Patent Application
20160130350
This application generally relates to methods and compositions for inhibiting inflammation. In particular, the application relates to the use of anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 agents, and/or other anti-inflammatory agents for prevention and treatment of inflammatory diseases.
Despite recent advances in studies related to the inflammation process, therapies for treatment of chronic inflammatory diseases have remained largely elusive. This is perhaps a result of the many and complex factors in the host that initiate and maintain inflammatory conditions. Current therapies have disadvantages associated with them, including the suppression of the immune system that can render the host more susceptible to bacterial, viral and parasitic infections. For example, use of steroids is a traditional approach to chronic inflammation treatment. Such treatment can lead to changes in weight and suppression of protective immunity. Advances in biotechnology have promoted the development of targeted biologicals with fewer side effects. To improve inflammatory disease treatment, technologies that alter and control the factors generated by cells of both innate and adaptive immunity systems need to be developed.
Host cells have surface receptors that associate with ligands to signal and regulate host cell activities. Administration of anti-TNF-α antibody or soluble TNF-α receptor has been shown to inhibit inflammatory diseases. Unfortunately, the side effects associated with this treatment can result in an increased risk of infections (e.g., tuberculosis) and other adverse reactions by mechanisms not fully understood. Similarly, antibody therapies focused on membrane bound molecules like CD40 have a propensity for inhibiting inflammation and graft-host diseases. While other targeted host cell therapies to prevent inflammatory diseases are being developed, there is no known single surface or secreted factor that will stop all inflammatory diseases. Consequently, the development of therapies to exploit newly identified specific host cell targets is required.
A variety of pathogens or toxins activate macrophages, neutrophils, T cells, B cells, monocytes, NK cells, Paneth and crypt cells, as well as epithelial cells shortly after entry into the mucosa. Chemokines represent a superfamily of small, cytokine-like proteins that are resistant to hydrolysis, promote neovascularization or endothelial cell growth inhibition, induce cytoskeletal rearrangement, activate or inactivate lymphocytes, and mediate chemotaxis through interactions with G-protein-coupled receptors. Chemokines can mediate the growth and migration of host cells that express their receptors. The cellular mechanisms responsible for the function of chemokines are often, but not entirely, Ca2+ flux dependent and pertussis toxin-sensitive. However, the precise mechanisms for chemokine-mediated events are not known.
The present invention relates to methods and compositions for treating or preventing inflammatory diseases or conditions. In one embodiment, the method comprises the step of administering to a subject diagnosed with an inflammatory disease or condition an effective amount of an anti-inflammatory agent that (1) inhibits the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (2) inhibits the interaction between any one of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (3) inhibits a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5.
In another embodiment, the method comprises the step of administering to a subject diagnosed with an inflammatory disease or condition a therapeutically effective amount of an anti-CXCL9 antibody, an anti-CXCL10 antibody, an anti-CXCL11 antibody, an anti-CXCL13 antibody, and anti-CXCR3 antibody, an anti-CXCR5 antibody, or combination thereof.
In one embodiment, the agent or antibody is administered in a dosage range from about 10 μg/kg body weight/day to about 10 mg/kg body weight/day.
The agent may comprise an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, or agent-encoding expression vector.
In another aspect, a method for enhancing effect of anti-inflammatory therapy, comprises administering to a subject who is receiving or has received anti-inflammatory therapy an effective amount of an anti-inflammatorying that (1) inhibits the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (2) inhibits the interaction between CXCR3 and CXCL9, CXCL10 or CXCL11, and the interaction between CXCR5 and CXCL13, or (3) inhibits a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, wherein the agent comprises an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, or agent-encoding expression vector.
In one embodiment, the subject is receiving anti-inflammatory therapy. In another embodiment, the subject has received anti-inflammatory therapy and has exhibited anti-inflammatory drug-resistance to an anti-inflammatory agent.
In a further aspect the present invention provides a pharmaceutical composition, comprising an anti-inflammatory agent capable of (1) inhibiting the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5; (2) inhibiting the interaction between any one of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (3) inhibiting a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, wherein the anti-inflammatory agent is an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, or agent-encoding expression vector; and a pharmaceutically acceptable carrier.
The following detailed description is presented to enable any person skilled in the art to make and use the invention. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that these specific details are not required to practice the invention. Descriptions of specific applications are provided only as representative examples. The present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
As used herein, the following terms shall have the following meanings:
The terms “treat,” “treating” or “treatment” as used herein, refers to a method of alleviating or abrogating a disorder and/or its attendant symptoms. The terms “prevent”, “preventing” or “prevention,” as used herein, refer to a method of barring a subject from acquiring a disorder and/or its attendant symptoms. In certain embodiments, the terms “prevent,” “preventing” or “prevention” refer to a method of reducing the risk of acquiring a disorder and/or its attendant symptoms.
As used herein, the terms “anti-inflammatory activity” or “anti-inflammatory response” refer to a reduction or prevention of inflammation manifested in a change in cells, such as proliferation, activation, gene expression, and the like. A reduction in inflammation may include, for example, reducing the secretion or expression of inflammatory cytokines, chemokines, cytokine/chemokine receptors; adhesion molecules, proteases, and/or immunoglobulins; reducing chemotaxis or migration of cells; reducing the blood concentration of monocytes and/or local accumulation thereof at the sites of inflammation; increasing apoptosis of immune cells; suppressing class-II MHC presentation; reducing the number of autoreactive cells; increasing immune tolerance, reducing autoreactive cell survival, combinations thereof, and the like.
As used herein, the term “anti-inflammatory agent” refers to a biologic agent which, upon binding to a protein reduces or prevents inflammatory activity or upon binding to a nucleic acid encoding an inflammatory protein product reduces or blocks expression of an mRNA or protein corresponding to the inflammatory protein product. Anti-inflammatory agents are to be distinguished from anti-inflammatory small molecule chemical compounds as further described herein. Exemplary anti-inflammatory agents include antibodies, antibody fragments, short interfering RNAs (siRNAs), aptamers, synbodies, binding agents, peptides, aptamer-siRNA chimeras, single stranded antisense oligonucleotides, triplex forming oligonucleotides, ribozymes, external guide sequences, agent-encoding expression vectors, and the like.
As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site or epitope binding domain that specifically binds (immunoreacts with) an antigen. The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit specific binding to a target antigen. By “specifically bind” or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity with other polypeptides. The term “antibody” also includes antibody fragments that comprise a portion of a full length antibody, generally the antigen binding or variable region thereof.
The term “anti-inflammatory antibody” refers to an antibody or antibody fragment agent.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
“Humanized” forms of non-human antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and/or capacity. Methods for making humanized and other chimeric antibodies are known in the art.
“Bispecific antibodies” are antibodies that have binding specificities for at least two different antigens.
The use of “heteroconjugate antibodies” is also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells. It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving the use of crosslinking agents. Alternatively, they may be prepared by fusing two antibodies or fragments thereof by recombinant DNA techniques known to those of skill in the art.
As used herein, the term “nucleic acid” refers to a polydeoxyribonucleotide (DNA or an analog thereof) or polyribonucleotide (RNA or an analog thereof) made up of at least two, and preferably ten or more bases linked by a backbone structure. In DNA, the common bases are adenine (A), guanine (G), thymine (T) and cytosine (C), whereas in RNA, the common bases are A, G, C and uracil (U, in place of T), although nucleic acids may include base analogs (e.g., inosine) and abasic positions (i.e., a phosphodiester backbone that lacks a nucleotide at one or more positions). Exemplary nucleic acids include single-stranded (ss), double-stranded (ds), or triple-stranded polynucleotides or oligonucleotides of DNA and RNA.
The term “polynucleotide” refers to nucleic acids containing more than 10 nucleotides.
The term “oligonucleotide” refers to a single stranded nucleic acid containing between about 15 to about 100 nucleotides.
The term “promoter” is to be taken in its broadest context and includes transcriptional regulatory elements (TREs) from genomic genes or chimeric TREs therefrom, including the TATA box or initiator element for accurate transcription initiation, with or without additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) which regulate activation or repression of genes operably linked thereto in response to developmental and/or external stimuli, and trans-acting regulatory proteins or nucleic acids. The promoter may be constitutively active or it may be active in one or more tissues or cell types in a developmentally regulated manner. A promoter may contain a genomic fragment or it may contain a chimera of one or more TREs combined together.
In a pharmacological sense, in the context of the present invention, a “therapeutically effective amount” of an anti-inflammatory antibody, agent or small molecule inhibitor, or combination thereof refers to an amount effective in the prevention or treatment of a disorder for the treatment of which the anti-inflammatory agent or combination thereof is effective. A “disorder” or “disease” is any inflammatory condition that would benefit from treatment with the antibody, agent or small molecule inhibitor.
The term “inflammatory bowel disease” or “IBD” refers to the group of disorders that cause the intestines to become inflamed, generally manifested with symptoms including abdominal cramps and pain, diarrhea, weight loss and intestinal bleeding. The main forms of IBD are ulcerative colitis (UC) and Crohn's disease.
The term “ulcerative colitis” or “UC” is a chronic, episodic, inflammatory disease of the large intestine and rectum characterized by bloody diarrhea. Ulcerative colitis is characterized by chronic inflammation in the colonic mucosa and can be categorized according to location: “proctitis” involves only the rectum, “proctosigmoiditis” affects the rectum and sigmoid colon, “left-sided colitis” encompasses the entire left side of the large intestine, “pancolitis” inflames the entire colon.
The term “Crohn's disease,” also called “regional enteritis,” is a chronic autoimmune disease that can affect any part of the gastrointestinal tract but most commonly occurs in the ileum (the area where the small and large intestine meet). Crohn's disease, in contrast to ulcerative colitis, is characterized by chronic inflammation extending through all layers of the intestinal wall and involving the mesentery as well as regional lymph nodes. Whether or not the small bowel or colon is involved, the basic pathologic process is the same.
Ulcerative colitis and Crohn's disease can be distinguished from each other clinically, endoscopically, pathologically, and serologically in more than 90% of cases; the remainder are considered to be indeterminate IBD.
The term “mucosal tissue” refers to any tissue in which mucosal cells are found, such tissues, include, for example, gastro-intestinal tissues (e.g., stomach, small intestine, large intestine, rectum), uro-genital tissue (e.g., vaginal tissue, penile tissue, urethra), nasal-larynx tissue (e.g., nasal tissue, larynx tissue), mouth (buccal tissue) to name a few. Other mucosal tissues are known and easily identifiable by one of skill in the art.
The term “inhibits” is a relative term, an agent inhibits a response or condition if the response or condition is quantitatively diminished following administration of the agent, or if it is diminished following administration of the agent, as compared to a reference agent. Similarly, the term “prevents” does not necessarily mean that an agent completely eliminates the response or condition, so long as at least one characteristic of the response or condition is eliminated. Thus, a composition that reduces or prevents an inflammatory response, can, but does not necessarily completely eliminate such a response, so long as the response is measurably diminished, for example, by at least about 50%, such as by at least about 70%, or about 80%, or even by about 90% of (that is to 10% or less than) the response in the absence of the agent, or in comparison to a reference agent.
The term “increased level” refers to a level that is higher than a normal or control level customarily defined or used in the relevant art. For example, an increased level of immunostaining in a tissue is a level of immunostaining that would be considered higher than the level of immunostaining in a control tissue by a person of ordinary skill in the art.
The term “biological sample,” as used herein, refers to material of a biological origin, which may be a body fluid or body product such as blood, plasma, urine, saliva, spinal fluid, stool, sweat or breath. A biological sample may include tissue samples, cell samples, or combination thereof.
Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed.
Methods for Inhibiting Inflammation Using Anti-Inflammatory Agents that Inhibit the Expression or Activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 or CXCR5
CXCL9, CXCL10, and CXCL11 chemokines are ligands for the CXCR3 chemokine receptor. CXCL13 chemokine is the ligands for the CXCR5 chemokine receptor. Each of these chemokine ligands and their receptor are locally upregulated and play a role in various inflammatory diseases, including inflammatory bowel diseases. Additionally, CXCL9, -CXCL10, CXCL11 and CXCL13 chemokines enhance inflammation both in vivo and in vitro. CXCR3 and CXCR3 are members of the chemokine receptor family of G protein coupled receptors (GPCRs). Interaction of CXCR3 with CXCL9, CXCL10, and CXCL11 and/or interaction of CXCR5 with CXCL13 activate inflammation.
One aspect of the present application relates to methods for inhibiting inflammation using agents that inhibit the expression or activity of CXCL9, CXCL10, CXCL11 CXCL13, CXCR3 or CXCR5. “Activities” include, for example, transcription, translation, intracellular translocation, secretion, signal transduction, phosphorylation by kinases, cleavage by proteases, homophilic and heterophilic binding to other proteins, ubiquitination, and the like.
In some embodiments, a method for treating or preventing an inflammatory condition in a subject comprises administering to a subject diagnosed with an inflammatory disease an effective amount of an anti-inflammatory agent that: (1) inhibits the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (2) inhibits the interaction between CXCR3 and any one of CXCL9, CXCL10, and CXCL11 or interaction between CXCR5 and CXCL13, or (3) inhibits a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5.
In certain embodiments, a therapeutically effective amount of at least one anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is administered to a subject in need thereof as a sole anti-inflammatory agent. In other embodiments, a therapeutically effective amount of at least one anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is administered to a subject in need thereof as a primary anti-inflammatory agent, in conjunction with the treatment of the subject beforehand, at the same time, or afterward with a therapeutically effective amount of a secondary anti-inflammatory agent.
An anti-inflammatory agent is a biologic agent capable of reducing or preventing inflammation. Exemplary anti-inflammatory agent include anti-inflammatory antibodies, short interfering RNAs (siRNAs), CXCL9-binding agents, CXCL10-binding agents, CXCL11-binding agents, CXCL13-binding agents, CXCR5-binding agents and CXCR3-binding agents, antisense oligonucleotides, ribozymes, triplex forming oligonucleotides, external guide sequences, agent-encoding expression vectors and anti-inflammatory small molecule chemical compounds.
In a preferred embodiment, the method comprises administering to a subject diagnosed with an inflammatory disease a therapeutically effective amount of an anti-CXCL9 antibody, an anti-CXCL10 antibody, an anti-CXCL11 antibody, an anti-CXCR3 antibody, an anti-CXCL13 antibody, an anti-CXCR5 antibody or a combination thereof, resulting in reduced inflammation.
Exemplary inflammatory diseases or conditions include, but are not limited to, anaphylaxis, septic shock, osteoarthritis, rheumatoid arthritis, psoriasis, asthma, allergies (e.g., drug, insect, plant, food), atherosclerosis, delayed type hypersensitivity, dermatitis, diabetes mellitus, juvenile onset diabetes, graft rejection, inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, enteritis, and interstitial cystitis; multiple sclerosis, myasthemia gravis, Grave's disease, Hashimoto's thyroiditis, pneumonitis, prostatitis, psoriasis, nephritis, pneumonitis, chronic obstructive pulmonary disease, chronic bronchitis rhinitis, spondyloarthropathies, scheroderma, systemic lupus erythematosus, and thyroiditis. In a preferred embodiment, the inflammatory condition is an inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis, enteritis, and interstitial cystitis (including drug-induced cystitis and spontaneous cystitis).
In some embodiments, the subject is diagnosed with an inflammatory condition that results in elevated CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5 expression. In other embodiments, the method of treatment further comprises the step of determining whether the level of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR53 expression is elevated in a tissue from the subject, and, if so, administering to the subject a therapeutically effective amount of an anti-inflammatory agent that: (1) inhibits the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (2) inhibits the interaction between CXCR3 and any one of CXCL9, CXCL10, and CXCL11, or the interation between CXCR5 and CXCL13, or (3) inhibits a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5.
In some embodiments, the therapeutically effective amount of an anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 anti-inflammatory agent augments the effectiveness of one or more additional therapeutically effective agents or small molecule agents in inhibiting inflammation. In a more particular embodiment, the therapeutically effective amount of the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 anti-inflammatory agent reduces the amount of the one or more additional therapeutically effective agents or small molecule agents required for inhibiting inflammation.
In particular embodiments, treatment of a subject with an anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR5 and/or anti-CXCR3 anti-inflammatory agent is carried out in conjunction with the treatment of the subject beforehand, at the same time, or afterward with a therapeutically effective amount of at least one secondary agent directed against a chemokine, cytokine, receptor thereof, or derivatives thereof, including soluble receptors and the like.
In one embodiment, a method for enhancing effect of anti-inflammatory therapy comprises administering to a subject who is receiving or has received anti-inflammatory therapy an effective amount of an anti-inflammatory agent that (1) inhibits the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, or (2) inhibits the interaction between any one of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5 or (3) inhibits a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, wherein the agent comprises an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, or agent-encoding expression vector.
In a particular embodiment, the subject is receiving anti-inflammatory therapy. In another embodiment, the subject has received anti-inflammatory therapy, but has exhibited anti-inflammatory drug-resistance to an anti-inflammatory agent.
In a preferred embodiment, the subject is administered an effective amount of an anti-CXCL9 antibody, an anti-CXCL10 antibody, an anti-CXCL11 antibody, an anti-CXCL13 antibody, anti-CXCR3 antibody, an anti-CXCR5 antibody, or combination thereof for.
An anti-inflammatory agent may include any inhibitor of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5 activity and/or expression. Exemplary anti-inflammatory agents include antibodies, short interfering RNA (siRNA), aptamer-siRNA chimeras, single stranded antisense oligonucleotides, triplex forming oligonucleotides, ribozymes, external guide sequences, agent-encoding expression vectors, and combination thereof.
An anti-inflammatory antibody may be an anti-chemokine antibody, an anti-chemokine receptor antibody, anti-cytokine antibody, an anti-cytokine receptor antibody, an anti-proinflammatory peptide antibody, or a combination thereof (e.g., bispecific antibody).
A preferred anti-inflammatory antibody of the present application is one which binds to human CXCL9, CXCL10, CXCL11 or CXCL13 and preferably blocks (partially or completely) the ability of CXCL9, CXCL10, CXCL11 or CXCL13 to bind and/or activate the CXCR3 or CXCR5 receptor. Another preferred antibody of the present invention is one which binds to human CXCR3 or CXCR5 and preferably blocks (partially or completely) the ability of a cell carrying the receptor, such as an epithelial, endothelial or lymphoid cell, from binding to and/or being activated by CXCL9, CXCL10 CXCL11 and/or CXCL13.
In one embodiment, the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is a monoclonal antibody. In another embodiment, the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is a humanized antibody. In another embodiment, the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is an antibody fragment. In yet another embodiment, the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody is a humanized antibody fragment.
In other embodiments, the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 or anti-CXCR5 antibody binds to CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 or CXCR5, respectively, with a kd value in the range of 0.01 pM to 10 μM, 0.01 pM to 1 μM, 0.01 pM to 100 nM, 0.01 pM to 10 nM, 0.01 pM to 1 nM, 0.1 pM to 10 μM, 0.1 pM to 1 μM, 0.1 pM to 100 nM, 0.1 pM to 10 nM, 0.1 pM to 1 nM, 1 pM to 10 μM, 1 pM to 1 μM, 1 pM to 100 nM, 1 pM to 10 nM, 1 pM to 1 nM, 10 pM to 10 μM, 10 pM to 1 μM, 10 pM to 100 nM, 10 pM to 10 nM, 10 pM to 1 nM, 100 pM to 10 μM, 100 pM to 1 μM and 100 pM to 100 nM. In some other embodiments, the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 or anti-CXCR5 antibody binds to non-target proteins with a kd value of greater than 100 nM. In certain embodiments, the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 or anti-CXCR5 antibody binds to the target protein (i.e., CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 or CXCR5, respectively, with a Kd value in the range of 0.01 pM to 100 nM or 0.01 pM to 10 nM, and binds to non-target proteins with a Kd value of greater than 100 nM.
An anti-inflammatory antibody may be administered in any form suitable for neutralizing CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5 activity. Exemplary antibody or antibody derived fragments may include any member of the group consisting of: IgG, antibody variable region; isolated CDR region; single chain Fv molecule (scFv) comprising a VH and VL domain linked by a peptide linker allowing for association between the two domains to form an antigen binding site; bispecific scFv dimer; minibody comprising a scFv joined to a CH3 domain; diabody (dAb) fragment; single chain dAb fragment consisting of a VH or a VL domain; Fab fragment consisting of VL, VH, CL and CH1 domains; Fab′ fragment, which differs from a Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region; Fab′-SH fragment, a Fab′ fragment in which the cysteine residue(s) of the constant domains bear a free thiol group; F(ab)2, bivalent fragment comprising two linked Fab fragments; Fd fragment consisting of VH and CH1 domains; derivatives thereof; and any other antibody fragment(s) retaining antigen-binding function. Fv, scFv, or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains. When using antibody-derived fragments, any or all of the targeting domains therein and/or Fc regions may be “humanized” using methodologies well known to those of skill in the art. In some embodiments, the anti-inflammatory antibody is modified to remove the Fc region.
In particular embodiments, an anti-CXCR3 antibody or antibody fragment thereof is conjugated to or fused to a second antibody or antibody binding fragment to enhance its binding to target cells carrying the CXCR3 receptor.
In addition, an anti-inflammatory agent may be conjugated to one or more secondary anti-inflammatory agent(s), such as an anti-inflammatory small molecule(s) to provide a further level of anti-inflammatory activity.
Short Interfering RNAs (siRNAs).
An siRNA is a double-stranded RNA that can be engineered to induce sequence-specific post-transcriptional gene silencing of mRNAs corresponding to any one of the above-described chemokine, cytokine or receptors thereof.
siRNAs exploit the mechanism of RNA interference (RNAi) for the purpose of “silencing” gene expression of targeted chemokine-, cytokine- or receptor genes. This “silencing” was originally observed in the context of transfecting double stranded RNA (dsRNA) into cells. Upon entry therein, the dsRNA was found to be cleaved by an RNase III-like enzyme, Dicer, into double stranded small interfering RNAs (siRNAs) 21-23 nucleotides in length containing 2 nucleotide overhangs on their 3′ ends. In an ATP dependent step, the siRNAs become integrated into a multi-subunit RNAi induced silencing complex (RISC) which presents a signal for AGO2-mediated cleavage of the complementary mRNA sequence, which then leads to its subsequent degradation by cellular exonucleases.
In one embodiment, the anti-inflammatory agent comprises a synthetic siRNA. Synthetically produced siRNAs structurally mimic the types of siRNAs normally processed in cells by the enzyme Dicer. Synthetically produced siRNAs may incorporate any chemical modifications to the RNA structure that are known to enhance siRNA stability and functionality. For example, in some cases, the siRNAs may be synthesized as a locked nucleic acid (LNA)-modified siRNA. An LNA is a nucleotide analogue that contains a methylene bridge connecting the 2′-oxygen of the ribose with the 4′ carbon. The bicyclic structure locks the furanose ring of the LNA molecule in a 3′-endo conformation, thereby structurally mimicking the standard RNA monomers.
In other embodiments, the anti-inflammatory agent may comprise an expression vector engineered to transcribe a short double-stranded hairpin-like RNA (shRNA) that is processed into a targeted siRNA inside the cell. The shRNAs can be cloned in suitable expression vectors using kits, such as Ambion's SILENCER® siRNA Construction Kit, Imgenex's GENESUPPRESSOR™ Construction Kits, and Invitrogen's BLOCK-IT™ inducible RNAi plasmid and lentivirus vectors.
Synthetic siRNAs and shRNAs may be designed using well known algorithms and synthesized using a conventional DNA/RNA synthesizer. A variety of chemokine-, cytokine- and receptor-targeted siRNAs may be commercially obtained from Origen (Rockville, Md.).
In some embodiments, the anti-inflammatory agent is a CXCL9-, CXCL10-, CXCL11-, CXCL13-, CXCR3- or CXCR5-binding agent. The binding agent may comprise any non-antibody protein, peptide, or synthetic binding molecule, such as an aptamer or synbody, which is capable of specifically binding directly or indirectly to CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 or CXCR5 so as to inhibit the interaction and/or activation between CXCR3 and CXCL9, CXCL10 or CXCL11; or the interaction and/or activation between CXCR5 and CXCL13, or which inhibits a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5, which is associated with reducing or preventing an inflammatory response.
The CXCL9-, CXCL10-, CXCL11-, CXCL13, CXCR3 and/or CXCR5-binding agents may be produced by any conventional method for generating high-affinity binding ligands, including SELEX, phage display, and other methodologies, including combinatorial chemistry- and/or high throughput methods known to those of skill in the art.
An aptamer is a nucleic acid version of an antibody that comprises a class of oligonucleotides that can form specific three dimensional structures exhibiting high affinity binding to a wide variety of cell surface molecules, proteins, and/or macromolecular structures. Aptamers are commonly identified by an in vitro method of selection sometimes referred to as Systematic Evolution of Ligands by EXponential enrichment or “SELEX”. SELEX typically begins with a very large pool of randomized polynucleotides which is generally narrowed to one aptamer ligand per molecular target. Typically, aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
An aptamer can be chemically linked or conjugated to the above described nucleic acid inhibitors to form targeted nucleic acid inhibitors, such as aptamer-siRNA chimeras. An aptamer-siRNA chimera contains a targeting moiety in the form of an aptamer which is linked to an siRNA. When using an aptamer-siRNA chimera, it is preferable to use a cell internalizing aptamer. Upon binding to specific cell surface molecules, the aptamer can facilitate internalization into the cell where the nucleic acid inhibitor acts. In one embodiment both the aptamer and the siRNA comprises RNA. The aptamer and the siRNA may comprise any nucleotide modifications as further described herein. Preferably, the aptamer comprises a targeting moiety specifically directed to binding cells expressing the chemokine-, cytokine- and/or receptor target genes, such as lymphoid, epithelial cell, and/or endothelial cells.
Synbodies are synthetic antibodies produced from libraries comprised of strings of random peptides screened for binding to target proteins of interest. Synbodies are described in US 2011/0143953 and Diehnelt et al., PLoS One, 5(5):e10728 (2010).
CXCL9-, CXCL10-, CXCL11-, CXCL13-, CXCR3-, CXCR5-binding agents, including aptamers and synbodies, can be engineered to bind target molecules very tightly with Kds between 10−10 to 10−12 M. In some embodiments, the CXCL9-, CXCL10-, CXCL11-, CXCL13-, CXCR3- or CXCR5-binding agent bind the target molecule with a Kd less than 10−6, less than 10−8, less than 10−9, less than 10−10, or less than 10−12 M.
In another embodiment, the anti-inflammatory inhibitor agent may comprise an antisense oligonucleotide or polynucleotide capable of inhibiting the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5. The antisense oligonucleotide or polynucleotide may comprise a DNA backbone, RNA backbone, or chemical derivative thereof. In one embodiment, the antisense oligonucleotide or polynucleotide comprises a single stranded antisense oligonucleotide or polynucleotide targeting for degradation. In preferred embodiments, the anti-inflammatory inhibitor agent comprises a single stranded antisense oligonucleotide complementary to a CXCL9, CXCL10, CXCL11, CXCL12, CXCR3 or CXCR5 mRNA sequence. The single stranded antisense oligonucleotide or polynucleotide may be synthetically produced or it may be expressed from a suitable expression vector. The antisense nucleic acid is designed to bind via complementary binding to the mRNA sense strand so as to promote RNase H activity, which leads to degradation of the mRNA. Preferably, the antisense oligonucleotide is chemically or structurally modified to promote nuclease stability and/or increased binding.
In some embodiments, the antisense oligonucleotides are modified to produce oligonucleotides with nonconventional chemical or backbone additions or substitutions, including but not limited to peptide nucleic acids (PNAs), locked nucleic acids (LNAs), morpholino backboned nucleic acids, methylphosphonates, duplex stabilizing stilbene or pyrenyl caps, phosphorothioates, phosphoroamidates, phosphotriesters, and the like. By way of example, the modified oligonucleotides may incorporate or substitute one or more of the naturally occurring nucleotides with an analog; internucleotide modifications incorporating, for example, uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.); modifications incorporating intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), or alkylators, and/or modified linkages (e.g., alpha anomeric nucleic acids, etc.).
In some embodiments, the single stranded oligonucleotides are internally modified to include at least one neutral charge in its backbone. For example, the oligonucleotide may include a methylphosphonate backbone or peptide nucleic acid (PNA) complementary to the target-specific sequence. These modifications have been found to prevent or reduce helicase-mediated unwinding. The use of uncharged probes may further increase the rate of hybridization to polynucleotide targets in a sample by alleviating the repulsion of negatively-charges nucleic acid strands in classical hybridization.
PNA oligonucleotides are uncharged nucleic acid analogs for which the phosphodiester backbone has been replaced by a polyamide, which makes PNAs a polymer of 2-aminoethyl-glycine units bound together by an amide linkage. PNAs are synthesized using the same Boc or Fmoc chemistry as are use in standard peptide synthesis. Bases (adenine, guanine, cytosine and thymine) are linked to the backbone by a methylene carboxyl linkage. Thus, PNAs are acyclic, achiral, and neutral. Other properties of PNAs are increased specificity and melting temperature as compared to nucleic acids, capacity to form triple helices, stability at acid pH, non-recognition by cellular enzymes like nucleases, polymerases, etc.
Methylphosphonate-containing oligonucleotides are neutral DNA analogs containing a methyl group in place of one of the non-bonding phosphoryl oxygens. Oligonucleotides with methylphosphonate linkages were among the first reported to inhibit protein synthesis via anti-sense blockade of translation.
In some embodiments, the phosphate backbone in the oligonucleotides may contain phosphorothioate linkages or phosphoroamidates. Combinations of such oligonucleotide linkages are also within the scope of the present invention.
In other embodiments, the oligonucleotide may contain a backbone of modified sugars joined by phosphodiester internucleotide linkages. The modified sugars may include furanose analogs, including but not limited to 2-deoxyribofuranosides, α-D-arabinofuranosides, α-2′-deoxyribofuranosides, and 2′,3′-dideoxy-3′-aminoribofuranosides. In alternative embodiments, the 2-deoxy-β-D-ribofuranose groups may be replaced with other sugars, for example, β-D-ribofuranose. In addition, β-D-ribofuranose may be present wherein the 2-OH of the ribose moiety is alkylated with a C1-6 alkyl group (2-(O—C1-6 alkyl) ribose) or with a C2-6 alkenyl group (2-(O—C2-6 alkenyl) ribose), or is replaced by a fluoro group (2-fluororibose).
Related oligomer-forming sugars include those used in locked nucleic acids (LNA) as described above. Exemplary LNA oligonucleotides include modified bicyclic monomeric units with a 2′-O-4′-C methylene bridge, such as those described in U.S. Pat. No. 6,268,490, the disclosures of which are incorporated by reference herein.
Chemically modified oligonucleotides may also include, singly or in any combination, 2′-position sugar modifications, 5-position pyrimidine modifications (e.g, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-[2-(1H-indole-3yl)ethyl]carboxyamide)-2′-deoxyuridine, 5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridine chloride, 5-(N-napthylcarboxyamide)-2′-deoxyuridine, and 5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine), 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo- or 5-iodo-uracil, methylations, unusual base-pairing combinations, such as the isobases isocytidine and isoguanidine, and the like.
Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena ribozymes. There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo. Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates, such as CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 or CXCR5 mRNAs. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence.
Triplex forming oligonucleotides (TFOs) are molecules that can interact with either double-stranded and/or single-stranded nucleic acids, including CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 or CXCR5 genomic DNA regions or their corresponding mRNAs. When TFOs interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. TFOs can bind target regions with high affinity and specificity. In preferred embodiments, the triplex forming molecules bind the target molecule with a Kd less than 10−6, 10−8, 10−10, or 10−12. Exemplary TFOs for use in the present invention include PNAs, LNAs, and LNA modified PNAs, such as Zorro-LNAs.
External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target an mRNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells.
In one embodiment, a method for treating or preventing an inflammatory condition in a subject, comprises administering to a subject diagnosed with an inflammatory disease an effective amount of an expression vector expressing an anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13 agent, anti-CXCR3 agent and/or anti-CXCR5 agent. In a particular embodiment, the method comprises administering to a subject diagnosed with an inflammatory disease an effective amount of an expression vector expressing an anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 antibody. In another embodiment, the method comprises administering to a subject diagnosed with an inflammatory disease an effective amount of an expression vector expressing an an anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5 siRNA. The expression vector can be any expression vector capable of delivering and expressing a polynucleotide encoding an anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR5 and/or anti-CXCR3 agent including antibodies, siRNAs, antisense oligonucleotides or polynucleotides, and the like.
As used herein, the term “expression vector” includes any nucleic acid capable of directing expression of a nucleic acid. Expression vectors may be delivered to cells using two primary delivery schemes: viral-based delivery systems using viral vectors and non-viral based delivery systems using, for example, plasmid vectors. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, these methods can be used to target certain diseases and cell populations by using the targeting characteristics inherent to the carrier or engineered into the carrier.
The nucleic acids that are delivered to cells contain one or more transcriptional regulatory elements, including promoters and/or enhancers, for directing the expression of siRNAs. A promoter comprises a DNA sequence that functions to initiate transcription from a relatively fixed location in regard to the transcription start site. A promoter contains TRE elements required for basic interaction of RNA polymerase and transcription factors, and may operate in conjunction with other upstream elements and response elements. Preferred promoters are those capable of directing expression in a target cell of interest. The promoters may include constitutive promoters (e.g., HCMV, SV40, elongation factor-1α (EF-1α)) or those exhibiting preferential expression in a particular cell type of interest. Enhancers generally refer to DNA sequences that function away from the transcription start site and can be either 5′ or 3′ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase and/or regulate transcription from nearby promoters.
The promotor and/or enhancer may be specifically activated either by light or specific chemical inducing agents. In some embodiments, inducible expression systems regulated by administration of tetracycline or dexamethasone, for example, may be used. In other embodiments, gene expression may be enhanced by exposure to radiation, including gamma irradiation and external beam radiotherapy (EBRT), or alkylating chemotherapeutic drugs.
Cell or tissue-specific transcriptional regulatory elements (TREs) can be incorporated into expression vectors to allow for transcriptional targeting of expression to desired cell types. Expression vectors generally contain sequences for transcriptional termination, and may additionally contain one or more elements positively affecting mRNA stability. An expression vector may further include an internal ribosome entry site (IRES) between adjacent protein coding regions to facilitate expression two or more proteins from a common mRNA in an infected or transfected cell. Additionally, the expression vectors may further include nucleic acid sequence encoding a marker product. This marker product may be used to determine if the gene has been delivered to the cell and is being expressed. Preferred marker genes are the E. coli lacZ gene, which encodes β-galactosidase, and green fluorescent protein (GFP).
Viral-Based Expression Vectors.
In some embodiments, the anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL13, anti-CXCR3 and/or anti-CXCR5-antibody- or siRNA encoding sequences (or shRNAs) are delivered from viral-derived expression vectors. Exemplary viral vectors may include or be derived from adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, poxvirus, HIV virus, lentivirus, retrovirus, Sindbis and other RNA viruses, and the like. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Moloney Leukemia virus (MMLV), HIV and other lentivirus vectors. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Poxviral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. Viral delivery systems typically utilize viral vectors having one or more genes removed and with and an exogenous gene and/or gene/promotor cassette being inserted into the viral genome in place of the removed viral DNA. The necessary functions of the removed gene(s) may be supplied by cell lines which have been engineered to express the gene products of the early genes in trans.
Non-Viral Expression Vectors.
In other embodiments, nonviral delivery systems are utilized for delivery of plasmid vectors or other bioactive non nucleic acid agents using lipid formulations comprising, for example, liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) and anionic liposomes. Liposomes can be further conjugated to one or more proteins or peptides to facilitate targeting to a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Furthermore, an anti-inflammatory agent can be administered as a component of a microcapsule or nanoparticle that can be targeted to a cell type of interest using targeting moieties described herein or that can be designed for slow release of one or more anti-inflammatory agent (s) in accordance with a predetermined rate of release or dosage.
In other embodiments, the nucleic acids may be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.). The nucleic acids may be in solution or suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. Vehicles such as “stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to cells of interest), receptor mediated targeting of DNA through cell specific ligands or viral vectors targeting e.g., lymphoid, epithelial or endothelial cells. In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration.
In some embodiments, a therapeutically effective amount of at least one anti-CXCL9, anti-CXCL10, anti-CXCL11, and/or anti-CXCR3 antibody is administered to a subject in need thereof in conjunction with a secondary anti-inflammatory agent. The a secondary anti-inflammatory agent may be given before, at the same time, or after the administration of the antibody or antibodies. Preferably, the secondary anti-inflammatory agent is directed against a chemokine, cytokine, receptor thereof, or combination thereof.
The secondary anti-inflammatory agent may comprise an anti-inflammatory antibody, short interfering RNA (siRNA), chemokine and chemokine receptor binding agents, antisense oligonucleotides, triplex forming oligonucleotides, ribozymes, external guide sequences, agent-encoding expression vectors or an anti-inflammatory small molecule chemical compound. In some embodiments, the secondary anti-inflammatory agent comprise another an anti-inflammatory antibody directed to determinants on CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and/or CXCR5. In other embodiments, the secondary anti-inflammatory agent comprises an antibody or an agent directed against a secondary chemokine, cytokine, or receptor thereof.
In some embodiments, the secondary anti-inflammatory agent is an anti-inflammatory agent directed against a chemokine, cytokine or receptor thereof. Exemplary chemokine or chemokine receptor targeted in accordance with the present invention, including protein and cDNA sequences, respectively, from NIH-NCBI GenBank, are described in Table 1.
In some embodiments, the secondary anti-inflammatory agent binds specifically to a cytokine or cytokine receptor. Exemplary cytokine or cytokine receptor targets and/or their reactive inhibitory products include, but are not limited to, interferon-α, -β, or -γ; tumor necrosis factor (TNF)-alpha, e.g., (infliximab (REMICADE), adalimumab (HUMIRA®), D2E7 (BASF Pharma), and HUMICADE® (Celltech)); soluble forms of the TNF receptor (etanercept (ENBREL®)); CD20, including rituximab (RITUXAN®), humanized 2H7, 2F2 (Hu-Max-CD20), human CD20 antibody (Genmab), and humanized A20 antibody (Immunomedics); TNF-beta; interleukin-2 (IL-2), including daclizumab; IL-2 receptor, interleukin-4 (IL-4) and IL-4 receptor; interleukin-6 (IL-6) and IL-6 receptor; interleukin-1 (IL-1) receptor, including IL-1 receptor agents, such as anakinra (KINERET®); LFA-1, including anti-CD11a, anti-CD18 antibodies, and soluble peptides containing a LFA-3 binding domain; anti-L3T4 antibodies; interleukin-1β (IL-1β); interleukin-8 (IL-8); interferon-γ (IFN-γ); vascular endothelial growth factor (VEGF); leukemia inhibitory factor (LIF); monocyte chemoattractant protein-1 (MCP-1); RANTES; interleukin-10 (IL-10); interleukin-12 (IL-12); matrix metalloproteinase 2 (MMP2); IP-10; macrophage inflammatory protein 1α (MIP1α); macrophage inflammatory protein 1β (MIP1β); pan-T, including anti-CD3 or anti-CD4/CD4a antibodies; BAFF (zTNF4, BLyS) and BAFF receptor, BR3; anti-idiotypic antibodies for MHC antigens and MHC fragments; CD40 receptor and anti-CD40 ligand (CD154); CTLA4-Ig; T-cell receptor antibodies, such as T10B9; heterologous anti-lymphocyte globulin; streptokinase; transforming growth factor-beta (TGF-beta); streptodomase; RNA or DNA from the host; chlorambucil; deoxyspergualin; T-cell receptor; and T-cell receptor fragments.
Anti-Inflammatory Small Molecule Chemical Compounds.
Exemplary small molecule anti-inflammatory agents that can be used as secondary anti-inflammatory agent include, but are not are not limited to, small molecule compounds or medicaments selected from the group consisting of analgesics, such as aspirin or TYLENOL® (Acetaminophen); 2-amino-6-aryl-5-substituted pyrimidines; nonsteroidal anti-inflammatory drugs (NSAIDs), such as acemetacin, amtolmetin, azapropazone, benorilate, benoxaprofen, benzydamine hydrochloride, bromfenal, bufexamac, butibufen, carprofen, celecoxib, choline salicylate, diclofenac dipyone, droxicam, etodolac, etofenamate, etoricoxib, felbinac, fentiazac, floctafenine, ibuprofen, indoprofen, isoxicam, lomoxicam, loxoprofen, licofelone, fepradinol, magnesium salicylate, meclofenamic acid, meloxicam, morniflumate, niflumic acid, nimesulide, oxaprozen, piketoprofen, priazolac, pirprofen, propyphenazone, proquazone, rofecoxib, salalate, sodium salicylate, sodium thiosalicylate, suprofen, tenidap, tiaprofenic acid, trolamine salicylate, zomepirac, aclofenac, aloxiprin, naproxen, aproxen, aspirin, diflunisal, fenoprofen, indomethacin, mefenamic acid, piroxicam, phenylbutazone, salicylamide, salicylic acid, sulindac, desoxysulindac, tenoxicam, tramadol, ketoralac, clonixin, fenbufen, benzydamine hydrochloride, meclofenamic acid, flufenamic acid, or tolmetin; ganciclovir; glucocorticoids such as cortisol or aldosterone; anti-inflammatory agents, such as cyclooxygenase inhibitors; 5-lipoxygenase inhibitors; leukotriene receptor agents; purine agents, such as azathioprine and mycophenolate mofetil (MMF); alkylating agents, such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde; cyclosporine; 6 mercaptopurine; corticosteroids, including oral glucocorticosteroids or glucocorticoid analogs, e.g., prednisone; methylprednisolone, including SOLU-MEDROL® and methylprednisolone sodium succinate, triamcinolone, and betamethasone, dexamethasone; aminosalicylate; azathioprine, calcineurin inhibitors, such as cyclosporine, tacrolimus (FK-506), and sirolimus (rapamycin); RS-61443 (mycophenolate mofetil); dihydrofolate reductase inhibitors, such as methotrexate (oral or subcutaneous); anti-malarial agents, such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; sulfasalazine (AZULFIDINE); hydroxychloroquine (PLAQUENIL); mitoxantrone (NOVANTRONE®; Immunex Corporation), interferon β-1α (AVONEX®; Ares-Sorono Group), interferonβ-1b (BETASERON®; Berlex Laboratories, Inc.); glatiramer acetate (COPAXONE®; Teva Pharmaceuticals); antibiotics, such as FLAGYL® (metronidazole) or CIPRO® (Ciprofloxacin); and combinations and derivatives thereof.
In some embodiments, the primary anti-inflammatory agent and the secondary anti-inflammatory agent are directed against the same chemokine/chemokine receptor. In other embodiments, the primary anti-inflammatory agent and the secondary anti-inflammatory agent are directed against different chemokine/chemokine receptor. Table 2 describes the association between inflammatory disease and certain chemikine and chemikine receptors.
The anti-inflammatory agents may be administered to the subject with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In certain embodiments, the anti-inflammatory agent(s) may be administered directly to an inflammed tissue. For example, in the case of inflammatory bowel disorders, mucosal tissue may be directly contacted with the anti-inflammatory agent(s). For skin inflammatory diseases such as psoriasis, dermal tissue may be contacted directly with the anti-inflammatory agent(s) in a cream, lotion, or ointment. For asthma, pulmonary tissue, e.g., bronchoalveolar tissue may be contacted by inhalation of a liquid or powder aspirate. The anti-inflammatory agent may also be placed on a solid support such as a sponge or gauze for administration against the target chemokine to the affected tissues.
The anti-inflammatory agents of the instant application can be administered in the usually accepted pharmaceutically acceptable carriers. Acceptable carriers include, but are not limited to, saline, buffered saline, and glucose in saline. Solid supports, liposomes, nanoparticles, microparticles, nanospheres or microspheres may also be used as carriers for administration of the anti-inflammatory agents.
The appropriate dosage (“therapeutically effective amount”) of the anti-inflammatory agents will depend, for example, on the condition to be treated, the severity and course of the condition, the mode of administration, whether the antibody or agent is administered for preventive or therapeutic purposes, the bioavailability of the particular agent(s), previous therapy, the age and weight of the patient, the patient's clinical history and response to the antibody, the type of the anti-inflammatory agent used, discretion of the attending physician, etc. The anti-inflammatory agent is suitably administered to the patent at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards. The anti-inflammatory agent may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.
As a general proposition, the therapeutically effective amount of the anti-inflammatory agent (e.g., antibodies and/or anti-inflammatory small molecule compounds) is administered will be in the range of about 1 ng/kg body weight/day to about 100 mg/kg body weight/day whether by one or more administrations. In a particular embodiments, each anti-inflammatory agent is administered in the range of from about 1 ng/kg body weight/day to about 10 mg/kg body weight/day, about 1 ng/kg body weight/day to about 1 mg/kg body weight/day, about 1 ng/kg body weight/day to about 100 μg/kg body weight/day, about 1 ng/kg body weight/day to about 10 μg/kg body weight/day, about 1 ng/kg body weight/day to about 1 μg/kg body weight/day, about 1 ng/kg body weight/day to about 100 ng/kg body weight/day, about 1 ng/kg body weight/day to about 10 ng/kg body weight/day, about 10 ng/kg body weight/day to about 100 mg/kg body weight/day, about 10 ng/kg body weight/day to about 10 mg/kg body weight/day, about 10 ng/kg body weight/day to about 1 mg/kg body weight/day, about 10 ng/kg body weight/day to about 100 μg/kg body weight/day, about 10 ng/kg body weight/day to about 10 μg/kg body weight/day, about 10 ng/kg body weight/day to about 1 μg/kg body weight/day, 10 ng/kg body weight/day to about 100 ng/kg body weight/day, about 100 ng/kg body weight/day to about 100 mg/kg body weight/day, about 100 ng/kg body weight/day to about 10 mg/kg body weight/day, about 100 ng/kg body weight/day to about 1 mg/kg body weight/day, about 100 ng/kg body weight/day to about 100 μg/kg body weight/day, about 100 ng/kg body weight/day to about 10 μg/kg body weight/day, about 100 ng/kg body weight/day to about 1 μg/kg body weight/day, about 1 μg/kg body weight/day to about 100 mg/kg body weight/day, about 1 μg/kg body weight/day to about 10 mg/kg body weight/day, about 1 μg/kg body weight/day to about 1 mg/kg body weight/day, about 1 μg/kg body weight/day to about 100 μg/kg body weight/day, about 1 μg/kg body weight/day to about 10 μg/kg body weight/day, about 10 μg/kg body weight/day to about 100 mg/kg body weight/day, about 10 μg/kg body weight/day to about 10 mg/kg body weight/day, about 10 μg/kg body weight/day to about 1 mg/kg body weight/day, about 10 μg/kg body weight/day to about 100 μg/kg body weight/day, about 100 μg/kg body weight/day to about 100 mg/kg body weight/day, about 100 μg/kg body weight/day to about 10 mg/kg body weight/day, about 100 μg/kg body weight/day to about 1 mg/kg body weight/day, about 1 mg/kg body weight/day to about 100 mg/kg body weight/day, about 1 mg/kg body weight/day to about 10 mg/kg body weight/day, about 10 mg/kg body weight/day to about 100 mg/kg body weight/day.
In other embodiments, the anti-inflammatory agent (e.g., antibodies and/or anti-inflammatory small molecule compounds) is administered at a dose of 500 μg to 20 g every three days, or 25 mg/kg body weight every three days.
In other embodiments, each anti-inflammatory agent is administered in the range of about 10 ng to about 100 ng per individual administration, about 10 ng to about 1 μg per individual administration, about 10 ng to about 10 μg per individual administration, about 10 ng to about 100 μg per individual administration, about 10 ng to about 1 mg per individual administration, about 10 ng to about 10 mg per individual administration, about 10 ng to about 100 mg per individual administration, about 10 ng to about 1000 mg per injection, about 10 ng to about 10,000 mg per individual administration, about 100 ng to about 1 μg per individual administration, about 100 ng to about 10 μg per individual administration, about 100 ng to about 100 μg per individual administration, about 100 ng to about 1 mg per individual administration, about 100 ng to about 10 mg per individual administration, about 100 ng to about 100 mg per individual administration, about 100 ng to about 1000 mg per injection, about 100 ng to about 10,000 mg per individual administration, about 1 μg to about 10 μg per individual administration, about 1 μg to about 100 μg per individual administration, about 1 μg to about 1 mg per individual administration, about 1 μg to about 10 mg per individual administration, about 1 μg to about 100 mg per individual administration, about 1 μg to about 1000 mg per injection, about 1 μg to about 10,000 mg per individual administration, about 10 μg to about 100 μg per individual administration, about 10 μg to about 1 mg per individual administration, about 10 μg to about 10 mg per individual administration, about 10 μg to about 100 mg per individual administration, about 10 μg to about 1000 mg per injection, about 10 μg to about 10,000 mg per individual administration, about 100 μg to about 1 mg per individual administration, about 100 μg to about 10 mg per individual administration, about 100 μg to about 100 mg per individual administration, about 100 μg to about 1000 mg per injection, about 100 μg to about 10,000 mg per individual administration, about 1 mg to about 10 mg per individual administration, about 1 mg to about 100 mg per individual administration, about 1 mg to about 1000 mg per injection, about 1 mg to about 10,000 mg per individual administration, about 10 mg to about 100 mg per individual administration, about 10 mg to about 1000 mg per injection, about 10 mg to about 10,000 mg per individual administration, about 100 mg to about 1000 mg per injection, about 100 mg to about 10,000 mg per individual administration and about 1000 mg to about 10,000 mg per individual administration. The chemotherapeutic agent contained in the PBM nanoparticles may be administered daily, or every 2, 3, 4, 5, 6 and 7 days, or every 1, 2, 3 or 4 weeks.
In other particular embodiments, the amount of the anti-inflammatory agent administered at a dose of about 0.0006 mg/day, 0.001 mg/day, 0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day, 0.06 mg/day, 0.1 mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6 mg/day, 10 mg/day, 30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600 mg/day, 1000 mg/day, 2000 mg/day, 5000 mg/day or 10,000 mg/day. As expected, the dosage will be dependant on the condition, size, age and condition of the patient.
Dosage can be tested in several animal models that can partially mimic chronic ulcerative colitis. The most widely used model is the 2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model, which induces chronic inflammation and ulceration in the colon. When TNBS is introduced into the colon of susceptible mice via intra-rectal instillation, it induces T-cell mediated immune response in the colonic mucosa, in this case leading to a massive mucosal inflammation characterized by the dense infiltration of T-cells and macrophages throughout the entire wall of the large bowel. Moreover, this histopathologic picture is accompanied by the clinical picture of progressive weight loss (wasting), bloody diarrhea, rectal prolapse, and large bowel wall thickening.
Another colitis model uses dextran sulfate sodium (DSS), which induces an acute colitis manifested by bloody diarrhea, weight loss, shortening of the colon and mucosal ulceration with neutrophil infiltration. DSS-induced colitis is characterized histologically by infiltration of inflammatory cells into the lamina propria, with lymphoid hyperplasia, focal crypt damage, and epithelial ulceration. These changes are thought to develop due to a toxic effect of DSS on the epithelium and by phagocytosis of lamina propria cells and production of TNF-alpha and IFN-gamma. Despite its common use, several issues regarding the mechanisms of DSS about the relevance to the human disease remain unresolved. DSS is regarded as a T cell-independent model because it is observed in T cell-deficient animals such as SCID mice.
The administration of the anti-inflammatory agent of the present application can be evaluated in the TNBS or DSS models for amelioration of gastrointestinal disease. CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and CXCR5 are believed to play a role in the inflammatory response in inflammatory bowel disorders, including colitis, and the neutralization of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and CXCR5 activity by administrating the anti-inflammatory agent of the present application can provide a potential therapeutic approach for gastrointestinal inflammatory diseases, including IBD.
As shown in the Table 2, the particular chemokines which give rise to inflammatory diseases differ with the disease. They also differ among individuals. Hence, it is wise, when treating an individual, to identify the particular chemokines which are increased in the tissues of the patient. By exposing patient tissue samples to the particular antibodies against each of the chemokines and evaluating the amount of antibody/chemokine binding, it is possible to evaluate the level of expression for each chemokine to enable a determination of the appropriate type and amount of antibodies to administer for a given inflammatory disease.
The antibody may be administered, as appropriate or indicated, a single dose as a bolus or by continuous infusion, or as multiple doses by bolus or by continuous infusion. Multiple doses may be administered, for example, multiple times per day, once daily, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeks or monthly. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.
Another aspect of the present application relates to compositions and kits for treating or preventing inflammatory conditions. In one embodiment, the composition comprises an anti-inflammatory agent capable of (1) inhibiting the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR5 and/or CXCR3; (2) inhibiting the interaction between any one of CXCL9, CXCL10, CXCL11, CXCL13, CXCR5 and/or CXCR3, or (3) inhibiting a biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR5 and/or CXCR3, wherein the anti-inflammatory agent is an antibody, antibody fragment, short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide, aptamer-siRNA chimera, single stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, external guide sequence, agent-encoding expression vector, and a pharmaceutically acceptable carrier.
The composition of the present invention may contain a single type of antibody directed against any one of CXCL9, CXCL10, CXCL11, CXCL13, CXCR5 and CXCR3, or two or more antibodies directed against the same chemokine or chemokine receptor, different chemokines or chemokine receptors, or combinations thereof as described above. The composition may also contain therapeutically effective amounts of other anti-inflammatory agents as described above.
As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, lubricants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions. In certain embodiments, the pharmaceutically acceptable carrier comprises serum albumin.
The pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intrathecal, intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the injectable composition should be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the requited particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a neuregulin) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the pharmaceutical compositions are formulated into ointments, salves, gels, or creams as generally known in the art.
In certain embodiments, the pharmaceutical composition is formulated for sustained or controlled release of the active ingredient. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and Tables, are incorporated herein by reference.
Primer Design. Messenger RNA sequences for CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 were obtained from the NIH-NCBI gene bank database (Table 1). Primers were designed using the BeaconJ 2.0 computer program. Thermodynamic analysis of the primers was conducted using computer programs: Primer Premier) and MIT Primer 3. The resulting primer sets were compared against the entire human genome to confirm specificity.
Real Time PCR Analysis.
Lymphocytes or inflamed tissues were cultured in RMPI-1640 containing 10% fetal calf serum, 2% human serum, supplemented with non-essential amino acids, L-glutamate, and sodium pyruvate (complete media). Additionally, primary inflammatory and normal-paired matched tissues were obtained from clinical isolates (Clinomics Biosciences, Frederick, Md. and UAB Tissue Procurement, Birmingham, Ala.). Messenger RNA (mRNA) was isolated from 106 cells using TriReagent (Molecular Research Center, Cincinnati, Ohio) according to manufacturers protocols. Potential genomic DNA contamination was removed from these samples by treatment with 10 U/μl of RNase free DNase (Invitrogen, San Diego, Calif.) for 15 minutes at 37° C. RNA was then precipitated and resuspended in RNA Secure (Ambion, Austin, Tex.). cDNA was generated by reverse transcribing approximately 2 μg of total RNA using Taqman7 reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) according to manufacturers protocols. Subsequently, cDNAs were amplified with specific human cDNA primers, to CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2, using SYBR7 Green PCR master mix reagents (Applied Biosystems) according to manufacturers protocol. The level of copies of mRNA of these targets were evaluated by real-time PCR analysis using the BioRad Icycler and software (Hercules, Calif.).
Anti-Sera Preparation.
The 15 amino acid peptides from chemokines CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 (Table 1) were synthesized (Sigma Genosys, The Woodlands, Tex.) and conjugated to hen egg lysozyme (Pierce, Rockford, Ill.) to generate the antigens for subsequent immunizations for anti-sera preparation or monoclonal antibody generation. The endotoxin levels of chemokine peptide conjugates were quantified by the chromogenic Limulus amebocyte lysate assay (Cape Cod, Inc., Falmouth, Miss.) and shown to be <5 EU/mg. 100 μg of the antigen was used as the immunogen together with complete Freund's adjuvant Ribi Adjuvant system (RAS) for the first immunization in a final volume of 1.0 ml. This mixture was administered in 100 ml aliquots on two sites of the back of the rabbit subcutaneously and 400 ml intramuscularly in each hind leg muscle. Three to four weeks later, rabbits received 100 μg of the antigen in addition to incomplete Freund's adjuvant for 3 subsequent immunizations. Anti-sera were collected when antibody titers reached 1:1,000,000. Subsequently, normal or anti-sera were heat-inactivated and diluted 1:50 in PBS.
Monoclonal Antibody Preparation.
The 15 amino acid peptides from chemokines CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 (Sequences 1 through 30) were synthesized (Sigma Genosys) and conjugated to hen egg lysozyme (Pierce) to generate the Antigen@ for subsequent immunizations for anti-sera preparation or monoclonal antibody generation. The endotoxin levels of chemokine peptide conjugates were quantified by the chromogenic Limulus amebocyte lysate assay (Cape Cod, Inc., Falmouth, Miss.) and shown to be <5 EU/mg. 100 μg of the antigen was used as the immunogen together with complete Freund's adjuvant Ribi Adjuvant system (RAS) for the first immunization in a final volume of 200 μl. This mixture was subcutaneously administered in 100 μl aliquots at two sites of the back of a rat, mouse, or immunoglobulin-humanized mouse. Two weeks later, animals received 100 μg of the antigen in addition to incomplete Freund's adjuvant for 3 subsequent immunizations. Serum were collected and when anti-CXCL9, -CXCL10, -CXCL11, -CCRL1, -CCRL2, -CCR5, -CCL1, -CCL2, -CCL3, -CCL4, -CCL4L1, -CCL5, -CCL7, -CCL8, -CCL14-1, -CCL14-2, -CCL14-3, -CCL15-1, -CCL15-2, -CCL16, -CCL19, -CCL23-1, -CCL23-2, -CCL24, -CCL26, -CCR6, -CCL20, and -CCL25, -CCL25-1, -CCL25-2 antibody titers reached 1:2,000,000, hosts were sacrificed and splenocytes were isolated for hybridoma generation.
B cells from the spleen or lymph nodes of immunized hosts were fused with immortal myeloma cell lines (e.g., YB2/0). Hybridomas were next isolated after selective culturing conditions (i.e., HAT-supplemented media) and limiting dilution methods of hybridoma cloning. Cells that produce antibodies with the desired specificity were selected using ELISA. Hybridomas from normal rats or mice were humanized with molecular biological techniques in common use. After cloning a high affinity and prolific hybridoma, antibodies were isolated from ascites or culture supernatants and adjusted to a titer of 1:2,000,000 and diluted 1:50 in PBS.
Anti-Sera or Monoclonal Antibody Treatment.
Knockout or transgenic mice (8 to 12 weeks old, Charles River Laboratory, Wilmington, Mass.) that spontaneous—or when treated—develop inflammatory diseases were treated with 200 μl intraperitoneal injections of either anti-sera or monoclonal antibodies specific for each of the chemokines every three days. The inflammatory disease state of the host was next monitored for progression or regression of disease.
Cytokine Analysis by ELISA.
The serum level of IL-2, -IL-6, -TNF-α, and -IFN-γ were determined by ELISA, following the manufacturers instructions (E-Biosciences, San Diego, Calif.). Plates were coated with 100 μl of the respective capture antibody in 0.1 M bicarbonate buffer (pH 9.5) and incubated O/N at 4° C. After aspiration and washing with wash buffer, the wells were blocked with assay diluent for 1 hour at RT. Samples and standards were added and incubated for 2 hours at RT. Next, 100 μl of detection antibody solutions were added and incubated for 1 hour. 100 μl of avidin-HRP solution was added and incubated for 30 minutes. Subsequently, 100 μl Tetramethylbenzidine (TMB) substrate solution was added and allowed to react for 20 minutes. 50 μl of the stop solution was added and plates were read at 450 nm. The cytokine ELISA assays were capable of detecting >15 pg/ml for each assay.
Cytokine Analysis by Multiplex Cytokine ELISA.
The T helper cell derived cytokines, IL-1α, IL-1β, IL-2, IL-12, IFN-γ, TNF-α, in serum were also determined by Beadlyte mouse multi-cytokine detection system kit provided by BioRad, following manufacturer instructions. Filter bottom plates were rinsed with 100 μl of bio-plex assay buffer and removal using a Millipore Multiscreen Separation Vacuum Manifold System (Bedford, Mass.), set at 5 in Hg. IL-1α, IL-1β, IL-2; IL-12, IFN-γ, TNF-α beads in assay buffer were added into wells. Next, 50 μl of serum or standard solution were added and the plates were incubated for 30 minutes at RT with continuous shaking (setting 3) using a Lab-Line Instrument Titer Plate Shaker (Melrose, Ill.), after sealing the plates. The filter bottom plates were washed 2 times, as before, and centrifuged at 300×g for 30 seconds. Subsequently, 50 μl of anti-mouse IL-1α, IL-1β, IL-2, IL-12, IFN-γ, TNF-α antibody-biotin reporter solution was added in each well followed by incubation with continuous shaking for 30 minutes followed by centrifugation at 300×g for 30 seconds. The plates were washed 3 times with 100 μl of bio-plex assay buffer as before. Next, 50 μl streptavidin-phycoerythrin solution was added to each well and incubated with continuous shaking for 10 minute at RT. 125 μl of bio-plex assay buffer was added and Beadlyte readings were measured using a Luminexl instrument (Austin, Tex.). The resulting data was collected and calculated using Bio-plexl software (Bio-Rad). The cytokine Beadlyte assays were capable of detecting >5 pg/ml for each analyte.
Serum Amyloid Protein A (BAA) ELISA.
The SAA levels were determined by ELISA using a kit supplied by Biosource International, (Camarillo, Calif.). Briefly, 50 μl of SAA-specific monoclonal antibody solution was used to coat micro-titer strips to capture SAA. Serum samples and standards were added to wells and incubated for 2 hours at RT. After washing in the assay buffer, the HRP-conjugated anti-SAA monoclonal antibody solution was added and incubated for 1 hour at 37° C. After washing, 100 μl Tetramethylbenzidine (TMB) substrate solution was added and the reaction was stopped after incubation for 15 minutes at RT. After the stop solution was added, the plates were read at 450 nm.
Histology and Pathology Scoring.
Fixed tissues were sectioned at 6 μm, and stained with hematoxylin and eosin for light microscopic examination. The intestinal lesions were multi-focal and of variable severity, the grades given to any section of intestine took into account the number of lesions as well as their severity. A score (0 to 4) was given, based on the following criteria: (Grade 0) no change from normal tissue. (Grade 1) 1 or a few multi-focal mononuclear cell infiltrates, minimal hyperplasia and no depletion of mucus. (Grade 2) lesions tended to involve more of the mucosa and lesions had several multi-focal, yet mild, inflammatory cell infiltrates in the lamina propria composed of mononuclear cells, mild hyperplasia, epithelial erosions were occasionally present, and no inflammation was noticed in the sub-mucosa. (Grade 3) lesions involved a large area of mucosa or were more frequent than Grade 2, where inflammation was moderate and often involved in the sub-mucosa as well as moderate epithelial hyperplasia, with a mixture of mononuclear cells and neutrophils. (Grade 4) lesions usually involved most of the section and were more severe than Grade 3 lesions. Additionally, Grade 4 inflammations were more severe and included mononuclear cell and neutrophils; epithelial hyperplasia was marked with crowding of epithelial cells in elongated glands. The summation of these score provide a total inflammatory disease score per mouse. The disease score could range from 0 (no change in any segment) to a maximum of 12 with Grade 4 lesions of segments.
Data Analysis.
SigmaStat 2000 (Chicago, Ill.) software was used to analyze and confirm the statistical significance of data. The data were subsequently analyzed by the Student's t-test, using a two-factor, unpaired test. In this analysis, treated samples were compared to untreated controls. The significance level was set at p<0.05.
Semiquantitative RT-PCR Identification of Molecular Targets.
RT-PCR products obtained using CXCL9-, CXCL10-, CXCL11-, CCRL1-, CCRL2-, CCR5-, CCL1-, CCL2-, CCL3-, CCL4-, CCL4L1-, CCL5-, CCL7-, CCL8-, CCL14-1-, CCL14-2-, CCL14-3-, CCL15-1-, CCL15-2-, CCL16-, CCL19-, CCL23-1-, CCL23-2-, CCL24-, CCL26-, CCR6-, CCL20-, and CCL25-, CCL25-1-, CCL25-2-specific primer sets did not cross react with other gene targets due to exclusion of primers that annealed to host sequences. The primers used produced different size amplicon products relative the polymorphisms that resulted in CCL4 versus CCL4L1, CCL14-1, CCL14-2, versus CCL14-3, CCL15-1 versus CCL15-2, CCL23-1 versus CCL23-2, and CCL25, CCL25-1, versus CCL25-2. To this end, RT-PCR analysis of tissue from subjects exhibiting anaphylaxis, arthritis (e.g., rheumatoid, psoriatic), asthma, allergies (e.g., drug, insect, plant, food), atherosclerosis, delayed type hypersensitivity, dermatitis, diabetes (e.g., mellitus, juvenile onset), graft rejection, inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis, enteritis), multiple sclerosis, myasthemia gravis, pneumonitis, psoriasis, nephritis, rhinitis, spondyloarthropathies, scheroderma, systemic lupus, or thyroiditis revealed that CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, CCL25-2 were differentially expressed by inflammatory host cells.
In Vivo Inflammatory Disease Inhibition.
Mammals that develop anaphylaxis, septic shock, arthritis (e.g., rheumatoid, psoriatic), asthma, allergies (e.g., drug, insect, plant, food), atherosclerosis, bronchitis, chronic pulmonary obstructive disease, delayed type hypersensitivity, dermatitis, diabetes (e.g., mellitus, juvenile onset), graft rejection, Grave's disease, Hashimoto's thyroiditis, inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis, enteritis), interstitial cystitis, multiple sclerosis, myasthemia gravis, pneumonitis, psoriasis, nephritis, rhinitis, spondyloarthropathies, scheroderma, systemic lupus erythematosus, or thyroiditis were allowed to develop the inflammatory disease of interest. Antibodies directed against CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, or CCL25, CCL25-1, CCL25-2 differentially affected the progression and regression of inflammatory disease as determined by histological scoring and comparing pre- and post-treatment serum levels of IFN-γ, IL-1α, IL-1β, IL-6, IL-12, TNF-α, amyloid protein A. Antibodies directed towards CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, or CCL25, CCL25-1, CCL25-2 effectively lead to the both regression and impeding progression of inflammatory disease as determined by histological scoring and comparing pre- and post-treatment serum levels of IFN-γ, IL-1α, IL-1β, IL-6, IL-12, TNF-α, amyloid protein A.
As indicated previously, the chemokines used in the methods of the invention are known. Their accession numbers for the protein sequences are identified in Table 1.
As shown in the table, the particular chemokines which give rise to inflammatory diseases differ with the disease. They also differ among individuals. Hence, it is wise, when treating an individual, to identify the particular chemokines which are increased in the tissues of the patient. Using the antibodies produced against each of the chemokines and exposing the tissue samples from the patient to the particular antibodies, then evaluating the amount of antibody/chemokine binding, it is possible to evaluate the level of expression for each chemokine and to administer to the patient the particular antibodies that will bind the excessive chemokine. This tailored approach to treatment of inflammatory disease is novel, and a particularly valuable aspect of the invention.
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This analysis shows that CXCR3+ CD4+ T cells, which consisted of both CD45RB populations induced induction of colitis in TCR β×δ−/− mice (Panel C).
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Total fecal IgG and IgA levels were determined to correlate changes in intestinal Abs during CD. As shown in
Control groups showed moderately higher levels of serum IL-12 p40, compared with IP-10 Ab-treated mice (
Observed pathologic changes included small multifocal infiltrates in the lamina propria of the ascending and transverse colon. These infiltrates consisted of lymphocytes and occasional small numbers of neutrophils. Epithelial cells were not hypertrophied in the IP-10-inhibited group. Multinucleated, enlarged epithelial, and elongated glandular cells were also present in control mice. However, colitis progression was more aggressive in control groups, as noted by multifocal lesions in all regions of the large intestine, especially in colon. The results show a marked improvement in colitis associated with CXCL10 blockade.
Chronic colitis in the IL-10 corresponded with an increase in SAA levels (>300 μg/mL)(
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The mice that received anti-CXCL10 Ab showed a significant reduction in intestinal inflammation. An increase in leukocyte infiltrates (
The colon pathology of control samples showed hypertrophied epithelial layers at multiple sites, with only a few inflammatory infiltrates and low expression of CXCL9, CXCL10, CXCL11 and CXCR3 (
Following M. avium subsp. paratuberculosis challenge, IFN-γ and TNF-α levels were significantly higher (˜6-fold) in sera of IL-10−/− challenged with live M. avium subsp. paratuberculosis than in control mice; mice exposed to heat-killed M. avium subsp. paratuberculosis had ˜2-fold greater TNF-α and IFN-γ responses than those of controls, but these differences were not significant (
While total IgG1, IgG2, IgG3, and IgG4 subclass Abs were significantly higher in the sera of IBD patients compared to healthy donors (data not shown), the profile of the IgG humoral response in IBD patients also revealed increases in Mycobacteria-specific IgG1 and IgG2 Abs (
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The intestinal tissues of mice challenged with Mycobacteria showed higher increases in leukocyte infiltrates, which consisted of lymphocytes and occasionally polymorphonuclear cells as well as a higher frequency of lymphoid follicles in live versus heat-killed Mycobacteria-challenged groups (
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CYP-induced cystitis in mice led to substantial increases in serum levels of CXCL10>>CXCL9 when compared with the levels in unaffected controls (
Control Ab-treated mice given CYP showed pathological signs of cystitis (i.e., urinary bladder inflammation, discontinuous uroepitheium). However, affected mice treated with anti-CXCL10 Ab displayed a reduction in cystitis, as noted by a decrease in urinary bladder leukocyte infiltrates (
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To investigate local and peripheral changes in Th1 and inflammatory cytokine expression during CYP-induced cystitis, the levels of IFN-γ, IL-12p40, and TNF-α mRNAs expressed by leukocytes isolated from the spleen, iliac lymph nodes and urinary bladder were measured by quantitative RT-PCR analysis. CYP-induced mice receiving control Ab exhibited substantial decreases in the expression of IFN-γ, IL-12p40, and TNF-α mRNAs by splenocytes; however, this treatment significantly increased the expression of cytokines by urinary bladder leukocytes than compared to unaffected mice (
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The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and is not intended to detail all those obvious modifications and variations of it that will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the components and steps in any sequence that is effective to meet the objectives there intended, unless the context specifically indicates the contrary. All the references cited in the specification are herein incorporated by reference in their entirely.
This application is a Continuation of U.S. patent application Ser. No. 13/535,202 filed on Jun. 27, 2012, which is a continuation-in-part of U.S. patent application Ser. No. 13/105,406 filed on May 11, 2011, now U.S. Pat. No. 8,318,170, which is a continuation of U.S. patent application Ser. No. 10/712,393, filed on Nov. 14, 2003, now U.S. Pat. No. 7,964,194, which claims priority to U.S. Provisional Patent Application No. 60/426,350, filed Nov. 15, 2002. The entirety of all of the aforementioned applications is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60426350 | Nov 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13535202 | Jun 2012 | US |
| Child | 14843811 | US | |
| Parent | 10712393 | Nov 2003 | US |
| Child | 13105406 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13105406 | May 2011 | US |
| Child | 13535202 | US |
