Patent Grant
7091310
Not applicable.
Not applicable.
The instant application contains a Sequence Listing which has been submitted via triplicate CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. The CD-R, recorded on Sep. 8, 2003 are labeled “CRF”, “Copy 1” and “Copy 2”, respectively, and each contains only one identical 1.83 MB file 5929600003.APP.
Chemokines (chemoattractant cytokines) are a family of homologous serum proteins of between 7 and 16 kDa, which were originally characterized by their ability to induce migration of leukocytes. Most chemokines have four characteristic cysteines (Cys), and depending on the motif displayed by the first two cysteines, they have been classified into CXC or alpha, CC or beta, C or gamma, and CX3C or delta chemokine classes. Two disulfide bonds are formed between the first and third cysteine and between the second and fourth cysteine. In general, it was thought that the disulfide bridges were required, and Clark-Lewis and co-workers reported that, at least for IL-8, the disulfide bridges are critical for chemokine activity (Clark-Lewis et al., J. Biol. Chem. 269:16075–16081, 1994). The only exception to having four cysteines is lymphotactin, which has only two cysteine residues. Thus, lymphotactin manages to retain a functional structure with only one disulfide bond.
In addition, the CXC, or alpha, subfamily has been divided into two groups depending on the presence of the ELR motif (Glu-Leu-Arg) preceding the first cysteine: the ELR-CXC chemokines and the non-ELR-CXC chemokines (see, e.g., Clark-Lewis, supra, and Belperio et al., “CXC Chemokines in Angiogenesis,” J. Leukoc. Biol. 68:1–8, 2000).
ELR-CXC chemokines, such as IL-8, are generally strong neutrophil chemoattractants while non-ELR chemokines, such as IP-10, and SDF-1, predominantly recruit lymphocytes. CC chemokines, such as RANTES, MIP-1-alpha, MCP-1, generally function as chemoattractants for monocytes, basophils, eosinophils, and T-cells but not neutrophils. In general, chemokines are chemotactic agents that recruit leukocytes to the sites of injuries.
Specific Chemokines
IL-8
Interleukin-8 (IL-8 or CXCL8) was first identified in 1987 as a chemokine with the ability to specifically activate neutrophils. Upon exposure to IL-8, neutrophils are activated, and change their shape. Neutrophils are activated by a process that is probably mediated by an increase in intracellular calcium levels. This activation allows neutrophils to migrate across the vascular wall. Secretion of IL-8 can occur from a wide variety of cells, including other leukocytes, fibroblasts, endothelial cells, and epithelial cells in response to ischemia and trauma.
IP-10
Interferon-inducible protein-10 (IP-10 or CXCL10) is induced by interferon-gamma and TNF-alpha, and is produced by keratinocytes, endothelial cells, fibroblasts and monocytes. IP-10 is thought to play a role in recruiting activated T cells to sites of tissue inflammation (Dufour, et al., “IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking,” J Immunol., 168:3195–204, 2002). In addition, IP-10 may play a role in hypersensitivity. It may also play a role in the genesis of inflammatory demyelinating neuropathies (Kieseier, et al., “Chemokines and chemokine receptors in inflammatory demyelinating neuropathies: a central role for IP-10,” Brain 125:823–34, 2002).
MIP-1α
Macrophage inflammatory protein-1-alpha (MIP-1α, MIP-1-alpha or CCL3) is a factor produced by macrophages in response to their stimulation by bacterial endotoxins. It activates neutrophils, eosinophils, and basophils and appears to play a role in inflammation. Additionally, it is especially potent as a basophil agonist, and appears to act through a rapid rise in intracellular calcium, and causes the release of histamine, sulfido-leukotrienes, and also plays a role in chemotaxis. MIP-1α may also act to inhibit stem cell proliferation.
RANTES
RANTES (Regulated upon Activation, Normal T-cell Expressed, and presumably Secreted or CCL5) is a chemokine that acts on T-cells, eosinophils and basophils and assists in recruiting leukocytes to inflammatory sites. In particular, it increases the adherence of monocytes to endothelial cells, and selectively supports the migration of certain types of leukocytes. In some cases, RANTES has been shown to activate basophils and causes the release of histamines. It may also be involved in the proliferation and activation of certain types of killer cells.
I-309
I-309 refers to the name of a cDNA clone encoding a chemokine (Miller, et al., “A novel polypeptide secreted by activated human T lymphocytes,” J Immunol., 143(9):2907–16, 1989). I-309 is chemotactic for human monocytes, and additionally activates them. However, it appears to have no effect on neutrophils.
MCP-1
Monocyte chemoattractant (or chemotactic) protein-1 (monocyte chemotactic protein-1, MCP-1 or CCL2) is another CCL chemokine. It is expressed by monocytes, endothelial cells, smooth muscle cells, and certain types of epithelial cells in culture. The expression of MCP1 is induced in human peripheral blood mononuclear leukocytes by phytohemagglutinin (PHA), lipopolysaccharide, and IL1. MCP-1 functions as a chemoattractant for monocytes but not neutrophils. There have been reports that two point mutations are sufficient for MCP-1 to become chemotactic for neutrophils. MCP-1 activates monocytes and macrophages in vivo, as well as basophils. Additionally, it can induce the proliferation and activation of certain types of killer cells.
CCL28
CCL28 (hMEC) is a recently described CC chemokine which may play a particularly important role in homeostasis or inflammatory responses in the gastrointestinal system (Wang et al., J. Biol. Chem. 275:22313–23, 2000).
SDF-1
Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a CXC chemokine that demonstrates in vitro activity with respect to lymphocytes and monocytes but not neutrophils. It is a highly potent in vivo chemoattractant for mononuclear cells. SDF-1 has been shown to induce intracellular actin polymerization in lymphocytes, and to induce a transient elevation of cytoplasmic calcium in some cells.
Chemokine Receptors
The receptors for chemokines are G-protein coupled seven-transmembrane receptors. Based on the chemokine class they bind, the receptors have been named CXCR1, CXCR2, CXCR3, CXCR4, and CXCR5 (all of which bind CXC chemokines); CCR1 through CCR9 (all of which bind CC chemokines); XCR1 (which binds the C chemokine, Lptn); and CX3CR1 (which binds the CX3C chemokine, fractalkine or neurotactin (See Table 1)).
The chemokines and their receptors have received increasing attention in the last few years. In addition to their role in HIV pathogenesis, it is now clear that chemokines participate in many pathological conditions such as inflammation and diseases or conditions associated with autoimmune responses. They also play a very important role in normal homeostasis, including lymphoid development and migration, and the growth of bone. As a result of their role in various physiological processes and pathological conditions and diseases, chemokines have important potential therapeutic applications.
This invention relates in one aspect to the design, preparation, derivation, and use of peptide agonists and antagonists of chemokines referred to herein as chemokine analogs. In preferred embodiments, this invention relates to the design, preparation, derivation, or use of peptide agonists or antagonists of a chemokine selected from the group consisting of IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, and CCL28 (hMEC). In other preferred embodiments, the invention relates to the design, preparation, derivation, or use of chemokine analogs derived from one or more of the seven chemokines: IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, and CCL28. Particularly preferred embodiments are set forth infra in the Detailed Description of the Invention, Examples and Claims.
Another aspect of the invention is directed towards a method for treating disease, disorder or abnormal condition comprising administering to a patient in need of such treatment a therapeutically effective amount of a chemokine analog having a structure selected from the group consisting of sequence a1 (SEQ ID NO:9) to sequence a154 (SEQ ID NO:162), inclusive; sequence b1 (SEQ ID NO:163) to sequence b575 (SEQ ID NO:728), inclusive; sequence c1 (SEQ ID NO:729) to sequence c160 (SEQ ID NO:881), inclusive; sequence d1 (SEQ ID NO:882) to sequence d90 (SEQ ID NO:971), inclusive; sequence e1 (SEQ ID NO:972) to sequence e382 (SEQ ID NO:1350), inclusive; sequence g2 (SEQ ID NO:1351) to sequence g97 (SEQ ID NO:1446), inclusive; and sequence h1 (SEQ ID NO:1447) to sequence h184 (SEQ ID NO:1631), inclusive, in a pharmaceutically acceptable carrier.
In preferred embodiments, said disease, disorder or abnormal condition is selected from the group consisting of autoimmune diseases, acute chronic inflammation, cancer, cardiovascular disease, infectious disease, and inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, atherosclerosis, psoriasis, rhinitis, autoimmunity, and organ transplant rejection. In other preferred embodiments, the administration of the compound of the invention serves to increase the hemocrit, assist in mobilizing and recovering stem cells, stimulate the production of blood cells, assist in vaccine production, or assist in gene therapy.
A further aspect of this invention relates to therapeutic uses of chemokine analogs to cure, to manage, or to prevent a disease or disorder selected from the group consisting of autoimmune diseases, acute chronic inflammation, cancer, cardiovascular disease, infectious disease, and inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, atherosclerosis, psoriasis, rhinitis, autoimmunity, and organ transplant rejection. A further aspect of this invention relates to therapeutic uses of chemokine analogs to increase the hemocrit, assist in mobilizing and recovering stem cells, stimulate the production of blood cells, or assist in vaccine production.
Another aspect of the invention is directed towards providing pharmaceutical compositions of chemokine analogs in order to treat a mammal by enhancing or inhibiting the action of a chemokine on its receptor. An additional aspect of the invention relates to the use of pharmaceutical compositions of analogs of human IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, or CCL28 to treat a human by enhancing or inhibiting the action of IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, or CCL28 on its respective receptor.
A still further aspect of the invention is a method for modulating the activity of a chemokine receptor by contacting this chemokine receptor with a compound comprising a structure selected from the group consisting sequence a1 (SEQ ID NO:9) to sequence a154 (SEQ ID NO:162), inclusive; sequence b1 (SEQ ID NO:163) to sequence b575 (SEQ ID NO:728), inclusive; sequence c1 (SEQ ID NO:729) to sequence c160 (SEQ ID NO:881), inclusive; sequence d1 (SEQ ID NO:882) to sequence d90 (SEQ ID NO:971), inclusive; sequence e1 (SEQ ID NO:972) to sequence e382 (SEQ ID NO:1350), inclusive; sequence g2 (SEQ ID NO:1351) to sequence g97 (SEQ ID NO:1446), inclusive; and sequence h1 (SEQ ID NO:1447) to sequence h184 (SEQ ID NO:1631), inclusive.
Another aspect of the invention consists of using the chemokine analogs of the invention to treat a patient so as to (a) mobilize intracellular calcium in the patient, (b) mobilize leukocytes or more specifically, neutrophils, or (c) decrease the toxic effects of a cytotoxic agent on white blood cells, leukocytes and/or hematopoietic cells.
The invention relates to the design, preparation, derivation, and use of chemokine analogs. In one aspect, this invention is directed to the synthesis or use of chemokine analogs which bind to receptors for any of seven human chemokines: IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, and CCL28 (hMEC). In another aspect, the invention is directed to the synthesis, design, derivation, or use of chemokine analogs or derivatives of one or more of the seven human chemokines: IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, and CCL28 (hMEC). In a further aspect, the invention is directed to the synthesis, design, derivation, or use of agonist or antagonist analogs of one or more of the following seven human chemokines: IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, and CCL28, and derivatives thereof. The invention is not limited in its application to the details of structures and the arrangements of components set forth in the following description or illustrated in the drawings and the figures. Further, it should be understood that in any claimed list or claimed Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the list or Markush group. Additionally, any individual member of the claimed list or the claimed Markush group can be removed from the list or Markush group without affecting the patentability of the remaining members.
The sequences of the seven aforementioned human chemokines are shown below. First are two CXC chemokines: IL-8, and IP-10; second are five CC chemokines: MIP-1α, MCP-1, RANTES, I-309, and CCL28.
The N-terminal region of chemokines is involved in the binding and activating site of its receptor, as well as is the carboxy terminal region. The beta sheet structure that connects the two termini appears to play a role in the stabilization of the CXCR and assuring that the termini are in the proper conformation.
Examples of these analogs are compounds containing structures corresponding to various regions or portions of the chemokines. In preferred embodiments, the chemokine analog comprises an N-terminal region and a C-terminal region joined together by means of a linker. In other preferred embodiments, the amino acid residues of the chemokine or chemokine analog are cyclized, e.g., by etherification of lysine and serine residues or by other means described infra or known in the art. In still other preferred embodiments, the chemokine analog comprises a sequence derived from the wild-type chemokine sequence but with one or more of the cysteines replaced with another amino acid including natural and non-natural amino acids). Other preferred embodiments include chemokine analogs comprising an N-terminal region, an internal region containing up to three anti-parallel β-sheets, a C-terminal region containing an α-helical structure, a combination of the N- and C-terminal regions linked together directly, a combination of a N-terminal and internal region, or a combination of an internal and C-terminal region, or finally a combination of N-terminal, internal and C-terminal regions. The regions selected from the N-terminal, internal and C-terminal regions may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, 30, 35, 40, 41, or 45 amino acids in length.
Examples of such analogs also include a cross combination of one chemokine region to a different region from a different chemokine in the same or different family. These examples include, but are not limited to, regions of two CXC chemokines: IL-8, IP-10, and five CC chemokines: MIP-1α, MCP-1, RANTES, I-309, and CCL28.
Chemokine analogs of the invention are useful for treating or preventing inflammatory conditions, autoimmune disorders, cancer, graft rejection, bacterial infection, viral infection, vascular conditions (for example, atherosclerosis, restenosis, systemic lupus erythematosis, and ischemia-reperfusion), sepsis, tumorigenesis, and angiogenesis; stem cell mobilization as well as vaccine production and blood cell recovery following chemotherapy. Inflammatory conditions contemplated by the present invention include both acute and chronic inflammatory diseases. Chemokine analogs of the inventions may also prove useful in conducting gene therapy; one manner they may assist in the methods of gene therapy is through an arrest of the cell cycle.
Examples of uses of the chemokine analogs in some aspects of the invention include, but are not limited to, treatment or management of arthritis, asthma, colitis/illeitis, psoriasis, atherosclerosis and the like. Examples of uses of the chemokine analogs in some aspects of the invention to treat or manage autoimmune conditions include, but are not limited to, rheumatoid arthritis, multiple sclerosis and other autoimmunological diseases. Examples of uses of the chemokine analogs in some aspects of the invention to treat or manage cancer include, but are not limited to, treatment or management of human malignancy/cancer cell metastasis and relapses. Examples of uses of the chemokine analogs in some aspects of the invention to assist in blood cell recovery include, but are not limited to, blood cell elevation after chemotherapy/radiotherapy and stem cell mobilization for transplantations. Examples of uses of the chemokine analogs in some aspects of the invention for vaccine production include, but are not limited to, enhancement in humoral antibody production, increases in antigen presenting T-cells, increases in dendritic cells and immunological features known as vaccine induction. Chemokines may also play a role in osteoporosis and thus osteoporosis may be treated by chemokine analogs of the invention. Chemokine analogs of the invention may also prove useful in treating genetic disease through gene therapy.
As defined by the present invention a chemokine analog acts as an agonist or an antagonist to a native chemokine. The agonistic activity of the chemokine analogs of the present invention includes mimicking of biological activity induced by corresponding native chemokines. The antagonistic activity of the chemokine analogs of the present invention includes inhibition of biological activity induced by native chemokines. The instant invention also encompasses a chemokine analog that acts as an agonist or an antagonist to a different native chemokine.
Peptides
In this application, the products of the present invention are referred to by various terms, including “analogs” of the present invention, “chemokine mimetics,” “chemokine analogs,” and “chemokine derivatives.” These terms are used interchangeably and denote equivalent compounds. The term “polypeptides of the present invention,” may also be used herein to refer to chemokine analogs. Chemokine analogs of the present invention comprise a structure which comprises a sequence selected from the group set forth as SEQ ID NO:9 through SEQ ID NO:1631, and thus may comprise additional elements such as R-group substituents and a linker selected from the possibilities set forth in the instant invention.
As defined by the present invention, biological activity refers to the biological activity of the native chemokines, as defined and measured by the scientific reports known to those of skill in the art, and exemplified in the following review articles (Daugherty, B. L. et al., “Radiolabeled Chemokine binding assays,” Methods in Molecular Biology (2000) vol. 138, pp129–134, Buser, R. et al. “Calcium Mobilization,” Methods in Molecular Biology (2000) vol. 138, pp143–148, Ponath, P. D. et al., “Transwell Chemotaxis,” Methods in Molecular Biology (2000) vol. 138, pp113–120 Humana Press. Totowa, N.J.). Aspects of biological activity include, but are not limited to, receptor binding, chemotaxis, calcium mobilization, along with other activities recognized by those of skill in the art.
The amino acids are identified in the present application by the conventional one-letter and three-letter abbreviations as indicated below, and are preceded by “L-” to indicate their L-form and by “D-” to refer to their D form. These abbreviations are generally accepted in the peptide art as recommended by the IUPAC-IUB commission in biochemical nomenclature:
All of the peptide sequences set out herein are written according to the generally accepted convention whereby the N-terminal amino acid is on the left and the C-terminal amino acid is on the right.
Chemokine mimetics of the invention may include chemokine derivatives or chemokine analogs and their derivatives, such as C-terminal hydroxymethyl derivatives, O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides and compounds in which a C-terminal phenylalanine residue is replaced with a phenethylamide analogue (e.g., Ser-Ile-phenethylamide as an analog of the tripeptide Ser-Ile-Phe), glycosylated chemokine derivatives, polyethylene glycol modified derivatives, or biotinylated derivatives. Chemokine analogs of the invention include pharmaceutically acceptable salts of the chemokine analogs.
Modifying Groups
In one aspect of the invention, the chemokine analogs of the invention, (such as peptides derived from IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, or CCL28) may be coupled directly or indirectly to at least one modifying group. In some aspects of the invention, the term “modifying group” is intended to include structures that are directly attached to the peptidic structure (e.g., by covalent bonding or covalent coupling), as well as those that are indirectly attached to the peptidic structure (e.g., by a stable non-covalent bond association or by covalent coupling through a linker to additional amino acid residues). In other aspects of the invention the term “modifying group” may also refer to mimetics, analogues or derivatives thereof, which may flank the IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, or CCL28 core peptidic structure. For example, the modifying group can be coupled to the amino-terminus or carboxy-terminus of an IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, or CCL28 peptidic structure, or to a peptidic or peptidomimetic region flanking the core structure. Alternatively, the modifying group can be coupled to a side chain of at least one amino acid residue of an IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, or CCL28 peptidic structure, or to a peptidic or peptido-mimetic region flanking the core domain (e.g., through the epsilon amino group of a lysyl residue(s); through the carboxyl group of an aspartic acid residue(s) or a glutamic acid residue(s); through a hydroxy group of a tyrosyl residue(s), a serine residue(s) or a threonine residue(s); or any other suitable reactive group on an amino acid side chain). In other aspects, modifying groups covalently coupled to the peptidic structure can be attached by means and using methods well known in the art for linking chemical structures, including, for example, amide, alkylamino, sulfide, carbamate or urea bonds.
In some embodiments, the modifying group may comprise a cyclic, heterocyclic or polycyclic group. The term “cyclic group,” as used herein, includes cyclic saturated or unsaturated (i.e., aromatic) group having from 3 to 10; from 4 to 8; or 5, 6, or 7 carbon atoms. Exemplary non-aromatic cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. The term “heterocyclic group” includes optionally substituted, saturated or unsaturated, three- to eight-membered cyclic structures in which one or more skeletal atoms is oxygen, nitrogen, sulfur, or combinations thereof. Cyclic groups may be unsubstituted or substituted at one or more ring positions. A cyclic group may for example be substituted with halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, —CF3, —CN. The cyclic group may also be linked to a substituent, such as halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, —CF3, or —CN, by means of a saturated or unsaturated chain of 1, 2, 3, 4, 5, 6, 7, 8, or more carbon atoms; additionally one or more of the carbon atoms may be replaced with an oxygen, nitrogen, or sulfur atom. Other means of linking these groups are also possible.
In one embodiment of the invention, chemokines and chemokine analogs are designed by replacing all or part of the beta-sheet domain with a linker. In a different embodiment, all or a portion of the amino-terminal domain and all or a portion of the carboxy-terminal domain of a chemokine or chemokine analog are connected with a linker. In another embodiment, the chemokines and chemokine analogs are designed so that there are cyclized by covalent modification between residues of the peptide. In still other embodiments, the cysteines of the chemokines are replaced by other amino acids. In further embodiments, chemokines and chemokine analogs are modified by attaching modifying groups to the amino terminus.
Definitions
The term “heterocyclic group” includes cyclic saturated, unsaturated and aromatic groups having from 3 to 10; from 4 to 8; or 5, 6, or 7 carbon atoms, wherein the ring structure includes about one or more heteroatoms. Heterocyclic groups include pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine, piperazine, morpholine. The heterocyclic ring may be substituted at one or more positions with such substituents as, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, other heterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, —CF3, —CN. Heterocycles may also be bridged or fused to other cyclic groups as described below. A linker may also link the heterocyclic group to such substituents as, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, —CF3, —CN.
The term “polycyclic group” as used herein is intended to refer to two or more saturated, unsaturated or aromatic cyclic rings in which two or more carbons are common to two adjoining rings, so that the rings are “fused rings.” Rings that are joined through non-adjacent atoms are termed “bridged” rings. Each of the rings of the polycyclic group may be substituted with such substituents as described above, as for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, —CF3, or —CN.
The term “alkyl” refers to a saturated aliphatic groups, including straight chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone (C1–C20 for straight chain, C3–C20 for branched chain), or 10 or fewer carbon atoms. In some embodiments, cycloalkyls may have from 4–10 carbon atoms in their ring structure, such as rings made from 5, 6 or 7. Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, having from one to ten carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have chain lengths of ten or less carbons.
The term “alkyl” (or “lower alkyl”) as used throughout the specification and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including alkylcarbonyl and arylcarbonyl groups)), and esters (including alkyloxycarbonyl and aryloxycarbonyl groups), thiocarbonyl, acyloxy, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. The moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and phosphinates), sulfonyls (including sulfates, sulfonamidos, sulfamoyls and sulfonates), or silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), —CF3, —CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, —CF3, —CN, and the like.
The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
The term “aralkyl,” as used herein, refers to an alkyl or alkylenyl group substituted with at least one aryl group. Exemplary aralkyls include benzyl (i.e., phenylmethyl), 2-naphthylethyl, 2-(2-pyridyl)propyl, 5-dibenzosuberyl, and the like.
The term “alkylcarbonyl,” as used herein, refers to —C(O)-alkyl. Similarly, the term “arylcarbonyl” refers to —C(O)-aryl. The term “alkyloxycarbonyl,” as used herein, refers to the group —C(O)—O-alkyl, and the term “aryloxycarbonyl” refers to —C(O)—O-aryl. The term “acyloxy” refers to —O—C(O)—R7, in which R7 is alkyl, alkenyl, alkynyl, aryl, aralkyl or heterocyclyl.
The term “amino,” as used herein, refers to —N(Rα)(Rβ), in which Rα and Rβ are each independently hydrogen, alkyl, alkyenyl, alkynyl, aralkyl, aryl, or in which Rα and Rβ together with the nitrogen atom to which they are attached form a ring having 4–8 atoms. Thus, the term “amino,” as used herein, includes unsubstituted, monosubstituted (e.g., monoalkylamino or monoarylamino), and disubstituted (e.g., dialkylamino or alkylarylamino) amino groups. The term “amido” refers to —C(O)—N(Rα)(Rβ), in which Rα and Rβ are as defined above. The term “acylamino” refers to —N(R′α)C(O)—R7, in which R7 is as defined above and Rα is alkyl.
As used herein, the term “nitro” means —NO2; the term “halogen” designates —F, —Cl, —Br or —I; the term “sulflydryl” means —SH; and the term “hydroxyl” means —OH.
The term “aryl” as used herein includes 5-, 6- and 7-membered aromatic groups that may include from zero to four heteroatoms in the ring, for example, phenyl, pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole, thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics.” The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or the like. Aryl groups can also be part of a polycyclic group. For example, aryl groups include fused aromatic moieties such as naphthyl, anthracenyl, quinolyl, indolyl, and the like.
Modifying groups may also include groups comprising biochemical labels or structures, such as biotin, fluorescent-label-containing groups, light scattering or plasmon resonant particle, a diethylene-triaminepentaacetyl group, a (O)-menthoxyacetyl group, a N-acetylneuraminyl group, a cholyl structure or an iminobiotinyl group. A chemokine analog or chemokine mimetic compound may be modified at its carboxy terminus with a cholyl group according to methods known in the art. Cholyl derivatives and analogs may also be used as modifying groups. For example, a preferred cholyl derivative is Aic (3-(O-aminoethyl-iso)-cholyl), which has a free amino group that can be used to further modify the chemokine mimetic compound. A modifying group may be a “biotinyl structure,” which includes biotinyl groups and analogues and derivatives thereof (such as a 2-iminobiotinyl group). In another embodiment, the modifying group may comprise a fluorescent-label group, e.g., a fluorescein-containing group, such as a group derived from reacting an IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, and CCL28 derived peptidic structure with 5-(and 6-)-carboxyfluorescein, succinimidyl ester or fluorescein isothiocyanate. The chemokine analogs may also be modified by attaching other fluorescent labels including rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin and energy transfer fluorescent dyes or fluorescent ion indicators. In various other embodiments, the modifying group(s) may comprise an N-acetylneuraminyl group, a trans-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetyl group, an (S)-(−)-indoline-2-carboxyl group, a (−)-menthoxyacetyl group, a 2-norbornaneacetyl group, a γ-oxo-5-acenaphthenebutyryl, a (−)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a 2-iminobiotinyl group, a diethylenetriaminepentaacetyl group, a 4-morpholinecarbonyl group, a 2-thiopheneacetyl group or a 2-thiophenesulfonyl group. In other embodiments, light scattering groups, magnetic groups, nanogold, other proteins, a solid matrix, radiolabels, or carbohydrates may be attached.
In still other aspects, the modifying group may be an oligomer, for example, polyethylene glycol, an oligonucleotide, a polypeptide (which may or may not be derived from a chemokine) or one moiety of a binding pair.
Functional Enhancement
A chemokine analog compound of the invention may be further modified to alter the specific properties of the compound while retaining the desired functionality of the compound. For example, in one embodiment, the compound may be modified to alter a pharmacokinetic property of the compound, such as in vivo stability, solubility, bioavailability or half-life. The compound may be modified to label the compound with a detectable substance. The compound may be modified to couple the compound to an additional therapeutic moiety. To further chemically modify the compound, such as to alter its pharmacokinetic properties, reactive groups can be derivatized. For example, when the modifying group is attached to the amino-terminal end of the IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, and CCL28 core domain, the carboxy-terminal end of the compound may be further modified. Potential C-terminal modifications include those that reduce the ability of the compound to act as a substrate for carboxypeptidases. Examples of C-terminal modifiers include an amide group, an ethylamide group and various non-natural amino acids, such as D-amino acids, β-alanine, C-terminal decarboxylation, and a C-terminal alcohol. Alternatively, when the modifying group is attached to the carboxy-terminal end of the aggregation core domain, the amino-terminal end of the compound may be further modified, for example, to reduce the ability of the compound to act as a substrate for aminopeptidases.
Chemokines and chemokine analogs of the invention may be modified by the addition of polyethylene glycol (PEG). PEG modification may lead to improved circulation time, improved solubility, improved resistance to proteolysis, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation (For a review see, Francis et al., International Journal of Hematology 68:1–18, 1998). PEGylation may also result in a substantial reduction in bioactivity.
The chemokine analogs of the invention may also be coupled to a radioisotope such as yttrium-90 or iodine-131 for therapeutic purposes (see, e.g., DeNardo et al., “Choosing an optimal radioimmunotherapy dose for clinical response,” Cancer 94(4 Suppl): 1275–86, 2002; Kaltsas et al., “The value of radiolabelled MIBG and octreotide in the diagnosis and management of neuroendocrine tumours,” Ann Oncol 12 Suppl 2:S47–50, 2001).
Detection Enhancement
A chemokine mimetic compound can be further modified to label the compound by reacting the compound with a detectable substance. In some aspects of the invention, suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, light scattering or plasmon resonant materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic groups which are members of a binding pair and are capable of forming complexes include streptavidin/biotin, avidin/biotin and an antigen/antibody complex (e.g., rabbit IgG and anti-rabbit IgG). Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin and energy transfer fluorescent dyes. An example of a luminescent material includes luminol. Examples of light scattering or plasmon resonant materials include gold or silver particles and quantum dots. Examples of suitable radioactive material include 14C, 123I, 124I, 125I, 131I, Tc99m, 35S or 3H. A chemokine mimetic compound may be radioactively labeled with 14C, either by incorporation of 14C into the modifying group or one or more amino acid structures in the chemokine mimetic compound. Labeled chemokine mimetic compounds may be used to assess the in vivo pharmacokinetics of the compounds, as well as to detect disease progression or propensity of a subject to develop a disease, for example for diagnostic purposes. Tissue distribution chemokine receptors can be detected using a labeled chemokine mimetic compound either in vivo or in an in vitro sample derived from a subject. For use as an in vivo diagnostic agent, a chemokine mimetic compound of the invention may be labeled with radioactive technetium or iodine. A modifying group can be chosen that provides a site at which a chelation group for the label can be introduced, such as the Aic derivative of cholic acid, which has a free amino group. For example, a tyrosine residue within the IL-8, IP-10, MIP-1α, MCP-1, RANTES, I-309, and CCL28 sequence may be substituted with radioactive iodotyrosyl. Any of the various isotopes of radioactive iodine may be incorporated to create a diagnostic or therapeutic agent. 123I (half-life=13.2 hours) may be used for whole body scintigraphy, 124I (half life=4 days) may be used for positron emission tomography (PET), 125I (half life=60 days) may be used for metabolic turnover studies and 131I (half life=8 days) may be used for whole body counting and delayed low resolution imaging studies.
Prodrug
In an alternative chemical modification, a chemokine analog compound of the invention may be prepared in a “prodrug” form, wherein the compound itself does not act as a chemokine analog agonist, but rather is capable of being transformed, upon metabolism in vivo, into a chemokine analog agonist or antagonist compound as defined herein. For example, in this type of compound, the modifying group can be present in a prodrug form that is capable of being converted upon metabolism into the form of an active chemokine analog agonist. Such a prodrug form of a modifying group is referred to herein as a “secondary modifying group.” A variety of strategies are known in the art for preparing peptide prodrugs that limit metabolism in order to optimize delivery of the active form of the peptide-based drug.
Synthesis
Chemokine analog compounds of the invention may be prepared by standard techniques known in the art. A peptide or polypeptide component of a chemokine analog may comprise, at least in part, a peptide synthesized using standard techniques (such as those described by Clark-Lewis, I., Dewald, B., Loetscher, M., Moser, B., and Baggiolini, M., (1994) J. Biol. Chem., 269, 16075–16081). Automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600, Appliedbiosystems/Pioneer). Peptides and polypeptides may be assayed for chemokine receptor agonist or antagonist activity in accordance with standard methods. Peptides and polypeptides may be purified by HPLC and analyzed by mass spectrometry. Peptides and polypeptides may be dimerized. In one embodiment, peptides and polypeptides are dimerized via a disulfide bridge formed by gentle oxidation of the cysteines using 10% DMSO in water. Following HPLC purification, dimer formation may be verified, by mass spectrometry. One or more modifying groups may be attached to a MCP-1, RANTES, IL-8, IP-10, MIP-1α, I-309, or CCL28-derived peptidic component by standard methods, for example, using methods for reaction through an amino group (e.g., the alpha-amino group at the amino-terminus of a peptide), a carboxyl group (e.g., at the carboxy terminus of a peptide), a hydroxyl group (e.g., on a tyrosine, serine or threonine residue) or other suitable reactive group on an amino acid side chain.
In alternative embodiments, analogs derived from the C-terminal and N-terminal joined by a linker could be cyclized in their C-terminal moiety using side-chain to side-chain; side-chain to scaffold or, scaffold to scaffold cyclization. In some embodiments, lactamization, etherification, or RCM (Ring Closing Methatesis) are used to carry out this reaction.
For instance, chemokine analogs may be cyclized using a lactam formation procedure by joining the γ-carboxy side chain or the α-carboxy moiety of glutamate (Glu) residue to the ε-amino side chain of lysine (Lys) residue, as indicated in the following sequences by underlining of linked residues. Lactams may for example be formed between glutamic acid and lysine (Lys) in the C-terminal portion of the polypeptide (which does not correspond necessarily with the numbering of that residue in the native sequence). In further alternatives, a lysine (Lys) may be substituted by ornithine (Orn) or any other (Lor D) natural or (L or D) non-natural amino acid having an amino group on its side chain. Similarly, glutamate (Glu) may for example be substituted with aspartate (Asp), denoted by nomenclature such as (Glu->Asp) indicating a substitution in a given position in the peptide wherein aspartate replaces glutamate.
The chemokine analogs of the invention include chemokine polypeptide sequences wherein one or more of the amino acids have been replaced by a conservative amino acid substitution. The term “conservative amino acid substitution” refers to a polypeptide chain in which one of the amino acid residues is replaced with an amino acid residue having a side chain with similar properties. Families of amino acid residues having side chains with similar properties are well known in the art. These families include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, an amino acid residue in a chemokine is replaced with another amino acid residue from the same side chain family.
Recombinant Synthesis
Chemokines, chemokine fragments, or chemokine analogs may also be synthesized, in whole or in part, by recombinant methods using expression vectors encoding all or part of a chemokine.
Vectors, or preferably expression vectors, may contain a gene encoding a polypeptide of the invention, a functional derivative thereof, or another useful polypeptide. These vectors may be employed to express the encoded polypeptide in either prokaryotic or eukaryotic cells.
The term “vector” in this application refers to a DNA molecule into which another DNA of interest can be inserted by incorporation into the DNA of the vector. One skilled in the art is familiar with the term. Examples of classes of vectors can be plasmids, cosmids, viruses, and bacteriophage. Typically, vectors are designed to accept a wide variety of inserted DNA molecules and then used to transfer or transmit the DNA of interest into a host cell (e.g., bacterium, yeast, higher eukaryotic cell). A vector may be chosen based on the size of the DNA molecule to be inserted, as well as based on the intended use. For transcription into RNA or transcription followed by translation to produce an encoded polypeptide, an expression vector would be chosen. For the preservation or identification of a specific DNA sequence (e.g., one DNA sequence in a cDNA library) or for producing a large number of copies of the specific DNA sequence, a cloning vector would be chosen. If the vector is a virus or bacteriophage, the term vector may include the viral/bacteriophage coat.
Following entry into a cell, all or part of the vector DNA, including the insert DNA, may be incorporated into the host cell chromosome, or the vector may be maintained extrachromosomally. Those vectors that are maintained extrachromosomally are frequently capable of autonomous replication in a host cell into which they are introduced (e.g., many plasmids having a bacterial origin of replication). Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
The term “expression vector” refers to a DNA construct which allows one to place a gene encoding a gene product of interest, usually a protein, into a specific location in a vector from which the selected gene product can be expressed by the machinery of the host cell, or alternately, by in vitro expression system. This type of vector is frequently a plasmid, but other forms of expression vectors, such as bacteriophage vectors and viral vectors (e.g., adenoviruses, replication defective retroviruses, and adeno-associated viruses), may be employed. The selection of expression vectors, control sequences, transformation methods, and the like, are dependent on the type of host cell used to express the gene.
Prokaryotic Hosts
Prokaryotic hosts are, in generally, very efficient and convenient for the production of recombinant polypeptides and are, therefore, one type of preferred expression system. Prokaryotes most frequently are represented by various strains of E. coli, but other microbial strains may be used, including other bacterial strains. Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, recombinantly-produced polypeptides will not be glycosylated.
In prokaryotic systems, vectors that contain replication sites and control sequences derived from a species compatible with the host may be used. Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColEl, pSC101, pACYC 184, pVX, pUC118, pUC119 and the like). Suitable phage or bacteriophage vectors may include λgt10, λgt11, vectors derived from filamentous bacteriophage such as m13, and the like. Suitable Streptomyces plasmids include p1J101, and streptomyces bacteriophages such as fC31. Bacillus plasmids include pC194, pC221, pT127, and the like. Suitable Pseudomonas plasmids have been reviewed by Izaki (Jpn. J. Bacteriol. 33:729–742, 1978) and John et al. (Rev. Infect. Dis. 8:693–704, 1986).
To express a protease of the invention (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link the sequence encoding the protease of the invention to a functional prokaryotic promoter. Such promoters are either constitutive or inducible promoters, but commonly inducible promoters are used. Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene sequence of pBR322, and the cat promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (PL and PR), the trp, recA, lacZ, lac, and gal promoters of E. coli, the α-amylase and the V-28-specific promoters of B. subtilis, the promoters of the bacteriophages of Bacillus, and Streptomyces promoters. Prokaryotic promoters are reviewed by Glick (Ind. Microbiot. 1:277–282, 1987), Cenatiempo (Biochimie 68:505–516, 1986), and Gottesman (Ann. Rev. Genet. 18:415–442, 1984). Additionally, proper expression in a prokaryotic cell also requires the presence of a ribosome-binding site upstream of the encoding sequence. Such ribosome-binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365–404, 1981).
Fusion Protein
Proteins may be expressed as fusion proteins. Genes for proteins expressed as fusion proteins ligated into expression vectors that add a number of amino acids to a protein encoded and expressed, usually to the amino terminus of the recombinant protein. Such a strategy of producing fusion proteins is usually adopted for three purposes: (1) to assist in the purification by acting as a ligand in affinity purification, (2) to increase the solubility of the product, and (3) to increase the expression of the product. Often, expression vectors for use in fusion protein production, a proteolytic cleavage site is included at the junction of the fusion region and the protein of interest to enable purification of the recombinant protein away from the fusion region following affinity purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase, and may also include trypsin or chymotrypsin. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31–40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Improving Yield
Maximizing recombinant protein expression in E. coli can be assisted by expressing the protein or fusion protein in a host bacteria with an impaired proteolytic system so as to reduce the post-synthesis degradation of the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119–128). Another strategy is to alter the mix of codons used in the coding sequence to reflect the usage of the individual codons for each amino acid in the host (e.g., E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111–2118)). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques and may prove useful for a variety of prokaryotic and eukaryotic expression systems.
Eukaryotic Hosts
Suitable hosts may include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells both in vivo and in tissue culture. Useful mammalian cell hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, and cells of lymphoid origin and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332, which may provide better capacities for correct post-translational processing. In general, eukaryotic organisms such as yeast provide substantial advantages in that they can also carry out post-translational modifications.
A large number of yeast expression systems may be potentially utilized which incorporate promoter and termination elements from the actively expressed sequences coding for glycolytic enzymes. These expression systems produce in large quantities of proteins when yeast are grown in mediums rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. A number of recombinant DNA strategies exist utilizing strong promoter sequences and high copy number plasmids which can be utilized for production of the desired proteins in yeast. Examples of vectors suitable for expression in S. cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229–234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933–943), pJRY88 (Schultz et al., (1987) Gene 54:113–123), pYES2 (In Vitrogen Corporation, San Diego, Calif.), and picZ (In Vitrogen Corp, San Diego, Calif.).
In another embodiment, the protein of interest may be expressed in insect cells for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter may be used (Rubin, Science 240:1453–1459, 1988). Additionally, baculovirus vectors can be engineered to express large amounts of the protein of interest in cultured insect cells (e.g., Sf9 cells)(Jasny, Science 238:1653, 1987; Miller et al., in: Genetic Engineering, Vol. 8, Plenum, Setlow et al., eds., pp. 277–297, 1986). Vectors which may be used include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156–2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31–39).
Plant cells may also be utilized as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S promoters, and nopaline synthase promoter and polyadenylation signal sequences. Furthermore, the protein of interest may be expressed in plants which have incorporated the expression vector into their germ line.
In yet another embodiment, a nucleic acid of the invention may be expressed in mammalian cells using a mammalian expression vector. Possibilities and techniques for expression in mammalian cells has recently been summarized (Colosimo, et al., “Transfer and expression of foreign genes in mammalian cells,” Biotechniques 29(2):314–8, 320–2, 324 passim, 2000; which is hereby incorporated by reference in its entirety including any drawings, tables, and figures.). Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187–195). For use in mammalian cells, the regulatory sequences of the expression vector are often derived from viral regulatory elements. For example, commonly used promoters are derived from Simian Virus 40 (SV40), polyoma, Adenovirus 2, and cytomegalovirus (CMV) viruses. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273–288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355–365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304–31, 1981); and the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971–6975, 1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951–5955, 1984). Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, and the like, may be employed. Regulatory elements may also be derived from adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like.
Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation. Expression of proteins of interest in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
The recombinant mammalian expression vector may also be designed to be capable of directing expression of the nucleic acid preferentially in a particular cell type (i.e., tissue-specific regulatory elements are used to control the expression). Such tissue-specific promoters include the liver-specific albumin promoter (Pinkert et al. (1987) Genes Dev. 1:268–277); lymphoid-specific promoters (e.g., Calame and Eaton (1988) Adv. Immunol. 43:235–275), and in particular promoters of immunoglobulins and T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729–733, Banerji et al. (1983) Cell 33:729–740; Queen and Baltimore (1983) Cell 33:741–748); mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166); and pancreas-specific promoters (Edlund et al. (1985) Science 230:912–916). Developmentally-regulated promoters may also be utilized, for example, the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537–546), and the murine hox promoters (Kessel and Gruss (1990) Science 249:374–379).
Preferred eukaryotic plasmids include, for example, SV40, BPV, pMAM-neo, pKRC, vaccinia, 2-micron circle, and the like, or their derivatives. Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265–274, 1982; Broach, In: “The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445–470, 1981; Broach, Cell 28:203–204, 1982; Bollon et al., J. Clin. Hematol. Oncol. 10:39–48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563–608, 1980).
Once the vector or nucleic acid molecule containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, DEAE-dextran-mediated transfection, lipofection, calcium phosphate-precipitation, direct microinjection, and the like. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (2001). After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene(s) results in the production of a protein of interest, or fragments thereof.
For other suitable expression systems for both prokaryotic and eukaryotic cells see Sambrook, et al., “Molecular Cloning: A Laboratory Manual,” 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001, which is hereby incorporated by reference in its entirety, including any drawings, figures, and tables.
For transformation of eukaryotic cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, neomycin, methotrexate, glyphosate, and bialophos. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the protein of interest or can be introduced on a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) the protein of interest. Accordingly, the invention further provides methods for producing the protein of interest using the host cells of the invention. In one embodiment, the method comprises culturing the host cell into which a recombinant expression vector encoding the protein of interest has been introduced in a suitable medium such that the protein of interest is produced, and may be purified by one skilled in the art.
In some aspects of the chemokine analogs of the invention, the analogs contain a linker, having the denoted structure [linker] (shown in bold), wherein the linker has the following structure: H2N-ZA-COOH as defined below.
IL-8 Compounds
In one aspect of this invention, the chemokine analogs contain linear IL-8 analogs of the present invention corresponding to a portion of the N-terminal of IL-8 having the following structures:
Preferred embodiments of linear IL-8 analogs of the present invention corresponding to a portion of the internal-region of IL-8 having the following structures:
Preferred embodiments of linear IL-8 analogs of the present invention corresponding to a portion of the C-terminal of IL-8 having the following structures:
Preferred embodiments of linear IL-8 analogs of the present invention corresponding to a portion of the N-terminal joined with a linker to the C-terminal region of IL-8 having the following structures:
Preferred embodiments of cyclic IL-8 analogs of the present invention corresponding to a cyclic portion of the N-terminal-region of IL-8 having the following structures:
Preferred embodiments of cyclic IL-8 analogs of the present invention corresponding to a cyclic portion of the internal-region of IL-8 having the following structures (the underlined residues below are cyclized):
Preferred embodiments of cyclic IL-8 analogs of the present invention corresponding to a cyclic portion of the C-terminal region of IL-8 having the following structures (the underlined residues below are cyclized):
Preferred embodiments of cyclic IL-8 analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to a cyclic portion of the C-terminal region of IL-8 having the following structures (the underlined residues below are cyclized):
In the above structures:
A wide variety of amino acid substitutions may be made in polypeptide sequences, such as lysine to glutamic acid, lysine to aspartic acid, Orn to Glu, Orn to Asp. Moieties other than naturally occurring amino acids may also be substituted, such as Btd:
The linker is a bifunctional group covalently attached to the N-terminal and C-terminal portions of the analog having the structure: H2N-ZA-COOH wherein ZA is selected from the group consisting of: (1) alkyl, alkenyl, aralkyl, alkynyl; (2) —(CH2)n— wherein n is an integer n=9 to 14; (3) any combination of four natural amino acids or non-natural amino acids; and (4) -(Gly)4- (SEQ ID NO: 1640).
IP-10 Compounds:
Preferred embodiments of linear IP-10 analogs of the present invention corresponding to a portion of N-terminal have the following structures:
Preferred embodiments of linear IP-10 analogs of the present invention corresponding to a portion of the internal region of IP-10 having the following structures:
Preferred embodiments of linear IP-10 analogs of the present invention corresponding to a portion of the N-terminal region and the internal region of IP-10 having the following structures:
Preferred embodiments of linear IP-10 analogs of the present invention corresponding to a portion of the C-terminal region of IP-10 having the following sequence:
Preferred embodiments of cyclic IP-10 analogs of the present invention corresponding to a portion of the N-terminal region joined with a linker to a cyclic portion of the C-terminal region of IP-10 having the following structures:
Preferred embodiments of cyclic IP-10 analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to a cyclic portion of the C-terminal region of IP-10 having the following structures (underlined residues are cyclized):
In some embodiments IP-10 analogs were cyclized by etherification between Lys66 and Ser69 (underlined residues are cyclized).
In the above structures:
A wide variety of amino acid substitutions may be made in polypeptide sequences, such as lysine to glutamic acid, lysine to aspartic acid, Orn to Glu, Orn to Asp. Moieties other than naturally occurring amino acids may also be substituted, such as Btd:
The linker is a bifunctional group covalently attached to the N-terminal and C-terminal portions of the analog having the structure: H2N-ZA-COOH wherein ZA is selected from the group consisting of: (1) alkyl, alkenyl, aralkyl, alkynyl; (2) —(CH2)n— wherein n is an integer n=9 to 14; (3) any combination of four natural amino acids or non-natural amino acids; and (4) -(Gly)4- (SEQ ID NO: 1640).
MIP-1α Compounds:
Preferred embodiments of linear MIP-1α analogs of the present invention corresponding to a portion of the N-terminal region of MIP-1α having the following sequence:
Preferred embodiments of linear MIP-1α analogs of the present invention corresponding to a portion of the internal region of MIP-1α having the following sequences:
Preferred embodiments of linear MIP-1α analogs of the present invention corresponding to a portion of the N-terminal and the internal region of MIP-1α having the following structures:
Preferred embodiments of linear MIP-1α analogs of the present invention corresponding to a portion of the C-terminal region of MIP-1α having the following structures:
Preferred embodiments of linear MIP-1α analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to the C-terminal region of MIP-1α having the following structures:
In some preferred embodiments, glutamine (Gln57) of MIP-1α analogs was replaced by lysine (Lys).
Preferred embodiments of cyclic MIP-1α analogs of the present invention corresponding to a cyclic portion of the internal region of MIP-1α having the following structures:
Preferred embodiments of cyclic MIP-1α analogs of the present invention corresponding to a portion of the N-terminal region joined with a linker to a cyclic the C-terminal region of MIP-1α having the following structures:
In the above structures:
A wide variety of amino acid substitutions may be made in polypeptide sequences, such as lysine to glutamic acid, lysine to aspartic acid, Orn to Glu, Orn to Asp. Moieties other than naturally occurring amino acids may also be substituted, such as Btd:
The linker is a bifunctional group covalently attached to the N-terminal and C-terminal portions of the analog having the structure: H2N-ZA-COOH wherein ZA is selected from the group consisting of: (1) alkyl, alkenyl, aralkyl, alkynyl; (2) —(CH2)n— wherein n is an integer n=9 to 14; (3) any combination of four natural amino acids or non-natural amino acids; and (4) -(Gly)4- (SEQ ID NO: 1640).
RANTES Compounds
Preferred embodiments of linear RANTES analogs of the present invention corresponding to a portion of the N-terminal region of RANTES having the following structures:
Preferred embodiments of linear RANTES analogs of the present invention corresponding to a portion of the internal region of RANTES having the following structures:
Preferred embodiments of linear RANTES analogs of the present invention corresponding to a portion of the N-terminal and a portion the internal region of RANTES having the following structures:
Preferred embodiments of linear RANTES analogs of the present invention corresponding to a portion of the C-terminal region of RANTES having the following structures:
Preferred embodiments of linear RANTES analogs of the present invention corresponding to a portion of the N-terminal region joined with a linker to the C-terminal region of RANTES having the following structures:
Preferred embodiments of cyclic RANTES analogs of the present invention corresponding to cyclic a portion of the internal region of RANTES having the following structures:
Preferred embodiments of cyclic RANTES analogs of the present invention corresponding to the N-terminal region and a cyclic a portion of the internal region of RANTES having the following structures:
Preferred embodiments of cyclic RANTES analogs of the present invention corresponding to a portion of the N-terminal region joined with a linker to cyclic portion of the C-terminal region of RANTES having the following structures:
Lys-Trp-Val-Arg-Glu-Tyr-Ile-Asn-Ser-Leu-Glu-Met-Ser-(OH)NH2
In the above structures:
A wide variety of amino acid substitutions may be made in polypeptide sequences, such as lysine to glutamic acid, lysine to aspartic acid, Orn to Glu, Orn to Asp. Moieties other than naturally occurring amino acids may also be substituted, such as Btd:
The linker is a bifunctional group covalently attached to the N-terminal and C-terminal portions of the analog having the structure: H2N-ZA-COOH wherein ZA is selected from the group consisting of: (1) alkyl, alkenyl, aralkyl, alkynyl; (2) —(CH2)n— wherein n is an integer n=9 to 14; (3) any combination of four natural amino acids or non-natural amino acids; and (4) -(Gly)4- (SEQ ID NO: 1640).
I-309 Compounds
Preferred embodiments of linear I-309 analogs of the present invention corresponding to a portion of the N-terminal region of I-309 having the following structures:
Preferred embodiments of linear I-309 analogs of the present invention corresponding to a portion of the internal region of I-309 having the following structures:
Preferred embodiments of linear I-309 analogs of the present invention corresponding to a portion of the N-terminal region and a portion of the internal region of I-309 having the following structures:
Preferred embodiments of linear I-309 analogs of the present invention corresponding to a portion of the C-terminal region of I-309 having the following structures:
Preferred embodiments of linear I-309 analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to the C-terminal region of I-309 having the following structures:
Preferred embodiments of cyclic I-309 analogs of the present invention corresponding to a cyclic portion of the internal region of I-309 having the following structures:
Lys-Arg-Gly-Lys-Glu-Ala-(OH)NH2
Preferred embodiments of cyclic I-309 analogs of the present invention corresponding to a cyclic portion of the N-terminal region and the internal region of I-309 having the following structures:
Glu-Ile-Pro-Leu-Arg-Ala-Ile-Leu-(OH)NH2
Preferred embodiments of cyclic I-309 analogs of the present invention corresponding to a cyclic portion of the C-terminal region of I-309 having the following structures:
Preferred embodiments of cyclic I-309 analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to a cyclic portion of the C-terminal region of I-309 having the following structures (underlined residues are cyclized):
In the above structures:
A wide variety of amino acid substitutions may be made in polypeptide sequences, such as lysine to glutamic acid, lysine to aspartic acid, Orn to Glu, or Orn to Asp. Moieties other than naturally occurring amino acids may also be substituted, such as Btd:
Btd* is:
The linker is a bifunctional group covalently attached to the N-terminal and C-terminal portions of the analog having the structure: H2N-ZA-COOH wherein ZA is selected from the group consisting of: (1) alkyl, alkenyl, aralkyl, alkynyl; (2) —(CH2)n— wherein n is an integer n=9 to 14; (3) any combination of four natural amino acids or non-natural amino acids; and (4) -(Gly)4- (SEQ ID NO: 1640).
MCP-1 Compounds:
Preferred embodiments of linear MCP-1 analogs of the present invention corresponding to a portion of the N-terminal and the internal region of MCP-1 having the following structures:
From the chemokine MCP-1 the following compounds:
Preferred embodiments of linear MCP-1 analogs of the present invention corresponding to a portion of the N-terminal and the internal region of MCP-1 having the following structures:
Preferred embodiments of linear MCP-1 analogs of the present invention corresponding to a portion of the internal region of MCP-1 having the following structures:
Preferred embodiments of linear MCP-1 analogs of the present invention corresponding to a portion of the C-terminal region of MCP-1 having the following structures:
Preferred embodiments of linear MCP-1 analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to the C-terminal region of MCP-1 having the following structures:
Preferred embodiments of cyclic MCP-1 analogs of the present invention corresponding to a cyclic portion of the N-terminal and the internal region of MCP-1 having the following structures:
Preferred embodiments of cyclic MCP-1 analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to a cyclic portion of the C-terminal region of MCP-1 having the following structures:
In the above structures:
A wide variety of amino acid substitutions may be made in polypeptide sequences, such as lysine to glutamic acid, lysine to aspartic acid, Orn to Glu, or Orn to Asp. Moieties other than naturally occurring amino acids may also be substituted, such as Btd:
The linker is a bifunctional group covalently attached to the N-terminal and C-terminal portions of the analog having the structure: H2N-ZA-COOH wherein ZA is selected from the group consisting of: (1) alkyl, alkenyl, aralkyl, alkynyl; (2) —(CH2)n— wherein n is an integer n=9 to 14; (3) any combination of four natural amino acids or non-natural amino acids; and (4) -(Gly)4- (SEQ ID NO: 1640).
CCL28 Compounds
Preferred embodiments of linear CCL28 analogs of the present invention corresponding to a portion of the N-terminal region of CCL28 having the following structures:
Preferred embodiments of linear CCL28 analogs of the present invention corresponding to a portion of the internal region of CCL28 having the following structures:
Preferred embodiments of linear CCL28 analogs of the present invention corresponding to a portion of the C-terminal region of CCL28 having the following structures:
Preferred embodiments of linear CCL28 analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to the C-terminal region of CCL28 having the following structures:
Preferred embodiments of cyclic CCL28 analogs of the present invention corresponding to a portion the N-terminal region joined with a linker to a cyclic portion of the C-terminal region of CCL28 having the following structures:
In the above structures:
A wide variety of amino acid substitutions may be made in polypeptide sequences, such as lysine to glutamic acid, lysine to aspartic acid, Orn to Glu, or Orn to Asp. Moieties other than naturally occurring amino acids may also be substituted, such as Btd:
The linker is a bifunctional group covalently attached to the N-terminal and C-terminal portions of the analog having the structure: H2N-ZA-COOH wherein ZA is selected from the group consisting of: (1) alkyl, alkenyl, aralkyl, and alkynyl; (2) —(CH2)n— wherein n is an integer, n=9 to 14; (3) any combination of four natural amino acids or non-natural amino acids; and (4) -(Gly)4- (SEQ ID NO: 1640).
Compositions
The invention further provides pharmaceutical compositions containing chemokine receptor agonists or antagonists. In one embodiment, such compositions include a chemokine analog compound in a therapeutically, diagnostically or prophylactically effective amount sufficient to be used in treating diseases or disorders selected from the group consisting of autoimmune diseases, acute chronic inflammation, cancer, cardiovascular disease, infectious disease, and inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, atherosclerosis, psoriasis, rhinitis, autoimmunity, and organ transplant rejection. In another embodiment, such compositions include a chemokine analog compound in a therapeutically or prophylactically effective amount sufficient to be used to increase the hemocrit, assist in mobilizing and recovering stem cells, stimulate the production of blood cells, assist in vaccine production, or assist in gene therapy.
An “effective amount” of a compound of the invention includes a therapeutically effective amount or a prophylatically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The term “therapeutically effective amount” may also refer to that amount of active compound, prodrug or pharmaceutical agent that elicits a biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician in order to provide a therapeutic effect.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting a cytotoxic effect of a cytotoxic agent. Typically, a prophylactic dose is used in organisms prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. The term “preventing” refers to decreasing the probability that an organism contracts or develops an abnormal condition.
In particular embodiments, a preferred range for therapeutically or prophylactically effective amounts of chemokine analogs may be 0.1 nM-0.1 M, 0.1 nM-0.05 M, 0.05 nM-15 μM or 0.01 nM-10 μM. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.
The amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” as used herein refers to physically discrete units suited as unitary dosages for subjects 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 (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
The terms “administration” or “administering” refer to a method of incorporating a compound into the cells or tissues of an animal, preferably a mammal, and still more preferably a human, in order to treat or prevent an abnormal condition. When the compound or prodrug of the invention is provided in combination with one or active agents, the terms “administration” or “administering” include sequential or concurrent introduction of the compound or prodrug with the other agent(s). For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, injection, parenteral, dermal, and aerosol applications.
The term “therapeutic effect” refers to the inhibition or activation of factors causing or contributing to the abnormal condition (including a disease or disorder). A therapeutic effect relieves or prevents to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase or decrease in the number of lymphocytic cells present at a specified location, (b) an increase or decrease in the ability of lymphocytic cells to migrate, (c) an increase or decrease in the response of lymphocytic cells to a stimulus, (d) an increase or decrease in the proliferation, growth, and/or differentiation of cells; (e) inhibition (i.e., slowing or stopping) or acceleration of cell death; (f) relieving, to some extent, one or more of the symptoms associated with an abnormal condition; (g) enhancing or inhibiting the function of the affected population of cells; (h) activating an enzyme activity present in cells associated with the abnormal condition; and (i) inhibiting an enzyme activity present in cells associated with the abnormal condition.
The term “abnormal condition” refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism and includes, but is not limited to, conditions commonly referred to as diseases or disorders. An abnormal condition can relate to cell proliferation, cell differentiation, cell survival, cell migration or movement, or the activities of enzymes within a cell. Diseases and disorders may include inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, atherosclerosis, psoriasis, rhinitis, autoimmunity, organ transplant rejection, and genetic diseases.
As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. 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 pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutically acceptable carrier “may comprise pharmaceutically acceptable salts.”
Pharmaceutical formulations for parenteral administration may include liposomes. Liposomes and emulsions are well known examples of delivery vehicles or carriers that are especially useful for hydrophobic drugs. Depending on biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with target-specific antibody. The liposomes will bind to the target protein and be taken up selectively by the cell expressing the target protein.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene 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 required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or 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, monostearate salts and gelatin. Moreover, the chemokine analogs may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
Additionally, suspensions of the compounds of the invention may be prepared as appropriate oily suspensions for injection. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes. Suspensions to be used for injection may also contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Sterile injectable solutions can be prepared by incorporating the active compound 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 that 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. In accordance with an alternative aspect of the invention, a chemokine analog may be formulated with one or more additional compounds that enhance the solubility of the chemokine analog.
If the compounds of the invention are to be administered by inhalation, they may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, together with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin, for example, for use in an inhaler may be formulated containing a powder mix of the compound and a suitable powder base such as starch or lactose.
The term “modulates” refers to altering the function or activity of a chemokine receptor by contacting it with a chemokine or chemokine analog and thus increasing or decreasing the probability that a complex forms between the receptor and a natural binding partner. A chemokine or chemokine analog preferably increases the probability that such a complex forms between the chemokine receptor and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the chemokine receptor and the natural binding partner depending on the concentration of the chemokine or chemokine analog exposed to the receptor, and most preferably decreases the probability that a complex forms between the chemokine receptor and the natural binding partner depending on the concentration of the chemokine or chemokine analog exposed to the polypeptide.
The term “chemokine receptor” refers to a chemokine receptor as the term is used by one skilled in the art and also refers to any other polypeptide capable of binding a chemokine or chemokine analog.
In preferred embodiments, a modulator preferably activates the catalytic activity of a chemokine receptor, more preferably activates or inhibits the catalytic activity of a chemokine receptor depending on the concentration of the chemokine or chemokine analog exposed to the chemokine receptor, or most preferably inhibits the catalytic activity of a chemokine receptor depending on the concentration of the chemokine or chemokine analog exposed to the chemokine receptor.
The term “natural binding partner” refers to G proteins, polypeptides, lipids, small molecules, or nucleic acids that bind to chemokine receptors in cells or in the extracellular environment. The term natural binding partner includes a substrate to be acted upon by the chemokine receptor. A change in the interaction between a chemokine receptor and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of chemokine receptor/natural binding partner complex. This can result in a decreased or increased activity of the chemokine receptor.
The terms “activated,” “activating,” and “activation” refer to an increase in the cellular or extracellular function of a chemokine receptor. The chemokine receptor function is preferably the interaction with a natural binding partner, and most preferably catalytic activity. The term “inhibits” refers to decreasing the cellular or extracellular activity of the chemokine receptor. The cellular or extracellular activity of a chemokine receptor is preferably the interaction with a natural binding partner, and most preferably catalytic activity.
The term “complex” refers to an assembly of at least two molecules bound to one another. A signal transduction complex often contains at least two protein molecules bound to one another. For instance, a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand. Another example is a chemokine bound to a chemokine receptor. Still another example is a G protein bound to a chemokine receptor.
The term “contacting” as used herein refers to adding together a solution or a composition comprising the chemokine or chemokine analog with a liquid medium bathing the polypeptide or cells comprising a chemokine receptor. The solution comprising the chemokine or chemokine analog may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the chemokine or chemokine analog into the cells of the methods. The solution comprising the chemokine or chemokine analog may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipette-based device or syringe-based device.
As discussed supra, compounds of the present invention may prove useful in increasing the hemocrit, mobilizing stem cells, or in assisting in vaccine production or otherwise stimulating the immune system to effectuate tumor destruction. For example, the chemokine SDF-1 has been shown to enhance platelet production (Lane et al., Blood 96:4152–59, 2000) and B-cell production (Nagasawa, T., Int. J. Hematol. 72:408–11, 2000), inter alia. Analogs of chemokines may also be useful in improving the engraftment of stem cells following transplantation (Nagasawa, 2000). Chemokine analogs of the invention may also prove useful in mobilizing stem cells (Gazitt, Y., J. Hematother Stem Cell Res 10:229–36, 2001; Hattori et al., Blood 97:3354–59, 2001). They may also prove useful in enhancing anti-tumor immunity (Nomura et al., Int. J. Cancer 91:597–606, 2001; Mach and Dranoff, Curr. Opin. Immunol. 12:571–75, 2000). Other aspects and roles of modulating chemokine function are reviewed in Schwarz and Wells (Schwarz and Wells, Nat. Rev. Drug Discov. 1:347–58, 2002). Chemokine analogs of the present invention may also prove useful in facilitating gene therapy. Glimm and colleagues reported that one chemokine, SDF-1, arrests hematopoietic stem cell cycling, thus allowing a better transfection of these cells with gene constructs for the purpose of gene therapy (Glimm H. et al., “Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor-1 enhances their ability to engraft NOD/SCID mice,” Blood 99(9):3454–57, 2002). All of the above references are incorporated by reference herein their entirety, including any drawings, tables, and figures.
The following examples illustrate, but do not limit, the present invention.
The efficacy of chemokine analogs of the invention as chemokine receptor agonists is demonstrated through receptor binding assays. A competitive dose response for binding to a chemokine receptor by the natural binding chemokine, another chemokine, and chemokine analogs of the invention may be demonstrated by the method set forth in Daugherty et al., Methods in Molecular Biology v138 “Chemokine Protocols” edited by Proudfoot et al., Human Press, Totowa, N.J. p129–148, 2000, which is hereby incorporated by reference in its entirety, including any figures, tables and drawings.
The efficacy of chemokine analogs of the invention as chemokine receptor agonists is demonstrated through chemotaxis assays. The effect of a native chemokine and chemokine analogs of the invention may be compared by the method set forth in Ponath et al., Methods in Molecular Biology v138 “Chemokine Protocols” edited by Proudfoot et al., Human Press, Totowa, N.J. p113–120, 2000, which is hereby incorporated by reference in its entirety, including any figures, tables and drawings.
(IL-8)
The efficacy of IL-8 and IL-8 peptide analogs as CXCR1 and CXCR2 agonists was demonstrated through CXCR1 and CXCR2 receptor binding assays. A competitive dose response for binding to the IL-8 receptor by native IL-8 and the CXCR1 and CXCR2 agonists against 125I-IL-8 is shown in
(I-309)
This example illustrates the efficacy of I-309 peptide analogs in mediating intracellular calcium mobilization ([Ca2+]i). To illustrate that the binding of I-309 peptide analogs results in the agonistic activation of the CCR8 receptor, [Ca2+]i mobilization assays were conducted, the results of which are shown in
Peptides of the invention may be synthesized chemically using the Fmoc/tBu strategy on a continuous flow peptide synthesizer, as for example has been carried out using the following protocols:
Main Solvent: N,N-Dimethylformamide (DMF): certified ACS spectroanalyzed from Fisher (D131-4) M.W=73.10. The DMF is treated with activated molecular sieves, type 4A (from BDH: B54005) for at least two weeks then tested with FDNB (2,4-Dinitrofluorobenzene from Eastman).
Procedure: Mix equal volumes of FDNB solution (1 mg/ml in 95% EtOH) and DMF; Let stand 30 minutes; read the absorbance at 381 nm over a FDNB blank (0.5 ml FDNB+0.5 ml 95% EtOH). If the absorbance˜0.2, the DMF is suitable to be used for the synthesis.
Deblocking Agent: 20% Piperidine (from Aldrich Chemical company, catalog No: 10,409-4) in DMF containing 0.5% Triton X100 v/v (from Sigma, catalog No: T-9284).
Activating Agents: 2-(H-benzotriazol-1yl) 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU: M.W.=321.09. from Quantum Richilieu, catalog No: R0139)/Hydroxybenzotriazole (HOBt M.W.=135.1 from Quantum Richilieu, catalog No.: R0166-100) respectively, 0.52 M in DMF and 4-Methylmorpholine (NMM; M.W.=101.15, d=0.926 from Aldrich, catalog No.: M5,655-7): 0.9 M in DMF or in the case of sensitive amino acids to racemization like Cys, we use 2,4,6-Collidine, 99% (M.W.=121.18, d=0.917, from Aldrich, catalog No: 14,238-7): 0.78M in DMF/DCM, 1/1 v/v.
Support: TentaGel R RAM (90 μm), RinK-type Fmoc (from Peptides International, catalog No.: RTS-9995-PI): 0.21 mmol/g, 0.5 g for 0.1 mmol of peptide.
Fmoc-L-amino derivative, side-chains protected with: Boc; tBu; Trt groups: with 4 fold excess (from Peptides International, Bachem, Novabiochem, Chem-Impex Inc). Glu24 and Lys24 are Allyl-protected (from Millipore/Perseptive Biosystems).
The first amino acid Asn31 and the remaining residues are double coupled at room temp. or at 45° C. automatically with 4-fold excess in each coupling. The synthesis is interrupted after residue Leu19. The peptide-bound support is removed from the synthesizer column and placed in a react-vial containing a small magnetic bar for gentle stirring.
Removal of the Allyl Groups
A solution of tetrakis(triphenylphosphine)Palladium(0) Pd(PPh3)4 (from Sigma-Aldrich, catalog No: 21,666-6); M.W.=1155.58×0.1 mmol peptide×3 fold=347 mg dissolved in 5% Acetic Acid; 2.5% NMM in CHCl3 to 0.14 M, under argon. The solution is added to the support-bound peptide previously removed from the column in a reactvial containing a small magnetic bar for gentle stirring. The mixture is flushed with argon, sealed and stirred at room temperature for 6 hours. The support-bound peptide is transferred to a filter funnel, washed with 30 ml of a solution made of 0.5% Sodium Diethyldithiocarbonate/in DMF, then DCM; DCM/DMF (1:1) and DMF. A positive Kaiser test indicate the deprotection of the amino side chain of the Lys20.
Lactam Formation:
Activating agent: 7-Azabenztriazol-1-yloxytris (pyrrolindino) phosphonium-hexafluorophosphate (PyAOP: M.W.=521.7 from PerSeptive Biosystems GmbH, catalog No: GEN076531), 1.4-fold: 0.105 mmol×1.4×521.7=76.6 mg and NMM 1.5-fold: 0.105×1.4×1.5=0.23 mmol v=0.23/0.9 M NMM solution=263 μl).
The cyclisation may be carried out in an amino acid vial at room temperature overnight (˜16 hours) with gentle agitation. The completion of cyclization may be indicated by a negative kaiser test. The support-bound peptide may be poured into the column, washed with DMF and the synthesis continues to completion, with a cyclic amide bridge thereby introduced into the peptide.
Final Product Removal from the Support:
The support-bound peptide is removed from the synthesizer in to a medium filter funnel, washed with DCM to replace the non-volatile DMF and thoroughly dried under high vacuum for at least two hours, or preferably, overnight.
The cleavage may be performed at room temperature for 4 hours with gentle agitation on a rocker.
Precipitation of the Peptide
The cleaved peptide solution is filtered through a filter funnel in a 50 ml round bottom flask. The support is rinsed twice with 4 ml TFA. The TFA solution is concentrated on a rotavap and added drop wise into a cold diethyl ether previously treated with activated neutral aluminum oxide to make it free of peroxide. Approximately 10-fold excess of ether are used. The beads are stored until the yield is determined and peptide characterized. The precipitate is collected at room temperature in screw capped 50 ml polypropylene vial by centrifugation at 2K rpm using a top bench centrifuge (4 minutes run time). The pellet is washed 3× with cold ether, centrifuged and dried with a flow of argon. The precipitate is dissolved in 20% acetonitrile, 0.1% TFA and lyophilized.
Crude Product Characterization:
The product is characterized by analytical HPLC.
An aliquot of the product is weighed and dissolved in 20% acetonitrile 0.1% TFA at a concentration of 2 mg/ml. The solution is microfuged and 20 μl is applied onto the column. The main peak or the major peaks are collected, SpeedVac dried and molecular weight determined by mass spectrometry.
Structure of some of the compounds used in this study is shown below.
The invention illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations that is not specifically disclosed herein, Thus, for example, the terms “comprising,” “including,” “containing,” etc., shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalent of the invention shown or portion thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the inventions embodied herein disclosed can be readily made by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form the part of these inventions. This includes within the generic description of each of the inventions a proviso or negative limitation that will allow removing any subject matter from the genus, regardless or whether or not the material to be removed was specifically recited. In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further, when a reference to an aspect of the invention lists a range of individual members, as for example, ‘SEQ ID NO:9 to SEQ ID NO:162, inclusive,’ it is intended to be equivalent to listing every member of the list individually, and additionally it should be understood that every individual member may be excluded or included in the claim individually.
The steps depicted and/or used in methods herein may be performed in a different order than as depicted and/or stated. The steps are merely exemplary of the order these steps may occur. The steps may occur in any order that is desired such that it still performs the goals of the claimed invention.
From the description of the invention herein, it is manifest that various equivalents can be used to implement the concepts of the present invention without departing from its scope. Moreover, while the invention has been described with specific reference to certain embodiments, a person of ordinary skill in the art would recognize that changes can be made in form and detail without departing from the spirit and the scope of the invention. The described embodiments are considered in all respects as illustrative and not restrictive. It should also be understood that the invention is not limited to the particular embodiments described herein, but is capable of many equivalents, rearrangements, modifications, and substitution without departing from the scope of the invention. Thus, additional embodiments are within the scope of the invention and within the following claims.
Further, all patents and publications described herein are hereby incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.
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