacodynamic half life may be due, at least in part, to differences in the ability of various lipoproteins and components thereof to inactivate (e.g. , suppress the activity of) the compound of formula (A).

Lipid A analogs are typically lipophilic molecules. As such, they are able to bind to or otherwise associate with hydrophobic surfaces such as lipoproteins (e.g. , as a part of a lipid layer). Without wishing to be bound by any particular theory, it is believed that Lipid A analogs, such as the compounds described herein, exhibit little or no preference for a specific class of lipoproteins (e.g. , HDL, LDL, VLDL or other lipoproteins). However, because HDL provides the predominant lipoprotein surface area in blood, a large portion of the Lipid A analog becomes bound to or otherwise associated with the HDL or a component thereof. As discussed in more detail above, it is suggested that this phenomenon is at least partially responsible for the short pharmacodynamic life of the Lipid A analog. Without wishing to be bound by any particular theory, it is also believed that Lipid A analogs, once bound to or otherwise associated with the lipoprotein or component thereof, do not typically migrate to other lipoproteins. It has been shown herein that binding or otherwise associating with anti- sequestration components (such as LDL or components thereof) does not inactivate the Lipid A analog, as is the case with native HDL. Accordingly, the present invention is also based, at least in part, on the finding that the pharmacodynamics of compounds of formula (A) can be improved by pre-incubation or pre- association with one or more anti- sequestration components.

In order to more clearly and concisely describe the subject matter of the claims, the following definitions are intended to provide guidance as to the meaning of terms used herein.

As used herein, the articles "a" and "an" mean "one or more" or "at least one," unless otherwise indicated. That is, reference to any element of the present invention by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present.

Certain values and ranges are recited in connection with various embodiments of the present invention, e.g. , amount of a compound of formula (I) present in a composition. It is to be understood that all values and ranges which fall between the values and ranges listed are intended to be encompassed by the present invention unless explicitly stated otherwise.

The term "about" as used herein in association with parameters, ranges and amounts, means that the parameter or amount is within ±1% of the stated parameter or amount.

As used herein, the term "inactivation", when used in reference to Lipid A analogs, refers to the phenomenon where the Lipid A analog is rendered unavailable for inhibition of TLR4 activation. For example, in some embodiments, inactivation can occur via the encapsulation or sequestration of the Lipid A analog within a

macrostructure, such as HDL. That is, inactivation does not refer to the metabolism or breaking down of the compound, but rather to its inability to achieve a biological endpoint (for example, due to association with a component that renders the compound unavailable or "hidden"). Typically, inactivated Lipid A analogs are detectable in the bloodstream at levels that would be efficacious under normal (i. e. , non-inactivated) circumstances, but are inactive or of marginal activity when compared to their activity immediately post infusion as assessed by an "ex vivo" assay (e.g. , Wong et al. J. Clin. Pharmacol., 43:735-742 (2003)).

J. Compositions

In some embodiments, the present invention provides compositions which include a Lipid A analog (e.g. , a compound of formula (A) or a pharmaceutically acceptable salt thereof) and an anti- sequestration component.

In some embodiments, compositions of the present invention include a compound of formula (A) or a pharmaceutically acceptable salt thereof. Compounds of formula (A) have the

and pharmaceutically acceptable salts thereof

wherein R¹ is selected from:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to C15 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R is selected from:

where A is straight or branched C7 to C12 alkyl; and each B and D,

independently, is straight or branched C4 to C9 alkyl;

R⁴ is selected from:

straight or branched C8 to C

where U is straight or branched C2 to C4 alkyl; V is straight or branched C5 to

C9 alkyl; and W is hydrogen or -C¾;

RA is R⁵-0-CH₂-, where R⁵ is hydrogen or straight or branched CI to C5 alkyl;

> 6 ·

R is hydroxy; and

A¹ and A² are each independently

O

-O P OH

OH

As used herein, "alkyl" groups include saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g. , methyl, ethyl, propyl, butyl, pentyl, hexyl, methylene, ethylene, propylene, butylene, pentylene, hexylene, etc.), cyclic alkyl groups (or "cycloalkyl" or "alicyclic" or "carbocyclic" groups) (e.g. , cyclopropyl, cyclopentyl, cyclohexyl, etc.), branched-chain alkyl groups (isopropyl, tert- butyl, sec -butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g. , alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups). The term "CI to C6" as in "CI to C6 alkyl" means alkyl groups containing 1 to 6 carbon atoms.

In some embodiments, R¹ is:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to C15 alkyl; and Q is straight or branched CI to C3 alkyl. In some embodiments, J is a CI alkyl, e.g. , -CH2-. In some embodiments, K is a CIO to C12 alkyl, e.g. , a Cl l alkyl.

In some embodiments, R² is straight or branched C8 to C12 alkyl, e.g. , a C9 to Cl l alkyl, e.g. , a CIO alkyl.

In some embodiments, R is:

where A is straight or branched C7 to C12 alkyl; and B is straight or branched C4 to C9 alkyl. In some embodiments, A is a C8 to CI 1 alkyl, e.g. , a C9 alkyl. In some embodiments, B is a C5 to C8 alkyl, e.g. , a C6 alkyl.

In some embodiments, R is:

where U is straight or branched C2 to C4 alkyl; V is straight or branched C5 to C9 alkyl; and W is hydrogen or -CH₃. In some embodiments, U is C2 alkyl, e.g. , -CH₂CH₂-. In some embodiments, V is a C6 to C8 alkyl, e.g. , a C7 alkyl. In some embodiments, W is

In some embodiments, RA is R⁵-0-CH₂-, where R⁵ is straight or branched CI to C5 alkyl. In some embodiments, R5 is a CI alkyl, e.g. , -CH₃.

In some embodiments, the compositions of the present invention include one or more compounds of formula (A):

or pharmaceutically acceptable salts thereof

where R¹ is:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R³ is:

where A is straight or branched C7 to C12 alkyl; and B is straight or branched C4 to C9 alkyl;

R⁴ is:

where U is straight or branched C2 to C4 alkyl; V is straight or branched C5 to C9 alkyl; and W is hydrogen or -CH₃;

R_A is R⁵-0-CH₂-, where R⁵ is straight or branched CI to C5 alkyl;

R⁶ is hydroxy; and

A¹ and A² are each independently

O O P OH

OH

In some embodiments, the compositions of the present invention include one or more compounds of formula (I). Compounds of formula (I) have the following structure:

or pharmaceutically acceptable salts thereof. The compound of formula I may also be known as E5564, 1287, eritoran, SGEA or (a-D-Glucopyranose, 3-0-decyl-2-deoxy-6- 0-[2-deoxy-3-0-[(3R)-3-methoxydecyl)-6-0-methyl-2-[[(- HZ)-l-oxo-l l- octadecenyl)amino]-4-0-phosphono- -D-glucopyranosyl]-2-[(l,3- dioxotetradecyl)amino]-l-(dihydrogen phosphate). The compound of formula I is described as compound 1 in U.S. Pat. No. 5,681,824, which is incorporated herein by reference. The compound of formula I may be prepared in the form of a micelle, as described in U.S. Pat. No. 6,906,042, which is incorporated herein by reference in its entirety for the description of such micelles and methods for preparing same.

As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et ah , describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977), which is incorporated herein by reference. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting a free base or free acid function with a suitable reagent, as described generally below. For example, a free base function can be reacted with a suitable acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may, include metal salts such as alkali metal salts, e.g. , sodium or potassium salts; and alkaline earth metal salts, e.g. , calcium or magnesium salts. Sodium salts of compounds within the scope of Compound I are described, for example, in U.S. Patent Application No. 12/516,082 and U.S. Patent Publication No. 2008/0227991. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. In some embodiments, the compound of formula I is a sodium salt, e.g. , a tetrasodium salt.

In some embodiments, the compositions of the present invention include an anti- sequestration component. As used herein, the phrase "anti-sequestration component" refers to a component which, when pre-incubated or otherwise associated with a Lipid A analog (e.g. , a compound of formula (A)), causes no or minimal inactivation of the Lipid A analog. For example, an anti-sequestration component resists interactions with the Lipid A analog that would suppress the activity of the Lipid A analog (e.g. ,

encapsulation of the Lipid A analog). Without wishing to be bound by any particular theory, it is believed that anti- sequestration components may function by binding to the compound of formula (A) and carrying it through the bloodstream with no or minimal encapsulation, sequestration or other type of interaction which hides or masks the compound. It is to be understood that "anti-sequestration" does not necessarily mean a total lack of encapsulation or sequestration, but rather enough to allow the compound of formula (A) to remain at least partially active as described herein.

In some embodiments the anti-sequestration component can associate with and carry a Lipid A analog through the bloodstream with no or minimal inactivation. In some embodiments, the combination of a Lipid A analog (e.g., a compound of formula (A) or pharmaceutically acceptable salt thereof) with an anti-sequestration component results in less than about 80% inhibition, less than about 85% inhibition, less than about 90% inhibition, less than about 95% inhibition of activity of the Lipid A analog by blood in an 18 hour period. In some embodiments, the combination of a Lipid A analog with an anti-sequestration component results in less than about 80% inhibition, less than about 85% inhibition, less than about 90% inhibition, less than about 95% inhibition of Lipid A analog activity by blood in a 24 hour period. In some embodiments, the combination of a Lipid A analog with an anti-sequestration component results in less than about 80% inhibition, less than about 85% inhibition, less than about 90% inhibition, less than about 95% inhibition of activity of the Lipid A analog by blood in a 36 hour period, a 48 hour period or a 72 hour period. In some embodiments, the combination of a Lipid A analog with an anti-sequestration component results in less than about 99% inhibition in an 18 hour period. In some embodiments, the combination of a Lipid A analog with an anti-sequestration component results in less than about 99% inhibition in a 24 hour period. In some embodiments, the combination of a Lipid A analog with an anti-sequestration component results in less than about 99% inhibition in a 36 hour period, a 48 hour period or a 72 hour period.

That is, in some embodiments, the combination of a Lipid A analog with an anti- sequestration component allows for the preservation of more than about 85%, more than about 90%, more than about 95% or more than about 99% of the activity of the circulating Lipid A analog in the blood in an 18 hour period, e.g. , in a 24 hour period, a 36 hour period, a 48 hour period or a 72 hour period.

In some embodiments, the anti-sequestration component results in less than 25% inactivation of the Lipid A analog. In some embodiments, the anti- sequestration component results in less than 20% inactivation of the Lipid A analog. In some embodiments, the anti-sequestration component results in less than 15% inactivation of the Lipid A analog. In some embodiments, the anti-sequestration component results in less than 10% inactivation of the Lipid A analog. In some embodiments, the anti- sequestration component results in less than 8% inactivation of the Lipid A analog. In some embodiments, the anti- sequestration component results in less than 6%

inactivation of the Lipid A analog. In some embodiments, the anti- sequestration component results in less than 4% inactivation of the Lipid A analog. In some embodiments, the anti-sequestration component results in less than 2% inactivation of the Lipid A analog.

In some embodiments, the anti-sequestration component is an anti-sequestration lipoprotein component (i.e. , includes a lipoprotein or a component of a lipoprotein). In some embodiments, the anti- sequestration lipoprotein component includes a component of HDL. The present inventors have determined that certain components of HDL are capable of inactivating compounds of formula (A), while other components are anti- sequestration, i.e. , do not inactivate. In some embodiments, the anti-sequestration lipoprotein component is a lower density lipoprotein component. As used herein, the phrase "lower density lipoprotein component" refers to a component of a lipoprotein selected from LDL, IDL or VLDL (i.e. , components of lipoproteins having a density less than HDL). In some embodiments, the anti- sequestration lipoprotein component is a component of HDL, LDL, IDL, VLDL or a mixture thereof. In some embodiments, the anti-sequestration lipoprotein component is LDL, IDL, VLDL or a mixture thereof. In some embodiments, the anti- sequestration lipoprotein component is LDL, VLDL or a mixture thereof.

As used herein, the term "lipoprotein" refers to a biochemical assembly which includes proteins and lipids and which can transport water-insoluble lipids in the water- based bloodstream. Lipoproteins are generally classified by size and density and include, from largest to smallest: chylomicrons, VLDL, IDL, LDL and HDL. "High density lipoproteins" or "HDLs" are the smallest lipoproteins with the highest density because they typically contain the highest proportion of protein. HDL particles generally are less than about 11 nm in diameter, but can fluctuate in size and mass depending upon the contents. "Low density lipoproteins" or "LDLs" are slightly larger and lower in density than HDLs. They are generally about 22 nm in diameter and have a mass of about 3 million Daltons, however, because the components of LDL particles may change, their mass and size can also vary. "Intermediate-density lipoproteins" or "IDLs" are formed from the degradation of very low-density lipoproteins. IDLs are, in general, about 25 to about 35 nm in diameter, and they typically contain primarily a range of triacylglycerols and cholesterol esters. IDLs are somewhat similar to LDLs. "Very-low-density lipoproteins" or "VLDLs" are assembled in the liver from

triglycerides, cholesterol, and apolipoproteins. VLDL is converted in the bloodstream to low-density lipoprotein (LDL). VLDL particles generally have a diameter of 30-80 nm. In some embodiments, anti-sequestration lipoprotein components include components of HDL, LDL, IDL, VLDL, fragments and/or mixtures thereof. In some embodiments, anti-sequestration lipoprotein components include components of LDL, IDL, VLDL, fragments and/or mixtures thereof. In some embodiments, anti- sequestration lipoprotein components include components of LDL, VLDL, fragments and/or mixtures thereof. Components of HDL, LDL, IDL or VLDL are generally known in the art and include, but are not limited to apolipoproteins, triglycerides, cholesterols, cholesterol esters, phospholipids and fragments thereof. In some embodiments, the anti- sequestration lipoprotein component is an apolipoprotein. In some embodiments, the anti-sequestration lipoprotein component is apolipoprotein C2 or apolipoprotein C3. In some embodiments, the anti-sequestration lipoprotein component is a fragment of an apolipoprotein, such as a fragment of apolipoprotein C2 or a fragment of apolipoprotein C3.

In some embodiments, the anti-sequestration component does not include intact normal HDL (e.g. , native HDL found in human blood). In some embodiments, the anti- sequestration component does not include free apolipoprotein Al. In some

embodiments, the anti-sequestration component does not include free apolipoprotein A2. In some embodiments, the anti-sequestration component does not include free apolipoprotein E.

Components of HDL, LDL, IDL, or VLDL also include lipid bound proteins or lipid bound protein fragments, such as lipid bound apolipoproteins. In some

embodiments, the anti-sequestration component is a lipid bound apolipoprotein or a lipid bound apolipoprotein fragment. As used herein, the term "lipid bound protein" refers to a protein which is bound to, incorporated into or otherwise associated with a lipid (e.g. , a single lipid or a lipid macrostructure such as a liposome). In some embodiments, the lipid is a purified lipid. In other embodiments, the lipid is a naturally occurring lipid in situ. The association between the apolipoprotein may be via a linker, a bond, hydrophobic interactions or ionic interactions. In some embodiments, the anti- sequestration component is a lipid bound apolipoprotein A2, a lipid bound

apolipoprotein A2 fragment, a lipid bound apolipoprotein CI or a lipid bound apolipoprotein CI fragment. In some embodiments, the lipid bound apolipoprotein is an apolipoprotein bound to, incorporated into or associated with a reconstituted lipoprotein, such as reconstituted HDL (rHDL) or reconstituted LDL (rLDL). In some embodiments, the lipid bound apolipoprotein is apolipoprotein A2 or a fragment thereof bound to, incorporated into or associated with rHDL (rHDL-A2) or apolipoprotein CI or a fragment thereof bound to, incorporated into or associated with rHDL (rHDL-Cl) or serum amyloid A or a fragment thereof bound to, incorporated into or associated with rHDL (rHDL-SAA). In some embodiments, the lipid bound apolipoprotein is apolipoprotein A2 bound to, incorporated into or associated with rLDL (rLDL-A2) or apolipoprotein CI bound to, incorporated into or associated with rLDL (rLDL-Cl) or serum amyloid A bound to, incorporated into or associated with rLDL (rLDL-SAA).

In some embodiments, the anti-sequestration component is selected from rHDL- A2, rHDL-Cl, rLDL-A2, rLDL-Cl, rHDL-SAA, rLDL-SAA and mixtures thereof. In some embodiments the anti-sequestration component does not include apolipoprotein Al bound to, incorporated into or associated with rHDL or rLDL.

A skilled artisan can determine which components are anti-sequestration components using routine experimentation and the teachings provided herein. A non- limiting example is a cytokine assay such as described in the Examples. A test component can be pre-incubated with a compound of formula (A). An assay to determine the release of a cytokine (such as TNF-a) in whole blood in the presence of a TLR-4 ligand (such as LPS) would than be performed using this composition. The results from this assay would then be compared to results from a similar assay performed (e.g. , concurrently) using a composition comprising a compound of formula (A) pre-incubated with normal, intact HDL. A composition comprising an anti- sequestration component would result in less TNF-a release than a composition comprising the compound of formula (A) pre-incubated with normal, intact HDL.

//. Pharmacodynamics

In some embodiments, the present invention provides a method for extending the pharmacodynamic half-life of a compound of formula (A). The method includes contacting the compound of formula (A) with an anti-sequestration component such that the pharmacodynamic half-life of the compound of formula (A) is extended relative to the pharmacodynamic half-life of free compound of formula (A). For example, in some embodiments, the method includes contacting a compound of formula (I) gpo(OH)₂

or a pharmaceutically acceptable salt thereof with an anti-sequestration component .

The compound of formula (A) and the anti-sequestration component (e.g. , the component of LDL) may be combined in any ratio, depending on a number of factors, including the molecular weight of the anti- sequestration component. In some embodiments, the ratio of anti- sequestration component to compound of formula (A) is between about 50000: 1 and about 1 :50000 by weight. In some embodiments, the ratio of anti-sequestration component to compound of formula (A) is between about 40000: 1 and about 1 :40000, between about 30000: 1 and about 1 :30000, between about 25000: 1 and about 1 :25000, between about 20000: 1 and about 1 :20000, between about 10000: 1 and about 1 : 10000, between about 5000: 1 and about 1 :5000, between about 2500: 1 and about 1 :2500 by weight. In some embodiments, the ratio of anti-sequestration component to compound of formula (A) is between about 1000: 1 and about 1 : 1000 by weight. In some embodiments, the ratio of anti- sequestration component to compound of formula (A) is between about 100: 1 and about 1 : 1000 by weight. In some embodiments, the ratio of anti- sequestration component to compound of formula (A) is between about 10: 1 and about 1 : 1000 by weight. In some embodiments, the ratio of anti-sequestration component to compound of formula (A) is between about 1000: 1 and about 1 : 100 by weight. In some embodiments, the ratio of anti- sequestration component to compound of formula (A) is between about 1000: 1 and about 1 : 10 by weight. In some embodiments, the ratio of anti-sequestration component to compound of formula (A) is between about 100: 1 and about 1 : 100 by weight. In some embodiments, the ratio of anti-sequestration component to compound of formula (A) is between about 100: 1 and about 1 : 1 by weight. In some embodiments, the ratio of anti- sequestration component to compound of formula (A) is between about 100: 1 and about 10: 1 by weight. In some embodiments, the formulations described herein provide compounds of formula (A) with long-lasting activity in the bloodstream. That is, in some

embodiments, the formulations described herein provide a long pharmacodynamic half life of compounds of formula (A) while in circulation. In some embodiments, the formulations described herein provide a pharmacodynamic half life of about 8 hours to about 72 hours. In some embodiments, the formulations described herein provide a pharmacodynamic half life of about 8 hours to about 48 hours. In some embodiments, the formulations described herein provide a pharmacodynamic half life of about 8 hours to about 36 hours. In some embodiments, the formulations described herein provide a pharmacodynamic half life of about 8 hours to about 24 hours. In some embodiments, the formulations described herein provide a pharmacodynamic half life of about 10 hours to about 18 hours. In some embodiments, the formulations described herein provide a pharmacodynamic half life of about 12 hours. In some embodiments, the formulations described herein provide a pharmacodynamic half life of about equal to the pharmacokinetic half life of the compound of formula (A).

///. Methods

In some aspects, the present invention provides methods for treating E5564 responsive states. As used herein, the phrase "E5564 responsive state" refers to diseases, disorders, states and/or conditions which can be treated, prevented, or otherwise ameliorated by the administration of a compound of formula (A), e.g. , a compound of formula (I). Without wishing to be bound by any particular theory, it is believed that compounds of formula (A) function, at least partially, by inhibiting activation of TLR4 such as the activation seen by LPS and/or other ligands of TLR4 such as heat shock proteins or fibronectin fragments. Accordingly, in some

embodiments, E5564 responsive states are states that arise from activation of TLR4.

Examples of E5564 responsive state include, for example, endotoxemia (including surgery-related endotoxemia), sepsis, septic shock, trauma or severe tissue injury, HIV infection, immunological disorders, allograft rejection, graft-versus-host disease and damage to the gastrointestinal tract (e.g. , mucositis) due to chemotherapy or radiation.

Additional disorders can be found, for example, in U.S. Patent No. 5,750,664, U.S.

Patent No. 5,935,938, U.S. Patent No. 6,417, 172, U.S. Patent No. 5,952,309, U.S. Patent

No. 7,348,316, U.S. Application Publication No. 2005/0215517, U.S. Application

Publication No. 2006/0276431, U.S. Application Publication No. 2005/0101549, U.S. Application Publication No. 2008/0096841 and U.S. Application Publication No.

2007/0072824, each of which is incorporated herein by reference for their descriptions of diseases, disorders, states and/or conditions which can be treated, prevented, or otherwise ameliorated by the administration of a compound of formula (A), e.g. , E5564.

In some embodiments, the present invention provides methods for treating sepsis. The methods include administering a composition as described herein, such that sepsis is treated. In some embodiments, the present invention provides methods for treating GVHD. The methods include administering a composition as described herein, such that GVHD is treated. In some embodiments, the present invention provides methods for treating mucositis. The methods include administering a composition as described herein, such that mucositis is treated.

"Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent (e.g. , a compound of formula (A) in the compositions of the present invention) to a subject, or to an isolated tissue or cell line from a subject. The subject generally has a disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder. The purpose of treatment is generally to cure, heal, alleviate, relieve, remedy, ameliorate, or improve such disease, disorder, symptoms or predisposition. "Treated", as used herein, refers to the disease or disorder being cured, healed, alleviated, relieved, remedied, ameliorated, or improved.

As used herein, the term "subject" refers to animals such as mammals, including, but not limited to, humans, primates, cows, sheep, goats, horses, pigs, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species. In some embodiments, the subject is a human.

In some embodiments, the compositions of the present invention are able to inhibit activation of cells as measured by changes in the release of cytokines or other cell signaling molecules, e.g. , TNF-a or interleukins. In some embodiments, activation of cells is inhibited by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, about 100%. In some embodiments, TNF-a release (e.g. , from immunological cells) is inhibited by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, about 100%. In some embodiments, activation of cells is inhibited for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 4 days, at least about 5 days. In some embodiments, TNF-a release is inhibited for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 4 days, at least about 5 days.

In some embodiments, the compositions of the present invention include a compound of formula (A) in an effective amount. As used herein, the term "effective amount" refers to the amount of the compound of formula (A) necessary to achieve a desired effect. The term "desired effect" refers generally to any result that is anticipated by the skilled artisan when the compounds described herein are administered to a subject. In some embodiments, the desired effect is treatment of an E5564 responsive state. In some embodiments, the desired effect is treatment of sepsis. In some embodiments, the desired effect is treatment of mucositis. In some embodiments, the desired effect is treatment of allograft rejection or GVHD. The exact amount of the compounds described herein (e.g. , the compound of formula (A)) required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the diseases, its mode of administration, and the like.

It will be appreciated that, according to the methods of the present invention, the compounds/compositions of the present invention (e.g. , a composition which includes a compound of formula (A) or formula (I)) may be administered using any amount and any route of administration effective for treating an E5564 responsive state or for extending pharmacodynamic half-life as described herein. It will be understood, however, that the administration of the compounds/compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see, for example, Goodman and Oilman's, "The Pharmacological Basis of Therapeutics," Tenth Edition, A. Oilman, J. Hardman and L. Limbird, eds., McGraw- Hill Press, 155- 173, 2001, which is incorporated herein by reference in its entirety). In some embodiments, the compound of formula I is administered systemically. As used herein, "systemic administration" refers to any means by which the compound of formula I can be made systemically available. In some embodiments, systemic administration encompasses intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration (e.g. , into a carotid artery), intradermal administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation (e.g. , aerosol), intracerebral or intravesicular, nasal, oral, intraocular, pulmonary administration, impregnation of a catheter, by suppository and direct injection into a tissue, or systemically absorbed topical or mucosal administration. Mucosal administration includes administration to the respiratory tissue, e.g. , by inhalation, nasal drops, ocular drop, etc. ; anal or vaginal routes of administration, e.g. , by suppositories; and the like.

In some embodiments, the compounds described herein are administered intravenously. In other embodiments, the compounds described herein are administered orally. In some embodiments, the compounds described herein are administered pre- operatively, peri-operatively, and/or post-operatively.

In some embodiments, the compositions described herein are administered in a single bolus. Such a bolus administration may be followed by one or more maintenance doses (e.g. , bolus or via infusion). In some embodiments, the compositions described herein are administered in an intermittent intravenous infusion. In some embodiments, the compositions described herein are administered in a continuous intravenous infusion. Such infusion may be preceded by a bolus dose, or may be followed by one or more maintenance doses (bolus or via infusion). In some embodiments, maintenance doses are administered as an intermittent intravenous infusion. Maintenance dosage may be administered at regular intervals, e.g. , daily, every other day, bi-weekly, weekly, etc. In some embodiments, maintenance doses are administered intermittently, such as weekly or bi-weekly.

Without wishing to be bound by any particular theory, it is believed that extending the pharmacodynamic half-life of the compounds of formula (A) can, in turn, allow for more infrequent dosing (e.g. , versus administration of the compound of formula (A) without an anti- sequestration component). In some embodiments, the compositions described herein are administered no more than once about every 8 hours, about every 10 hours, about every 12 hours, about every 14 hours, about every 16 hours, about every 18 hours, about every 20 hours, about every 22 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 60 hours, about every 72 hours, about every 84 hours, about every 96 hours, about every five days or about once a week. In some embodiments, the compositions described herein are administered no more than once about every 12 hours. In some embodiments, the compositions described herein are administered no more than once about every 18 hours. In some embodiments, the compositions described herein are administered no more than once about every 24 hours. In some embodiments, the compositions described herein are administered no more than once about every 48 hours.

In some embodiments, the compounds of formula (A) or the compounds of formula (I) may be administered at dosage levels of about 0.001 mg/kg to about 50 mg/kg, from about 0.01 mg/kg to about 25 mg/kg, or from about 0.1 mg/kg to about 10 mg/kg of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. For example, in some embodiments, the compounds described herein may be administered at dosages listed above in a bolus or in a continuous infusion. It will also be appreciated that dosages smaller than 0.001 mg/kg or greater than 50 mg/kg (for example 50-100 mg/kg) can be administered to a subject. In some embodiments, the compounds may be administered in a continuous or intermittent infusion of between about 0.01 mg/hour and about 3 mg/hour, e.g. , between about 0.03 mg/hour and about 1.0 mg/hour.

Formulations for use in this invention may be a solid, liquid, paste, or gel comprising a compound as descried above. In a simple form, a formulation for use in this invention may consist of a compound as described herein, e.g. , a compound of formula (A) or a compound of formula (I) dissolved in a sterile aqueous liquid vehicle, suitable for infusion during surgery or for injection thereafter.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein may mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, dextrose (e.g. , 5%) in water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. It will be appreciated that the compounds/compositions described herein may be administered systemically in dosage forms, formulations or suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants such that the compound effectiveness is optimized. For example, the compounds described herein may be formulated together with appropriate excipients into a pharmaceutical composition, which, upon administration of the composition to the subject, systemically releases the active substance in a controlled manner. Alternatively, or additionally, compound dosage form design may be optimized so as to increase the effectiveness of the compound of formula I upon administration. The above strategies (i.e. , dosage form design and rate control of drug input), when used alone or in combination, can result in a significant increase in compound effectiveness and are considered part of the invention.

In some embodiments, the compounds/compositions described herein are administered in a formulation having long lasting circulation in the blood. For example, in some embodiments, the compounds described herein are administered in a formulation which has a circulating half life of at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, or at least about 50 hours. In one embodiment, the compounds described herein are administered in a formulation which has a circulating half life of at least about 30 hours. In some embodiments, the compounds described herein are administered in a formulation which includes micelles having a mean hydrodynamic diameter of between about 7 nm and about 15 nm. In some embodiments, the compounds described herein are administered in a formulation which includes micelles having a mean hydrodynamic diameter of between about 7 nm and about 14 nm, between about 7 nm and about 13 nm, between about 7 nm and about 12 nm, or between about 7 nm and about 11 nm. In some embodiments, the compounds described herein are administered in a formulation which includes micelles having a mean hydrodynamic diameter of between about 7 nm and about 10 nm. In some embodiments, the compounds described herein are administered in a formulation which includes micelles having a mean hydrodynamic diameter of between about 7 nm and about 9 nm. In some embodiments, the compositions described herein are administered in a formulation which includes particles (e.g. , micelles) having a mean hydrodynamic diameter of approximately the size of an LDL particle, an IDL particle or a VLDL particle. In some embodiments, the compositions described herein are administered in a formulation which includes particles (e.g. , micelles) having a mean hydrodynamic diameter of approximately the size of an HDL particle.

It will also be appreciated that compounds/compositions described herein, e.g. , compositions which include compounds of formula (A), may be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, the compound of formula I may be administered concurrently with an agent that treats sepsis), or they may achieve different effects (e.g., control of any adverse effects).

Exemplification of the Invention

The methods of this invention can be understood further by the following examples. It will be appreciated, however, that these examples do not limit the invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the present invention as described herein and as hereinafter claimed.

Example 1

Materials and Methods

An Eritoran (a compound of formula (I)) solution was prepared at a

concentration of 500nM by dissolving lyophilized powder in deionized water. A solution of purified lipopolysaccharide (LPS) was prepared at a concentration of 1 μg/mL by reconstituting LPS from 055 :B5 Escherichia coli (Sigma- Aldrich®) in deionized water. Both Eritoran and LPS were sonicated prior to use in a water bath sonicator for 1-2 minutes.

Lyophilized apolipoproteins A2, CI, C2, C3, and E (Meridian Life Sciences®, Inc.) and apolipoprotein Al were reconstituted in deionized water as provided below. Crosslinking reagent, bis[sulfosuccinimidyl] suberate, also known as BS3 was purchased from Pierce (Thermo Fisher Scientific, Inc.). Reagents for SDS-PAGE include β- mercaptoethanol (Sigma- Aldrich®), Laemmli sample buffer (Bio-Rad®), molecular weight marker (Fermentas) and 4-20% pre-cast gradient gels (Bio-Rad®). For native gels, a high molecular weight marker kit (GE Healthcare) containing proteins with defined diameters were used to estimate particle size and native sample buffer (Bio- Rad®), which contained no SDS, was used to load protein.

Plasma lipoprotein separation by ultracentrifugation

Human normolipidemic plasma (Bioreclamation) was separated into lipoprotein fractions by density gradient ultracentrifugation for the collection of HDL. Briefly, 1.02g sodium bromide was added to 3mL plasma in clear ultracentrifuge tubes and was cooled on ice for 2-3 hours at 4°C alongside sodium bromide density solutions

(1.006g/mL, 1.063g/mL, 1.21g/mL) before setting the gradient. Density solutions, 2.8mL each, were layered on top of cooled plasma in the order 1.21g/mL, 1.063 g/mL and 1.006g/mL (top layer). The tubes were placed into individual titanium buckets and capped. Buckets were centrifuged at 40,000 rpm and 15°C for 18 hours.

The lipoprotein fractions were removed from the centrifuge tubes. The HDL fraction from each tube was pooled into a 50mL BD Falcon™ tube and stored at 4°C while other lipoprotein fractions were discarded. Subsequently, HDL was desalted using desalting columns. The protein content in the eluted fractions was measured using the DC-protein assay (Bio-Rad®) and bovine serum albumin as a standard, substantially according to manufacturer's specifications. Total cholesterol (TC) was measured using the Wako Cholesterol E kit (Wako Chemicals USA), substantially according to manufacturer's specifications. Fractions with the highest concentration of protein and TC were pooled together and adjusted to a 0.9% NaCl solution for isotonicity.

Generation of reconstituted HDL (rHDL) with apolipoproteins

To create spherical particles that effectively mimic the physical structure of human HDL3, the whole lipid fraction from delipidated human HDL was combined with protein in a molar ratio of 80:1 phospholipid:protein when using individual

apolipoproteins. When creating rHDL with a combination of apolipoproteins, molar ratios of apolipoproteins used were based on the circulating plasma concentration of each protein and its association with HDL. Initially, the whole lipid fraction was added to a glass vial and evaporated completely under nitrogen gas after which ImL of reconstitution buffer (NaCl 150 mM, Tris lOmM, diethylenetriaminepentaacetic acid O. lmM, pH 8.0) was added to the tube and sonicated until all lipids were dissolved into the buffer solution. Sodium cholate (cholate:phospholipid molar ratio, 1 : 1) was then added to the tube and mixed vigorously. Subsequently, the desired apolipoprotein(s) were added to the tube and mixed. The mixture was stirred under argon gas at room temperature overnight and centrifuged the following day in a 3 mL tube at 99,000 rpm and 4°C for 4 hours to remove any free lipids. Once the upper lipid layer was removed, the density of the mixture was adjusted to 1.21 g/mL using KBr and the centrifugation was repeated for an additional 4 hours. The upper fraction of the mixture containing the rHDL was collected and dialyzed against 2L of reconstitution buffer for approximately one day. Protein content was then measured as described above. At least 1.5-2.5mg of protein was used for starting material and final concentrations ranged from 2.5 - 4 mg/mL. All rHDL and HDL were stored under nitrogen gas to prevent oxidation.

Particle size analysis

Characterizing the rHDL consisted of determining protein number per particle and its size. Protein content was estimated using a water-soluble crosslinking reagent, BS3, which links proteins together on the particle surface by reacting with primary amino groups in the side chain of lysine residues and/or the N-terminus of the polypeptide. After crosslinking, linked products {i.e. , dimers, trimers, etc.) were differentiated by SDS-PAGE. Particle size of rHDL was estimated by running samples on a non-denaturing gel in the presence of protein standards with a known stokes radius.

The relative mobility or Rf of smears were evaluated in Lab Works imaging software (UVP, Inc., Upland, CA). Particle diameter was then approximated by comparing the distance migrated by the bands of the sample of interest to the distance migrated by standards. Human HDL was also included for comparison.

TNF-a assay in human whole blood

A whole blood assay was chosen to test the inhibitory activity of eritoran, an exemplary compound described herein, using LPS as a TLR4 agonist to provide the best simulated administration environment. TNF-a was chosen as an exemplary marker for cellular activation by LPS because it is typically released by macrophages during an immune response and plays a role in mediating deleterious effects on the host.

Apolipoproteins and rHDL-containing apolipoproteins were incubated in sterile 48-well plates in the presence of 50nM eritoran or Ca²⁺ and Mg²⁺-free Hank's Balanced Salt Solution (HBSS) (Invitrogen™) for a total volume of 100 for 18 hours at 37°C with shaking. A range of concentrations for each free apolipoprotein were tested. The low concentrations were chosen to reflect physiological levels present in a sepsis patient whereas high concentrations reflect levels in healthy persons. A single concentration of rHDL-containing apolipoprotein was tested, which was based on the lowest

concentration that could maximally inhibit drug activity in a concentration-response curve based on native HDL. Controls during the 18-hour incubation include HBSS with HDL alone and HBSS with HDL and 50nM eritoran. The desired final concentration of HDL added was 0.8mg/mL based on a dose response in which inactivation of eritoran was optimal.

After the incubation, additional controls were added to the plate as follows: 5.05μί of lμg/ L· LPS was added to all wells and treatment and control groups were diluted 5-fold by the addition 400μί EDTA-treated freshly drawn human blood from healthy volunteers to a final volume of 500μ Final concentrations of eritoran and LPS per well were 14ng/mL (ΙΟηΜ) and lOng/mL, respectively. Plates were incubated with gentle shaking for an additional 3 hours at 37°C and then centrifuged at 1,000 x g for 10 minutes at 4°C, after which plasma samples were collected and frozen at -80°C. The final concentrations of eritoran were ΙΟηΜ (14ng/mL), and lOng/mL LPS was chosen for this study even though clinically relevant levels of LPS in sepsis patients are typically below Ing/mL and plasma concentrations of eritoran as high as 29 micromolar can be safely administered in sepsis patients. All plasma samples were diluted 1/10 in dH20 and vortexed thoroughly before being analyzed for TNF-a using an enzyme-linked immunosorbent assay (ELISA) for human TNF-a (R&D Systems®), substantially according to manufacturer's specifications.

Results

Apolipoproteins A2, CI, C2, C3, and E were incubated in the free form overnight with eritoran to allow for potential drug binding before being added to human whole blood and treated with LPS to induce the release of cytokines. Apolipoproteins Al and SAA were not tested in the free form. In addition to the groups of interest, several quality controls were added to each plate to ensure that there was no inherent release of TNF-a without LPS (Blood only group) and that the whole blood assay was consistent and working (HDL controls).

The release of TNF-a, a pro-inflammatory cytokine, was used as a marker for drug activity. Stimulation by LPS causes an increase in the release of TNF-a from peripheral blood mononuclear cells in the blood and drug activity is inferred by its ability to effectively reduce this response. Since TNF-a levels can vary widely from person to person after stimulation with LPS, all values were normalized within subjects to their own LPS (only) control samples in order to compare effects between subjects. Although absolute values of TNF-a may differ, overall trends are typically the same from person to person. Quality controls were performed for the assays, but have been omitted from graphs as they were not included in the statistical analyses.

TNF-a release from human whole blood after stimulation with lOng/mL LPS in the presence of eritoran (ΙΟηΜ final) and apolipoprotein A2 at concentrations of 83μg/mL, 25C^g/mL and 40C^g/mL was detected. Apolipoprotein A2, the second most abundant apoprotein on healthy HDL, was shown to significantly inactivate eritoran at the low (mean=180.9%, p=0.041) and intermediate (69.6%, p=0.036) concentrations, but was less effective at the highest concentration (31.5%, p>0.05), as shown in Figure 1. Furthermore, at the highest concentration, apolipoprotein A2 begins to reduce cytokine release without the presence of the drug (A2/LPS). Without wishing to be bound by any theory, it is believed that this is due to the fact that, at higher levels of the protein, a threshold concentration may be achieved where LPS response is diminished by the protein itself, as some lipoproteins have been shown to inhibit activity of LPS

(Thompson PA, Kitchens RL. /. Immunol. 2006; 177:4880-7). Likewise, at the lowest concentration, Apo A2 presence alone causes an unforeseen increase in TNF-a release in which levels exceed that of the LPS only group. This result is not fully understood, but may indicate that fraction A2 contained some TLR4 agonistic activity.

TNF-a release from human whole blood after stimulation with lOng/mL LPS in the presence of eritoran (ΙΟηΜ final) and apolipoprotein CI at concentrations of

5. ^g/mL, 25μg/mL and 58^g/mL was detected. The effect of apolipoprotein CI on drug activity at the lower, septic concentration was not statistically significant despite appearance of inhibitory activity (see Figure 2). In this case, lack of significant difference is a consequence of the large deviation from the mean observed in one subject at this particular concentration. Conversely, at the intermediate and high concentrations, Apolipoprotein CI was shown to significantly inactivate the drug (87.3%, p=0.025; 45.4%, p=0.036).

TNF-a release from human whole blood after stimulation with lOng/mL LPS in the presence of eritoran (ΙΟηΜ final) and apolipoprotein C2 at concentrations of 0.64μg/mL, 9μg/mL and 18μg/mL was detected. Apolipoprotein C2 caused minimal drug inactivation as compared to other apolipoproteins. Although graphically, eritoran appears to lose some activity across the concentration range in the presence of apolipoprotein C2 (13.9% at low, 29% at high), as shown in Figure 3, apolipoprotein C2 was not found to be significantly different from the LPS/Eri group at all three concentrations.

TNF-a release from human whole blood after stimulation with lOng/mL LPS in the presence of eritoran (ΙΟηΜ final) and apolipoprotein C3 at concentrations were 9μg/mL, 36μg/mL and 72μg/mL was detected. As shown in Figure 4, the results for apolipoprotein C3 were similar to the results for apolipoprotein C2. These data indicate that apolipoprotein C3 also causes minimal inactivation of eritoran.

TNF-a release from human whole blood after stimulation with lOng/mL LPS in the presence of eritoran (ΙΟηΜ final) and apolipoprotein E at concentrations were 0.5^g/mL, 12^g/mL and 25μg/mL was detected. Thus, Apolipoprotein E caused substantial inactivation of eritoran at the low (66.5%), intermediate (141.8%) and high (75.1%) concentrations tested, as shown in Figure 5. However, due to variability between subjects, statistical significance from the LPS/Eri group at the low and intermediate concentrations could not be detected. On the other hand, apolipoprotein E was shown to cause a significant reduction in drug activity (75.1%, p=0.004) at the high concentration.

In order to test apolipoprotein Al and SAA, the most abundant apolipoproteins on HDL in both the healthy and septic state, rHDL constructs containing individual apolipoproteins were formed from purified components and tested in the TNF-a assay. A preliminary whole blood assay performed using rHDL-Al illustrated that rHDL at a protein concentration as low as 0.7 mg/mL greatly reduced TNF-a release by itself without the presence of drug. Because of this, the ability to measure apolipoprotein effects on drug activity was difficult at certain concentrations. Thus, an HDL concentration-response curve was repeated at a range of protein concentrations below about 0.7 mg/mL, and is provided in Figure 6. From this data, it was evident that, although HDL inactivates eritoran optimally at 0.8 mg/mL protein, there is still 20-30% inactivation below 0.8 mg/mL. In light of this information, 0.1 mg/mL was chosen as the final protein concentration that was to be used for all rHDL studies. At this concentration, rHDL could be made with less protein, while still maintaining the desired level of inactivation. Each set of rHDL were characterized for the number of protein molecules/particle using crosslinking with BS3 and run on a native gel alongside a high molecular weight marker (HMW) to estimate particle size. Reconstituted HDLs were used within one week of preparation to limit oxidation and/or contamination.

As shown in Figures 7-10, rHDL containing apolipoprotein Al and SAA caused greater reductions in drug activity as compared to rHDL containing apolipoprotein A2 or apolipoprotein CI. Without wishing to be bound by any particular theory, it is believed that rHDL containing apolipoprotein A2 and rHDL containing apolipoprotein CI would thus be good anti- sequestration components because they likely bind the compounds of formula (A), but do not inactivate or only weakly inactivate. Figure 7 shows that lipid- bound Al (rHDL-Al) causes a loss in drug activity (mean= 47.4%, p=0.02) as shown by the inability of eritoran to fully suppress TNF-a release. Likewise, eritoran antagonistic activity against LPS-induced TNF-a release in whole blood is inhibited in the presence of rHDL-SAA. As shown in Figure 8, at 0.1 mg/mL, lipid-bound SAA (rHDL-SAA) causes a greater than 25% decrease in drug activity (26.4%, p<0.001).

On the other hand, significant reductions in drug efficacy were not observed when eritoran activity was tested after pre-incubation with rHDL-Cl (13.0%, p>0.05), see Figure 9. Although, a minor inhibition was detected, it was not considered statistically significant from the LPS/Eri group, in which TNF-a was zero. Similarly, there is a less than 15% drop in drug activity when eritoran was tested for its ability to block TNF-a in the presence of rHDL-A2 (13.1%, p>0.05), see Figure 10. It is noted that one subject from treatment group rHDL-A2 and one subject from treatment group rHDL-Cl had zero release of TNF-a indicating no effect of the rHDL-apolipoprotein construct on inhibitory activity of eritoran.

Normal HDL contains an assortment of apolipoproteins in varied amounts.

Being able to manipulate apolipoprotein composition is a powerful method by which to probe how changes in the apolipoprotein class and quantity affect drug inactivation by HDL. A model rHDL containing all apolipoproteins in the molar ratios they would be found associated with HDL in the healthy state (dubbed normal rHDL or NrHDL) was tested in the TNF-a assay.

Molar ratios for NrHDL, 63: 31 : 12: 3: 10: 1 (Al : A2 : CI : C2 : C3 : E), were established from Havel and Kane, in The Metabolic & Molecular Bases of Inherited Disease, 2001. Reconstituted HDL containing all apolipoproteins were made in a similar fashion to the process described above, and were characterized by SDS-PAGE and transmission electron microscopy (TEM). To reproduce the degree of inactivation observed with native HDL, a concentration of 0.8mg/mL by protein was used in the assay. As shown in Figure 11, when treated with 0.8 mg/mL NrHDL, there is a considerable loss of eritoran activity against LPS-induced TNF-a release. Due to inter- subject variability, the change is not considered statistically different from the

LPS/Eritoran group (mean= 46.9%, p>0.05).

Another set of model rHDL were created to simulate the apolipoprotein composition as would be found on a septic HDL (dubbed septic rHDL or SrHDL). Serum amyloid A almost entirely replaces apolipoprotein Al on the HDL particle during sepsis, and the molar ratio was established by estimating apolipoprotein composition from confirmed plasma concentrations of apolipoproteins during sepsis and the percentage of that concentration circulating as part of a HDL: 1461: 17: 22: 2: 2: 8: 1 (SAA: Al: A2: CI : C2: C3: E). Unfortunately, when the SrHDL preparation was made, the sample was laden with lipid and it appeared cloudy and white-gray. This suggested that SrHDL particles did not form properly and interference from floating lipid prevented an accurate reading of the protein concentration. Thus, SrHDL was unable to be tested in the TNF-a assay.

Prospective Example

A compound of formula (A) will be pre-incubated or otherwise associated or complexed with an anti-sequestration lipoprotein component (e.g. , LDL, free apolipoprotein(s) or rHDL-Cl or rHDL-C2, rHDL-A2, etc.). The pre-incubated sample will be compared to a composition without an anti-sequestration lipoprotein component (e.g. , free E5564) in an in vivo sepsis model. It is believed that the sample associated with the anti-sequestration lipoprotein component will exhibit activity for a longer period of time than the sample without the anti-sequestration lipoprotein component.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

Claims

We claim

1. A composition comprising a compound of formula (A) and an anti- sequestration component,

or a pharmaceutically acceptable salt thereof

where R¹ is selected from:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R is selected from:

where A is straight or branched C7 to C12 alkyl; and each B and D, independently, is straight or branched C4 to C9 alkyl;

R⁴ is selected from:

straight or branched C8 to C12 alkyl, and

where U is straight or branched C2 to C4 alkyl; V is straight or branched C5 to C9 alkyl; and W is hydrogen or -CH₃;

R_A is R⁵-0-CH₂-, where R⁵ is hydrogen or straight or branched CI to C5 alkyl; R⁶ is hydroxy; and

A¹ and A² are each independently

O

-O- -P OH

OH

The composition of claim 1 , wherein

R¹ is:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

straight or branched C8 to C12 alkyl;

R³ is:

where A is straight or branched C7 to C12 alkyl; and B is straight or branched C4 to C9 alkyl;

R⁴ is:

where U is straight or branched C2 to C4 alkyl; V is straight or branched

C5 to C9 alkyl; and W is hydrogen or -CH₃;

R_A is R⁵-0-CH₂-, where R⁵ is straight or branched CI to C5 alkyl;

R⁶ is hydroxy; and

A¹ and A² are each independently

O O P OH

OH

3. A composition comprising a compound of formula (I) and an anti-sequestration component, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

4. The composition of any one of claims 1-3, wherein the anti-sequestration

component is an anti-sequestration lipoprotein component.

5. The composition of any one of claims 1-4, wherein the anti-sequestration

component is a component of HDL, LDL, IDL or VLDL.

6. The composition of any one of claims 1-4, wherein the anti-sequestration

component is a component of LDL or VLDL.

7. The composition of any one of claims 1-6, wherein the anti-sequestration

component is at least one component selected from an apolipoprotein, a triglyceride, a cholesterol, a cholesterol ester, a phospholipids, a lipoprotein fragment and mixtures thereof.

8. The composition of any one of claims 1-7, wherein the anti-sequestration

component is at least one component selected from apolipoprotein C2, apolipoprotein C3, a fragment of apolipoprotein C2, a fragment of apolipoprotein C3 and mixtures thereof.

9. The composition of any one of claims 1-6, wherein the anti-sequestration

component is a lipid bound apolipoprotein or a lipid bound fragment of an apolipoprotein.

10. The composition of any one of claims 1-6, wherein the anti-sequestration component is selected from lipid bound apolipoprotein A2, lipid bound apolipoprotein CI and mixtures thereof.

11. The composition of claim 9, wherein the lipid bound apolipoprotein is an

apolipoprotein bound to or associated with reconstituted HDL.

12. The composition of claim 11, wherein the lipid bound apolipoprotein is selected from rHDL-A2, rHDL-Cl, rHDL-SAA and mixtures thereof.

13. The composition of claim 11, wherein the lipid bound apolipoprotein is selected from rHDL-A2, rHDL-Cl, and mixtures thereof.

14. The composition of any one of claims 1-13, wherein the ratio of anti- sequestration component to compound of formula (A) is between about 1: 10 and about 1000:1 by weight.

15. A method for treating sepsis in a subject in need thereof, the method comprising administering a composition of any one of the preceding claims to the subject, such that sepsis is treated.

16. The method of claim 15, wherein the composition is administered in a single bolus.

17. The method of claim 15, wherein the composition is administered in a bolus followed by one or more maintenance doses.

18. The method of any one of claims 15-17, wherein the composition is administered no more than once about every 12 hours.

19. The method of any one of claims 15-17, wherein the composition is administered no more than once about every 18 hours.

20. The method of any one of claims 15-17, wherein the composition is administered no more than once about every 24 hours.

21. The method of any one of claims 15-17, wherein the composition is administered no more than once about every 48 hours.

22. The method of claim 15, wherein the composition is administered in an

intermittent intravenous infusion.

23. The method of claim 15, wherein the composition is administered in a

continuous intravenous infusion.

24. The method of any one of claims 15-23, wherein the compound of formula (A) in the composition is administered in a dosage of about 0.1 mg/kg to about 10 mg/kg per day.

25. The method of any one of claims 15-24, wherein TNF-a release in the subject is inhibited by at least about 80% for at least about 24 hours.

26. The method of any one of claims 15-24, wherein TNF-a release in the subject is inhibited by at least about 90% for at least about 72 hours.

27. A method for extending the pharmacodynamic half-life of a compound of

formula (A), the method comprising contacting the compound of formula (A) with an anti-sequestration component, wherein the compound of formula (A) is:

or a pharmaceutically acceptable salt thereof

where R¹ is selected from:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R is selected from:

where A is straight or branched C7 to C12 alkyl; and each B and D, independently, is straight or branched C4 to C9 alkyl;

R⁴ is selected from:

straight or branched C8 to C12 alkyl, and

where U is straight or branched C2 to C4 alkyl; V is straight or branched

C5 to C9 alkyl; and W is hydrogen or -CH₃;

RA is R⁵-0-CH₂-, where R⁵ is hydrogen or straight or branched CI to C5 alkyl; R⁶ is hydroxy; and

A¹ and A² are each independently

O

-O- -P OH

OH

such that the pharmacodynamic half-life of the compound of formula (A) is extended relative to the pharmacodynamic half-life of free compound of formula (A).

The method of claim 27, wherein

R¹ is:

where J is straight or branched CI to C3 alkyl; K is straight or branched C8 to CI 5 alkyl; and Q is straight or branched CI to C3 alkyl; ;

R² is straight or branched C8 to C12 alkyl;

R³ is:

where A is straight or branched C7 to C12 alkyl; and B is straight or branched C4 to C9 alkyl;

R⁴ is:

where U is straight or branched C2 to C4 alkyl; V is straight or branched

C5 to C9 alkyl; and W is hydrogen or -CH₃;

R_A is R⁵-0-CH₂-, where R⁵ is straight or branched CI to C5 alkyl;

R⁶ is hydroxy; and

A¹ and A² are each independently

O

-O P OH

OH

A method for extending the pharmacodynamic half-life of a compound of formula (I), the method comprising contacting the compound of formula (I) with an anti- sequestration component, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof; such that the pharmacodynamic half-life of the compound of formula (I) is extended relative to the pharmacodynamic half-life of free compound of formula (I).

30. The method of any one of claims 27-29, wherein the anti-sequestration

component is an anti- sequestration lipoprotein component.

31. The method of any one of claims 27-30, wherein the anti-sequestration

component is a component of HDL, LDL, IDL or VLDL.

32. The method of any one of claims 27-30, wherein the anti-sequestration

component is a component of LDL or VLDL.

33. The method of any one of claims 27-32, wherein the anti-sequestration

component is at least one component selected from an apolipoprotein, a triglyceride, a cholesterol, a cholesterol ester, a phospholipid, a lipoprotein fragment and mixtures thereof.

34. The method of any one of claims 27-33, wherein the anti-sequestration

component is at least one component selected from apolipoprotein C2, apolipoprotein C3, a fragment of apolipoprotein C2, a fragment of apolipoprotein C3 and mixtures thereof.

35. The method of any one of claims 27-32, wherein the anti-sequestration

component is a lipid bound apolipoprotein or a lipid bound fragment of an apolipoprotein.

36. The method of any one of claims 27-32, wherein the anti-sequestration

component is selected from lipid bound apolipoprotein A2, lipid bound apolipoprotein CI and mixtures thereof.

37. The method of claim 35, wherein the lipid bound apolipoprotein is an

apolipoprotein bound to or associated with reconstituted HDL. The method of claim 37, wherein the lipid bound apolipoprotein is selected from rHDL-A2, rHDL-Cl, rHDL-SAA and mixtures thereof.

The method of claim 37, wherein the lipid bound apolipoprotein is selected from rHDL-A2, rHDL-Cl, and mixtures thereof.

The method of any one of claims 27-39, wherein the ratio of anti-sequestration component to compound of formula (I) is between about 1:10 and about 1000:1 by weight.