HK1236439A1 - Methods of lowering serum cholesterol - Google Patents

Description

Method for lowering serum cholesterol

Cross reference to related applications

This application claims priority to U.S. provisional application serial No. 62/006,651, filed on 2/6/2014, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to methods of treating or preventing hypercholesterolemia and a diverse array of associated diseases, disorders, and conditions by administering agents that modulate lipoprotein homeostasis.

Introduction to the design reside in

Hypercholesterolemia is a common form of hyperlipidemia and hyperlipoproteinemia, where high levels of cholesterol are present in the blood. Cholesterol is transported within lipoproteins in plasma, which are classified according to their density as: VLDL (very low density lipoprotein), IDL (medium density lipoprotein), LDL (low density lipoprotein) and HDL (high density lipoprotein). Elevated levels of VLDL, IDL, and LDL, particularly LDL, are associated with an increased risk of cardiovascular disorders, including atherosclerosis and heart disease. Conversely, higher HDL levels are thought to exert a protective effect. In subjects with hypercholesterolemia that is not controlled by dietary restrictions, pharmaceutical intervention is usually authorized.

Increased innate immune system activity has been linked to the development of obesity-related dyslipidemia and insulin resistance as well as type II diabetes. Bone marrow-derived monocyte macrophages play a key role in the innate immune system. They are recruited to tissues in response to infection, tissue injury, or other trauma, and are particularly enriched in tissues that are frequently exposed to exogenous and endogenous toxins, such as the liver. Recent studies have shown that macrophages are involved in diet-induced changes in liver metabolism and insulin sensitivity, and that they play a role in type II Diabetes and obesity (Huang et al, Diabetes59:347-57 (2010)). Thus, modulation of hepatic macrophage homeostasis may provide an alternative approach to the treatment and prevention of metabolic abnormalities.

While conventional lipid lowering agents that generally exert their activity by reducing cholesterol production or absorption are effective in treating a large portion of the patient population, alternative agents, particularly agents that act through different mechanisms of action, would provide valuable therapeutic options, both as monotherapy and as a complement to existing drug regimens.

Summary of The Invention

The present disclosure contemplates methods of treating and/or preventing a variety of diseases, disorders and conditions, and/or symptoms thereof, using IL-10, modified (e.g., pegylated) IL-10, and related agents and compositions thereof described herein. Particular embodiments relate to the treatment and/or prevention of abnormally high levels of cholesterol and/or hypercholesterolemia in a subject. Other particular embodiments relate to modulating kupffer cells (e.g., by increasing activity and/or increasing number) to achieve treatment and/or prevention of abnormally high levels of cholesterol and/or hypercholesterolemia performance in a subject.

Hypercholesterolemia itself is generally asymptomatic. However, chronic elevation of serum cholesterol contributes to the formation of atherosclerotic plaques in the arteries. Relatively small plaques may rupture and cause clots to form and block blood flow. In contrast, larger plaques may cause arterial stenosis or blockage of the involved arteries. Sudden occlusion of the coronary arteries can lead to myocardial infarction, while occlusion of the arteries supplying the brain can lead to stroke.

The progressive development of stenosis or obstruction results in a gradual reduction in the blood supply to tissues and organs, often resulting in impaired activity. Tissue ischemia may manifest as one or more symptoms. For example, transient ischemia of the brain (transient ischemic attack) may manifest as transient loss of vision, dizziness or impaired balance, aphasia, paresis, and paresthesia. Insufficient blood supply to the heart may be manifested as chest pain; ocular ischemia may manifest as transient visual loss in one eye; while insufficient blood supply to the legs may be manifested as calf pain.

Hypercholesterolemia can be classified into various types according to characteristic manifestations. For example, type IIa hyperlipoproteinemia may be associated with blepharoma (yellowish plaque under the skin around the eyelids), xanthoma (deposition of yellowish cholesterol-rich material) of the senile ring (white or grey discoloration of the periphery of the cornea) and tendons (usually fingers). In contrast, type III hyperlipidemia may be associated with xanthoma of the palm, knee and elbow.

According to the lipid hypothesis, abnormal cholesterol levels in the blood (typically higher concentrations of LDL particles and lower concentrations of functional HDL particles) are strongly associated with cardiovascular disease due to increased atheromatous plaque development in arteries (atherosclerosis). Because high circulating LDL concentrations have been correlated with atheromatous plaque formation, LDL is often referred to as "bad cholesterol"; in contrast, high concentrations of HDL can remove cholesterol from cells, thereby reducing atheromatous plaque formation, and thus HDL is commonly referred to as "good cholesterol". However, recent evidence suggests that total cholesterol is the most relevant indicator of cardiovascular abnormalities.

To date, therapeutic control of systemic cholesterol levels has focused primarily on inhibiting the absorption of dietary cholesterol and inhibiting endogenous hepatocyte cholesterol synthesis. For example, ezetimibe (ZETIA) inhibits dietary absorption in the small intestine, and it has been shown to significantly reduce serum cholesterol in gene deficient mice strains APOE-/-and LDLR-/-mice fed a high fat diet (Davis, H.R., Jr. et al, Arterioscler Thromb Vasc Biol,2001.21(12): 2032-38). As another example, statin cholesterol lowering therapeutics act by inhibiting HMG-CoA mediated cholesterol synthesis through the mevalonate pathway which is primarily active in hepatocytes.

Therapeutic modalities for the treatment of hypercholesterolemia that act through other mechanisms of action have been developed or are in later development. These modalities include: a PCSK9 inhibitor that enhances the recirculation of LDL receptors to the cell surface so as to increase the rate of removal of LDL particles from the blood; nipagin (KYNAMERO), an antisense oligonucleotide used in the treatment of Familial Hypercholesterolemia (FH), which acts by hybridizing to apoB-100 and thus limiting the amount of LDL-C that can form; and lomitapide (JUXTAPID), also used for the treatment of FH, which prevents the formation and secretion of VLDL by inhibiting microsomal triglyceride transfer protein in the liver. Like other cholesterol lowering agents, these morphologies have been associated with adverse effects that limit their efficacy in certain patient populations (e.g., mifepristone and lomitapide have been associated with fatty liver disease caused by the accumulation of cholesterol in the liver).

As discussed further herein, macrophages play a major role in cholesterol homeostasis by absorbing LDL cholesterol as well as the Ac-LDL and Ox-LDL forms of cholesterol. Although Kupffer Cells (KCs) account for only about 10% to 15% of 100 to 300 billion total hepatocytes, KCs are 18 times more efficient in cholesterol catabolism than hepatocytes. Thus, modulating KC function and/or increasing KC numbers represents a novel means of treating and preventing hypercholesterolemia and associated diseases, disorders and conditions. Embodiments of the present disclosure include administering to a subject an agent (e.g., a small molecule, polypeptide, or antibody) that modulates kupffer cells (e.g., by increasing activity and/or increasing number) to effect treatment and/or prevention of abnormally high levels of cholesterol and/or hypercholesterolemia performance in the subject. In particular embodiments, modulating kupffer cell function is used to treat and/or prevent Familial Hypercholesterolemia (FH). In certain embodiments, the agent is an IL-10 agent (e.g., PEG-IL-10).

As discussed further below, human IL-10 is a homodimer and each monomer comprises 178 amino acids, the first 18 of which comprise a signal peptide. Specific embodiments of the present disclosure include mature human IL-10 polypeptides lacking a signal peptide (see, e.g., U.S. Pat. No. 6,217,857), or mature human PEG-IL-10. In other specific embodiments, the IL-10 agent is a variant of mature human IL-10. The variants may exhibit less than, comparable to, or greater than the activity of mature human IL-10; in certain embodiments, the activity is comparable to or greater than the activity of mature human IL-10.

The terms "IL-10", "IL-10 polypeptide", "agent", and the like are intended to be broadly construed and include, for example, human and non-human IL-10 related polypeptides, including homologs, variants (including muteins) and fragments thereof, as well as IL-10 polypeptides having, for example, a leader sequence (e.g., a signal peptide), and modified forms of the foregoing. In other embodiments, the terms "IL-10", "IL-10 polypeptide", "agent" is an agonist. Numerous specific embodiments relate to pegylated IL-10, which is also referred to herein as "PEG-IL-10".

The present disclosure contemplates methods wherein the IL-10 agent comprises at least one modification to form a modified IL-10 agent, wherein the modification does not alter the amino acid sequence of the IL-10 agent. Certain embodiments of the present disclosure contemplate such modifications in order to enhance one or more properties (e.g., pharmacokinetic parameters, efficacy, etc.). In other embodiments, modification of IL-10 does not have a therapeutically relevant adverse effect on immunogenicity, and in still other embodiments, the modified IL-10 is less immunogenic than unmodified IL-10. In some embodiments, the modified IL-10 agent is a PEG-IL-10 agent. In other embodiments, the PEG-IL-10 agent can include at least one PEG molecule covalently attached to at least one amino acid residue of at least one subunit of IL-10, or a mixture of mono-pegylated IL-10 and di-pegylated IL-10. The PEG component of the PEG-IL-10 agent may have a molecular mass of greater than about 5kDa, greater than about 10kDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is about 5kDa to about 10kDa, about 5kDa to about 15kDa, about 5kDa to about 20kDa, about 10kDa to about 15kDa, about 10kDa to about 20kDa, about 10kDa to about 25kDa, or about 10kDa to about 30 kDa.

Other modified IL-10 agents are discussed in detail below. In some embodiments, the modified IL-10 agent comprises at least one Fc fusion molecule, at least one serum albumin (e.g., HSA or BSA), an HSA fusion molecule, or an albumin conjugate. In other embodiments, the modified IL-10 agent is glycosylated, is hesylated, or includes at least one albumin binding domain. Some modified IL-10 agents may include more than one type of modification. In particular embodiments, the modification is site-specific, and in still other embodiments, it comprises a linker.

The present disclosure also contemplates nucleic acid molecules encoding the above. Certain embodiments envision the use of gene therapy in conjunction with the teachings herein. For gene therapy uses and methods, cells of a subject can be transformed in vivo with a nucleic acid encoding an IL-10-related polypeptide as set forth herein. Alternatively, cells can be transformed with a transgene or polynucleotide in vitro and then transplanted into a tissue of the subject in order to effect treatment. Alternatively, isolated primary cells or established cell lines can be transformed with transgenes or polynucleotides encoding IL-10-related polypeptides, and then optionally transplanted into a tissue of a subject.

As depicted in the experimental section, PEG-rMuIL-10 was found to reduce physiological plasma cholesterol levels by up to 70% in a phagocyte-dependent manner in invasively challenged LDLR-/-mice fed a high fat diet. This finding is consistent between mouse and human, and illustrates the relationship between IL-10 regulation of KC scavenger receptor modulation and enhanced cholesterol absorption. In addition, phagocytic cells play a consistent and powerful role in the normal endogenous regulation of total plasma cholesterol.

In particular embodiments, the present disclosure features a method of identifying an agent that induces phosphorylation of STAT3 in KC, the method comprising: a) contacting a candidate agent with KC; b) determining STAT3 phosphorylation levels in the KC; and c) comparing the level of STAT3 phosphorylation in b) to the level of STAT3 phosphorylation induced by a reference standard, wherein a higher level of STAT3 phosphorylation in the KC compared to the level of STAT3 phosphorylation in the reference standard identifies the candidate agent as an agent capable of inducing phosphorylation.

In some embodiments, an in vitro model is used to identify agents that induce phosphorylation of STAT3 in KC. In other embodiments, the KC is from the sinusoids of the liver.

In other embodiments of the present disclosure, the candidate agent comprises a small molecule, polypeptide, or antibody.

The present disclosure also contemplates embodiments wherein the reference standard is an interleukin, interferon, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Leukemia Inhibitory Factor (LIF), bone morphogenic protein 2(BMP-2), oncostatin M (OSM), or leptin. In particular embodiments, the interleukin is IL-5, IL-6, or IL-10.

The disclosure also includes methods of assessing whether an agent capable of inducing phosphorylation reduces at least one of serum cholesterol levels and triglyceride levels. In some methods, such assessment is performed using a biochemical assay, an in vitro assay, an ex vivo assay, or an in vivo model.

The present disclosure also contemplates a method of identifying an agent that reduces serum cholesterol in a subject, comprising: a) administering a candidate agent to the subject, wherein the candidate agent induces STAT3 phosphorylation in KC; b) determining the subject's serum cholesterol level; and c) comparing the serum cholesterol level in b) to a serum cholesterol level measured after administration of a reference standard (e.g., a statin) to the subject, wherein the reference standard is known to reduce serum cholesterol; wherein a candidate agent that reduces serum cholesterol to an extent that exceeds or is comparable to the reference standard is identified as an agent that reduces serum cholesterol in the subject. In some embodiments of the disclosure, the candidate agent comprises a small molecule, polypeptide, or antibody. Other embodiments of the disclosure further include assessing whether the agent that reduces serum cholesterol in the subject induces hepatocyte proliferation.

Embodiments are contemplated wherein the subject is a human. The serum cholesterol level in a human may be 200 to 239mg/dL or at least 240 mg/dL. Other embodiments are contemplated wherein the animal model is a mouse model (e.g., an LDLR-/-mouse model).

The present disclosure also contemplates a method of lowering serum cholesterol in a subject in need thereof comprising administering a therapeutically effective amount of an agent capable of modulating KC homeostasis. The KC homeostasis may comprise increasing the ability of the KC to remove lipoproteins from serum, and/or it may comprise increasing the number of KCs that remove lipoproteins from serum.

The agent mentioned in the previous paragraph may be any agent identified using one of the methods described above. In particular embodiments, the agent is an IL-10 agent. These agents may be administered to the subject parenterally (e.g., subcutaneously), orally, or by any other means described herein or known to the skilled artisan.

Combination therapy is contemplated herein such that a therapeutically acceptable amount of at least one other cholesterol lowering agent is administered.

The present disclosure contemplates a method of treating or preventing hypercholesterolemia or a hypercholesterolemia-associated disease, disorder, or condition in a subject (e.g., a human) comprising administering (e.g., via parenteral (e.g., SC) or oral administration) to the subject a therapeutically effective amount of an agent identified using any of the methods described herein. The hypercholesterolemia-associated disease, disorder or condition can be a cardiovascular disorder (e.g., atherosclerosis), a thrombosis or thrombotic condition, an inflammatory disorder (e.g., vasculitis), or a fibrotic disorder. Particular embodiments are contemplated wherein the fibrotic disorder is liver-related, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis.

In particular embodiments, the methods may further comprise administering a therapeutically acceptable amount of at least one other therapeutic or prophylactic agent, such as a cholesterol homeostasis agent (e.g., a statin, bile acid resin, ezetimibe, fibric acid, niacin, or PCSK9 inhibitor), an anti-obesity agent, or an anti-inflammatory agent. Other agents are contemplated herein for use in combination therapy and are known to those of ordinary skill in the art.

The present invention contemplates pharmaceutical compositions comprising a pharmaceutically effective amount of one or more of the agents described above and a pharmaceutically acceptable diluent, carrier or excipient. In general, such compositions are suitable for human administration. These pharmaceutical compositions may comprise one or more other prophylactic or therapeutic agents, examples of which are described herein.

In certain embodiments, sterile containers (e.g., vials or syringes) may contain these pharmaceutical compositions. The sterile container can be contained in a kit, which can also contain one or more other prophylactic or therapeutic agents, reconstitution means, instructions for use, and the like.

The present disclosure also contemplates a method of treating or preventing a liver disease, disorder or condition in a subject (e.g., a human) comprising administering (e.g., parenterally, including subcutaneously) to the subject a therapeutically effective amount of an IL-10 agent capable of modulating KC homeostasis, wherein the liver disease, disorder or condition is non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

Other embodiments of the present disclosure contemplate methods of treating or preventing a liver disease, disorder or condition in a subject (e.g., a human) comprising administering (e.g., parenterally, including subcutaneously) to the subject a therapeutically effective amount of an IL-10 agent capable of modulating KC homeostasis, wherein the amount is sufficient to achieve a mean IL-10 serum trough concentration of 1pg/mL to 10.0 ng/mL; and wherein the liver disease, disorder or condition is NASH or NAFLD.

In still other embodiments, the present disclosure contemplates a method of treating or preventing a liver disease, disorder or condition in a subject (e.g., a human) comprising administering (e.g., parenterally, including subcutaneously) to the subject a therapeutically effective amount of a cytokine (e.g., an IL-10 agent) capable of modulating KC homeostasis, wherein the amount is sufficient to maintain an average cytokine (e.g., IL-10) serum trough concentration over a period of time; wherein the mean cytokine (e.g., IL-10) serum trough concentration is 1.0pg/mL to 10.0 ng/mL; wherein the mean cytokine (e.g., IL-10) serum trough concentration is maintained for at least 95% of the time period; and wherein the liver disease, disorder or condition is NASH or NAFLD. As used herein, the term "cytokine" is intended to have its ordinary meaning in the art.

In certain methods, the mean cytokine (e.g., IL-10) serum trough concentration is within the following range: 0.1ng/mL to 10ng/mL, 0.1ng/mL to 5.5ng/mL, 0.5ng/mL to 10ng/mL, 0.5ng/mL to 5.5ng/mL, 0.75ng/mL to 10.0ng/mL, 0.75ng/mL to 5.5ng/mL, 0.9ng/mL to 10.0ng/mL, 0.9ng/mL to 5.5ng/mL, 0.9ng/mL to 5.1ng/mL, 0.9ng/mL to 5.0ng/mL, 0.9ng/mL to 4.5ng/mL, 0.9ng/mL to 4.0ng/mL, 0.9ng/mL to 3.5ng/mL, 0.9ng/mL to 3.0ng/mL, 1.0ng/mL to 5.1ng/mL, 1.0ng/mL to 5.0ng/mL, 1.0ng/mL to 4.5ng/mL, 1.0ng/mL to 4.0ng/mL, 1.0ng/mL to 3.5ng/mL, or 1.0ng/mL to 3.0 ng/mL. The present disclosure contemplates methods wherein the cytokine (e.g., IL-10 agent) is administered to the subject at least twice daily, at least once every 48 hours, at least once every 72 hours, at least once weekly, at least once every 2 weeks, at least once every month, at least once every 2 months, or at least once every 3 months, or less frequently. In some embodiments of the methods described herein, the mean IL-10 serum trough concentration is maintained for at least 90% of the time period, for at least 95% of the time period, for at least 97% of the time period, for at least 99% of the time period, or for 100% of the time period.

The present disclosure contemplates embodiments wherein the IL-10 agent is mature human IL-10 or a variant of mature human IL-10. In particular embodiments, the variants exhibit activity comparable to that of mature human IL-10.

In some embodiments, the disease, disorder, or condition is NASH, and in other embodiments, it is NAFLD.

In some embodiments, modulating kupffer cell homeostasis comprises increasing the ability of KC to remove lipoproteins from serum, and in other embodiments increasing the number of KCs in order to remove lipoproteins from serum.

Embodiments are contemplated herein wherein the cytokine (e.g., IL-10 agent) reduces hepatic cholesterol and/or triglycerides, reduces or reverses portal perivenous collagen deposition, or increases the number of hepatocytes.

Some embodiments further comprise administering the cytokine (e.g., IL-10 agent) with at least one other prophylactic or therapeutic agent. In certain embodiments of the present disclosure, the prophylactic or therapeutic agent is a cholesterol homeostasis agent. In some embodiments, the cholesterol homeostasis agent comprises a statin, a bile acid resin, ezetimibe, fibric acid, niacin, or a PCSK9 inhibitor. The cholesterol homeostasis agents are generally capable of directly or indirectly ameliorating a cardiovascular disorder. In particular embodiments, a prophylactic or therapeutic agent is an agent that can be used to prevent or treat atherosclerosis. In other embodiments, the prophylactic or therapeutic agent is an antidiabetic agent or an antiobesity agent, while in other embodiments it is an immunological agent or an anti-inflammatory agent. Other exemplary prophylactic and therapeutic agents are set forth below.

Other embodiments of the present disclosure are described herein, and still other embodiments will be envisioned by the skilled artisan after reviewing the present disclosure.

Brief Description of Drawings

FIGS. 1A-1L show the modulating effect of PEG-rMuIL-10 on plasma cholesterol levels in wild type and LDLR-/-mice fed normal and high fat diets.

FIGS. 1M to 1N show the effect of PEG-rHuIL-10 on plasma cholesterol levels in oncology patients.

FIGS. 2A to 2L show the effect of PEG-rMuIL-10 on the expression of genes associated with liver function and cholesterol regulation.

Fig. 3A-3H show the effect of PEG-rMuIL-10 on the number of scavenger receptors and kupffer cells in treated and untreated liver tissue.

Fig. 4A to 4G show the results of the evaluation performed to determine whether cells associated with the myeloid lineage are responsible for the control of plasma cholesterol by PEG-rmuel-10.

FIGS. 5A-5D show the results of an evaluation performed to determine which cells in the liver will respond to PEG-rHuIL-10.

FIGS. 6A-6F show that PEG-rHuIL-10 increases the uptake of acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) in monocytes and LDL turnover in Kupffer cells.

Fig. 7A-7C show additive effects of PEG-rMuIL-10 and ezetimibe on cholesterol reduction.

Figures 8A to 8I show that kupffer cells were observed to play a role in reducing lipid, cholesterol and triglyceride accumulation with the introduction of PEG-rMuIL-10.

Fig. 9A to 9I show that treatment of animals with the indicated background with PEG-rMuIL-10 caused hepatocyte proliferation.

Detailed Description

Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments set forth herein, but is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

When a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where stated ranges include one or both of the stated limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. In addition, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

SUMMARY

The present disclosure contemplates the use of the agents described herein and compositions thereof for the treatment and/or prevention of various metabolic-related diseases (e.g., hypercholesterolemia), disorders and conditions, and/or symptoms thereof.

The present disclosure also contemplates the use of IL-10 agents (e.g., IL-10 polypeptides) and other cytokines for treating or preventing a liver disease, disorder or condition, comprising administering an IL-10 agent (or other cytokine agent) that modulates kupffer cell homeostasis. In particular embodiments, the liver disease, disorder, or condition is non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

In certain aspects of the disclosure, such treatment or prevention is achieved by utilizing specific dosing parameters. The present disclosure is based on the following findings: there is an optimal mean IL-10 (or other cytokine) serum trough concentration range and an optimal dosing range that can achieve a therapeutically relevant reduction in serum cholesterol.

In some embodiments of the disclosure, an IL-10 agent is administered to a subject having or at risk of having a disease or disorder treatable with an IL-10 agent (or other cytokine agent) in an amount sufficient to achieve a serum trough concentration above about 1ng/mL but below about 10ng/mL, while in other embodiments, the serum trough concentration is above about 2ng/mL but below about 10 ng/mL.

Some of the embodiments and descriptions set forth herein are described in the context of IL-10 agents (e.g., PEG-IL-10 agents). It is to be understood that, in view of the use case, recitation of an IL-10 agent may also refer more broadly to a cytokine agent, where appropriate.

It should be noted that any reference to "human" in connection with the polypeptides and nucleic acid molecules of the present disclosure is not meant to be limiting as to the manner or source in which the polypeptide or nucleic acid is obtained, but is merely made with reference to the following sequence as it may correspond to the sequence of a naturally occurring human polypeptide or nucleic acid molecule. In addition to human polypeptides and nucleic acid molecules encoding them, the present disclosure also contemplates IL-10 related polypeptides and corresponding nucleic acid molecules (and, in some cases, cytokine polypeptides and corresponding nucleic acid molecules) from other species.

Definition of

Unless otherwise indicated, the following terms are intended to have the meanings indicated below. Other terms are defined elsewhere in this specification.

The terms "patient" or "subject" are used interchangeably to refer to a human or non-human animal (e.g., a mammal).

The terms "administration", "administering", and the like, as they apply to, for example, a subject, cell, tissue, organ, or biological fluid, refer to, for example, contacting of IL-10 or PEG-IL-10), a nucleic acid (e.g., a nucleic acid encoding native human IL-10), a pharmaceutical composition or a diagnostic agent comprising the foregoing with the subject, cell, tissue, organ, or biological fluid. In the case of cells, administration includes contact of the agent with the cell (e.g., in vitro or ex vivo) and contact of the agent with a bodily fluid with which the cell is in contact.

The terms "treat", "treating", "treatment", and the like refer to a course of action (such as administration of IL-10 or a pharmaceutical composition comprising IL-10) that begins after a disease, disorder, or condition, or a symptom thereof, has been diagnosed, observed, or the like, so as to temporarily or permanently eliminate, reduce, inhibit, ameliorate, or ameliorate at least one underlying cause of the disease, disorder, or condition afflicting a subject, or at least one symptom associated with the disease, disorder, or condition afflicting a subject. Thus, treatment includes inhibiting (e.g., arresting the development or further development of the disease, disorder or condition or clinical symptoms associated therewith) active disease. The term can also be used in other situations, such as where IL-10 or PEG-IL-10 contacts the IL-10 receptor, for example, in a bulk or colloidal phase.

The term "in need of treatment" as used herein refers to the judgment made by a physician or other caregiver that a subject is in need of or will benefit from treatment. The determination is made based on a variety of factors in the field of expertise of the doctor or caregiver.

The terms "prevent", "preventing", "prevention", and the like refer to a course of action (such as administration of IL-10 or a pharmaceutical composition comprising IL-10) that begins, typically in the context of a subject predisposed to a particular disease, disorder, or condition, in a manner that temporarily or permanently prevents, suppresses, inhibits, or reduces the risk of the subject developing the disease, disorder, condition, or the like (as determined, for example, by the absence of clinical symptoms) or delays the onset thereof (e.g., prior to the onset of the disease, disorder, condition, or symptoms thereof). In certain instances, the term also refers to slowing the progression of a disease, disorder, or condition or inhibiting its progression to a deleterious or otherwise undesirable state.

As used herein, the term "in need of prevention" refers to the judgment made by a physician or other caregiver that a subject needs or will benefit from preventive care. The determination is made based on a variety of factors in the field of expertise of the doctor or caregiver.

The phrase "therapeutically effective amount" refers to an amount of an agent administered to a subject, either alone or as part of a pharmaceutical composition, and in a single dose form or as part of a series of doses, in an amount capable of having any detectable positive effect on any symptom, aspect, or feature of a disease, disorder, or condition when administered to the subject. A therapeutically effective amount can be determined by measuring relevant physiological effects and can be adjusted in conjunction with dosing regimens and diagnostic assays for the condition of the subject, and the like. For example, measuring the amount of inflammatory cytokines produced after administration can indicate whether a therapeutically effective amount has been used.

The phrase "in an amount sufficient to effect an alteration" means that there is a detectable difference between the indicator level measured before (e.g., the baseline level) and after administration of the particular therapy. The index includes any objective parameter (e.g., serum concentration of IL-10) or subjective parameter (e.g., the subject's health well-being).

The term "small molecule" refers to a chemical compound having a molecular weight of less than about 10kDa, less than about 2kDa, or less than about 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, and synthetic molecules. Therapeutically, small molecules may be more permeable to cells, less susceptible to degradation, and less likely to elicit an immune response than large molecules.

The term "ligand" refers to a peptide, polypeptide, membrane-associated or membrane-bound molecule or complex thereof, which may act, for example, as a receptor agonist or antagonist. "ligand" encompasses natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and binding compositions derived from antibodies. "ligands" also encompass small molecules, e.g., peptide mimetics of cytokines and antibodies. The term also encompasses agents that are neither agonists nor antagonists, but that can bind to the receptor without significantly affecting its biological properties (e.g., signaling or adhesion). In addition, the term includes membrane-bound ligands that have been rendered soluble in form of the membrane-bound ligand, for example, by chemical or recombinant means. The ligand or receptor may be entirely intracellular, i.e., it may reside in the cytoplasm, nucleus, or some other intracellular compartment. The complex of ligand and receptor is referred to as a "ligand-receptor complex".

The terms "inhibitor" and "antagonist" or "activator" and "agonist" refer to inhibitory or activating molecules that are used, for example, to activate, e.g., a ligand, receptor, cofactor, gene, cell, tissue, or organ, respectively. An inhibitor is a molecule that reduces, blocks, prevents, delays activation, inactivates, desensitizes, or down regulates, for example, a gene, protein, ligand, receptor, or cell. An activator is a molecule that increases, activates, promotes, enhances activation, sensitizes, or upregulates, for example, a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a molecule that reduces, blocks or inactivates a constitutive activity. An "agonist" is a molecule that interacts with a target to cause or promote increased activation of the target. An "antagonist" is a molecule that opposes the action of an agonist. Antagonists prevent, reduce, inhibit, or neutralize the activity of an agonist, and antagonists may also prevent, inhibit, or reduce the constitutive activity of a target, e.g., a target receptor, even in the absence of an identified agonist.

The terms "modulate", "modulation" and the like refer to the ability of a molecule (e.g., activator or inhibitor) to directly or indirectly enhance or attenuate the function or activity of an agent (e.g., an IL-10 agent) (or a nucleic acid molecule encoding the same); or enhance the ability of the molecule to produce an effect comparable to the effect of an agent (e.g., an IL-10 agent). The term "modulator" is intended to refer broadly to a molecule capable of achieving the above-described activity. For example, a modulator of a gene, receptor, ligand or cell is a molecule that alters the activity of the gene, receptor, ligand or cell, where its regulatory properties can be used to activate, inhibit or alter the activity. The modulator may act alone, or it may use a cofactor, for example, a protein, metal ion, or small molecule. The term "modulator" includes agents that operate by the same mechanism of action as an agent (e.g., an IL-10 agent) (i.e., an agent that modulates the same signaling pathway as an agent (e.g., an IL-10 agent) in a similar manner thereto) and are capable of eliciting a biological response comparable to (or greater than) that of the agent (e.g., an IL-10 agent).

Examples of modulators include small molecule compounds and other bio-organic molecules. Numerous libraries (e.g., combinatorial libraries) of small molecule compounds are commercially available and can serve as starting points for the identification of modulators. The skilled artisan is able to develop one or more assays (e.g., biochemical assays or cell-based assays) in which libraries of such compounds can be screened in order to identify one or more compounds having a desired property; thereafter, the skilled pharmaceutical chemist can optimize such compound or compounds by, for example, synthesizing and evaluating analogs and derivatives thereof. Synthetic and/or molecular modeling studies can also be used for the identification of activators.

The "activity" of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor; catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; an antigenic activity; modulating the activity of other molecules, etc. The term may also refer to activity in modulating or maintaining cell-cell interaction (e.g., adhesion), or in maintaining cell structure (e.g., cell membrane). "activity" may also mean specific activity, e.g., [ catalytic activity ]/[ mg protein ], or [ immunological activity ]/[ mg protein ], concentration in a biological compartment, etc. The term "proliferative activity" encompasses activities that promote, for example, normal cell division as well as cancer, tumor, dysplasia, cell transformation, metastasis and angiogenesis, are essential for, or are particularly relevant to, normal cell division as well as cancer, tumor, dysplasia, cell transformation, metastasis and angiogenesis.

As used herein, "equivalent," "equivalent activity," "activity equivalent to … …," "equivalent effect," "effect equivalent to … …," and the like are relative terms that may be considered quantitatively and/or qualitatively. The meaning of the terms is often dependent on the situation in which they are used. For example, two agents that are both capable of activating a receptor can be considered to have comparable effects from a qualitative perspective, but can be considered to lack comparable effects from a quantitative perspective if one agent is only capable of achieving 20% of the activity of the other agent, as determined in art-accepted assays (e.g., dose-response assays) or art-accepted animal models. "comparable" when comparing one result to another (e.g., one result to a reference standard) often means that one result has a standard deviation from the reference of less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In particular embodiments, a result is comparable to a reference standard if it has a standard deviation from the reference of less than 15%, less than 10%, or less than 5%. By way of example, but not limitation, the activity or effect may refer to efficacy, stability, solubility, or immunogenicity.

The term "response" of a term, e.g. a cell, tissue, organ or organism, encompasses biochemical or physiological behavior, such as changes in concentration, density, adhesion or migration within a biological compartment, gene expression rate or differentiation state, wherein the changes are associated with activation, stimulation or therapy or with internal mechanisms such as gene programming. In certain instances, the terms "activate", "stimulate", and the like refer to activation of a cell, e.g., by internal mechanisms, as well as by external or environmental factors; however, the terms "inhibit", "downregulate" and the like refer to the opposite effect.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include genetically-encoded and non-genetically-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified polypeptide backbone. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences; a fusion protein having heterologous and homologous leader sequences; a fusion protein with or without an N-terminal methionine residue; a fusion protein having an immunologically tagged protein; and so on.

It is understood that throughout this disclosure, reference to amino acids is made according to the single or three letter code. For the convenience of the reader, the single and three letter amino acid codes are provided below:

as used herein, the term "variant" encompasses naturally occurring variants and non-naturally occurring variants. Naturally occurring variants include homologs (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, between one species and another) and allelic variants (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, between one individual and another within a species). Non-naturally occurring variants include polypeptides and nucleic acids that comprise changes in the amino acid or nucleotide sequence, respectively, wherein the changes in the sequence are artificially introduced (e.g., muteins); the changes are produced, for example, in the laboratory by human intervention ("human manual"). Thus, herein, a "mutein" broadly refers to a recombinant protein that has been mutated, typically carrying a single or multiple amino acid substitution, and often derived from a cloned gene that has undergone site-directed or random mutagenesis, or derived from a completely synthetic gene.

The terms "DNA," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein to refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) or analogs thereof of any length. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

As used herein in the context of polypeptide structures, "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the amino-most and carboxyl-most termini, respectively, of a polypeptide, while the terms "N-terminus" (N-terminal) "and" C-terminus "(C-terminal)" refer to the relative positions in the polypeptide amino acid sequence with respect to the N-terminus and C-terminus, respectively, and may include residues at the N-terminus and C-terminus, respectively. "immediately N-terminal" or "immediately C-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue, wherein the first amino acid residue is covalently bound to the second amino acid residue to provide a contiguous amino acid sequence.

"derived from" in the context of an amino acid sequence or nucleotide sequence (e.g., "derived from" the amino acid sequence of an IL-10 polypeptide) is intended to indicate that the polypeptide or nucleic acid has a sequence based on the sequence of a reference polypeptide or nucleic acid (e.g., a naturally occurring IL-10 polypeptide or IL-10 encoding nucleic acid), and is not intended to limit the source or method by which the protein or nucleic acid is produced. For example, the term "derived from" includes homologs or variants of a reference amino acid or DNA sequence.

In the context of polypeptides, the term "isolated" means that the polypeptide of interest, if found in nature, is present in an environment different from the environment in which it may be found in nature. By "isolated" is meant to include polypeptides within a sample that are substantially enriched for a polypeptide of interest and/or in which the polypeptide of interest is partially or substantially purified. When a polypeptide does not occur naturally, "isolated" means that the polypeptide has been separated from the environment in which it was produced by synthetic or recombinant means.

By "enriched" is meant that the sample is manipulated non-naturally (e.g., by a scientist) such that the polypeptide of interest is present at a concentration a) higher (e.g., at least 3-fold higher, at least 4-fold higher, at least 8-fold higher, at least 64-fold or more higher) than the concentration of the polypeptide in a starting sample, such as a biological sample (e.g., a sample in which the polypeptide is naturally present or is present after administration), or b) higher than the environment in which the polypeptide is produced (e.g., as in a bacterial cell).

By "substantially pure" is meant that a component (e.g., a polypeptide) constitutes more than about 50% of the total content of the composition, and typically more than about 60% of the total polypeptide content. More typically, "substantially pure" refers to a composition in which at least 75%, at least 85%, at least 90%, or more of the total composition is the component of interest. In some cases, the polypeptide will constitute more than about 90% or more than about 95% of the total content of the composition.

The term "specifically binds" or "selectively binds" when referring to a ligand/receptor, antibody/antigen or other binding pair refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. Thus, under specified conditions, a defined ligand binds to a particular receptor and does not bind in significant amounts to other proteins present in the sample. The method envisagedThe antibody of the method or the binding composition of the antigen binding site derived from the antibody binds its antigen or a variant or mutein thereof with an affinity that is at least 2-fold higher, at least 10-fold higher, at least 20-fold higher or at least 100-fold higher than the affinity to any other antibody or binding composition derived therefrom. In a specific embodiment, the antibody will have a molecular weight greater than about 10 as determined by, for example, Scatchard analysis (Munsen et al, 1980Analyt. biochem.107:220-239)⁹Affinity of L/mol.

IL-10 and PEG-IL-10

The anti-inflammatory cytokine IL-10, also known as human Cytokine Synthesis Inhibitory Factor (CSIF), is classified as a type 2 cytokine, a group of cytokines including IL-19, IL-20, IL-22, IL-24(Mda-7) and IL-26, interferons (IFN- α, IFN- β, IFN- γ, IFN- κ, IFN- Ω and IFN- τ), and interferon-like molecules (restrictors, IL-28A, IL-28B and IL-29).

IL-10 is a cytokine with pleiotropic effects in immune regulation and inflammation. Although expressed primarily in macrophages, IL-10 expression has also been detected in activated T cells, B cells, mast cells and monocytes. It is produced by mast cells, counteracting the inflammatory effects these cells have at the site of anaphylaxis. Although IL-10 limits the production and secretion of proinflammatory cytokines primarily in response to toll-like receptor agonists, it is also stimulatory for certain T cells and mast cells and stimulates B cell maturation, proliferation, and antibody production. IL-10 can block NF- κ B activity and is involved in the regulation of the JAK-STAT signaling pathway. It also induces the cytotoxic activity of CD8+ T cells and antibody production by B cells, and it inhibits macrophage activity and tumor inflammatory responses. The regulation of CD8+ T cells is dose dependent, with higher doses inducing a stronger cytotoxic response.

Due to its pleiotropic activity, IL-10 has been implicated in a variety of diseases, disorders and conditions, including inflammatory conditions, immune-related disorders, fibrotic disorders, metabolic disorders (including cholesterol regulation), and cancer. The clinical and preclinical evaluation of IL-10 for a number of such diseases, disorders, and conditions has consolidated its therapeutic potential.

Human IL-10 is a homodimer with a molecular mass of 37kDa, where each 18.5kDa monomer comprises 178 amino acids, the first 18 of which constitute the signal peptide. Each monomer includes four cysteine residues that form two intramolecular disulfide bonds. IL-10 dimer becomes biologically inactive after disrupting the non-covalent interactions between two monomeric subunits. Data from the published crystal Structure of IL-10 indicate that the functional dimer exhibits some similarity to IFN- γ (ZDanov et al, (1995) Structure (Lond)3: 591-601). The description herein refers generally to homodimers; however, certain aspects discussed may also apply to monomers, as will be apparent from the context.

Various embodiments of the present disclosure contemplate human IL-10(NP _000563) and murine IL-10(NP _034678) exhibiting 80% homology and uses thereof. In addition, the scope of the present disclosure includes IL-10 orthologs from other mammalian species and modified forms thereof, including rat (accession number NP-036986.2; GI 148747382); cattle (accession number NP-776513.1; GI 41386772); sheep (accession NP-001009327.1; GI 57164347); dog (accession number ABY 86619.1; GI 166244598); and rabbits (accession number AAC 23839.1; GI 3242896).

As mentioned above, the terms "IL-10", "IL-10 polypeptide", "IL-10 molecule", "IL-10 agent", and the like, are intended to be broadly construed and include, for example, human and non-human IL-10 related polypeptides, including homologs, variants (including muteins) and fragments thereof, as well as IL-10 polypeptides having, for example, a leader sequence (e.g., a signal peptide), as well as modified forms of the foregoing. In other specific embodiments, the IL-10, IL-10 polypeptide, and IL-10 agent are agonists.

The IL-10 receptor is a type II cytokine receptor, consisting of alpha and beta subunits (also referred to as Rl and R2, respectively). Receptor activation requires binding to alpha and beta. One homodimer of an IL-10 polypeptide binds to alpha, while another homodimer of the same IL-10 polypeptide binds to beta.

The utility of recombinant human IL-10 is often limited by its relatively short serum half-life that may be caused by, for example, renal clearance, proteolytic degradation in the bloodstream, and monomerization. Thus, various approaches have been developed to improve the pharmacokinetic profile of IL-10 without disrupting its dimeric structure and thus adversely affecting its activity. Pegylation of IL-10 improves certain pharmacokinetic parameters (e.g., serum half-life) and/or enhances activity.

As used herein, the terms "pegylated IL-10" and "PEG-IL-10" refer to IL-10 molecules having one or more polyethylene glycol molecules covalently attached (typically through a linker, to stabilize attachment) to at least one amino acid residue of the IL-10 protein. The terms "mono-pegylated IL-10" and "mono-PEG-IL-10" mean that one polyethylene glycol molecule is covalently attached (typically through a linker) to a single amino acid residue on one subunit of the IL-10 dimer. As used herein, the terms "di-pegylated IL-10" and "di-PEG-IL-10" mean that at least one polyethylene glycol molecule is attached to a single residue on each subunit of the IL-10 dimer, typically through a linker.

In certain embodiments, the PEG-IL-10 used in the present disclosure is a mono-PEG-IL-10, wherein 1 to 9 PEG molecules are covalently attached by a linker to the alpha amino group of the amino acid residue at the N-terminus of one subunit of the IL-10 dimer. MonoPEGylation on one IL-10 subunit will generally result in a heterogeneous mixture of non-PEGylated, mono-PEGylated, and di-PEGylated IL-10 due to subunit shuffling. In addition, allowing the pegylation reaction to proceed to completion will generally result in non-specific polyglycolyzed IL-10, thus reducing its biological activity. Thus, particular embodiments of the present disclosure include administering a mixture of mono-pegylated and di-pegylated IL-10 produced by the methods described herein.

In some embodiments, an N-terminal pegylation chemistry strategy can be used such that the N-terminal is pegylated at about 99% specificity over a defined period of time (e.g., less than 18 hours). Allowing the chemical reaction to continue until beyond this time period will result in increased pegylation of lysine side chains. Several pegylation methods are described in the experimental section.

In particular embodiments, the PEG moiety has an average molecular weight between about 5kDa and about 50 kDa. While the method or site of attachment of PEG to IL-10 is not critical, in certain embodiments pegylation does not alter, or only minimally alters, the activity of the IL-10 agent. In certain embodiments, the increase in half-life exceeds any decrease in biological activity. The biological activity of PEG-IL-10 is typically measured by assessing the level of inflammatory cytokines (e.g., TNF-a or IFN- γ) in the serum of subjects challenged with bacterial antigens (lipopolysaccharide (LPS)) and treated with PEG-IL-10, as described in U.S. patent No. 7,052,686.

IL-10 variants (without modification, e.g., by pegylation or HAS conjugation) can be prepared with a variety of goals in mind, including increasing serum half-life, reducing immune response to IL-10, facilitating purification or preparation, reducing the conversion of IL-10 to its monomeric subunits, improving therapeutic efficacy, and reducing the severity or incidence of side effects during therapeutic use. Amino acid sequence variants are typically predetermined variants not found in nature, but some may be post-translational variants, such as glycosylation variants. Any variant of IL-10 may be used, provided that it retains a suitable level of IL-10 activity. Like wild-type IL-10, these IL-10 variants can be modified as described herein (by, e.g., pegylation or Fc fusion).

The phrase "conservative amino acid substitution" refers to a substitution that preserves the activity of a protein by replacing an amino acid in the protein with an amino acid having a side chain of similar acidic, basic, charge, polarity, or side chain size. Conservative amino acid substitutions generally entail the substitution of amino acid residues within the following groups: 1) l, I, M, V, F, respectively; 2) r, K, respectively; 3) f, Y, H, W, R, respectively; 4) g, A, T, S, respectively; 5) q, N, respectively; and 6) D, E. Guidance regarding substitutions, insertions or deletions may be based on alignments of the amino acid sequences of different variant proteins or proteins from different species. Thus, in addition to any naturally occurring IL-10 polypeptide, the present disclosure contemplates having 1,2,3,4, 5,6, 7,8, 9, or 10, typically no more than 20, 10, or 5 amino acid substitutions, wherein the substitutions are typically conservative amino acid substitutions.

The present disclosure also contemplates active fragments (e.g., subsequences) of mature IL-10 that contain contiguous amino acid residues derived from mature IL-10. The length of consecutive amino acid residues of a peptide or polypeptide subsequence may vary depending on the particular naturally occurring amino acid sequence from which the subsequence is derived. In general, peptides and polypeptides may be from about 20 amino acids to about 40 amino acids, from about 40 amino acids to about 60 amino acids, from about 60 amino acids to about 80 amino acids, from about 80 amino acids to about 100 amino acid acids, from about 100 amino acids to about 120 amino acids, from about 120 amino acids to about 140 amino acids, from about 140 amino acids to about 150 amino acids, from about 150 amino acids to about 155 amino acids, from about 155 amino acid acids up to full length peptides or polypeptides.

In addition, an IL-10 polypeptide can have a defined sequence identity over a defined length of contiguous amino acids (e.g., a "comparison window") as compared to a reference sequence. Methods of sequence alignment for comparison are well known in the art. The optimal sequence alignment can be performed for comparison, for example, by the local homology algorithm of Smith and Waterman, adv.appl.Math.2:482(1981), by the homology alignment algorithm of Needleman and Wunsch, J.Mol.biol.48:443(1970), by the similarity search method of Pearson and Lipman, Proc.Nat' l.Acad.Sci.USA 85:2444(1988), by computerized execution of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (eds. Ausubel et al., 1995 supplement).

As one example, a suitable IL-10 polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to about 20 amino acids to about 40 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 120 amino acids, about 120 amino acids to about 140 amino acids, about 140 amino acids to about 150 amino acids, about 150 amino acids to about 155 amino acids, up to a contiguous stretch of a full length peptide or polypeptide.

As discussed further below, IL-10 polypeptides may be isolated from non-natural sources (e.g., environments other than the environment in which they naturally occur), and may also be produced recombinantly (e.g., in genetically modified host cells, such as bacteria, yeast, pichia, insect cells, and the like), wherein the genetically modified host cells are modified with a nucleic acid comprising a nucleotide sequence encoding the polypeptide. IL-10 polypeptides may also be produced synthetically (e.g., by cell-free chemical synthesis).

The present disclosure contemplates nucleic acid molecules encoding IL-10 agents, including naturally occurring and non-naturally occurring isoforms, allelic and splice variants thereof. The present disclosure also encompasses nucleic acid sequences that differ from naturally occurring DNA sequences by one or more bases, but which, due to the degeneracy of the genetic code, are still translated into an amino acid sequence corresponding to an IL-10 polypeptide.

As previously indicated, the present disclosure also contemplates the use of gene therapy in conjunction with the teachings herein. Gene therapy is achieved by delivering genetic material, typically packaged in a vector, to endogenous cells in a subject in order to introduce novel genes, introduce additional copies of pre-existing genes, impair the function of existing genes, or repair existing but non-functional genes. Once inside the cell, the nucleic acid is expressed by the organelle, thereby producing the protein of interest. In the context of the present disclosure, gene therapy is used as a therapy to deliver nucleic acids encoding IL-10 agents for the treatment or prevention of the diseases, disorders, or conditions described herein.

As mentioned above, for gene therapy uses and methods, cells of a subject can be transformed in vivo with a nucleic acid encoding an IL-10 related polypeptide as set forth herein. Alternatively, cells can be transformed with a transgene or polynucleotide in vitro and then transplanted into a tissue of a subject in order to effect treatment. Alternatively, isolated primary cells or established cell lines can be transformed with transgenes or polynucleotides encoding IL-10-related polypeptides, and then optionally transplanted into a tissue of a subject.

Cholesterol homeostasis

Physiology of human body: cholesterol plays an essential role in a range of physiological processes, including cell membrane structure and biosynthesis of steroid hormones, bile acids and vitamin D. Cholesterol synthesis requires a complex 37-step process starting with the reduction of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) to mevalonate with the enzyme HGM-CoA reductase. This is a regulated, rate-limiting and irreversible step in cholesterol synthesis and is the site of action for statins (HMG-CoA reductase competitive inhibitors).

The liver is the primary regulator of cholesterol. It is not but the site of formation of VLDL, the precursor of circulating LDL, it is also the site where most receptor-mediated LDL clearance occurs.

The liver initially clears all cholesterol absorbed from the small intestine. Excess cholesterol absorption may increase the amount of cholesterol stored in the liver, leading to increased VLDL secretion (and thus LDL formation) and down-regulation of hepatic LDL receptor activity. On average, about half of all cholesterol entering the intestine is absorbed. The fractional absorption rates vary greatly from individual to individual, which may explain, at least in part, why some patients do not respond well or at all to statins and other classes of lipid lowering drugs. See, e.g., Turley, SD, (2004) Clin. Cardiol.6 supplement 3: III 16-21. The liver also recirculates cholesterol by excreting it in a non-esterified form (through the bile) into the digestive tract.

Blood lipid examination: total cholesterol is defined as the sum of LDL, HDL and VLDL. In general, total blood cholesterol levels<200mg/dL is considered normal, levels between 200-239mg/dL are considered borderline high, and levels>240mg/dL is considered high.

Since 1988, the National Cholesterol Education Program (NCEP) has issued guidelines for determining LDL as a primary target for cholesterol therapy. Current guidelines set forth in adult treatment version III (ATP-III) set targets for LDL <100mg/dL (2.6 mmol/L). Increased LDL is associated with atherosclerotic disease, thereby presenting a high risk of Coronary Heart Disease (CHD) related events, including clinical CHD, symptomatic carotid artery disease, peripheral artery disease, and abdominal aortic aneurysm. Diseases, disorders, and conditions associated with elevated cholesterol levels and the treatment and/or prevention thereof are described in detail below.

There is considerable evidence that low levels of high density cholesterol (HDL-C or HDL only) are contributing factors to the development of atherosclerosis and CHD. Low HDL is one of the most common lipid disorders in patients with premature coronary artery disease. Patients with hypertriglyceridemia often have lower HDL cholesterol. Certain drugs, including beta-blockers, progesterone, and testosterone, also lower HDL levels.

HDL cholesterol levels range from 40 to 50mg/dL in common men and 50 to 60mg/dL in common women. Studies have shown that the median HDL value associated with the lowest risk of atherosclerotic events is 62mg/dL in men and 81mg/dL in women. ATP-III guidelines for lipid lowering therapy determine that HDL levels below 40mg/dL are the primary positive risk factors and LDL levels ≧ 60mg/dL are negative risk factors (i.e., protective). A ratio of total cholesterol to HDL of less than 5:1 is considered desirable.

Triglycerides are transported in the bloodstream primarily by Very Low Density Lipoproteins (VLDL). There is considerable heterogeneity in the triglyceride-rich particles. Triglyceride-rich particles derived from dietary fat chylomicrons are not associated with CHD by themselves, but when very high (>1,000mg/dL) can lead to pancreatitis, venous and arterial thrombosis, acute heart attacks, and stroke. However, the size of these chylomicron particles is gradually reduced by lipoprotein lipase, thereby forming Intermediate Density Lipoprotein (IDL) which causes atheroma. Similarly, VLDL from liver is reduced in size by lipoprotein lipase, thereby producing IDL that causes atherogenesis. VLDL predicts the progression of coronary artery disease and CHD events and, therefore, has been increasingly considered as a risk factor for hypertriglyceridemia CHD.

High triglyceride levels are either caused by genetic predisposition or acquired. About 1/500 of people have a tendency to inherit from high plasma triglycerides for genetic predisposition. Acquired high triglycerides are most commonly associated with excessive alcohol intake, exogenous estrogens or estrogen agonists, poorly controlled diabetes, beta-blockers, corticosteroids, and uremia. Triglyceride levels above 1,000mg/dL reflect an acquired predisposition to high triglycerides plus a genetic predisposition. Less common causes of acquired hypertriglyceridemia include renal failure, nephrotic syndrome, proteinuria, hypothyroidism, many liver diseases, bronze diabetes, hyperparathyroidism, and glycogen storage disease.

According to the American Heart Association, triglyceride levels below 150mg/dL are normal; levels of 150 to 199mg/dL are borderline high values; levels of 200 to 499mg/dL are high; while the level of 500mg/dL or more is extremely high. Generally, triglyceride levels between 150 and 200mg/dL do not require drug treatment.

Testing: several general methods and systems have been used to assess the lipid profile of a subject. Any method or system now existing or later developed that may be used in conjunction with the teachings of the present disclosure.

The fasting cholesterol test, which typically utilizes a colorimetric assay system, is the traditional means of measuring total serum cholesterol. Such tests require blood to be drawn after a 12 hour fast to determine the lipoprotein profile. Typically, only total cholesterol, HDL, and triglycerides are measured; for cost reasons VLDL is not typically measured, but estimated as one fifth of triglyceride, and LDL is estimated using the friedwidth formula. While such tests are inexpensive and widely available (e.g., Sigma-Aldrich, st. louis, MO; BioVision, inc., Milpitas, CA), they require fasting and are not as sensitive as other tests because LDL is estimated rather than accurately determined.

When assessing hypercholesterolemia, it is often used to measure all lipoprotein subfractions (VLDL, IDL, LDL and HDL). Because the specific therapeutic goal is to lower LDL (while maintaining or increasing HDL), cholesterol tests that directly measure LDL levels are more accurate, and are particularly useful in those patients with elevated triglycerides. Although commercially available (e.g., beckmann coulter, inc.; break, CA), the use of these direct measurement tests is sometimes limited due to their cost.

Role of Kupffer cells in cholesterol homeostasis

Macrophages are generally classified by function and location; blood monocytes, liver kupffer cells (liver-specific macrophages), fixed tissue macrophages, and various dendritic cells are among the most prevalent types of macrophages.

Kupffer Cells (KCs), large fixed macrophages constituting 80-90% of the tissue macrophages present in the body, reside in the lumen of hepatic sinusoids and exhibit phagocytic activity against blood-borne substances entering the liver. KC plays a major role in the physiological maintenance of liver structure and is intimately involved in the response of the liver to infections (e.g. HCV), toxins (e.g. alcohol and drugs), ischemia, resection and other stresses (e.g. trauma).

Upon activation (e.g., by bacterial endotoxins), KC releases a variety of factors, including cytokines, prostanoids, nitric oxide, and reactive oxygen species, that modulate the phenotype of the KC itself as well as the phenotype of neighboring cells such as hepatocytes, astrocytes, endothelial cells, and other immune cells that pass through the liver. Macrophage scavenger receptors are expressed in KC, and such scavenger receptors are involved not only in the sterilization process, but also in lipid metabolism. Evidence suggests that KC represents a distinct cell population with distinct differentiation mechanisms, metabolic functions, and the ability to respond to inflammatory agents.

STAT3. The transcription factor STAT3 (signal transducer and activator of transcription 3) is a member of the STAT protein family. STAT family members are phosphorylated by receptor-associated kinases in response to cytokines and growth factors; thereafter, they form homodimers or heterodimers, which translocate to the nucleus where they serve as transcriptional activators. STAT3 plays a key role in many cellular processes, including cell growth and apoptosis. It is essential for the differentiation of TH17 helper T cells that play a role in a variety of autoimmune diseases. In addition, STAT3 is implicated in the regulation of lipid metabolism. (see Kinoshita et al, Kobe J.Med.Sci., Vol.54, No. 4, pp.E 200-E208, 2008).

STAT3 phosphorylation occurs in response to a variety of cytokines and growth factors, including certain interleukins (e.g., IL-5, IL-6, and IL-10), Leukemia Inhibitory Factor (LIF), Epidermal Growth Factor (EGF), certain interferons, Hepatocyte Growth Factor (HGF), bone morphogenic protein 2(BMP-2), the cytokine oncostatin M (OSM), and the hormone leptin. IL-6 reduces serum cholesterol in mice and humans, human LIF reduces serum cholesterol in hypercholesterolemic rabbits, and OSM reduces serum cholesterol in hypercholesterolemic hamsters; it is believed that this cholesterol reduction is achieved by upregulation of LDL receptors.

The present disclosure is based in part on the following findings: the KC's ability to remove lipoproteins from serum can be modulated (e.g., enhanced) in order to achieve a desired metabolic effect (e.g., cholesterol lowering). In particular aspects, the present disclosure features methods of identifying agents that induce phosphorylation of STAT3 in KC, thereby enhancing its ability to remove lipoproteins from serum. As noted herein, while it is not necessary to understand the potential mechanism of action of KC in involvement with cholesterol lowering in order to practice the present disclosure, it is believed that agents that induce phosphorylation of STAT3 in KC enhance the ability of KC to clear lipoproteins from serum. In this context, the phrase "an agent capable of inducing phosphorylation of STAT 3" is intended to broadly refer to any molecule (e.g., small molecules, polypeptides, and antibodies) that directly or indirectly, completely, or partially, results in an increase in phosphorylated STAT 3. In particular embodiments, such agents are factors that drive phosphorylation of STAT3 (e.g., IL-10, IL-6, LIF, and oncostatin M).

Scavenger receptor. Scavenger receptors are involved in the removal of many foreign substances and waste products from the body through a wide range of ligand specificities and a variety of receptor molecules. They constitute a group of receptors that recognize and absorb negatively charged macromolecules and LDL that has been modified by oxidation (oxLDL) or acetylation (acLDL).

Scavenger receptors are generally classified into three classes-class a, class B and class C, based on their structural characteristics. Class A scavenger receptors (type 1 scavenger receptor (SR-A1) and type 2 scavenger receptor (SR-A2)) are trimers that preferentially bind modified LDLs (e.g., oxLDL and acLDL) and have collagen-like domains necessary for ligand binding. Class a members include MSR1 (also known as SCARA1), MARCO (also known as SCARA2), SCARA3, COLEC12 (also known as SCARA4), and SCARA 5.

Class B scavenger receptors have been identified as oxidized LDL receptors and members thereof include CD36 and class BI scavenger receptor (SR-BI). Class B scavenger receptors are commonly referred to as SCARB1 (which also interacts with HDL); SCARB 2; and SCARB3 (also known as CD36), which is involved in phagocytosis of apoptotic cells and metabolism of long chain fatty acids. Class C scavenger receptors include other receptors that can bind oxidized LDL, including CD68, mucins, and lectin-like oxidized LDL receptor 1 (LOX-1).

As described herein, the uptake process of modified LDL by scavenger receptors is followed by intracellular degradation and/or efflux onto HLD particles. IL-10 has been shown to be involved in absorption and efflux processes and may also promote degradation processes.

Scavenger receptor function is evaluated herein (see experimental section), in particular against MSR1, MARCO, SCARB1, SCARB2 and CD 36. When the effect of IL-10(PEG-rMuIL-10) on the expression of genes associated with liver function and cholesterol regulation was evaluated, only two major gene groups were altered, one of which was the scavenger receptor (see example 2 and FIG. 2). As shown in fig. 2E to 2H, the a-type scavenger receptors Msr1 and Marco were induced 2 to 7-fold in wild-type and LDLR-/-mice maintained on a normal food diet and wild-type and LDLR-/-mice maintained on a high fat food diet. In addition, because the scavenger receptors are mainly expressed on macrophage-type cells, the difference in gene expression of the two cell surface proteins F4/80 and CD14, which are most frequently expressed on liver tissue-resident macrophages, was evaluated, and these genes were moderately induced under different gene backgrounds and dietary conditions (see fig. 2E to 2H). FIGS. 3A-3D show the effect of PEG-rMuIL-10 on Msr1 (see example 3). These data confirm the role of scavenger receptors in liver function and cholesterol regulation.

Effect of PEG-IL-10 and other cytokines on Cholesterol homeostasis and KC function

Role of IL-10 in cholesterol homeostasis. The extent to which PEG-IL-10 reduces plasma cholesterol is related to the total cholesterol level of the subject. This is observed in both mice and humans. As indicated in example 1 and figure 1, PEG-rMuIL-10 only reduces plasma cholesterol in hypercholesterolemic mice, while PEG-rMuIL-10 only reduces plasma cholesterol in patients with elevated plasma cholesterol levels. To illustrate, cancer patients with borderline high (-200 mg/dL) total cholesterol achieved approximately 40% cholesterol reduction after subcutaneous administration of PEG-rHuIL-10 per day, while patients with low (-100 mg/dL) total cholesterol were unaffected. These data indicate that PEG-IL-10 is more effective in the patient population that will benefit the most from cholesterol lowering.

It is believed that this response was triggered by the level of IL-10 ra expression, as high fat diet induced IL-10 ra up-regulation in the liver of mice (data not shown).

The present disclosure contemplates administration of a cytokine (e.g., PEG-IL-10) to a subject who will benefit from cholesterol lowering, regardless of its total cholesterol level. Thus, for example, in some embodiments, PEG-IL-10 is administered to a subject having the following total cholesterol levels: at least 150mg/dL, at least 160mg/dL, at least 170mg/dL, at least 180mg/dL, at least 190mg/dL, at least 200mg/dL, at least 210mg/dL, at least 220mg/dL, at least 230mg/dL, at least 240mg/dL, at least 250mg/dL, at least 260mg/dL, at least 270mg/dL, at least 280mg/dL, at least 290mg/dL, or at least 300 mg/dL. In other embodiments, PEG-IL-10 is administered to a subject having the following total cholesterol levels: at least 325mg/dL, at least 350mg/dL, at least 375mg/dL, at least 400mg/dL, at least 425mg/dL, at least 450mg/dL, at least 475mg/dL, or at least 500 mg/dL.

In particular embodiments, an IL-10 agent (or other cytokine agent) (e.g., PEG-IL-10) disclosed herein has anti-hyperlipidemia activity capable of reducing VLDL levels, IDL levels, LDL levels, or a combination thereof by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In yet other embodiments, an IL-10 agent (or other cytokine agent) (e.g., PEG-IL-10) disclosed herein has anti-hyperlipidemia activity capable of reducing VLDL levels, IDL levels, LDL levels, or a combination thereof by a range, e.g., from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, or from about 80% to about 100%; about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, or about 70% to about 90%; about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%; about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

In another embodiment of the disclosure, an IL-10 agent (or other cytokine agent) disclosed herein (e.g., PEG-IL-10) increases HDL levels. In some other (generally more common) embodiments, HDL levels themselves are not increased; in contrast, LDL levels and HDL levels are reduced, but LDL levels are reduced more than HDL levels, such that the final LDL to HDL ratio changes consistent with the HDL lipid hypothesis. In one aspect of these embodiments, the IL-10 agent (or other cytokine agent) increases HDL levels relative to LDL by, e.g., at least 2%, at least 3%, at least 10%, at least 12%, at least 15%, at least 17%, at least 20%, at least 22%, at least 25%, at least 27%, at least 30%, at least 32%, at least 35%, at least 37%, at least 40%, at least 42%, at least 45%, or at least 47%. In still other aspects of these embodiments, the IL-10 agent increases HDL levels relative to LDL by a range, e.g., from about 2% to about 100%; about 10% to about 50%, about 15% to about 50%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, or about 40% to about 50%; about 2% to about 45%, about 10% to about 45%, about 15% to about 45%, about 20% to about 45%, about 25% to about 45%, about 30% to about 45%, or about 35% to about 45%; about 2% to about 40%, about 10% to about 40%, about 15% to about 40%, about 20% to about 40%, about 25% to about 40%, or about 30% to about 40%; about 2% to about 35%, about 10% to about 35%, about 15% to about 35%, about 20% to about 35%, or about 25% to about 35%.

Role of IL-10 in KC function. As previously mentioned, although it is not necessary to understand the underlying mechanism by which IL-10 exerts its effects in order to practice the present disclosure, IL-10 activates the bone marrow immune system by activating the liver-resident KC, thereby significantly reducing systemic cholesterol levels in subjects with high cholesterol.

As detailed in the experimental section, the murine surrogate PEG-rmuel-10 was used to determine the regulatory effect of PEG-rHuIL-10 on total plasma cholesterol. FIGS. 2A-2D show that PEG-rMuIL-10 reduces two genes involved in cholesterol synthesis (Hmgcs1 and Hmgcs 2).

Administration of PEG-rMuIL-10 to LDLR-/-mice reduced total plasma cholesterol in a manner commensurate with increased scavenger receptor expression in KC, and demonstrated that KC is the predominant bone marrow cell type in response to increased clearance of PEG-rMuIL-10 with LDL. Because all phagocytic cells, most of which are myeloid, are removed, thereby significantly increasing plasma cholesterol, the phagocytic cell population plays an important role in regulating plasma cholesterol levels.

Treatment of NAFLD and NASH with IL-10 and other cytokines

Nonalcoholic steatohepatitis (NASH) is considered to be part of the range of nonalcoholic fatty liver disease (NAFLD), leading to inflammation and accumulation of adipose and fibrous tissue in the liver. Although the exact cause of NASH is unknown, risk factors include central obesity, type 2 diabetes, Insulin Resistance (IR), and dyslipidemia; combinations of the above are commonly described as metabolic syndrome. In addition, certain drugs have been associated with NASH, including tamoxifen, amiodarone, and steroids (e.g., prednisone and hydrocortisone). In the united states, nonalcoholic fatty liver disease is the most common cause of chronic liver disease, and the estimated incidence of NAFLD is 20-30%, and the estimated incidence of NASH is 3.5-5%. (see, e.g., Abrams, G.A., et al, Hepatology,2004.40(2): 475-83; Moreira, R.K., Arch Pathol Lab Med,2007.131(11): 1728-34).

NASH often presents no obvious symptoms, complicating its diagnosis. Liver function testing typically begins a diagnostic process in which individuals with NASH have about 90% elevated AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels. Other blood tests are commonly used to rule out other causes of liver disease, such as hepatitis. Imaging tests (e.g., ultrasound, CT scan, or MRI) can reveal fat accumulation in the liver, but often cannot distinguish NASH from other liver disease causes with similar appearance. Liver biopsy was required to confirm NASH.

The prognosis of individuals afflicted with NASH is difficult to predict, but features in liver biopsies may be helpful. The most serious complication of NASH is cirrhosis, which occurs when the liver becomes severely scarred. 8% to 26% of NASH individuals have been reported to develop cirrhosis, and NASH is predicted to become the primary indication for liver transplantation by 2020.

Currently, NASH treatment is primarily focused on the pharmaceutical and non-pharmaceutical management of those medical conditions associated with it, including hyperlipidemia, diabetes, and obesity. Although not curative, the pharmacological intervention of NASH itself involves treatment with vitamin E, pioglitazone, metformin, statins, omega-3 fatty acids and ursodeoxycholic acid (UDCA (ursodiol)). Other agents evaluated that are currently approved for different indications include losartan and telmisartan, exenatide, GLP-1 agonists, DPPIV inhibitors and carbamazepine. Combination therapy is expected to provide new opportunities for disease control.

Historically, kupffer cell activation has been correlated with the initiation and progression of liver disease (Kolios, g. et al, world j. gastroenterol,2006.12(46): 7413-20). In particular, Kupffer cells are believed to be involved in Alcoholic Liver Disease (ALD) (Eguchi, H. et al, Hepatology,1991.13(4):751-57), nonalcoholic fatty liver disease (NAFLD) (StienstraR. et al, Hepatology,2010.51(2):511-22) and nonalcoholic steatohepatitis (NASH) (Tomita, K. et al, Gut,2006.55(3): 415-24). In general, these diseases are thought to be associated with the inappropriate accumulation of liver cholesterol and triglycerides in both kupffer cells and hepatocytes. Thus, it is believed that loss of KC function may be of therapeutic relevance in treating metabolic disorders such as NASH. (Neyrinck et al, biochem. Biophys. Res. Comm.,385:351-56 (2009)).

The teachings of the present disclosure are directly contrary to this view. Although KC activation has been reported to be essential for optimizing Liver regeneration (Bilzer, M et al, (2006), Liver Int,26:1175-86), KC was not previously acknowledged to play an essential role in cholesterol and triglyceride regulation. Embodiments of the present disclosure are related to the following novel findings: many aspects of KC function require the absorption of large amounts of serum cholesterol and its subsequent removal and catabolism. The alterations to collagen deposition described herein are associated with the removal of triglycerides, which then results in the removal of pro-inflammatory stimuli, allowing the periportal injury and fibrosis to resolve.

As described in the experimental section, IL-10 (e.g., PEG-IL-10) activation of KC clearance is associated with reduced liver cholesterol and triglyceride accumulation, and certain embodiments of the present disclosure contemplate the use of PEG-IL-10 to induce removal of accumulated liver triglycerides and cholesterol. Inducing the removal of accumulated liver triglycerides and cholesterol in turn causes the reversal of early liver fibrosis and promotes the recovery of liver health by, for example, increasing the number of hepatocytes in the liver. These data support the use of IL-10 (e.g., PEG-IL-10) for the treatment of NAFLD and NASH. Thus, particular embodiments of the present disclosure contemplate the use of IL-10 (including human and non-human IL-10 related polypeptides, including homologs, variants (including muteins) and fragments thereof) in the treatment and/or prevention of NAFLD and NASH.

In addition, other cytokines may achieve loss of KC function and may have therapeutic relevance in treating liver-related disorders such as NAFLD and NASH. As used herein, the term "cytokine" is intended to have its ordinary meaning in the art. Cytokines are involved in cell signaling, i.e., cells of the immune system communicate with each other by releasing and responding to cytokines. Cytokines encompass a range of different proteins including interleukins, interferons, chemokines, lymphokines, and tumor necrosis factors. They are produced by a wide range of cells, including macrophages, B lymphocytes, T lymphocytes, mast cells, fibroblasts and endothelial cells.

Cytokines regulate the balance between humoral and cellular-based immune responses and regulate the maturation, growth, and responsiveness of specific cell populations. They also play an essential role in the host's response to, for example, infection, immune response, inflammation, and trauma. Although definitive classification is not readily possible, cytokines are sometimes classified as interleukins, lymphokines, monokines, interferons, colony stimulating factors, and chemokines.

In particular embodiments, the present disclosure contemplates the use of cytokines in the treatment and/or prevention of liver-related disorders, such as NAFLD and NASH. Dosage regimens suitable for cytokine agents in general and IL-10 agents in particular are described elsewhere herein.

Method for producing IL-10

The polypeptides of the disclosure may be produced by any suitable method, including non-recombinant methods (e.g., chemical synthesis) and recombinant methods.

A. Chemical synthesis

In the case of chemical synthesis of polypeptides, the synthesis may be carried out by liquid phase or solid phase. Solid Phase Peptide Synthesis (SPPS) allows the incorporation of unnatural amino acids and/or peptide/protein backbone modifications. Various forms of SPPS, such as 9-fluorenylmethyloxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc), can be used to synthesize the disclosed polypeptides. Details of chemical synthesis are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. chem.6: 3-10; and Camarero J.A. et al, (2005) Protein PeptLett.12: 723-8).

Solid phase peptide synthesis can be performed as described below. The α functional group (Ν α) and any reactive side chains are protected with acid-or base-labile groups. The protecting group is stable under the conditions used to attach the amide bond, but can be easily cleaved without damaging the peptide chain already formed. Suitable protecting groups for the alpha amino functional group include, but are not limited to, the following: boc, benzyloxycarbonyl (Z), O-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl, tert-pentyloxycarbonyl (Amoc), α -dimethyl-3, 5-dimethoxy-benzyloxycarbonyl, O-nitrothio, 2-cyano-tert-butoxy-carbonyl, Fmoc, 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl (Dde) and the like.

Suitable side chain protecting groups include, but are not limited to: acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl), benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, tert-butyl (tBu), tert-butyldimethylsilyl, 2-chlorobenzyl, 2-chlorobenzyloxycarbonyl, 2, 6-dichlorobenzyl, cyclohexyl, cyclopentyl, 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl (Dde), isopropyl, 4-methoxy-2, 3-6-trimethylbenzylsulfonyl (Mtr), 2,3,5,7, 8-pentamethylchroman-6-sulfonyl (Pmc), pivaloyl, tetrahydropyran-2-yl, tosyl (Tos), 2,4, 6-trimethoxybenzyl, trimethylsilyl and trityl (Trt).

In solid phase synthesis, the C-terminal amino acid is coupled to a suitable support material. Suitable support materials are those which are inert to the reagents and reaction conditions used for the stepwise condensation and cleavage reactions of the synthesis process and which are insoluble in the reaction medium to be used. Examples of commercially available support materials include styrene/divinylbenzene copolymers that have been modified with reactive groups and/or polyethylene glycol; chloromethylated styrene/divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers; and so on. When it is desired to prepare the peptide acids, polystyrene (1%) -divinylbenzene or derivatized with 4-benzyloxybenzyl-alcohol (Wang anchor) or 2-chlorotrityl chloride may be usedIn the case of peptide amides, polystyrene (1%) divinylbenzene or derivatized with 5- (4' -aminomethyl) -3',5' -dimethoxyphenoxy) pentanoic acid (PAL anchor) or p- (2, 4-dimethoxyphenyl-aminomethyl) phenoxy groups (Rink amide anchor) may be used

The linkage to the polymeric support is achieved by reacting the Fmoc-protected amino acid at the C-terminus with the support material at room or elevated temperature (e.g., between 40 ℃ and 60 ℃) and at a reaction time of, for example, 2 to 72 hours with the addition of ethanol, acetonitrile, N-Dimethylformamide (DMF), dichloromethane, tetrahydrofuran, N-methylpyrrolidone, or similar solvents containing activating reagents.

Coupling of an N α -protected amino acid (e.g., an Fmoc amino acid) to a PAL, Wang or Rink anchor may be carried out, for example, with the aid of a coupling reagent such as N, N '-Dicyclohexylcarbodiimide (DCC), N' -Diisopropylcarbodiimide (DIC) or other carbodiimides, 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU) or other urea salts, O-acyl-urea, benzotriazol-1-yl-tris-pyrrolidinyl-phosphonium hexafluorophosphate (PyBOP) or other phosphorus salts, N-hydroxysuccinimide, other N-hydroxyimides or oximes, in the presence or absence of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, for example by means of TBTU plus HOBt, with or without addition of a base such as for example Diisopropylethylamine (DIEA), triethylamine or N-methylmorpholine, for example diisopropylethylamine, using a reaction time of 2 to 72 hours (for example 3 hours, 1.5 to 3 fold excess of amino acid and coupling reagent, for example 2 fold excess, and at a temperature between about 10 ℃ and 50 ℃, for example 25 ℃, in a solvent such as dimethylformamide, N-methylpyrrolidone or dichloromethane, for example dimethylformamide).

It is also possible to use active esters (e.g. pentafluorophenyl, p-nitrophenyl, etc.), symmetrical anhydrides of N α -Fmoc-amino acids, acid chlorides or acid fluorides thereof in place of the coupling reagents under the conditions described above.

The na protected amino acid (e.g., Fmoc amino acid) can be coupled with the 2-chlorotrityl resin in dichloromethane with the addition of DIEA and with a reaction time of 10 to 120 minutes, e.g., 20 minutes, but is not limited to the use of the solvent and the base.

In peptide synthesis, protected amino acids are coupled sequentially according to conventional methods, typically in an automated peptide synthesizer. After cleavage of the na-Fmoc protecting group of the coupled amino acid on the solid phase by treatment with e.g. dimethylformamide containing piperidine (10% to 50%) for 5 to 20 minutes, e.g. 2 x 2 minutes with DMF containing 50% piperidine and 1 x 15 minutes with DMF containing 20% piperidine, the next protected amino acid is coupled with the previous amino acid in a3 to 10-fold excess, e.g. in a 10-fold excess, in an inert non-aqueous polar solvent such as dichloromethane, DMF or a mixture of both and at a temperature between about 10 ℃ and 50 ℃, e.g. at 25 ℃. The previously mentioned reagents for coupling the first N α -Fmoc amino acid to the PAL, Wang or Rink anchor are suitable for use as coupling reagents. It is also possible to use as substitutes active esters of protected amino acids or their chlorides or fluorides or symmetrical anhydrides.

At the end of the solid phase synthesis, the peptide is cleaved from the support material, while the side chain protecting groups are cleaved. Cleavage can be carried out with trifluoroacetic acid or other strong acid medium in 0.5 to 3 hours, e.g. 2 hours, with addition of 5% -20% V/V of a scavenger such as dimethyl sulfide, ethyl methyl sulfide, thioanisole, thiocresol, m-cresol, anisole dithioglycol, phenol or water, e.g. 15% V/V dimethyl sulfide/dithioglycol/m-cresol 1:1: 1. Peptides with fully protected side chains were obtained by cleaving the 2-chlorotrityl anchor with glacial acetic acid/trifluoroethanol/dichloromethane 2:2: 6. The protected peptide can be purified by chromatography on silica gel. If the peptide is linked to a solid phase by a Wang anchor and if a peptide with C-terminal alkyl amidation is intended, cleavage can be carried out by aminolysis with an alkylamine or fluoroalkylamine. The aminolysis is performed at a temperature between about-10 ℃ and 50 ℃ (e.g., about 25 ℃) and a reaction time between about 12 and 24 hours (e.g., about 18 hours). Alternatively, the peptide may be cleaved from the support by re-esterification, for example with methanol.

The acidic solution obtained may be mixed with a3 to 20 fold excess of cold diethyl ether or n-hexane, for example, a 10 fold excess of diethyl ether, in order to precipitate the peptide and thus separate the scavenger and cleaved protecting groups which remain in the diethyl ether. Further purification can be performed by reprecipitating the peptide several times from glacial acetic acid. The precipitate obtained can be dissolved in water or tert-butanol or a mixture of the two solvents, for example a tert-butanol/water 1:1 mixture, and freeze-dried.

The peptide obtained can be purified by various chromatographic methods, including ion exchange on a weakly basic resin in the form of acetate; in the case of non-derivatized polystyrene/divinylbenzene copolymers (e.g.,hydrophobic adsorption chromatography on XAD); performing adsorption chromatography on silica gel; ion exchange chromatography, e.g., on carboxymethyl cellulose; partition chromatography, e.g. inG-25; countercurrent distribution chromatography; or High Pressure Liquid Chromatography (HPLC), e.g., reverse phase HPLC on octyl or octadecyl silyl silica (ODS) phase.

B.Recombinant production

Methods describing the preparation of human and mouse IL-10 can be found, for example, in U.S. Pat. No. 5,231,012, which teaches methods for producing proteins having IL-10 activity, including recombinant and other synthetic techniques. IL-10 may be of viral origin, and cloning and expression of viral IL-10(BCRF1 protein) from EB virus is disclosed in Moore et al, (1990) Science 248: 1230. IL-10 can be obtained in numerous ways using standard techniques known in the art, such as those described herein. Recombinant human IL-10 is also commercially available, for example, from PeproTech, Inc.

Where recombinant techniques are used to produce the polypeptide, the polypeptide may be produced as an intracellular or secreted protein using any suitable construct and any suitable host cell, which may be prokaryotic or eukaryotic, such as a bacterial (e.g., e.coli) or yeast host cell, respectively. Other examples of eukaryotic cells that may be used as host cells include insect cells, mammalian cells, and/or plant cells. Where mammalian host cells are used, they may include human cells (e.g., HeLa, 293, H9, and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos 7, and CV 1); and hamster cells (e.g., Chinese Hamster Ovary (CHO) cells).

A variety of host-vector systems suitable for expressing the polypeptide can be used according to standard procedures known in the art. See, e.g., Sambrook et al, 1989Current Protocols in Molecular Biology Cold spring harbor Press, New York; and Ausubel et al, 1995Current Protocols in Molecular Biology, Wiley and Sons. Methods for introducing genetic material into a host cell include, for example, transformation, electroporation, conjugation, calcium phosphate methods, and the like. The transfer method may be selected so as to provide for stable expression of the introduced polypeptide-encoding nucleic acid. The polypeptide-encoding nucleic acid may be provided in the form of a heritable add-on element (e.g., a plasmid), or may be integrated by the genome. A variety of suitable vectors are commercially available for producing the polypeptide of interest.

The vector may provide for extrachromosomal maintenance in the host cell or may provide for integration into the host cell genome. Expression vectors provide transcriptional and translational control sequences, and may provide inducible or constitutive expression, wherein the coding regions are operably linked under the transcriptional control of a transcriptional initiation region and a transcriptional and translational termination region. In general, transcriptional and translational regulatory sequences can include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancer or activator sequences. The promoter may be constitutive or inducible, and may be a strong constitutive promoter (e.g., T7).

Expression constructs typically have appropriate restriction sites located near the promoter sequence to provide for insertion of the nucleic acid sequence encoding the protein of interest. Selectable markers operable to facilitate selection of cells containing the vector may be present in the expression host. In addition, the expression construct may include other elements. For example, an expression vector may have one or two replication systems, thus allowing it to be maintained in an organism, for example in mammalian or insect cells for expression and in prokaryotic hosts for cloning and amplification. In addition, the expression construct may contain a selectable marker gene to allow selection of transformed host cells. Alternative genes are well known in the art and will vary with the host cell used.

The isolation and purification of the protein may be achieved according to methods known in the art. For example, proteins can be isolated from lysates of cells genetically modified to constitutively and/or after induction, or from synthesis reaction mixtures by immunoaffinity purification, which generally involves contacting the sample with anti-protein antibodies, washing to remove non-specifically bound material, and eluting the specifically bound protein. The isolated protein may be further purified by dialysis and other methods normally used for protein purification. In one embodiment, the proteins may be separated using metal chelate chromatography. The protein may contain modifications to facilitate separation.

The polypeptide may be prepared in substantially pure or isolated form (e.g., free of other polypeptides). The polypeptide may be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). For example, a purified polypeptide can be provided such that the polypeptide is present in a composition that is substantially free of other expressed proteins, e.g., less than about 90%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1%.

IL-10 polypeptides can be produced using recombinant techniques known in the art for manipulating various IL-10 related nucleic acids to provide constructs capable of encoding IL-10 polypeptides. It will be appreciated that when a particular amino acid sequence is provided, in view of the background and experience of the skilled person in, for example, molecular biology, she will be able to identify a variety of different nucleic acid molecules encoding such an amino acid sequence.

Amide bond substitution

In some cases, IL-10 includes one or more linkages other than peptide bonds, e.g., at least two adjacent amino acids are joined via a linkage other than an amide bond. For example, one or more amide linkages within the backbone of IL-10 may be substituted in order to reduce or eliminate undesirable proteolytic or other degradation modes and/or increase serum stability and/or limit or increase conformational flexibility.

In another example, a linkage in the form of an isostere of an amide linkage may be used, such as-CH₂NH-、-CH₂S-、-CH₂CH₂-, -CH-CH- (cis and trans) -, -COCH₂-、-CH(OH)CH₂-or-CH₂SO-replaces one or more amide bonds (-CO-NH-) in IL-10. One or more amide linkages in IL-10 may also be replaced, for example, with reduced isosteric pseudopeptide bonds. See Couder et al, (1993) int.j.peptide Protein res.41: 181-184. Such permutations and how to implement them are known to those of ordinary skill in the art.

Amino acid substitutions

One or more amino acid substitutions may be made in the IL-10 polypeptide. The following are non-limiting examples:

a) the hydrophobic amino acid substituted by alkyl includes alanine, leucine, isoleucine, valine, norleucine, (S) -2-aminobutyric acid, (S) -cyclohexylalanine or aliphatic side chain C₁-C₁₀other simple α -amino acids substituted with carbon (including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions);

b) substitution of aromatic-substituted hydrophobic amino acids, including phenylalanine, tryptophan, tyrosine, thiotyrosine, biphenylalanine, 1-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, including the amino, alkylamino, dialkylamino, aza, halo (fluoro, chloro, bromo, or iodo) or alkoxy (C) groups of the aromatic amino acids listed above₁-C₄) Substituted forms, illustrative examples of which are: 2-aminophenylalanine, 3-aminophenylalanine or 4-aminophenylalanine, 2-chlorophenylalanine, 3-chlorophenylalanine or 4-chlorophenylalanine, 2-methylphenylalanine, 3-methylphenylalanine or 4-methylphenylalanine, 2-methoxyphenylalanine, 3-methoxyphenylalanine or 4-methoxyphenylalanine, 5-aminotrryptophan, 5-chlorotryptophan, 5-methyltryptophan or 5-methoxyphenylalanine, 2 '-aminoalanine, 3' -aminoalanine or 4 '-aminoalanine, 2' -chloropropanine, 3 '-chloropropanine or 4' -chloropropanine, 2-biphenylalanine, 3-biphenylalanine or 4-biphenylalanine, beta-amino-phenylalanine or 4-biphenylalanine, 2' -methylalanine, 3' -methylalanine or 4' -methylalanine, 2-biphenylalanine, 3-biphenylalanine or 4-biphenylalanine and 2-pyridylalanine or 3-pyridylalanine;

c) substitution of amino acids containing basic side chains, including arginine, lysine, histidine, ornithine, 2, 3-diaminopropionic acid, homoarginine, including alkyl, alkenyl or aryl-substituted (C) of the foregoing amino acids₁-C₁₀branched, linear or cyclic) derivatives, whether the substituent is on a heteroatom (such as the alpha nitrogen or one or more distal nitrogens) or on the alpha carbon, for example at the pro-R position, compounds serving as illustrative examples include Ν -isopropyl-lysine, 3- (4-tetrahydropyridyl) -glycine, 3- (4-tetrahydropyridyl) -alanine, Ν - γ, γ' -diethyl-homoarginine, also include compounds such as α -methyl-arginine, α -methyl-2, 3-diaminopropionic acid, α -methyl-histidine, α -methyl-ornithine, wherein the alkyl group occupies the pro-R position at the alpha carbon, also include amides formed from alkyl, aromatic, heteroaromatic, wherein the heteroaromatic group has one or more nitrogen, oxygen or sulfur atoms, alone or in combination, carboxylic acids or activated derivatives of many kinds, such as acid chlorides, active esters, active azo compounds and related derivatives, and any of lysine, ornithine or 2, 3-diaminopropionic acid;

d) substituted acidic amino acids including alkyl, aryl, aralkyl and heteroaryl sulfonamides of aspartic acid, glutamic acid, homoglutamic acid, tyrosine, 2, 4-diaminopropionic acid, ornithine or lysine, and tetrazolyl-substituted alkyl amino acids;

e) substituted side chain amide residues including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine; and

f) substituted hydroxyl-containing amino acids include serine, threonine, homoserine, 2, 3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine.

In some cases, IL-10 contains one or more naturally occurring non-gene encoded L-amino acids, synthetic L-amino acids, or D-enantiomers of amino acids. For example, IL-10 may comprise only D-amino acids. For example, an IL-10 polypeptide may comprise one or more of the following residues: hydroxyproline, beta-alanine, anthranilic acid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2, 3-diaminopropionic acid, alpha-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, naphthylalanine, pyridylalanine 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2, 4-diaminobutyric acid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine, -aminocaproic acid, omega-aminoheptanoic acid, omega-aminocaprylic acid, omega-aminododecanoic acid, omega-aminotetradecanoic acid, cyclohexylalanine, alpha, gamma-diaminobutyric acid, alpha, beta-diaminopropionic acid, -aminopentanoic acid and 2, 3-diaminobutyric acid.

Other modifications

Cysteine residues or cysteine analogs can be introduced into the IL-10 polypeptide to provide for linkage to another peptide by a disulfide bond, or to provide for cyclization of the IL-10 polypeptide. Methods of introducing cysteine or cysteine analogs are known in the art; see, for example, U.S. patent No. 8,067,532.

The IL-10 polypeptide may be cyclized. One or more cysteines or cysteine analogs can be introduced into the IL-10 polypeptide, wherein the introduced cysteines or cysteine analogs can form disulfide bonds with the second introduced cysteines or cysteine analogs. Other cyclization means include the introduction of oxime or lanthionine linkers; see, for example, U.S. patent No. 8,044,175. Any combination of amino acids (or non-amino acid moieties) that can form a cyclization bond can be used and/or introduced. Any combination of amino acids having functional groups that allow the introduction of a bridge (or amino acids and- (CH2) can be used_n-CO-or- (CH2)_n-C₆H₄-CO-) to produce a cyclised bond. Some examples are disulfide bonds, disulfide bond mimetics such as- (CH2)_n-carba bridges, thioacetals, thioether bridges (cystathionine or lanthionine) and bridges containing esters and ethers. In these examples, n can be any integer, but is typically less than 10.

Other modifications include, for example, N-alkyl (or aryl) substitutions (ψ [ CONR ]), or backbone crosslinks for the construction of lactams and other cyclic structures. Other derivatives include C-terminal hydroxymethyl derivatives, ortho-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminal modified derivatives, including substituted amides such as alkylamides and hydrazides.

In some cases, one or more L-amino acids are replaced with one or more D-amino acids in the IL-10 polypeptide.

In some cases, the IL-10 polypeptide is an inverse analog (see, e.g., Sela and Zisman (1997) FASEBJ.11: 449). Retro-inverso peptide analogs are linear polypeptide isomers in which the orientation of the amino acid sequence is reversed (retro) and in which the chirality (D-or L-) of one or more amino acids is reversed (retro), e.g., using D-amino acids rather than L-amino acids. (see, e.g., Jameson et al, (1994) Nature 368: 744; and Brady et al (1994) Nature 368: 692).

The IL-10 polypeptide may include a "protein transduction domain" (PTD), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic molecule that facilitates passage across a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the passage of the molecule across a membrane, for example from the extracellular space into the intracellular space, or from the cytosol into an organelle. In some embodiments, the PTD is covalently attached to the amino terminus of the IL-10 polypeptide, while in other embodiments, the PTD is covalently attached to the carboxy terminus of the IL-10 polypeptide. Exemplary protein transduction domains include, but are not limited to, the minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1TAT comprising YGRKKRRQRRR; SEQ ID NO://); a poly-arginine sequence comprising a number of arginine residues sufficient to direct entry into a cell (e.g., 3,4, 5,6, 7,8, 9, 10, or 10-50 arginines); the VP22 domain (Zender et al, (2002) Cancer Gene ther.9(6): 489-96); drosophila antennapedia protein transduction domain (Noguchi et al, (2003) Diabetes 52(7): 1732-1737); truncated human calcitonin peptide (Trehin et al (2004) pharm. research 21: 1248-1256); polylysine (Wender et al, (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLMKR (SEQ ID NO: /); transporter GWTLNSAGYLLGKI NLKALAALAKKIL (SEQ ID NO: /); KALAWEAKLAKALAKALAKHLAKALAKALK CEA (SEQ ID NO: /); and RQIKIWFQNRRMKWKK (SEQ ID NO: /). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: /), RKKRRQRRR (SEQ I D NO: /); an arginine homopolymer of 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (S EQ ID NO://); RKKRRQRR (SEQ ID NO://); YARAAARQARA (SEQ ID NO://); THRLPRRRRRR (SEQ ID NO://); and GGRRARRRRRR (SEQ ID NO://).

Carboxyl group COR of amino acid at C-terminal of IL-10 polypeptide₃Can be in free form (R)₃OH) or in the form of physiologically tolerable alkali or alkaline earth metal salts, such as, for example, sodium, potassium or calcium salts. The carboxyl group can also be replaced by a primary, secondary or tertiary alcohol,such as, for example, methanol, branched or unbranched C₁-C₆An alkyl alcohol, for example, ethanol or tert-butanol, is esterified. The carboxyl group can also be replaced by a primary or secondary amine, such as ammonia, branched or unbranched C₁-C₆Alkylamines or C₁-C₆Dialkylamines, such as methylamine or dimethylamine, are amidated.

Amino acid NR at the N-terminus of IL-10 polypeptides₁R₂The amino group of (A) may be in free form (R)₁Is H and R₂H) or in the form of a physiologically tolerable salt such as, for example, chloride or acetate. The amino group may also be acetylated with an acid to give R₁Is H and R₂Acetyl, trifluoroacetyl or adamantyl. The amino group may be present in a form protected by an amino protecting group such as those provided above, e.g., Fmoc, benzyloxycarbonyl (Z), Boc, and Alloc, commonly used in peptide chemistry. The amino group may be N-alkylated, wherein R₁And/or R₂＝C₁-C₆Alkyl or C₂-C₈Alkenyl or C₇-C₉An aralkyl group. The alkyl residue may be linear, branched or cyclic (e.g., ethyl, isopropyl and cyclohexyl, respectively).

Specific modifications aimed at enhancing and/or mimicking IL-10 function

It is often beneficial and sometimes necessary to improve one of the various physical properties of the therapeutic modalities disclosed herein (e.g., IL-10) and/or the manner in which they are administered. Improvements in physical properties include, for example, modulation of immunogenicity; methods of increasing aqueous solubility, bioavailability, serum half-life, and/or therapeutic half-life; and/or modulating biological activity. Certain modifications can also be used, for example, to generate antibodies (e.g., epitope tags) for detection assays and to provide ease of protein purification. Such improvements must generally be conferred without adversely affecting the biological activity of the therapeutic modality and/or increasing its immunogenicity.

PEGylation of IL-10 is one particular modification contemplated by the present disclosure, while other modifications include, but are not limited to, glycosylation (N-attachment and O-attachment); polysialylation; albumin fusion molecules comprising serum albumin (e.g., Human Serum Albumin (HSA), cynomolgus (cyno) serum albumin, or Bovine Serum Albumin (BSA)); albumin bound, for example, by a conjugated fatty acid chain (acylation); and Fc-fusion proteins.

Pegylation of polyethylene: clinical efficacy of protein therapeutics is often limited by short plasma half-life and susceptibility to protease degradation. Studies with various therapeutic proteins (e.g., filgrastim) have shown that such difficulties can be overcome by various modifications including conjugation or attachment of the polypeptide sequence to any of a variety of non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyalkylene oxide. This is often achieved by a linker moiety covalently bound to both the protein and the non-protein polymer (e.g., PEG). Such PEG conjugated biomolecules have been demonstrated to have clinically useful properties including better physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating half-life and reduced clearance, reduced immunogenicity and antigenicity, and reduced toxicity.

In addition to the beneficial effect of pegylation on pharmacokinetic parameters, pegylation itself may also enhance activity. For example, PEG-IL-10 has been shown to be more effective against certain cancers than non-pegylated IL-10 (see, e.g., EP 206636A 2). Certain embodiments of the present disclosure contemplate the use of a relatively small PEG (e.g., 5kDa) that can improve the pharmacokinetic profile of the IL-10 molecule without causing troublesome adverse effects; such PEG-IL-10 is particularly effective for long-term use.

PEG suitable for conjugation to a polypeptide sequence is generally soluble in water at room temperature and has the general formula R (O-CH)₂-CH₂)_nO-R, wherein R is hydrogen or a protecting group, such as an alkyl or alkanol group, and wherein n is an integer from 1 to 1000. When R is a protecting group, it typically has from 1 to 8 carbons. The PEG conjugated to the polypeptide sequence may be linear or branched. The present disclosure contemplates branch PEG derivatives, "star PEG" and multi-arm PEG. The molecular weight of PEG used in the present disclosure is not limited to any particular range, and examples are set forth elsewhere herein; for example, certain embodiments have a molecular weight between 5kDa and 20kDa, while other embodiments have a molecular weight between 4kDa and 10 kDa.

The present disclosure also contemplates compositions of conjugates in which the PEGs have different values of n, and thus, each different PEG is present in a particular ratio. For example, some compositions comprise a mixture of conjugates of n-1, 2,3, and 4. In some compositions, the percentage of n-1 conjugates is 18-25%, the percentage of n-2 conjugates is 50-66%, the percentage of n-3 conjugates is 12-16%, and the percentage of n-4 conjugates is at most 5%. Such compositions are produced by reaction conditions and purification methods known in the art. Exemplary reaction conditions are described throughout the specification. Cation exchange chromatography can be used to separate the conjugates, followed by identification of fractions containing conjugates with, for example, the desired number of attached PEGs, purified free of unmodified protein sequences, and free of conjugates with other numbers of attached PEGs.

Pegylation most commonly occurs at the N-terminal alpha amino group of the polypeptide, the amino group on the side chain of a lysine residue, and the imidazole group on the side chain of a histidine residue. Since most recombinant polypeptides have a single alpha and many amino groups and imidazole groups, depending on the linker chemistry, numerous positional isomers can be produced. General pegylation strategies known in the art may be applied herein. PEG can be conjugated to a polypeptide of the present disclosure through a terminal reactive group ("spacer") that mediates a bond between a free amino or carboxyl group of one or more polypeptide sequences and polyethylene glycol. PEG having a spacer that can be bound to a free amino group includes N-hydroxysuccinimide polyethylene glycol that can be prepared by activating polyethylene glycol succinate with N-hydroxysuccinimide. Another activated polyethylene glycol that can be bound to free amino groups is 2, 4-bis (O-methoxypolyethylene glycol) -6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride. Activated polyethylene glycols that bind to free carboxyl groups include polyoxyethylene diamines.

Conjugation of one or more polypeptide sequences of the present disclosure to PEG with a spacer can be performed by various conventional methods. For example, the conjugation reaction may be carried out at a temperature of 4 ℃ to room temperature for 30 minutes to 20 hours in a solution of pH 5 to 10 using a reagent to protein molar ratio of 4:1 to 30: 1. The reaction conditions may be selected so as to direct the reaction primarily to produce the desired degree of substitution. In general, low temperature, low pH (e.g., pH ≧ 5) and short reaction time tend to reduce the number of attached PEGs, while high temperature, neutral to high pH (e.g., pH ≧ 7) and longer reaction time tend to increase the number of attached PEGs. Various means known in the art can be used to terminate the reaction. In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing at, for example, -20 ℃. Pegylation of various molecules is discussed, for example, in U.S. Pat. nos. 5,252,714, 5,643,575, 5,919,455, 5,932,462, and 5,985,263. PEG-IL-10 is described, for example, in U.S. Pat. No. 7,052,686. The specific reaction conditions envisaged for use herein are set forth in the experimental section.

The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the PEG attribute (e.g., extended serum half-life) while conferring several other advantageous properties. For example, simple polypeptide chains (including, e.g., Ala, Glu, Gly, Pro, Ser, and Thr) capable of forming extended conformations similar to PEG can be recombinantly produced that have been fused to a peptide or protein drug of interest (e.g., Amunix's XTEN technology; Mountain View, CA). This avoids the need for an additional conjugation step in the manufacturing process. Furthermore, established molecular biology techniques enable control of the side chain composition of polypeptide chains, allowing optimization of immunogenicity and manufacturing properties.

Glycosylation: for the purposes of this disclosure, "glycosylation" is intended to broadly refer to the enzymatic process of attaching glycans to proteins, lipids, or other organic molecules. The term "sugar"glycosylation" when used in connection with the present disclosure is generally intended to mean the addition or deletion of one or more carbohydrate moieties (either by removal of potential glycosylation sites or by deletion of glycosylation by chemical and/or enzymatic means) and/or the addition of one or more glycosylation sites that may or may not be present in the native sequence. In addition, the phrase includes qualitative changes in the glycosylation of the native protein, including changes in the nature and proportions of the various carbohydrate moieties present.

Glycosylation can significantly affect the physical properties (e.g., solubility) of polypeptides, such as IL-10, and may also be very important in protein stability, secretion, and subcellular localization. Glycosylated polypeptides may also exhibit enhanced stability, or may improve one or more pharmacokinetic properties, such as half-life. In addition, the improved solubility may, for example, enable the production of formulations that are more suitable for pharmaceutical administration than formulations comprising non-glycosylated polypeptides.

The addition of glycosylation sites can be achieved by altering the amino acid sequence. Changes to the polypeptide can be made, for example, by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites). The structure of the N-linked and O-linked oligosaccharides and the sugar residues found in each type may be different. One carbohydrate type commonly found on both is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acids are usually the terminal residues of both N-linked and O-linked oligosaccharides and, by virtue of their negative charge, may confer acidic properties to the glycoprotein. One particular embodiment of the present disclosure includes the generation and use of N-glycosylation variants.

The polypeptide sequences of the present disclosure may optionally be altered by alterations at the nucleic acid level, in particular by mutating the nucleic acid encoding the polypeptide at preselected bases so as to generate codons that will translate into the desired amino acids.

Polysialylation: the present disclosure also contemplates polysialic acidsuse of conjugation of a polypeptide to a naturally occurring biodegradable α - (2 → 8) -linked polysialic acid ("PSA") for improving the stability and in vivo pharmacokinetic properties of the polypeptide.

Albumin fusion: other suitable components and molecules for conjugation include albumins, such as Human Serum Albumin (HSA), macaque serum albumin, and Bovine Serum Albumin (BSA).

In accordance with the present disclosure, albumin can be conjugated to drug molecules (e.g., polypeptides described herein) at the carboxy terminus, the amino terminus, both the carboxy terminus and the amino terminus, and internally (see, e.g., USP 5,876,969 and USP 7,056,701).

In the HSA-drug molecule conjugates contemplated by the present disclosure, various forms of albumin may be used, such as albumin secretion pre-sequences and variants thereof, fragments and variants thereof, and HSA variants. Such forms typically have one or more desired albumin activities. In other embodiments, the disclosure relates to fusion proteins comprising a polypeptide drug molecule fused directly or indirectly to albumin, albumin fragments, albumin variants, and the like, wherein the fusion protein has greater plasma stability than the unfused drug molecule and/or the fusion protein retains the therapeutic activity of the unfused drug molecule. In some embodiments, indirect fusion is achieved through a linker, such as a peptide linker or a modified form thereof.

As mentioned above, fusion of albumin to one or more polypeptides of the present disclosure can be achieved, for example, by genetic manipulation, so that a nucleic acid encoding HSA or a fragment thereof is joined to a nucleic acid encoding the one or more polypeptide sequences.

Alternative albumin binding strategies: several albumin-binding strategies have been developed as alternatives to direct fusion and may be used with the IL-10 agents described herein. For example, the present disclosure contemplates via conjugated fatty acid chains (acylation) and inclusion of an Albumin Binding Domain (ABD) polypeptide sequence and one or more polypeptides described hereinA fusion protein of the sequence of the peptide for albumin binding.

Conjugation to other molecules: other suitable components and molecules for conjugation include, for example, thyroglobulin; tetanus toxoid; diphtheria toxoid; polyamino acids such as poly (D-lysine: D-glutamic acid); rotavirus VP6 polypeptide; an influenza virus hemagglutinin; influenza virus nucleoprotein; keyhole Limpet Hemocyanin (KLH); and hepatitis b virus core protein and surface antigens; or any combination of the foregoing.

Thus, the present disclosure contemplates conjugation of one or more additional components or molecules at the N-terminus and/or C-terminus of a polypeptide sequence, such as another polypeptide (e.g., a polypeptide having an amino acid sequence heterologous to the subject polypeptide) or a carrier molecule. Thus, exemplary polypeptide sequences can be provided in the form of a conjugate with another component or molecule.

IL-10 polypeptides may also be conjugated to large slow metabolizing macromolecules such as proteins; polysaccharides such as sepharose, agarose, cellulose or cellulose beads; polymeric amino acids such as polyglutamic acid or polylysine; an amino acid copolymer; inactivated virus particles; inactivated bacterial toxins such as toxoid or leukotoxin molecules from diphtheria, tetanus, cholera; inactivated bacteria; and dendritic cells. Such conjugated forms can be used to generate antibodies against the polypeptides of the disclosure, if desired.

Other candidate components and molecules for conjugation include those suitable for isolation or purification. Specific non-limiting examples include binding molecules such as biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or molecules comprising a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.

Fc fusion molecules: in certain embodiments, the amino-terminus or carboxy-terminus of a polypeptide sequence of the present disclosure may be linked to an immunoglobulin Fc region (e.g., a c region)Human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, and thus biopharmaceutical products may require less frequent administration.

Fc binds to neonatal Fc receptors (FcRn) in endothelial cells lining the blood vessels, and after binding, the Fc fusion molecule is protected from degradation and is re-released into the circulation, thereby maintaining the molecule in circulation for a longer period of time. This Fc binding is believed to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc fusion techniques attach a single biopharmaceutical to the Fc region of an antibody in order to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to conventional Fc fusion conjugates.

Other modifications: the present invention contemplates the use of other IL-10 modifications, now known or to be developed, for improving one or more properties. One such method involves modifying the polypeptide sequence by hydroxyethyl starch, which utilizes hydroxyethyl starch derivatives linked to other molecules in order to modify the characteristics of the polypeptide sequence. Various aspects of hydroxyethyl starch are described, for example, in U.S. patent application Nos. 2007/0134197 and 2006/0258607.

The present disclosure also contemplates fusion molecules comprising a small ubiquitin-like modifier (SUMO) as a fusion tag (LifeSensors, inc.; Malvern, PA). The fusion of the polypeptides described herein to SUMO can deliver several beneficial effects, including enhanced expression, improved solubility, and/or development of auxiliary purification methods. SUMO proteases recognize the tertiary structure of SUMO and cleave the fusion protein at the C-terminus of SUMO, thus releasing the polypeptide described herein with the desired N-terminal amino acid.

The present disclosure also contemplates PASYlation^TM(XL-Protein Co., Ltd. (Fresin, Germany)). This technique expands the apparent molecular size of the target protein without: have a negative impact on the therapeutic biological activity of the protein; beyond the pore size of the glomeruli, thereby reducing renal clearance of the protein.

And (3) jointing:linkers and uses thereof have been described above. Any of the foregoing components and molecules used to modify the polypeptide sequences of the present disclosure may optionally be conjugated through a linker. Suitable linkers include "flexible linkers," which are generally of sufficient length to allow some movement between the modified polypeptide sequence and the attached components and molecules. Linker molecules are generally about 6-50 atoms long. The linker molecule can also be, for example, an arylacetyiidene group, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. Suitable linkers can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., Gly), 2,3,4, 5,6, 7,8, 9, 10-20, 20-30, 30-50, or more than 50 amino acids.

Exemplary flexible linkers include glycine polymers (G)_nGlycine-serine polymers (e.g., (GS)_n、GSGGS_n、GGGS_n、(G_mS_o)_n、(G_mS_oG_m)_n、(G_mS_oG_mS_oG_m)_n、(GSGGS_m)_n、(GSGS_mG)_nAnd (GGGS)_m)_nAnd combinations thereof, wherein m and o are each independently selected from integers of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and therefore can act as neutral tethers between components. Exemplary flexible joints include, but are not limited to: GGSG, GGSGG, GSGSG, GSGGG, GGGSG and GSSSG.

Therapeutic and prophylactic uses

The present disclosure contemplates the use of the IL-10 polypeptides (e.g., PEG-IL-10) described herein to treat and/or prevent diseases, disorders or conditions associated with or caused by, for example, hypercholesterolemia, abnormal lipid profiles, and/or symptoms thereof, as well as other disorders directly or indirectly associated with cholesterol homeostasis. Although specific uses are described in detail below, it should be understood that the disclosure is not so limited. Additionally, while specific categories of exemplary diseases, disorders, and conditions associated with or caused by hypercholesterolemia and abnormal lipid profiles are discussed below, it is to be understood that there is often overlap between one or more categories (e.g., certain cardiovascular diseases may have an inflammatory component).

Cardiovascular diseases. In particular embodiments, the present disclosure contemplates the use of IL-10 polypeptides (e.g., PEG-IL-10) described herein for the treatment and/or prevention of cardiovascular diseases, disorders, and conditions, as well as disorders associated therewith caused by hypercholesterolemia and abnormal lipid profiles.

As used herein, the terms "cardiovascular disease", "heart disease" and the like refer to any disease affecting the cardiovascular system, primarily heart disease, cerebral and renal vascular disease, and peripheral arterial disease. Cardiovascular disease is a range of diseases including coronary heart disease (e.g., ischemic heart disease or coronary artery disease), atherosclerosis, cardiomyopathy, hypertension, hypertensive heart disease, pulmonary heart disease, cardiac arrhythmia, endocarditis, cerebrovascular disease, and peripheral artery disease. Cardiovascular disease is the leading cause of death worldwide and although it usually affects the elderly, the antecedent cause of cardiovascular disease, particularly atherosclerosis, begins early in life.

Particular embodiments of the present disclosure relate to the use of IL-10 polypeptides described herein for the treatment and/or prevention of atherosclerosis, a chronic condition in which plaque is formed as a result of the accumulation of fatty substances such as cholesterol and triglycerides, thickening the arterial wall. As discussed further herein, atherosclerosis often involves a chronic inflammatory reaction of the arterial wall, which is largely caused by macrophage accumulation and promoted by LDL in cases where functional HDL does not adequately remove fat and cholesterol from macrophages. Chronic dilated atherosclerotic lesions may cause complete occlusion of the lumen, which may only be manifested when the stenosis of the lumen is so severe that insufficient blood supply to the downstream tissue results, leading to ischemia.

The present disclosure specifically contemplates embodiments in which cardiovascular disease includes hyperlipidemia (or hyperlipoproteinemia), i.e., a condition characterized by abnormally elevated levels of lipids and/or lipoproteins in the blood. Hyperlipidemia may be classified as familial (or primary) when caused by a particular genetic abnormality, as acquired (or secondary) when caused by another latent condition, or as idiopathic when the cause is unknown. Hyperlipidemia can also be classified based on which type of lipid and/or lipoprotein elevation. Non-limiting examples of hyperlipidemia include dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, hypercholesterolemia, and combined hyperlipidemia. Hyperlipoproteinemia includes, for example, type Ia hyperlipoproteinemia, type Ib hyperlipoproteinemia, type Ic hyperlipoproteinemia, type IIa hyperlipoproteinemia, type IIb hyperlipoproteinemia, type III hyperlipoproteinemia, type IV hyperlipoproteinemia and type V hyperlipoproteinemia.

In particular embodiments, the present disclosure contemplates the treatment and/or prevention of Familial Hypercholesterolemia (FH), a genetic disorder characterized by extremely high levels of LDL in the blood. FH is associated with early cardiovascular disease because accelerated deposition of cholesterol in the arterial wall leads to atherosclerosis. In some patient populations, total cholesterol levels are in the range of 350-550mg/dL, while in other patient populations they are in the range of 650-1000 mg/dL. Cholesterol levels may be significantly higher in obese patients with FH.

Efforts to treat cardiovascular disease by controlling lipid and/or lipoprotein levels in the blood have met with limited success. For example, while administration of statins reduces cardiovascular risk in some individuals, these therapeutic compounds do not reduce triglyceride levels. Members of the fibrate class of therapeutic agents may be administered in individuals at cardiovascular risk who exhibit undesirably high levels of triglycerides. However, despite lowering triglyceride and LDL levels, fibrates do not affect HDL levels. Furthermore, combination therapies involving statins and fibrates, while sometimes effective, often result in a significant increase in the risk of myopathy and rhabdomyolysis, and therefore can only be performed under very close medical supervision. In view of the limitations exemplified above, there is clearly a need for improved agents for the treatment of cardiovascular diseases, including those associated with high lipid and/or lipoprotein levels.

Thrombosis and thrombotic conditions. In other embodiments, the disclosure contemplates the use of IL-10 polypeptides (e.g., PEG-IL-10) described herein for the treatment and/or prevention of thrombotic and thrombotic diseases, disorders, and conditions, as well as disorders associated therewith that are caused by hypercholesterolemia and abnormal lipid profiles. Thrombosis, i.e., the formation of a thrombus within a blood vessel resulting in the obstruction of blood flow through the circulatory system, may be caused by abnormalities in one or more of the following (the wilcoxon triad): hypercoagulability or increased blood coagulation, endothelial cell damage or blood flow disturbances (stasis, turbulence).

Thrombosis is generally classified as venous thrombosis or arterial thrombosis, each of which may assume several subtypes. Venous thrombosis includes Deep Vein Thrombosis (DVT), portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Bugar's syndrome, Peyer's disease, and cerebral sinus thrombosis. Arterial thrombosis includes stroke and myocardial infarction.

Inflammatory disorders. When cholesterol and/or LDL is embedded in the vessel wall, an immune response may be triggered, which in turn may lead to chronic inflammation. In response to this inflammation, blood monocytes adhere to the endothelium, migrate into the subendothelial space, and differentiate towards macrophages. Macrophages, in turn, engulf cholesterol deposits and modified LDL by phagocytosis via scavenger receptors different from LDL receptors. However, the adaptation mechanism mediated by macrophages is insufficient to deal with uncontrolled cholesterol and/or LDL deposition seen in pathological conditions. Thus, the lipid-loaded macrophages are converted into "foam cells",usually accompanied by the release of inflammation inducing molecules. cholesterol/LDL deposition and the concomitant foam-cell mediated pro-inflammatory response in the vessel wall lead to the development of atherosclerotic lesions. Thus, lipid-laden macrophages are transformed into "foam cells", often accompanied by the release of inflammation inducing molecules. Both cholesterol/LDL deposition in the vessel wall and the concomitant foam-cell mediated pro-inflammatory response lead to the development of atherosclerotic lesions. Thus, one result of modulating the level of lipids or lipoproteins is the reduction or elimination of chronic inflammation.

The present disclosure includes embodiments in which an IL-10 agent (e.g., PEG-IL-10) described herein is used to treat and/or prevent vasculitis. Vasculitis is a diverse group of conditions that characterize the inflammation of blood vessel walls, including lymphatic and blood vessels, such as veins (phlebitis), arteries (arteritis), and capillaries, due to the migration and resultant injury of leukocytes . Inflammation may affect arteries and/or veins, regardless of size. It may be local or extensive, with areas of inflammation dispersed throughout a particular organ or tissue, or even affecting more than one organ system in the body. Vasculitis includes, but is not limited to, buerger's disease (thromboangiitis obliterans), cerebrovascular disease (vasculitis of the central nervous system), churg-schott's arteritis, cryoglobulinemia, primary cryoglobulinemia vasculitis, giant cell (temporal) arteritis, henoch-schoendo purpura, allergic vasculitis (allergic vasculitis), kawasaki disease, microaneuritis/polyangiitis, polyarteritis nodosa, polymyalgia rheumatica (PMR), rheumatoid vasculitis, takayasu's arteritis, thrombophlebitis, wegener's granulomatosis; and connective tissue disorders such as systemic lupus erythematosus, rheumatoid arthritis, recurrent polychondritis, Behcet's disease, or vasculitis secondary to other connective tissue disorders; and vasculitis secondary to viral infection.

Other embodiments relate to inflammatory heart diseases, which refer to conditions characterized by inflammation of the myocardium and/or surrounding tissues. Examples include, but are not limited to, endocarditis, inflammatory cardiac hypertrophy, and myocarditis.

Fibrotic disorders: the present disclosure also provides methods of treating or preventing fibrotic diseases, disorders and conditions. As used herein, the phrase "fibrotic diseases, disorders and conditions" and similar terms (e.g., "fibrotic disorders") and phrases should be broadly understood such that it includes any condition (e.g., fibrosis in one or more tissues) that may result in the formation of fibrotic tissue or scar tissue. For example, injuries (e.g., trauma) that may produce scar tissue include trauma to the skin, eyes, lungs, kidneys, liver, central nervous system, and cardiovascular system. The phrase also encompasses scar tissue formation resulting from stroke and tissue adhesion, for example, due to injury or surgery.

As used herein, the term "fibrosis" refers to the formation of fibrous tissue as a reparative or reactive process rather than as a normal component of an organ or tissue. Fibrosis is characterized by fibroblast accumulation and collagen deposition in any particular tissue over normal deposition.

Fibrotic disorders include, but are not limited to, fibrosis due to wound healing, systemic and local scleroderma, atherosclerosis, restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, cirrhosis, fibrosis due to chronic hepatitis b or c virus infection, kidney diseases (e.g., glomerulonephritis), heart diseases due to scar tissue, keloids, and hypertrophic scars, and ocular diseases such as macular degeneration and retinal and vitreoretinal diseases. Other fibrotic diseases include chemotherapy drug-induced fibrosis, radiation-induced fibrosis, and injuries and burns.

Fibrotic disorders are often liver-related and there is often a relationship between such disorders and the inappropriate accumulation of hepatic cholesterol and triglycerides in hepatocytes and kupffer cells. This accumulation appears to elicit a pro-inflammatory response, leading to liver fibrosis and cirrhosis. Liver disorders with a fibrotic component include nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). NAFLD occurs when there is steatosis (fat deposition in the liver) that is not due to excessive alcohol use. It is associated with insulin resistance and metabolic syndrome. NASH is the most extreme form of NAFLD and is considered to be the major cause of liver cirrhosis, an unknown cause.

Pharmaceutical composition

The IL-10 polypeptides of the present disclosure can be in the form of a composition suitable for administration to a subject. Generally, such compositions are "pharmaceutical compositions" comprising IL-10 and one or more pharmaceutically or physiologically acceptable diluents, carriers or excipients. In certain embodiments, the IL-10 polypeptide is present in a therapeutically acceptable amount. Pharmaceutical compositions may be used in the methods of the present disclosure; thus, for example, the pharmaceutical compositions can be administered to a subject ex vivo or in vivo to practice the therapeutic and prophylactic methods and uses described herein.

The pharmaceutical compositions of the present disclosure may be formulated so as to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein. Furthermore, the pharmaceutical compositions may be used in combination with other therapeutically active agents or compounds described herein in order to treat or prevent diseases, disorders, and conditions as contemplated by the present disclosure.

Pharmaceutical compositions typically comprise a therapeutically effective amount of an IL-10 polypeptide contemplated by the present disclosure and one or more pharmaceutically and physiologically acceptable formulating agents. Suitable pharmaceutically or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl paraben, ethyl paraben, or n-propyl paraben), emulsifiers, suspending agents, dispersants, solvents, fillers, bulking agents, detergents, buffers, vehicles, diluents, and/or adjuvants. For example, a suitable vehicle may be a physiological saline solution or citrate buffered saline, possibly supplemented with other substances commonly used in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. One skilled in the art will readily recognize a variety of buffers that may be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffering agents include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. As an example, the buffer component may be a water-soluble substance such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffers include, for example, Tris buffer, N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), 2- (N-morpholino) ethanesulfonic acid (MES), 2- (N-morpholino) ethanesulfonic acid sodium salt (MES), 3- (N-morpholino) propanesulfonic acid (MOPS), and N-Tris [ hydroxymethyl ] methyl-3-aminopropanesulfonic acid (TAPS).

After the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form, in a lyophilized form requiring reconstitution prior to use, in a liquid form requiring dilution prior to use, or in other acceptable forms. In some embodiments, the administration is provided in a single use container (e.g., a single use vial, ampoule, syringe, or auto-injector (similar to, for example)) While in other embodiments multiple use containers (e.g., multiple use vials) are provided. Any drug delivery device may be used to deliver IL-10, including implants (e.g., implantable pumps) and catheter systems, slow syringe pumps, and devices, all of which are well known to the skilled artisan. The polypeptides disclosed herein can also be released over a defined period of time using depot injections, typically administered subcutaneously or intramuscularly. Depot injections are typically solid or oil based and typically comprise at least one of the formulation components set forth herein. One of ordinary skill in the art is familiar with possible formulations and uses for depot injections.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. The suspensionThe suspensions may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which are mentioned herein. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable diluents, solvents and dispersion media which may be employed include water, ringer's solution, isotonic sodium chloride solution, Cremophor EL^TM(BASF, Parsippany, NJ) or Phosphate Buffered Saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Prolonged absorption of a particular injectable formulation can be brought about by the inclusion of an agent that delays absorption (e.g., aluminum monostearate or gelatin).

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, capsules, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups, solutions, microbeads or elixirs. In particular embodiments, the active ingredient of the agent that is co-administered with the IL-10 agent described herein is in a form suitable for oral use. Pharmaceutical compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more pharmaceutical agents, such as for example sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets, capsules and the like contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.

Tablets, capsules and the like suitable for oral administration may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated using techniques known in the art to form osmotic therapeutic tablets of the controlled release type. Other agents include biodegradable or biocompatible particles or polymeric substances, such as polyesters, polyanilic acids, hydrogels, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylene vinyl acetate copolymers, in order to control the delivery of the administered composition. For example, the oral agents may be embedded in microcapsules or colloidal drug delivery systems prepared by coacervation techniques or by interfacial polymerization, by using hydroxymethylcellulose or gelatin microcapsules or poly (methylmethacylate) microcapsules, respectively. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Methods for preparing the above formulations will be apparent to those skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin, or microcrystalline cellulose, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture thereof. Such excipients may be suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as naturally occurring phosphatides (e.g. lecithin), or condensation products of an alkylene oxide with fatty acids (e.g. polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g. for heptadecaethylene oxide cetyl alcohol), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g. polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g. polyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

The pharmaceutical compositions of the present disclosure may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin, or a mixture of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth; naturally occurring phospholipids, such as soy bean, lecithin and esters or partial esters derived from fatty acids; hexitol anhydrides, such as sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.

The formulations may also include carriers that protect the composition from rapid degradation or elimination from the body, such as controlled release formulations, including implants, liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate, alone or in combination with a wax, may be used.

The present disclosure contemplates administration of IL-10 polypeptides in the form of suppositories for rectal administration. Suppositories may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycols.

The IL-10 polypeptides contemplated by the present disclosure may be in the form of any other suitable pharmaceutical composition now known or later developed (e.g., sprays for nasal or inhalation use).

The concentration of the polypeptide or fragment thereof in the formulation can vary widely (e.g., less than about 0.1%, typically about 2% or at least about 2% up to 20% to 50% by weight or more) and will generally be selected based primarily on body fluid volume, viscosity, and subject-based factors, according to, for example, the particular mode of administration selected.

Route of administration

The present disclosure contemplates administering IL-10 (e.g., IL-10 polypeptides) and compositions thereof in any suitable manner. Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal), and intracerebroventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual, and inhalation. Depot injections, typically administered subcutaneously or intramuscularly, may also be utilized to release the IL-10 polypeptides disclosed herein over a defined period of time.

Many embodiments of the present disclosure contemplate parenteral administration. In some embodiments, the parenteral administration is intravenous administration, while in other embodiments, the parenteral administration is subcutaneous administration.

Combination therapy

The present disclosure contemplates the use of IL-10 (e.g., PEG-IL-10) in combination with one or more active therapeutic agents or other prophylactic or therapeutic modalities (e.g., radiation). In such combination therapies, the various active agents often have different mechanisms of action than IL-10. Such combination therapies may be particularly advantageous by allowing the dose of one or more agents to be reduced, thereby reducing or eliminating the adverse effects associated with the one or more agents; moreover, such combination therapies may have a synergistic therapeutic or prophylactic effect on a latent disease, disorder or condition.

In particular embodiments, the present disclosure provides methods of treating and/or preventing diseases, disorders, or conditions associated (directly or indirectly) with cholesterol homeostasis, including associated cardiovascular, thrombotic, and inflammatory disorders, using an IL-10 polypeptide (e.g., PEG-IL-10) described herein and at least one other therapeutic or diagnostic agent. It is to be understood that combination therapy is not limited to agents that treat and/or prevent the above-mentioned diseases, disorders, or conditions; for example, it is contemplated that agents used in combination with IL-10 polypeptides may have efficacy in treating or preventing other metabolic disorders, such as diabetes or obesity. Also contemplated herein are IL-10 polypeptides (e.g., PEG-IL-10) used in combination with improved dietary and/or exercise regimens.

As used herein, "combination" is meant to include therapies that can be administered separately, e.g., formulated separately for separate administration (e.g., as may be provided in a kit), as well as therapies that can be administered together in a single formulation (i.e., "co-formulation").

In certain embodiments, the IL-10 polypeptide is administered or applied sequentially, e.g., wherein one agent is administered followed by one or more other agents. In other embodiments, the IL-10 polypeptide is administered simultaneously, e.g., wherein two or more agents are administered simultaneously or approximately simultaneously; the two or more agents are present in two or more separate formulations or combined in a single formulation (i.e., a co-formulation). For the purposes of this disclosure, the two or more agents are considered to be administered in combination, whether they are administered sequentially or simultaneously.

In many cases the IL-10 polypeptides of the present disclosure can be used in combination with at least one active agent in any suitable manner. In one embodiment, treatment with the at least one active agent and at least one IL-10 polypeptide (i.e., homodimer) of the present disclosure is maintained over a period of time. In another embodiment, treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable) while treatment with an IL-10 polypeptide of the present disclosure is maintained at a constant dosing regimen. In another embodiment, treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable) while treatment with an IL-10 polypeptide of the present disclosure is reduced (e.g., lower dose, lower frequency of administration, or shorter treatment regimen). In yet another embodiment, treatment with the at least one active agent is reduced or discontinued (e.g., when the subject is stable), and treatment with an IL-10 polypeptide of the disclosure is increased (e.g., higher dose, more frequent dosing, or longer treatment regimen). In yet another embodiment, treatment with the at least one active agent is maintained, and treatment with an IL-10 polypeptide of the disclosure is discontinued or reduced (e.g., lower dose, lower frequency of administration, or shorter treatment regimen). In yet another embodiment, treatment with the at least one active agent and treatment with the IL-10 polypeptides of the disclosure is discontinued or reduced (e.g., lower dose, lower dosing frequency, or shorter treatment regimen).

Although the following sets forth a particular agent suitable for use in combination with the IL-10 polypeptides (e.g., PEG-IL-10) disclosed herein, it is to be understood that the disclosure is not so limited. Certain agents are set forth below in certain classes of exemplary diseases, disorders, and conditions; however, it should be understood that there is often overlap between one or more categories (e.g., certain agents may have both cardiovascular and anti-inflammatory effects).

Cholesterol homeostasis agents. Particular embodiments of the present disclosure relate to IL-10 polypeptidesA combination of agents associated with cholesterol homeostasis. Many of these agents target different pathways involved in cholesterol absorption, synthesis, transport, storage, catabolism and excretion, and are therefore particularly useful candidates for combination therapy.

Examples of therapeutic agents that can be used in combination therapy for the treatment of hypercholesterolemia (and thus are typically used, for example, in atherosclerosis) include statins (e.g., CRESTOR, LESCOL, LIPITOR, MEVACOR, pravacrol, and ZOCOR), which inhibit the enzymatic synthesis of cholesterol; bile acid resins (e.g., COLESTID, LO-CHOLEST, PREVALITE, QUESTRAN, and WELCHOL) which sequester cholesterol and prevent its absorption; ezetimibe (ZETIA), which blocks cholesterol absorption; fibric acid (e.g., TRICOR), which lowers triglycerides and may moderately increase HDL; niacin (e.g., NIACOR), which moderately lowers LDL cholesterol and triglycerides; and/or combinations of the foregoing (e.g., VYTORIN (ezetimibe and simvastatin)). Alternative cholesterol therapies that may be candidates for use in combination with the IL-10 polypeptides described herein include various supplements and herbs (e.g., garlic, sugarcane, and mukul). Several classes of the foregoing therapeutic agents are discussed further below.

Particular embodiments of the present disclosure include combinations of IL-10 agents and fibrates. Fibrates, a class of amphipathic carboxylic acids, can be used as antihyperlipidemic agents to lower levels of, for example, triglycerides and LDL, and to increase levels of HDL. Examples of suitable fibrates include, but are not limited to, bezafibrate, ciprofibrate, clofibrate, gemfibrozil and fenofibrate.

Other specific embodiments of the present disclosure include combinations of IL-10 agents with HMG-CoA reductase inhibitors (statins). HMG-CoA reductase inhibitors can lower LDL and/or cholesterol levels by inhibiting HMG-CoA reductase, an enzyme that plays an important role in cholesterol production in the liver. To compensate for the decreased cholesterol availability, the synthesis of hepatic LDL receptors is increased, resulting in increased clearance of LDL particles from the blood. Examples of suitable statins include, but are not limited to, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. Combinations of IL-10 polypeptides with statins are specifically contemplated herein.

Still other embodiments of the present disclosure comprise a combination of an IL-10 agent and niacin. Niacin can lower LDL levels by selectively inhibiting hepatic diacylglycerol acyltransferase 2, reducing triglyceride synthesis, and reducing VLDL secretion via receptors HM74 and HM74A or GPR 109A. A non-limiting use of niacin is as an anti-hyperlipidemic agent for inhibiting lipolysis in adipose tissue. By blocking lipolysis, niacin reduces free fatty acids in the blood and, as a result, reduces VLDL and cholesterol secretion by the liver. Niacin can also increase HDL levels in the blood by decreasing VLDL levels. Examples of suitable niacin include, but are not limited to, acipimox, niacin, niacinamide, and vitamin B3.

Other specific embodiments of the present disclosure include combinations of an IL-10 agent and a bile acid sequestrant. Bile acid sequestrants (also known as resins) bind certain components of bile in the gastrointestinal tract, disrupting the enterohepatic circulation of bile acids by sequestering them and preventing their resorption from the intestinal tract. Bile acid sequestrants are particularly effective at lowering LDL and cholesterol, and may also raise HDL levels. Examples of suitable bile acid sequestrants include, but are not limited to, colestyramine, colesevelam, and colestipol.

Other specific embodiments of the present disclosure include combinations of an IL-10 agent and a cholesterol absorption inhibitor. Cholesterol absorption inhibitors reduce the absorption of cholesterol from the intestine; this results in upregulation of LDL-receptors on the cell surface and increased uptake of LDL cholesterol into these cells, thus lowering LDL levels in the plasma. Examples of suitable cholesterol absorption inhibitors include, but are not limited to, ezetimibe, phytosterols, sterols, and stanols. Combinations of IL-10 polypeptides with ezetimibe are particularly contemplated herein. Ezetimibe selectively blocks cholesterol absorption and lowers plasma LDL levels by an average of 18%. When ezetimibe was co-administered with lower doses of statins, there was an additive decrease in LDL levels, which was equal to the decrease achieved with the maximum dose of statins alone. A reduced statin dose will cause fewer statin related adverse effects.

Still other particular embodiments of the present disclosure comprise a combination of an IL-10 agent and a fat absorption inhibitor. Fat absorption inhibitors reduce fat absorption from the intestine, thereby reducing caloric intake. In one aspect, the fat absorption inhibitor inhibits pancreatic lipase, an enzyme that breaks down triglycerides in the intestine. Examples of suitable fat absorption inhibitors include, but are not limited to orlistat.

In still other specific embodiments, the present disclosure contemplates the use of PEG-IL-10 agents described herein in combination with PCSK9 (proprotein convertase subtilisin/kexin type 9) modulators. PCSK9 plays an important regulatory role in cholesterol homeostasis. It is a serine protease expressed primarily in the liver, intestine and kidney. The encoded protein is synthesized as a soluble zymogen that undergoes autocatalytic molecular processing in the endoplasmic reticulum.

As part of the cholesterol homeostasis process, LDL cholesterol is removed from the blood when it binds to LDL receptors (LDLR) on the surface of hepatocytes and is absorbed by such cells. PCSK9 acts by binding to LDLR and inducing receptor degradation, thereby preventing LDLR recirculation to the cell surface to remove more LDL cholesterol, ultimately leading to its reduced metabolism. Preventing PCSK9 from binding LDLR allows the receptor to return to the cell surface and remove more cholesterol.

PCSK9 function inhibitors have been shown to cause a much stronger cholesterol reduction than traditional commercially available agents with an acceptable adverse effect profile. The present disclosure contemplates the use of PEG-IL-10 with any modulator that has direct or indirect inhibitory effect on PCSK 9. Several monoclonal antibodies that bind PCSK9 and interfere with its interaction with LDLR are being developed (e.g., Amgen (AMG145), Merck (1D05-IgG2), and Aventis/Regeneron (SAR236553/REGN 727)). In addition, peptides are being developed that mimic the EGFA domain of PCSK9 that can bind in LDLR, and gene silencing by administration of PCSK9 antisense oligonucleotides (ISIS Pharmaceuticals) has been demonstrated to increase LDLR expression and reduce circulating total cholesterol levels in mice. Other modulators of PCSK9 function contemplated for use in combination therapy with the PEG-IL-10 agents described herein are those that act by RNA interference (RNAi) (alaniam Pharmaceuticals) and those in the form of locked nucleic acids (LNA, also known as unreachable RNA) (Santaris Pharma).

The present disclosure encompasses pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

Immune and inflammatory conditions. The present disclosure provides methods of treating and/or preventing immune-related and/or inflammatory-related diseases, disorders, and conditions, and disorders related thereto, with an IL-10 polypeptide (e.g., PEG-IL-10) and at least one other agent having immune-related and/or inflammatory-related properties. For example, an IL-10 polypeptide can be administered with an agent that has efficacy in cardiovascular disorders with an inflammatory component.

Examples of therapeutic agents for use in combination therapy include, but are not limited to, non-sterol anti-inflammatory drugs (NSAIDs). NSAIDs, a large group of therapeutic compounds with analgesic, anti-inflammatory and anti-pyretic properties, reduce inflammation by blocking cyclooxygenase. Examples of such agents include: ibuprofen and other propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioprofen); acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fentiazac, isofenfenac, isoxepac, operanoic, sulindac, thionic acid, tolmetin, zidometacin, and zomepirac); fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid, and tolfenamic acid); biphenyl carboxylic acid derivatives (diflunisal and flufenisal); oxicams (isoxicam, piroxicam, sudoxicam and tenoxicam); salicylates (acetylsalicylic acid, sulfasalazine); and pyrazolones (azapropazone, beriptylon, feprazone, mofebuzone, oxyphenbutazone, phenylbutazone).

Other combinations include selective cyclooxygenase-2 (COX-2) inhibitors, selective cyclooxygenase-1 (COX1) inhibitors, and non-selective Cyclooxygenase (COX) inhibitors. Particular embodiments of the present disclosure contemplate the combination of an IL-10 polypeptide (e.g., PEG-IL-10) described herein with a suitable selective COX-2 inhibitor, such as celecoxib, etoricoxib, felicoxib, lumiracoxib, meloxicam, parecoxib, rofecoxib, and valdecoxib.

Other active agents for use in combination include steroids such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone, or hydrocortisone. Such combinations may be particularly advantageous because when treating a patient in combination with an IL-10 polypeptide of the invention, one or more side effects of steroids may be reduced or even eliminated by reducing the required steroid dose.

Other examples of agents that are used in combination to treat, for example, rheumatoid arthritis include cytokine inhibitory anti-inflammatory drugs (CSAIDs); antibodies or antagonists against other human cytokines or growth factors (e.g., TNF, LT, IL-1 β, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF or PDGF).

Specific combinations of active agents can interfere at different points in the autoimmune and subsequent inflammatory cascades, and include TNF antagonists such as chimeric, humanized or human TNF antibodies, REMICADE, anti-TNF antibody fragments (e.g., CDP870) and soluble p55 or p75TNF receptors, derivatives thereof, p75TNFRIgG (ENBREL.) or p55TNFR1gG (LENERCEPT), soluble IL-13 receptor (sIL-13), and also TNF α converting enzyme (TACE) inhibitors; similarly, an IL-1 inhibitor (e.g., an interleukin 1 converting enzyme inhibitor) may be effective. Other combinations include interleukin 11, anti-P7 s, and P-selectin glycoprotein ligand (PSGL). Other examples of agents useful in combination with the IL-10 polypeptides described herein include interferon- β 1A (AVONEX); interferon- β 1B (BETASERON); glatiramer acetate (copaxone); high pressure oxygen; intravenous immunoglobulin; cladribine; and antibodies or antagonists against other human cytokines or growth factors (e.g., antibodies against CD40 ligand and CD 80).

The present disclosure encompasses pharmaceutically acceptable salts, acids, or derivatives of any of the above.

Antidiabetic and antiobesity agentsthe combination therapies contemplated by the present disclosure utilize a number of anti-diabetic agents (and classes thereof) including 1) insulin, insulin mimetics, and agents that stimulate insulin secretion, including sulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide, glimepiride, glipizide), and meglitinides (e.g., repaglinide (PRANDIN) and nateglinide (STARLIX)), 2) biguanides (e.g., metformin (GLUCOPHAGE)) and other agents that act by promoting glucose utilization, reducing hepatic glucose production, and/or reducing intestinal glucose export, 3) alpha-glucosidase inhibitors (e.g., acarbose and miglitol) and other agents that can slow the digestion of carbohydrates and thus reduce absorption from the gut and reduce hyperglycemia, 4) agonists (e.g., insulin-stimulating analogs, such as insulin-stimulating analogs (VIPEPTA-1), insulin-stimulating agents (SALT-D), insulin-Stimulating Agents (SALT), SALT-S-5), and SALT-S), thereby increasing the effects of insulin-Stimulating Agents (SALT), insulin, SALT-E, SALT-E, SALT-E, and SALT-E-S-E (SALT), and other agents, SALT) and other agents, SALT inhibitors (SALT), such as inhibitors, SALT-E (SAE-E (SAE), and other agents, SAE-E (SAE) and other agents, such as inhibitors (SAE-E (SAEin yet other embodiments, the IL-10 agents described herein are used in combination with one or more suitable nuclear receptor binding agents (e.g., Retinoic Acid Receptor (RAR) binding agents, Retinol X Receptor (RXR) binding agents, Liver X Receptor (LXR) binding agents, and vitamin D binding agents).

In addition, the present disclosure contemplates combination therapies utilizing agents and methods that contribute to weight loss, such as agents that stimulate metabolism or reduce appetite, and improved dietary and/or exercise regimens that contribute to weight loss.

The present disclosure encompasses pharmaceutically acceptable salts, acids, or derivatives of any of the above.

Administration of drugs

An amount of an IL-10 polypeptide of the disclosure can be administered to a subject depending, for example, on the administration goal (e.g., the desired degree of regression); the age, weight, sex, and health and physical condition of the subject; the route of administration; and the nature of the disease, disorder, condition, or symptom thereof. The dosing regimen may also take into account the presence, nature and extent of any adverse effects associated with the administered agent. Effective dosages and dosing regimens can also be readily determined, for example, based on safety and dose escalation assays, in vivo studies (e.g., animal models), and other methods known to the skilled artisan.

In general, the dosing parameters dictate that the amount administered is less than the amount that is likely to produce irreversible toxicity to the subject (i.e., the maximum tolerated dose, "MTD"), and not less than the amount required to produce a measurable effect on the subject. Such amounts are determined by, for example, pharmacokinetic and pharmacodynamic parameters associated with ADME, taking into account route of administration and other factors.

An Effective Dose (ED) is the dose or amount of an agent that produces a therapeutic response or desired effect in a portion of the subject to whom it is administered. The "median effective dose" or ED50 of an agent is the dose or amount of the agent that produces a therapeutic response or desired effect in 50% of the population to which it is administered. While ED50 is often used as a reasonably expected measure of the effectiveness of an agent, it is not necessarily the dose that a clinician might consider appropriate by considering all relevant factors. Thus, in some cases, the effective amount is above the calculated ED50, in other cases, the effective amount is below the calculated ED50, and in other cases, the effective amount is the same as the calculated ED 50.

In addition, an effective dose of an IL-10 polypeptide of the disclosure may be an amount that produces a desired result relative to a healthy subject when administered to the subject in one or more doses. For example, an effective dose may be a dose that improves a diagnostic parameter, measure, marker, etc., of a particular disorder by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% for a subject experiencing the disorder, where 100% is defined as the diagnostic parameter, measure, marker, etc., exhibited by normal subjects.

When the IL-10 polypeptide is PEG-IL-10, the amount of PEG-IL-10 necessary to treat the diseases, disorders, or conditions described herein is based on the IL-10 activity of the conjugated protein, which, as noted above, can be determined by IL-10 activity assays known in the art. For example, in the case of tumors, suitable IL-10 activity includes, for example, infiltration into CD8+ T cells in the tumor site, expression of inflammatory cytokines (such as IFN-. gamma., IL-4, IL-6, IL-10, and RANK-L) from these infiltrating cells, and increased levels of TNF-. alpha.or IFN-. gamma.in the biological sample.

As with the drug, intravenous IL-10 administration is associated with a two-compartment kinetic model (see Rachmawai, H., et al, (2004) pharm. Res.21(11): 2072-78). The plasma drug concentration decays in a multi-exponential manner. Immediately after intravenous administration, the drug rapidly distributes throughout the initial space (defined minimally as plasma volume) and then slowly equilibrates to distribute to the extravascular space (e.g., certain tissues). The pharmacokinetics of subcutaneous recombinant hIL-10 have also been studied (Radwanski, E. et al, (1998) pharm. Res.15(12): 1895-1901). When evaluating appropriate parameters associated with IL-10 administration, volume of distribution and other pharmacokinetic considerations are appropriate. Furthermore, IL-10 pharmacokinetics and dosing principle leverage can prove invaluable for the achievements achieved by the effort to target IL-10 agents to specific cell types (see, e.g., rachmawai, H. (May 2007) Drug met.dist.35(5): 814-21).

The present disclosure contemplates administration of any dosage and dosing regimen that produces the desired therapeutic result. By way of example and not limitation, when the subject is a human, non-pegylated hIL-10 can be administered at a dose of greater than 0.5 μ g/kg/day, greater than 1.0 μ g/kg/day, greater than 2.5 μ g/kg/day, greater than 5 μ g/kg/day, greater than 7.5 μ g/kg, greater than 10.0 μ g/kg, greater than 12.5 μ g/kg, greater than 15 μ g/kg/day, greater than 17.5 μ g/kg/day, greater than 20 μ g/kg/day, greater than 22.5 μ g/kg/day, greater than 25 μ g/kg/day, greater than 30 μ g/kg/day, or greater than 35 μ g/kg/day. Also, by way of example and not limitation, when the subject is a human, the dose may be in excess of 0.5 μ g/kg/day, in excess of 0.75 μ g/kg/day, in excess of 1.0 μ g/kg/day, in excess of 1.25 μ g/kg/day, in excess of 1.5 μ g/kg/day, in excess of 1.75 μ g/kg/day, in excess of 2.0 μ g/kg/day, in excess of 2.25 μ g/kg/day, in excess of 2.5 μ g/kg/day, in excess of 2.75 μ g/kg/day, in excess of 3.0 μ g/kg/day, in excess of 3.25 μ g/kg/day, in excess of 3.5 μ g/kg/day, in excess of 3.75 μ g/kg/day, in excess of 4.0 μ g/kg/day, in excess of 4.25 μ g/kg/day, in excess of 4.5 μ g/kg/day, in excess of, More than 4.75 u g/kg/day or more than 5.0 u g/kg/day dose to administer containing relatively small PEG (e.g. 5kDa PEG-hIL-10, two PEG-hIL-10) polyethylene glycol hIL-10.

A therapeutically effective amount of PEG-IL-10 may be in the range of about 0.01 to about 100 μ g protein/kg body weight/day, about 0.1 to 20 μ g protein/kg body weight/day, about 0.5 to 10 μ g protein/kg body weight/day, or about 1 to 4 μ g protein/kg body weight/day. In some embodiments, PEG-IL-10 (e.g., about 1 to 16 μ g protein/kg body weight/day of PEG-IL-10) is administered by continuous infusion to deliver about 50 to 800 μ g protein/kg body weight/day. The infusion rate may be varied based on, for example, assessment of adverse effects and blood counts.

For administration of oral medicaments, the compositions may be provided in the form of tablets, capsules or the like containing from 1.0 to 1000 milligrams of the active ingredient, in particular 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0 and 1000.0 milligrams of the active ingredient.

In certain embodiments, the disclosed doses of an IL-10 polypeptide (e.g., PEG-IL-10) are included in a "unit dosage form". The phrase "unit dosage form" refers to physically discrete units, each unit containing a predetermined amount of an IL-10 polypeptide of the disclosure sufficient, alone or in combination with one or more other pharmaceutical agents, to produce the desired effect. It will be appreciated that the parameters of the unit dosage form will depend on the particular agent and the effect to be achieved.

Reagent kit

The present disclosure also contemplates kits comprising IL-10 polypeptides (e.g., PEG-IL-10) and pharmaceutical compositions thereof. The kits are generally in the form of physical structures containing various components, as described below, and can be used, for example, to practice the methods described above (e.g., administering an IL-10 polypeptide to a subject in need of restoring cholesterol homeostasis).

Kits may include one or more IL-10 polypeptides disclosed herein (provided, for example, in a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to a subject. The IL-10 polypeptide can be in a form that is easy to use or in a form that requires, e.g., reconstitution or dilution prior to administration. When the IL-10 polypeptide is in a form that requires reconstitution by a user, the kit may further comprise a buffer, a pharmaceutically acceptable excipient, or the like, packaged with or separately from the IL-10 polypeptide. When combination therapy is envisaged, the kit may contain several agents separately, or they may already be combined in a kit. Each component of the kit may be packaged in a separate container, and all of the different containers may be in a single package. The kits of the present disclosure may be designed to appropriately maintain the conditions necessary for the components contained therein (e.g., refrigeration or freezing).

The kit may contain a label or package insert including information identifying the components therein and instructions for their use (e.g., administration parameters, clinical pharmacology of the active ingredient (including mechanism of action, pharmacokinetics, and pharmacodynamics), adverse effects, contraindications, and the like). The label or insert may include manufacturer information such as lot number and expiration date. The label or package insert may be, for example, integrated into the physical structure containing the components, contained separately within the physical structure, or affixed to a component of the kit (e.g., an ampoule, tube, or vial).

The label or insert may additionally comprise, or be incorporated into, a computer-readable medium, such as a magnetic disk (e.g., hard disk, card, memory disk), optical disk (such as CD-or DVD-ROM/RAM), DVD, MP3, magnetic or electronic storage media (such as RAM and ROM) or hybrids of these (such as magnetic/optical storage media), FLASH media, or memory-type cards. In some embodiments, the actual instructions are not present in the kit, but provide a means for obtaining the instructions, e.g., over a network, from a remote source, e.g., over the internet.

Experiment of

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below have been performed and that all experiments can be performed. It should be understood that the exemplary descriptions written in this time are not necessarily made, but rather the descriptions may be made to generate the data and the like described herein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for.

Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (. degree. C.), and pressure is at or near atmospheric. Standard abbreviations are used, including the following: bp is base pair; kb is kilobase; pl to picoliter; s or sec is seconds; min is minutes; h or hr-hr; aa ═ amino acids; kb is kilobase; nt-nucleotide; pg is picogram; ng equals nanogram; μ g to μ g; mg ═ mg; g is gram; kg is kg; dL or dL-deciliter; μ L or μ L ═ microliter; mL or mL ═ mL; l or L ═ liter; μ Μ ═ micromolar; mM ═ millimole; m is mole; kDa ═ kilodaltons; i.m. intramuscularly; i.p. ═ intraperitoneally; SC or SQ, subcutaneous; QD is once daily; BID twice daily; once per week QW; QM is once a month; wt ═ wild type; HPLC ═ high performance liquid chromatography; BW is body weight; u is a unit; ns is not statistically significant; PBS ═ phosphate buffered saline; PCR ═ polymerase chain reaction; NHS ═ N-hydroxysuccinimide; cl ═ clodronate; HSA ═ human serum albumin; MSA ═ mouse serum albumin; IHC ═ immunohistochemistry; DMEM-dartbuck modified igger medium; GC-genomic copy; EDTA ═ ethylenediaminetetraacetic acid; PCSK9 ═ proprotein convertase subtilisin/kexin type 9; CYP7a1 ═ cytochrome P4507 a1, cholesterol 7 α -hydroxylase or cholesterol 7- α -monooxygenase; ABCG1 ═ ATP-binding cassette subfamily G member 1; MSR1 ═ macrophage scavenger receptor 1.

Materials and methods

The following general materials and methods may be used in the indicated circumstances, or may be used in the following examples:

standard methods in Molecular Biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Ausubel et al (2001) Current Protocols in Molecular Biology, Vol.1-4, John Wiley and Sons, Inc. New York, N.Y., which describes Cloning and DNA mutagenesis in bacterial cells (Vol.1), Cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol.3) and bioinformatics (Vol.4)).

The scientific literature describes methods for Protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins (see, e.g., Coligan et al (2000) Current Protocols in Protein Science, Vol.1-2, John Wileyand Sons, Inc., NY).

The generation, purification and fragmentation of polyclonal and monoclonal Antibodies is described (e.g., Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); standard techniques for characterizing ligand/receptor interactions can be used (see, e.g., Coligan et al (2001) Current protocols in Immunology, Vol.4, John Wiley, Inc., N.Y.); methods for Flow Cytometry, including Fluorescence Activated Cell Sorting (FACS), can be utilized (see, e.g., Shapiro (2003) Practical Flow Cytometry, john wiley and Sons, Hoboken, NJ); and fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and Probes), polypeptides, and antibodies for use as, for example, diagnostic reagents (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Software packages and databases for determining, for example, antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments can be utilized (see, e.g., GCG Wisconsin Package (Accelrys, Inc., san Diego, Calif.); and Decyphher^TM(TimeLogic Corp.，Crystal Bay，NV))。

Corrected mRNA units can be determined by calculating the relative quantification of gene expression changes in a sample based on gene expression in a reference sample.

Mouse: LDLR-/-mice were obtained from The Jackson Lab (Bar Harbor, ME). LDLR-/-mice have elevated serum cholesterol levels of 200-400mg/dL, and on average, cholesterol levels of-1,500 mg/dL when fed a high fat diet. Normal serum cholesterol in mice is 80-100 mg/dl.

Serum IL-10 concentration: for the experiments described herein, electrochemiluminescence assays were performed on MDS platforms for mIL-10The method determines the concentration of the test substance in the serum sample. LCB-33AEY (ARMO) anti-MIL-10 was used as capture antibody, and biotinylated anti-MIL-10 (R) was used&D) As a detection antibody. The upper and lower limits of 195,000pg-9pg were used to establish a PEG-rMuIL-10 standard curve. Plates were incubated for 2 hours at room temperature and after washing were detected by SulfoTag program on MSD2400 platform.

Alternatively, serum IL-10 concentration levels and exposure levels can be determined by other standard methods used in the art. For example, serum exposure level determination can be performed by collecting whole blood (50 μ l/mouse) from a mouse tail cutout into a flat capillary, separating serum and blood cells by centrifugation, and determining IL-10 exposure levels by standard ELISA kits and techniques.

Serum and tissue cholesterol quantification：

Method No. 1: sera were analyzed at a dilution of 1: 300. Mouse liver was crushed with a pestle (VWR) in 200. mu.L water. Twenty-five microliter aliquots were used to extract cholesterol/cholesteryl esters using the cholesterol/cholesteryl assay kit (BioVision) according to the manufacturer's instructions and measured with Spectra Max 340PC (Molecular Devices). Genomic DNA was extracted from an equivalent amount of sample using the DNeasy kit (QIAGEN) and measured with navueplus (GE Healthcare) to correct cholesterol/cholesteryl measurements.

Method No. 2: serum LDL-C or HDL-C quantification was performed by LDL-cholesterol assay using a two-reagent homogenization system on the Beckman Coulter AU system. The assay consists of two distinct phases. In phase 1, the unique detergent dissolves cholesterol from the non-LDL lipoprotein or non-HDL particles, depending on the desired assay output. The cholesterol is consumed by cholesterol esterase, cholesterol oxidase, peroxide and 4-aminoantipyrine (LDL-C) or DSBmT (HDL-C) to produce a colorless end product. In stage 2, the second detergent in the R2 reagent releases cholesterol from the remaining lipoproteins. The cholesterol is obtained byWith cholesterol esterase, cholesterol oxidase and a chromophore system to produce a blue complex, two-color measurements can be made at 540/660 nm. The resulting increase in absorbance is proportional to the concentration of LDL-C or HDL-C in the sample.

qPCR analysismice livers were crushed using pestle (VWR) in buffer RLT (QIAGEN) containing 10 μ Ι β -mercaptoethanol (Sigma-Aldrich), after which RNA was extracted using RNeasy kit (QIAGEN) according to manufacturer's instructions, using purified RNA as template for RT-PCR using RT2First Strand kit (QIAGEN), one microliter aliquots of the resulting cDNA samples were used for qpcr on ABI PRISM 7700 sequence detection system or ABI ViiA 7 real-time PCR machine (life technologies), CT values were corrected for the average CT values of Gapdh and Gusb.

Tissue triglyceride quantification: mouse liver was crushed with a pestle (VWR) in 200. mu.L water. Twenty-five microliter aliquots were used to extract triglycerides using the triglyceride assay kit (BioVision) according to the manufacturer's instructions and measured with Spectra Max 340PC (Molecular Devices). Protein concentrations were determined using the Pierce BCA protein assay (Thermo Scientific) according to the manufacturer's instructions and measured with Spectra Max 340 PC.

Immunohistochemistry：

Picric acid sirius red dyeing: the slides were heated in an oven at 60 ℃ for 45 minutes, dewaxed using xylene and a series of ethanol, and rehydrated in water; the solution was then kept in freshly prepared sirius kukojic acid red for 60 minutes according to the manufacturer's instructions, followed by two washes in acidified water. Nuclei were stained with weibert's hematoxylin for 8-10 minutes, dehydrated in three changes of 100% ethanol, cleaned and fixed.

Hematoxylin and eosin staining: two-time replacement of xyleneSlides were dewaxed (10 minutes each), dehydrated by absolute 95%, 70% ethanol (5 minutes each), and rehydrated in water. The slides were then placed in hematoxylin stain for 4 minutes, rinsed in water, differentiated in 1% acidic ethanol for 30 seconds, and stained in 0.01% eosin Y for 2 seconds, rinsed in 95% ethanol, dehydrated with absolute ethanol, and cleaned in xylene for 15 minutes, followed by coverslipping.

anti-F4/80, anti-Msr 1, anti-PCNA: liver tissue was fixed with 10% neutral buffered formaldehyde and embedded in paraffin. Tissue samples were cut into 5 μm thick sections, the sections were dewaxed in xylene, and hydrated in a gradient series of ethanol solutions (100%, 95%, 80%, 70%, 50% (3 changes, 5 minutes each)). Tissues on slides were subjected to heat-induced epitope repair (10mmol/L sodium citrate buffer, 98 ℃,20 min) followed by 3% H₂O₂Treatment to quench endogenous peroxidase. Sections were incubated in blocking solution (5% neutral goat serum) for 1 hour at room temperature. The selected primary antibody was applied to a slide and incubated overnight in a humidified chamber at 4 ℃. The secondary biotinylated antibody was then administered at a 1:250 dilution (Vector Lab, Burlingame, CA, USA) followed by incubation with streptavidin peroxidase. After each step sections were washed 3 times with PBS. Sections were stained with DAB substrate and counterstained with Mayer's hematoxylin for 2 minutes. Slides were dehydrated, cleaned and fixed in three changes of 100% ethanol.

Image quantification: for analysis of PEG-rMuIL-10 treated livers compared to vehicle treated livers, 5-10 mice per group were randomly selected and stained with sirius red (Polyscience Inc.), TUNEL (Roche), anti-PCNA (Abcam), hematoxylin (American MasterTech), anti-Msr 1(Abcam), anti-F4/80 (Abcam), or anti-CD 68 (Serotech). For each liver, 8-10 independent images were collected using a 20-fold objective lens. Meta Morph Imaging software (Molecular Devices) was then used to determine the color threshold by applying the color threshold to the representative field of view and adjusting the pixel divisionCloth to analyze the mean area of the signal corresponding to a positive signal.

In vitro absorption assayseparation of monocytes from Ficoll centrifuged peripheral blood mononuclear cells by Miltenyi magnetic bead Positive selection cells were stimulated in complete RPMI with or without PEG-rHuIL-10 for 24 h analysis of 1X 10⁶uptake of 20. mu.L DiLDL, OxLDL or AcLDL (Alfa Aesar) by individual cells within 4 hours macrophage was differentiated from positively selected peripheral blood mononuclear cells in cRPMI using 50ng/mL M-CSF (BioLegend) for 7 days cells were plated at 0.3-0.4 × 10⁶Individual cells/well were reseeded in 24-well plates and exposed to PEG-rHuIL-10 for 24 hours cells were washed once and exposed to 20. mu.L DiLDL, OxLDL or AcLDL (Alfa Aesar), absorbance was measured after 4 hours human primary hepatocytes (Triangle Research Labs) and Kupffer cells (Invitrogen) were thawed and thawed at 0.4 × 10⁶individual cells/well were seeded in 24-well plates and incubated overnight in hepatocyte culture medium (phenol red-free RPMI, penicillin/streptavidin, cell maintenance supplement B (Invitrogen)) and complete RPMI, respectively, cells were washed and exposed to PEG-rHuIL-10 for 24 hours after which the cells were washed once and exposed to 20 μ L of DiLDL, OxLDL or acll (Alfa Aesar), absorbance was measured after 4 hours all cells were washed once in 1 × PBS and lysed with 110 μ L of cell lysis buffer (Sigma-Aldrich), 45 μ L of cell lysate was transferred to clear-bottom black-wall plates (Greiner Bio-One) and fluorescence was read at 575 nm.

In vivo studies: mouse studies were conducted in Aragen Biosciences (Morgan Hill, CA) according to standard procedures and established guidelines approved by their research animal management and use committee (IACUC).

Hypercholesterolemia with high blood pressureModel: the in vivo fraction of the study was performed in Aragen Biosciences. Wild type and LDLR-/-C57BL/6 mice (7-8 weeks old) were maintained on normal diet or fed western style diet for 2 or 7 weeks prior to dosing. Pegylated recombinant mouse IL-10(PEG-rMuIL-10) or vehicle (10mM H) was administered subcutaneously dailyEPES, 100mM naci ph6.5, 0.05% mouse serum albumin) for 2 to 3 weeks; mice were maintained on their respective diets throughout the dosing period. For the clodronate depletion study, mice were given clodronate liposomes (5mg/mL clodronate) or vehicle liposomes (first dose: 0.2mL, subsequent dose: 0.1mL) intravenously every 3 days, starting the day before PEG-rMuIL-10 administration.

Pegylation of polyethylene：

The mono-/di-PEG-IL-10 mixtures described in the patent literature (e.g. US 2011/0250163) are used in preclinical and clinical analyses and in the examples set forth below. Two exemplary synthetic schemes are as follows:

exemplary protocol No. 1. IL-10 (e.g., rodent or primate) was dialyzed against 50mM sodium phosphate, 100mM sodium chloride (pH range 5-7.4). 5kDa PEG-propionaldehyde was reacted with IL-10 at a concentration of l-12mg/mL in a molar ratio of 1:1 to 1:7 in the presence of 0.75-30mM sodium cyanoborohydride. Alternatively, the reaction may be activated with picoline borane in a similar manner. The reaction was incubated at 5-30 ℃ for 3-24 hours. The pH of the pegylation reaction was adjusted to 6.3 and 7.5mg/mL hIL-10 was reacted with PEG to yield a IL-10 to PEG linker ratio of 1: 3.5. The final concentration of cyanoborohydride was about 25mM, and the reaction was allowed to proceed at 15 ℃ for 12-15 hours. The mono-PEG IL-10 and di-PEG IL-10 are the most predominant products of the reaction, each at a concentration of about 50% at the end. The reaction may be quenched using an amino acid (such as glycine or lysine) or alternatively a Tris buffer. Various purification methods, such as gel filtration, anion and cation exchange chromatography, and size exclusion HPLC (SE-HPLC) can be used to isolate the desired pegylated IL-10 molecule.

Exemplary protocol No. 2. IL-10 was dialyzed against 10mM sodium phosphate pH 7.0, 100mM NaCl. Dialyzed IL-10 was diluted 3.2-fold using dialysis buffer to achieve a concentration of about 0.5 to 12 mg/mL. SC-PEG-12kDa (Delmar Scientific Laboratories, Ma) was added before the linker was added ywood, Ill.) and one volume of 100mM sodium tetraborate pH 9.1 were added to 9 volumes of diluted IL-10 to raise the pH of the IL-10 solution to 8.6. The SC-PEG-12K linker was dissolved in dialysis buffer and an appropriate volume of linker solution (1.8 to 3.6 moles linker per mole IL-10) was added to the diluted IL-10 solution to initiate the pegylation reaction. The reaction was carried out at 5 ℃ to control the rate, and the reaction solution was gently stirred. When the mono-PEG-IL-10 yield was close to 40%, the reaction was stopped by adding 1M glycine solution to a final concentration of 30mM, as determined by size exclusion HPLC (SE-HPLC). The pH of the reaction solution was slowly adjusted to 7.0 using HCl solution and the reaction was 0.2 micron filtered and stored at-80 ℃.

PEG-IL-10 was formulated at 0.75-1.0mg/mL in 10mM HEPES pH 6.5, 100mM NaCl containing 0.05% MSA. Control (placebo) the same formulated vehicle without PEG-IL-10.

Example 1

PEG-rMuIL-10 reduces plasma cholesterol levels

The modulating effect of PEG-rMuIL-10 on plasma cholesterol levels in wild type mice and LDLR-/-mice fed normal diet and high fat diet was evaluated.

Referring to fig. 1A through 1C, wild type mice maintained on normal diet exhibited no reduction in total plasma cholesterol, LDL or HDL particles following daily subcutaneous administration of 1.0, 0.2 and 0.02mg/kg PEG-rMuIL-10 or vehicle control.

The effect of PEG-rMuIL-10 in LDLR-/-mice was then determined. When total plasma cholesterol levels reached approximately 200mg/dL in LDLR-/-mice maintained on a normal diet and wild type mice maintained on a high fat diet, 0.2mg/kg PEG-rMuIL-10, 0.02mg/kg PEG-rMuIL-10, or vehicle control was administered subcutaneously. Mice that received 0.2mg/kg PEG-rMuIL-10 showed 22% (FIG. 1D; LDLR-/-mice) and 25% (FIG. 1G; wild type mice) cholesterol reduction. Mice that received 0.2mg/kg PEG-rMuIL-10 showed a 30% (FIG. 1E; LDLR-/-mice) and 15% (FIG. 1H; wild type mice) reduction in LDL-C. Mice that received 0.2mg/kg PEG-rMuIL-10 showed a 36% (FIG. 1F; LDLR-/-mice) and 45% (FIG. 1I; wild type mice) reduction in HDL-C. When the initial levels of cholesterol and LDL-C were higher, LDLR-/-mice maintained on a high fat diet receiving 0.02mg/kg PEG-rMuIL-10 subcutaneously daily for two weeks showed a nearly 50% reduction in total plasma cholesterol levels, a 60% reduction in LDL-C levels, and a 35% reduction in HDL-C (see FIGS. 1J-1L, respectively). FIGS. 1A-1L also indicate the effect of 0.02mg/kg PEG-rMuIL-10 on each of the above parameters. Finally, longer dosing of LDLR-/-mice maintained on a high-fat diet resulted in a reduction of total plasma cholesterol by up to 70% (data not shown). In summary, the results indicate that the degree of plasma cholesterol reduction achieved with PEG-rMuIL-10 is dependent on total cholesterol levels.

To confirm the findings set forth above, total plasma cholesterol was evaluated in human patients (n ═ 4) treated with PEG-rHuIL-10 for cancer (mixed cancer of non-small cell lung cancer (NSCLC), Renal Cell Carcinoma (RCC), and colorectal cancer). The patient is administered 2.5. mu.g/kg or 5. mu.g/kg PEG-rHuIL-10 subcutaneously daily. As indicated in FIG. 1M, the greatest reduction in cholesterol was achieved with 2.5 μ g/kg PEG-rHuIL-10 administration in patients with borderline high (about 200mg/dL) total cholesterol; total plasma cholesterol was reduced by 40% in this patient population, consistent with the mouse data discussed previously. Patients with low (about 100mg/dL) total cholesterol were not affected by PEG-rHuIL-10, while patients with an initial plasma cholesterol concentration of about 140mg/dL achieved a reduction in cholesterol to about 100 mg/dL. Levels of 140mg/dL or lower are consistent with a time-of-non-life risk of a cardiovascular event. The use of 5 μ g/kg PEG-rHuIL-10 resulted in a decrease in cholesterol in a manner consistent with that observed when 2.5 μ g/kg PEG-rHuIL-10 was administered, and the amount of decrease also depends on the baseline cholesterol level of the patient (see FIG. 1N).

These data indicate that the mechanism by which PEG-IL-10 regulates serum cholesterol in mice is similar to that in humans.

Example 2

PEG-rMuIL-10 alters cholesterol synthesis and hepatic expression of scavenger receptor genes

The effect of PEG-rMuIL-10 on the expression of genes associated with liver function and cholesterol regulation was evaluated using Qiagen lipoprotein signaling and cholesterol metabolism, fibrosis, and innate and adaptive immune response tests. As shown in fig. 2A-2L, only two major groups of genes were altered in response to PEG-rMuIL-10 exposure.

The first group of regulatory genes is involved in endogenous cholesterol synthesis. 0.2mg/kg PEG-rMuIL-10, 0.02mg/kg PEG-rMuIL-10, or vehicle control was administered subcutaneously daily to wild-type mice maintained on a normal food diet (FIG. 2A), LDLR-/-mice maintained on a normal food diet (FIG. 2B), wild-type mice maintained on a high fat food diet (FIG. 2C), and LDLR-/-mice maintained on a high fat food diet (FIG. 2D). Two genes involved in endogenous cholesterol synthesis, Hmgcs1 and Hmgcs2, decreased moderately but consistently in the liver (FIGS. 2A-2D). These data are consistent with the report that IL-10 can reduce endogenous cholesterol synthesis by inhibiting the mevalonate pathway.

The second set of regulatory genes includes scavenger receptors. As shown in fig. 2E-2L, all further comparisons focused on differences between the vehicle control group and the group of mice administered 0.2mg/kg PEG-rMuIL-10, as the group of mice administered 0.02mg/kg PEG-rMuIL-10 exerted limited control over both plasma cholesterol control and gene regulation. 0.2mg/kg PEG-rMuIL-10 or vehicle control was administered subcutaneously daily to wild type mice maintained on a normal food diet (FIG. 2E), LDLR-/-mice maintained on a normal food diet (FIG. 2F), wild type mice maintained on a high fat food diet (FIG. 2G) and LDLR-/-mice maintained on a high fat food diet (FIG. 2H). As shown in fig. 2E to 2H, the a-type scavenger receptors Msr1 and Marco were induced 2 to 7 fold. To develop the scavenger receptor assay, differences in expression of other receptors known to regulate serum cholesterol were evaluated. LDL receptor expression was unchanged (data not shown) and there was no correlation in LDLR-/-and expression of CD36 and PCSK9 was also unchanged (see fig. 2E-2H). Because the scavenger receptors are predominantly expressed on macrophage-type cells, differences in the expression of the two cell surface proteins F4/80 and CD14, which are most frequently expressed on macrophages resident in liver tissue, were also evaluated. These genes were moderately induced under different gene backgrounds and dietary conditions (see fig. 2E to 2H). These data confirm the role of scavenger receptors in liver function and cholesterol regulation.

If PEG-rMuIL-10 induces an increase in lipoprotein uptake, this may be accompanied by an increase in efflux-related genes. Thus, the levels of efflux mediators ABCG1 and ABCA1 were evaluated. ABCG1 is considered to function primarily in the liver, while ABCA1 is considered to function primarily in the gastrointestinal tract. Both factors act as cholesterol efflux pumps for displacing cholesterol from the cell interior onto the low lipid HDL particles. 0.2mg/kg PEG-rMuIL-10 or vehicle control was administered subcutaneously daily to wild type mice maintained on a normal food diet (FIG. 2I), LDLR-/-mice maintained on a normal food diet (FIG. 2J), wild type mice maintained on a high fat food diet (FIG. 2K) and LDLR-/-mice maintained on a high fat food diet (FIG. 2L). As depicted in fig. 2I-2L, only ABCG1 showed a trend of moderately but consistently increasing expression.

Example 3

PEG-rMuIL-10 alters the expression of scavenger receptors and the presence of Kupffer cells

To assess whether PEG-rmuel-10 increases scavenger receptor and KC numbers, treated and untreated liver tissues were analyzed for differences in MSR1 expression by IHC.

0.2mg/kg PEG-rMuIL-10 or vehicle control was administered subcutaneously daily to wild type mice maintained on a normal food diet (FIG. 3A), LDLR-/-mice maintained on a normal food diet (FIG. 3B), wild type mice maintained on a high fat food diet (FIG. 3C) and LDLR-/-mice maintained on a high fat food diet (FIG. 3D). As shown in fig. 3A and 3B, administration of PEG-rMuIL-10 caused a slight increase in detectable MSR1 in wild type mice and LDLR-/-mice maintained on a normal diet. However, MSR1 levels in control tissues appeared to decrease with increasing dietary fat intake. Levels of detectable MSR1 were higher in wild type mice and LDLR-/-mice maintained on normal diet relative to mice of the same type maintained on high fat diet. The results were confirmed in the case of quantifying the amount of Msr1 using IHC (fig. 3A to 3D). In addition, treatment with PEG-rMuIL-10 appeared to correct Msr1 levels in liver tissue of LDLR-/-mice (data not shown).

Referring to fig. 2A-2L, scavenger receptors are most often expressed on macrophage lineage cells and identify increased expression of the message F4/80 and CD 14. Thus, 0.2mg/kg PEG-rMuIL-10 or vehicle control was administered subcutaneously daily to wild-type mice maintained on a normal food diet (fig. 3E), LDLR-/-mice maintained on a normal food diet (fig. 3F), wild-type mice maintained on a high fat food diet (fig. 3G) and LDLR-/-mice maintained on a high fat food diet (fig. 3H) in order to assess the protein expression level of F4/80 by IHC (data not shown). There was little to no difference in the presence of F4/80 positive cells in wild type mice and LDLR-/-mice maintained on normal diet (fig. 3E and 3F). However, there was a detectable increase in F4/80 positive cells in both wild type and LDLR-/-maintained high fat diet (fig. 3G and fig. 3H), indicating that PEG-rMuIL-10 increased the number of kupffer cells in the animal liver in the presence of increased dietary fat absorption.

In addition, expression analysis of IL-10R α indicated an approximately 2-fold difference in IL-10R α information between wild type mice maintained on normal versus high fat diet and LDLR-/-mice, suggesting that the difference in cholesterol regulation may be due in part to relative IL-10 receptor levels (data not shown).

Example 4

In vivo phagocyte depletion eliminates PEG-rMuIL-10 cholesterol lowering effects

To determine whether cells associated with the myeloid lineage are responsible for the control of plasma cholesterol by PEG-rMuIL-10, an evaluation was made.

Phagocytic cells were removed by administering animal clodronate (Cl) liposomes in the presence or absence of PEG-rMuIL-10 using standard techniques. After ensuring that all phagocytes in the liver were completely depleted and evaluating hepatocyte health by IHC (data not shown), the modulation of plasma cholesterol levels by PEG-rmail-10 was evaluated in wild type mice maintained on a normal food diet (fig. 4B), LDLR-/-mice maintained on a normal food diet (fig. 4C), wild type mice maintained on a high fat food diet (fig. 4D) and LDLR-/-mice maintained on a high fat food diet (fig. 4A).

As demonstrated in fig. 4A-4G, phagocyte depletion abrogated the regulation of plasma cholesterol by PEG-rMuIL-10. Mice given PEG-rMuIL-10(1 mg/kg) showed a 50% reduction in cholesterol, while there was a 25% increase in mice given PEG-rMuIL-10 and clodronate liposomes (FIG. 4A). Unexpectedly, mice receiving clodronate alone showed a 45% increase in total plasma cholesterol. Thereafter, phagocytes were depleted in wild-type mice and LDLR-/-mice maintained on a normal food diet (see fig. 4B and 4C, respectively) and wild-type mice maintained on a high-fat food diet (fig. 4D). Surprisingly, the removal of phagocytes consistently increased plasma cholesterol, indicating that phagocytes are generally involved in normal regulation of plasma cholesterol.

IHC evaluation indicated that PEG-rMuIL-10 did not act on the chlorophosphonate-mediated depletion of hepatic kupffer cells on day 7, but PEG-rMuIL-10 had begun to promote regrouping of hepatic kupffer cells on day 14 (data not shown). As shown in fig. 4E, the hepatic replenishment of hepatic kupffer cells by PEG-rMuIL-10 is consistent with a decrease in plasma cholesterol.

Administration of PEG-rMuIL-10 appears to enhance the entry of monocytes from the bone marrow into the systemic circulation. As shown in fig. 4F and 4G, peripheral monocytes initially decreased in mice after exposure to clodronate. PEG-rMuIL-10 then acts to promote recovery of this cell population. These data indicate that administration of PEG-rmuel-10 can slowly restore homeostasis, overcoming the effects of clodronate and enhancing regrouping of kupffer cells in the liver, thereby restoring their cholesterol-lowering capacity.

Example 5

PEG-rHuIL-10 induces scavenger receptor expression in human myeloid lineage

To clearly determine which cells in the liver responded to PEG-rHuIL-10, in vitro experiments were performed in which human monocytes (fig. 5A), human macrophages (fig. 5B), human kupffer cells (fig. 5C) and human hepatocytes (fig. 5D) were exposed to PEG-rHuIL-10. Referring to fig. 5A to 5D, only kupffer cells and peripheral monocytes showed up-regulation of scavenger receptor expression in response to PEG-rHuIL-10.

These data indicate that PEG-rHuIL-10 can upregulate scavenger receptor expression on myeloid cells but not hepatocytes. Notably, scavenger receptor regulation appears to be similar in mice and humans, suggesting conservation of the biological properties of IL-10 in terms of its effects on the bone marrow compartment.

Example 6

PEG-rHuIL-10 increases monocyte uptake of modified LDL and LDL uptake by Kupffer cells

To determine whether increased scavenger receptor expression correlates with increased lipoprotein uptake, peripheral human monocytes (fig. 6A, 6E, and 6F), human macrophages (fig. 6B), human kupffer cells (fig. 6C), and human hepatocytes (fig. 6D) were isolated and exposed to PEG-rHuIL-10.

Referring to fig. 6A-6D, the Y-axis is a quantification of the amount of labeled cholesterol that has been absorbed relative to a standard curve dilution of the same labeled cholesterol. The parameters on the X-axis can be briefly summarized as follows: cells were exposed to PEG-IL-10 for about 24 hours, then washed and exposed to the following conditions for 3 to 5 hours: i) no reagent, no labeled cholesterol, used to serve as a background control group; ii) no reagent, labeled cholesterol for use as a background control group; or iii) PEG-IL-10 with labeled cholesterol. Cells were washed, lysed, and total internalized labeled cholesterol was then quantified as indicated above. The data in fig. 6E and 6F are generated in a similar manner.

As shown in FIG. 6A, PEG-rHuIL-10 increases the uptake of acetylated LDL (Ac-LDL;, p <0.05) and oxidized LDL (Ox-LDL; (. x, p <0.001) by newly isolated peripheral blood mononuclear cells, but not unmodified LDL (LDL). FIGS. 6E and 6F show the effect of PEG-rHuIL-10 and PEG-rHuIL-10 plus mannose on acetylated LDL (FIG. 6E) and oxidized LDL (FIG. 6F) in human mononuclear cells.

In contrast, no difference was detected in macrophage differentiation of macrophage colony-stimulating factor (M-CSF) in response to PEG-rHuIL-10 uptake of any form of LDL (FIG. 6B). Referring to fig. 6C, PEG-rHuIL-10 increased kupffer cellular uptake of LDL (, p <0.05) but not Ac-LDL or Ox-LDL. These data support the involvement of kupffer cells in the regulation of plasma LDL cholesterol. The presence of changes in hepatocyte uptake of unmodified, oxidized and acetylated LDL was also assessed. No change in hepatocyte uptake of unmodified, oxidized and acetylated LDL was observed in response to PEG-rHuIL-10 (fig. 6D). Although myeloid cells are generally phagocytic, these data suggest that the nature of cholesterol absorption in response to PEG-rHuIL-10 differs between monocytes, macrophages, kupffer cells and hepatocytes.

When neutralizing antibodies against MARCO and CD36 were evaluated as potential inhibitors of the effect of PEG-rHuIL-10 on cholesterol absorption, no difference in absorption was observed (data not shown). In contrast, polydextrose inhibition of PEG-rHuIL-10 increased the uptake of Ac-LDL, but not Ox-LDL, by monocytes (data not shown). These data indicate that control of lipoprotein uptake by PEG-rHuIL-10 occurs through known and possibly unknown scavenger receptor modulation. Furthermore, consistent with in vivo efflux gene regulation, exposure of kupffer cells to IL-10 moderately increased cholesterol efflux onto HDL particles (data not shown).

Collectively, these data indicate that, while PEG-rHuIL-10 can enhance the uptake of Ac-LDL and Ox-LDL by peripheral monocytes, the Kupffer cell population in the liver is the PEG-rHuIL-10 mediated peripheral cholesterol-controlled target cell population. In addition, these data suggest that the myeloid lineage is under peripheral cholesterol control, suggesting that monocytes, macrophages and kupffer cells represent an undeveloped cellular reserve in the liver that actively removes plasma cholesterol. Finally, these data indicate that modulation of scavenger receptors on kupffer cells with PEG-rHuIL-10 and general modulation of myeloid lineage with other therapeutic compounds may represent an alternative means of lowering plasma cholesterol.

Example 7

PEG-rMuIL-10 and ezetimibe have additive effects on cholesterol reduction

To evaluate whether the PEG-rmulil-10 mediated effects observed in the previous examples were consistent with cell types not currently targeted with standard of care therapeutics, PEG-rmulil-10 was combined with an orally administered statin or an oral therapeutic agent ezetimibe that was able to block cholesterol absorption.

As shown in figure 7A, PEG-rMuIL-10(1.0mg/kg, subcutaneously, once daily) and ezetimibe (Ez) (10mg/kg, once daily by oral gavage) alone reduced plasma cholesterol by 58% and 55%, respectively, and by 86% when combined, when administered to LDLR-/-mice maintained on a high fat diet. The plasma cholesterol concentrations observed with PEG-rmuel-10 and ezetimibe administration were similar to normal plasma cholesterol levels observed in LDLR-/-mice.

Data relating to plasma LDL-C and plasma HDL-C was generated using an experimental method comparable to the method used to generate the data in FIG. 7A. As shown in FIGS. 7A and 7B, PEG-rMuIL-10 in combination with ezetimibe resulted in a decrease in LDL-C and HDL-C, respectively. While PEG-rmulil-10 in combination with statin (simvastatin) also reduced LDL-C and HDL-C (data not shown), oral administration of statin was toxic in mice and therefore limited the maximum level of plasma reduction that could be achieved with statin. The combination of PEG-rmuel-10 with both ezetimibe and statins suggests that the mechanism of action of each agent is non-redundant.

Example 8

PEG-rMuIL-10 reduces the accumulation of lipids, cholesterol and triglycerides

Evaluation was performed to determine the effect of PEG-rmuel-10 mediated activation of kupffer cells on plasma cholesterol clearance.

Treatment with PEG-rMuIL-10 reduced liver lipid droplets in LDLR-/-mice fed high fat diet as determined by evaluation of liver lipid concentrations by staining with oil red O (data not shown) (fig. 8A). In addition, the liver tissue concentrations of cholesterol and triglycerides were quantified. As shown in fig. 8B to 8E, liver cholesterol concentrations tended to be lower only in wild type mice and LDLR-/-mice fed a high fat diet; mice receiving a normal diet showed no appreciable effect. Fig. 8E to 8I show that liver triglyceride concentrations were lower in wild type mice fed a high fat diet and in LDLR-/-mice fed a normal diet and a high fat diet. Taken together, these data indicate that i) the scavenger system attack by which the kupffer cells remove plasma lipoproteins and ii) a modest enhancement of efflux onto HDL lipoproteins (data not shown) is sufficient to prevent inappropriate triglyceride and cholesterol accumulation within the kupffer cells and the liver cells.

Example 9

PEG-rMuIL-10 treatment results in hepatocyte proliferation

Administration of PEG-rMuIL-10 resulted in an increase in portal peri cell numbers in LDLR-/-mice (data not shown), reflecting a significant reduction in steatosis cells consistent with the reduction in liver triglycerides observed in the previous examples. As depicted in fig. 9A, liver weight gain was observed independent of diet (normal versus high fat diet) and strain (wild type versus LDLR-/-), but dose-related (data not shown). Ki67 gene expression was quantified to determine if the observations reflect liver proliferation. As shown in fig. 9B to 9E, when animals were treated with 0.2mg/kg PEG-rmui-10 (instead of 0.02mg/kg PEG-rmui-10), gene expression was increased more than 2-fold in wild type and LDLR-/-mice fed a normal diet and wild type and LDLR-/-mice fed a high fat diet.

To determine which cells were dividing, cells were stained for Proliferating Cell Nuclear Antigen (PCNA) by IHC and increased PCNA positive cells were observed in both wild type mice fed normal diet and LDLR-/-mice fed high fat diet (data not shown). As shown in fig. 9F-9I, PEG-rMuIL-10 administration resulted in an increase in PCNA positive cells in all mouse backgrounds. PCNA positive cells appear to be hepatocytes and cells that enter the liver through the portal vein.

Example 10

PEG-rMuIL-10 exhibits anti-fibrotic effects

Triglyceride accumulation in the liver is considered a predisposition to fatty liver disease, and long-term accumulation has been linked to the development of NASH (see, e.g., Liu, q. et al, Lipids Health Dis,2010.9: 42). Fatty liver disease is thought to progress to cirrhosis, which is associated with collagen deposition by activated hepatic stellate cells. IL-10 inhibits collagen secretion from these cells. Therefore, to determine with PEG-rMuIL-10 treatment of normal and hyperlipidemia wild type mice and LDLR-/-mice will cause liver collagen deposition changes and evaluation.

Comparison of representative periportal areas of liver from wild-type and LDLR-/-mice fed normal and high fat diet showed the greatest change in portal periportal collagen deposition in LDLR-/-mice fed high fat diet (data not shown). These data indicate that treatment with PEG-rMuIL-10 can inhibit portal peri-collagen deposition, consistent with the reported anti-fibrotic function of IL-10. Further evaluation showed that portal peri-venous collagen deposition in response to inflammatory stimuli derived from high plasma cholesterol could be restored to normal by PEG-rMuIL-10 administration. Consistent with these data, PEG-rMuIL-10 also reduced hepatic triglycerides in these animals (data not shown).

Collectively, these data indicate that PEG-rmuel-10 can restore liver health by inducing hepatocyte proliferation, reducing liver triglyceride concentrations, and remodeling of improperly deposited collagen. Hepatic triglyceride accumulation and subsequent collagen deposition are hallmarks of NAFLD and, if left unchecked, can progress to NASH. Because treatment with PEG-rMuIL-10 can reduce these predisposing factors, PEG-IL-10 represents one possible therapeutic modality for the treatment of NAFLD and NASH.

Example 11

Effect of PEG-rMuIL-10 on liver function

The possible link between exposure of mice to PEG-rMuIL-10 and hepatotoxicity was investigated. As illustrated in table 1, evaluation of changes in serum markers of liver function showed that PEG-rMuIL-10 did not significantly alter serum function markers of liver relative to a normal murine control group.

TABLE 1

Thus, while exposure of hypercholesterolemic mice to PEG-rMuIL-10 caused some changes in liver biology, those changes did not induce toxic major or minor effects. These data further support the potential use of PEG-rHuIL-10 in the control of high plasma cholesterol and the concomitant beneficial effects on NAFLD and NASH.

Specific embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the disclosed embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description, and it is contemplated that such variations may be used as appropriate by those skilled in the art. Accordingly, it is intended that the invention be practiced otherwise than as specifically described herein, and that the invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers and other references cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims (103)

1. A method of identifying an agent that induces phosphorylation of STAT3 in a kupffer cell, comprising:

a) contacting the candidate agent with the Kupffer cell,

b) determining STAT3 phosphorylation levels in said Kupffer cell, and

c) comparing the level of phosphorylation of STAT3 in b) with the level of phosphorylation of STAT3 induced by a reference standard,

wherein a higher level of phosphorylation of STAT3 in the Kupffer cell compared to the level of phosphorylation of STAT3 in the reference standard identifies the candidate agent as an agent that induces phosphorylation.

2. The method of claim 1, wherein an in vitro assay is used to identify an agent that induces phosphorylation of STAT3 in a kupffer cell.

3. The method of claim 2, wherein an ex vivo assay is used to identify an agent that induces phosphorylation of STAT3 in a kupffer cell.

4. The method of any one of claims 1 to 3, wherein the Kupffer cell is from a sinusoid of the liver.

5. The method of any one of claims 1-4, wherein the candidate agent comprises a small molecule, polypeptide, or antibody.

6. The method of any one of claims 1-5, wherein the reference standard is an interleukin, an interferon, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Leukemia Inhibitory Factor (LIF), bone morphogenetic protein 2(BMP-2), oncostatin M (OSM), or leptin.

7. The method of claim 6, wherein the interleukin is IL-5, IL-6, or IL-10.

8. The method of claim 1, further comprising assessing whether the agent capable of inducing phosphorylation reduces at least one of serum cholesterol levels and triglyceride levels.

9. The method of claim 8, wherein said assessing is performed using biochemical assays, in vitro and ex vivo assays, or in vivo models.

10. A method of identifying an agent that lowers serum cholesterol in a subject, comprising:

a) administering a candidate agent to the subject, wherein the candidate agent induces STAT3 phosphorylation in Kupffer cells,

b) determining the subject's serum cholesterol level, and

c) comparing the serum cholesterol level in b) to a serum cholesterol level measured after administration of a reference standard to the subject, wherein the reference standard is known to reduce serum cholesterol;

wherein a candidate agent that has a serum cholesterol lowering ability that exceeds or is comparable to the reference standard identifies an agent that lowers serum cholesterol in the subject.

11. The method of claim 10, wherein the subject is an animal model.

12. The method of claim 11, wherein the animal model is a mouse model.

13. The method of claim 12, wherein the mouse model is an LDLR-/-mouse model.

14. The method of claim 10, wherein the subject is a human.

15. The method of any one of claims 10-14, wherein the candidate agent comprises a small molecule, a polypeptide, or an antibody.

16. The method of any one of claims 10-14, wherein the reference standard is a statin.

17. The method of claim 11, wherein the serum cholesterol level is 200 to 239 mg/dL.

18. The method of claim 11, wherein the serum cholesterol level is at least 240 mg/dL.

19. The method of any one of claims 10-18, further comprising assessing whether the agent that reduces serum cholesterol in the subject induces hepatocyte proliferation.

20. A method of lowering serum cholesterol in a subject in need thereof comprising administering a therapeutically effective amount of an agent that modulates kupffer cell homeostasis.

21. The method of claim 20, wherein said modulating kupffer cell homeostasis comprises enhancing the ability of kupffer cells to remove lipoproteins from serum.

22. The method of claim 20, wherein said modulating kupffer cell homeostasis comprises increasing the number of kupffer cells to remove lipoproteins from serum.

23. The method of any one of claims 20 to 22, wherein the agent is identified using the method of any one of claims 1 to 9.

24. The method of any one of claims 20 to 22, wherein the agent is identified using the method of any one of claims 10 to 19.

25. The method of any one of claims 20-24, wherein the agent is an IL-10 agent.

26. The method of any one of claims 20-25, wherein the agent is administered parenterally.

27. The method of claim 26, wherein the parenteral administration is subcutaneous administration.

28. The method of any one of claims 20-24, wherein the agent is administered orally.

29. The method of any one of claims 20 to 28, further comprising administering a therapeutically acceptable amount of at least one other cholesterol lowering agent.

30. A method of treating or preventing hypercholesterolemia or a hypercholesterolemia-associated disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an agent identified using the method of any one of claims 1 to 19.

31. The method of claim 30, wherein the hypercholesterolemia-associated disease, disorder, or condition is a cardiovascular disorder.

32. The method of claim 31, wherein the cardiovascular disorder is atherosclerosis.

33. The method of claim 30, wherein the hypercholesterolemia-associated disease, disorder, or condition is thrombosis or a thrombotic condition.

34. The method of claim 30, wherein the hypercholesterolemia-associated disease, disorder, or condition is an inflammatory disorder.

35. The method of claim 34, wherein the inflammatory disorder is vasculitis.

36. The method of claim 30, wherein the disease, disorder, or condition is a fibrotic disorder.

37. The method of claim 36, wherein the fibrotic disorder is liver-related.

38. The method of claim 37, wherein the liver-related fibrotic disorder is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis.

39. The method of any one of claims 30-38, wherein the subject is a human.

40. The method of any one of claims 39-39, wherein the agent is administered parenterally.

41. The method of claim 40, wherein the parenteral administration is subcutaneous administration.

42. The method of any one of claims 30-39, wherein the agent is administered orally.

43. The method of any one of claims 30 to 39, further comprising administering a therapeutically acceptable amount of at least one additional therapeutic or prophylactic agent.

44. The method of claim 43, wherein the second therapeutic or prophylactic agent is a cholesterol homeostasis agent.

45. The method of claim 44, wherein the cholesterol homeostasis agent comprises a statin, a bile acid resin, ezetimibe, a fibric acid, niacin, or a PCSK9 inhibitor.

46. The method of claim 43, wherein the prophylactic or therapeutic agent is an antidiabetic agent or an antiobesity agent.

47. The method of claim 43, wherein the prophylactic or therapeutic agent is an immunological or anti-inflammatory agent.

48. A pharmaceutical composition comprising a pharmaceutically effective amount of the agent of any one of claims 1 to 19 and a pharmaceutically acceptable diluent, carrier or excipient.

49. The pharmaceutical composition of claim 48, wherein the composition is suitable for human administration.

50. The pharmaceutical composition of claim 48 or 49, further comprising at least one additional prophylactic or therapeutic agent.

51. The pharmaceutical composition of claim 50, wherein the prophylactic or therapeutic agent is a cholesterol homeostasis agent.

52. The pharmaceutical composition of claim 51, wherein the cholesterol homeostasis agent comprises a statin, a bile acid resin, ezetimibe, a fibric acid, a niacin, or a PCSK9 inhibitor.

53. The pharmaceutical composition of claim 50, wherein the prophylactic or therapeutic agent is an antidiabetic agent or an antiobesity agent.

54. The pharmaceutical composition of claim 50, wherein the prophylactic or therapeutic agent is an immunological agent or an anti-inflammatory agent.

55. A sterile container comprising the pharmaceutical composition of any one of claims 48-54.

56. The sterile container of claim 55, wherein said sterile container is a syringe.

57. A kit comprising the sterile container of claim 55 or 56.

58. The kit of claim 57, further comprising a second sterile container comprising at least one other prophylactic or therapeutic agent.

59. A method of treating or preventing a liver disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of an IL-10 agent capable of modulating Kupffer cell homeostasis,

wherein the liver disease, disorder or condition is non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

60. A method of treating or preventing a liver disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of an IL-10 agent capable of modulating Kupffer cell homeostasis,

wherein the amount is sufficient to achieve a mean IL-10 serum trough concentration of 1pg/mL to 10.0 ng/mL; and is

Wherein the liver disease, disorder or condition is NASH or NAFLD.

61. A method of treating or preventing a liver disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of a cytokine capable of modulating kupffer cell homeostasis, wherein the amount is sufficient to maintain an average cytokine serum trough concentration over a period of time;

wherein the mean cytokine serum trough concentration is 1.0pg/mL to 10.0 ng/mL;

wherein the mean cytokine serum trough concentration is maintained for at least 95% of the time period; and is

Wherein the liver disease, disorder or condition is NASH or NAFLD.

62. The method of claim 61, wherein the mean cytokine serum trough concentration is in the range of 0.1ng/mL to 10 ng/mL.

63. The method of claim 61, wherein the mean cytokine serum trough concentration is in the range of 0.1ng/mL to 5.0 ng/mL.

64. The method of claim 61, wherein the mean cytokine serum trough concentration is in the range of 1.0ng/mL to 10 ng/mL.

65. The method of claim 61, wherein the mean cytokine serum trough concentration is in the range of 1.0ng/mL to 5.0 ng/mL.

66. The method of claim 61, wherein the mean cytokine serum trough concentration is in the range of 0.9ng/mL to 5.1 ng/mL.

67. The method of any one of claims 61-66, wherein the cytokine is IL-10.

68. The method of any one of claims 61-67, wherein said period of time is at least 12 hours.

69. The method of any one of claims 61-67, wherein said period of time is at least 24 hours.

70. The method of any one of claims 61-67, wherein said period of time is at least 48 hours.

71. The method of any one of claims 61-67, wherein said period of time is at least 72 hours.

72. The method of any one of claims 61-67, wherein said period of time is at least 1 week.

73. The method of any one of claims 61-67, wherein said period of time is at least 2 weeks.

74. The method of any one of claims 61-67, wherein said period of time is at least 1 month.

75. The method of any one of claims 61-74, wherein the mean cytokine serum trough concentration is maintained for at least 97% of the time period.

76. The method of claim 75, wherein said mean cytokine serum trough concentration is maintained for at least 99% of said time period.

77. The method of claim 76, wherein said mean cytokine serum trough concentration is maintained for 100% of said time period.

78. The method of any one of claims 59, 60, and 67-77, wherein the IL-10 agent is mature human IL-10.

79. The method of any one of claims 59, 60, and 67-77, wherein the IL-10 agent is a variant of mature human IL-10, and wherein the variant exhibits activity comparable to mature human IL-10 activity.

80. The method of any one of claims 59-79, wherein the disease, disorder, or condition is NASH.

81. The method of any one of claims 59 to 79, wherein the disease, disorder or condition is NAFLD.

82. The method of any one of claims 59 to 81, wherein said modulating Kupffer cell homeostasis comprises enhancing the ability of Kupffer cells to remove lipoproteins from serum.

83. The method of any one of claims 59 to 81, wherein said modulating Kupffer cell homeostasis comprises increasing the number of Kupffer cells so as to remove lipoproteins from serum.

84. The method of any one of claims 59, 60, and 67-83, wherein the IL-10 agent reduces cholesterol.

85. The method of any one of claims 59, 60, and 67-83, wherein the IL-10 agent lowers triglycerides.

86. The method of any one of claims 59, 60, and 67-85, wherein the IL-10 agent reduces or reverses portal perivenous collagen deposition.

87. The method of any one of claims 59, 60, and 67-86, wherein the IL-10 agent increases hepatocyte number.

88. The method of any one of claims 59, 60, and 67-87, wherein the IL-10 agent is administered parenterally.

89. The method of claim 88, wherein the parenteral administration is subcutaneous administration.

90. The method of any one of claims 59-89, further comprising administering at least one additional prophylactic or therapeutic agent.

91. The method of any one of claims 59, 60, and 67-90, wherein the IL-10 agent comprises at least one modification to form a modified IL-10 agent, wherein the modification does not alter the amino acid sequence of the IL-10 agent.

92. The method of claim 91, wherein the modified IL-10 agent is a PEG-IL-10 agent.

93. The method of claim 92, wherein the PEG-IL-10 agent comprises at least one PEG molecule covalently attached to at least one amino acid residue of at least one subunit of IL-10.

94. The method of claim 91 or 92, wherein the PEG-IL-10 agent comprises a mixture of mono-pegylated IL-10 and di-pegylated IL-10.

95. The method of any one of claims 92-94, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of about 5kDa to about 20 kDa.

96. The method of any one of claims 92-94, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of greater than about 20 kDa.

97. The method of any one of claims 92-94, wherein the PEG component of the PEG-IL-10 agent has a molecular mass of at least about 30 kD.

98. The method of claim 91, wherein the modified IL-10 agent is an Fc fusion molecule.

99. The method of claim 91, wherein the modified IL-10 agent comprises serum albumin or an Albumin Binding Domain (ABD).

100. The method of claim 91, wherein the modified IL-10 agent is pulverized via glycosylation or hydroxyethylation.

101. The method of any one of claims 91-100, wherein the modification is site-specific.

102. The method of any one of claims 91 to 97 and 99 to 101, wherein the modification comprises a linker.

103. The method of any one of claims 59-102, wherein the subject is a human.