MODULATION OF CXCR4 AND RELATED METHODS AND COMPOSITIONS
A method for inhibiting the SDF1-CXCR4 signaling pathway in a subject, comprising administering to the subject an effective amount of a Beta Defensin (BD)-inducing agent. The BD-inducing agent may be a Fusobacterium associated defensin inducer (FAD-I) polypeptide or a double stranded RNA analog.
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This application claims benefit of prior filed U.S. Provisional Application No. 60/676,335, filed Apr. 28, 2005. This application is a continuation-in-part of U.S. application Ser. No. 10/538,811, filed Jun. 13, 2005, which is a National Stage of International Application No. PCT/US2003/040221, filed Dec. 15, 2003, which claims the benefit of U.S. Provisional Application No. 60/433,100, filed Dec. 13, 2002.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTWork described herein was funded, in part, by NIH grants RO-1 DE12589, RO-1 DE13992, RO-1 DE015510, AI 51649, and RO-1 GM36387. The United States government has certain rights in the invention.
BACKGROUNDSkin and mucosa have always been regarded as physical barriers to the outside environment; protecting the host from noxious intruders. Recent findings have led to the realization that these barriers are not only physical; they generate potent antimicrobial peptides (APs). These compounds, first described in Drosophila, are now known to be important for the “innate” immune system of a eukaryotic host. Antimicrobial peptides act on a broad spectrum of pathogens. The innate immune system works in conjunction with the adaptive immune system in mammals, by permitting the host to curb, delay, or avoid microbial growth shortly after an infection. Innate responses occur in a matter of hours, well before the acquired immune system can be sufficiently mobilized.
The defensin peptides are a superfamily of peptide antibiotics with a characteristic beta-sheet structure stabilized by two to three intramolecular disulfide bonds. They are strongly cationic by virtue of their numerous arginine and lysine residues. The amphipathic and cationic characteristics are important for antibacterial activity. Defensin peptides have been isolated from a number of phagocytes from mammals including humans, and various tissue and fluid sources such as mammalian trachea, intestine, tongue, human oral gingiva, human organs, plasma and urine.
The human defensin AP family is roughly divided into two subfamilies; alpha-defensins, found in azurophilic granules of PMNs and in the granules of Paneth cells found in the base of the crypts of Lieberkühn in the small intestine, and the beta-defensins, expressed generally by epithelial cells. The alpha- and beta-defensins differ in primary sequence and in the placement of the three disulfide bonds. The signature motif for beta-defensin genes includes two exons surrounding a variably sized intron. Exon 1 encodes the signal sequence, while exon 2 encodes the propeptide and mature peptide. This motif differs from that found in alpha-defensin genes in that the latter are organized with three exons and two introns. Other differentiating features between alpha- and beta-defensins include the fact that while the former are cytotoxic to mammalian cells when released from protective granules, the latter are not.
Defensins represent a family of small (3-5 kDa) cationic peptides having antimicrobial activities. To date, at least three subfamilies of defensins have been identified: alpha defensins, beta defensins, and the cyclic theta defensins, Alpha and beta defensins have a characteristic beta-sheet structure stabilized by two to three intramolecular disulfide bonds, but they differ in size and in the connectivity of their six cysteine residues. Alpha-defensins are 29 to 35-amino acid peptides having three intramolecular disulfide bonds through Cys1-Cys6, Cys2-Cys4, and Cys3-CysS, whereas mature beta defensins are up to 45-residue peptides having disulfide connectivity with Cys 1-Cys5, Cys2-Cys4, and Cys3-Cys6. Defensins are microbistatic or microbicidal against a wide spectrum of Gram-positive and Gram-negative bacteria, fungi, yeast, and some enveloped viruses. More specifically, human beta defensins-1 and -2 are predominantly active against Gram-negative bacteria, human beta-defensin -3 demonstrates a salt-insensitive broad spectrum of potent antimicrobial activity against pathogenic microbes including Gram-positive bacteria, and human beta defensin-4 has a specific salt-sensitive spectrum of antimicrobial activity. Alpha defensins also exhibit a tissue-specific expression pattern different from beta defensins, which are expressed predominantly in epithelia. Another important differentiating feature between alpha- and beta-defensins is that while the former are cytotoxic to mammalian cells when released from protective granules, the latter are not.
To date, more than 30 human beta-defensins have been discovered based on human genome mining. Human beta defensin (HBD)-1 is constitutively expressed by epithelia, while the expression of HBD-2, -3, and -4 is induced upon stimulation by inflammatory mediators, such as TNF-α and IL-1β, and contact with bacteria including mucoid forms of Psudomonas aeruginosa bacteria.
In addition to demonstrating antibacterial and antifungal properties, beta-defensins engage the CCR6 receptor on selected immune effector cells, such as immature dendritic cells and T cells and evoke a chemokine response, thereby recruiting these cells to the site of interest.
The growing problem of resistance to conventional antibiotics and the need for new antibiotics has stimulated interest in the development of antimicrobial peptides (APs) as therapeutics for humans and other animals. Unlike conventional antibiotics, acquisition of resistance by a sensitive organism against APs is surprisingly rare and difficult to generate.
Chemokines constitute a family of small molecular weight cytokines that induce migration and activation of leukocytes. Over 30 different human chemokines have been described. They vary in their specificity for different leukocyte types (neutrophils, monocytes, eosinophils, basophils, lymphocytes, dendritic cells, etc.), and in the types of cells and tissues where the chemokines are synthesized. These molecules are ligands for seven transmembrane G protein linked receptors that induce a signaling cascade costimulation for 1 cell activation in addition to participating in transendothelial migration of leukocytes. Over twelve different human chemokine receptors are known, including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR1, CXCR2, CXCR3, and CXCR4. The chemokines are divided between two main subfamilies, referred to as CC and CXC. CC and CXC chemokines are distinct from each other in their N terminal amino acid sequence which starts either with cysteine-cysteine or cysteine-X-cysteine where X is typically another L-amino acid. Another class of chemokine receptors is represented by CX3CR1. These receptors have a cysteine-X3-cysteine motif. The families are also distinct in their binding pattern to their receptors. For example, the CC chemokines bind to CC receptors and not to CXC receptors and vice versa.
Binding of a chemokine to its receptor typically induces intracellular signaling responses such as a transient rise in cytosolic calcium concentration, followed by cellular biological responses such as chemotaxis. Different chemokines regulate the trafficking of distinct populations of hemopoietic cells by activating specific transmembrane receptors expressed by these cells.
CXCR4, also known as CXCL12 and PBSF, is a G-protein coupled receptor. The natural ligand for CXCR4 is stromal cell-derived factor (SDF1). SDF1 alpha and 1 beta are small cytokines belonging to the intercrine CXC subfamily. In certain aspects, the present invention relates to the discovery that beta-defensins, and particularly beta-defensin-3 (“BD-3”), and related polypeptides, referred to herein as BD-polypeptides, antagonize the SDF1-CXCR4 signaling pathway.
CXCR4 is a positive regulator of angiogenesis, including tumor angiogenesis, and a negative regulator of mobilization of hematopoietic stem coils from the bone marrow into circulation. CXCR4 overactivation may also lead to defects in cargiogenesis. Accordingly, BD agents may be used to downregulate any of the various known and unknown CXCR4 activities.
SDF1 and its receptor CXCR4 have been shown to be critical for murine bone marrow engraftment by human SCID repopulating stem cells. Treatment of human cells with anti-CXCR antibodies prevented engraftment and promote mobilization. CD34(+)CD38(−/low) cells can be converted to CD34(+)CD38(−/low)CXCR4(+) stem cells by pretreatment with IL6 (147620) and stem cell factor (184745), which increased CXCR4 expression. This pretreatment potentiated migration to SDF1 and engraftment in primary and secondary transplanted mice.
CXCR4 is highly expressed in primary and metastatic human breast cancer cells but is undetectable in normal mammary tissue. High expression levels of the CXCR4 ligand, SDF1, are found in lymph nodes, lung, liver, and bone marrow. Analysis of malignant melanomas determined that in addition to CXCR4 and CCR7, these tumors also had high levels of CCR10; its primary ligand is CCL27, a skin-specific chemokine involved in the homing of memory T cells into the skin. These chemokines, as well as lung and liver extracts, also induce directional migration of breast cancer cells in vitro, which can be blocked by antibodies to CXCR4 or CCL21. Histologic and quantitative PCR analyses showed that metastasis of intravenously or orthotopically injected breast cancer cells could be significantly decreased in SCID mice by treatment with anti-CXCR4 antibodies. It has been proposed that the nonrandom expression of chemokine receptors in breast cancer and malignant melanoma, and probably in other tumor types, indicates that small molecule antagonists of chemokine receptors may be useful to interfere with tumor progression and metastasis in tumor patients.
CXCR4-SDF1 signaling is associated with primary intraocular B-cell lymphoma (PIOL). PIOL eyes show expression of CXCR4 and CXCR5 in the lymphoma cells.
Mobilization of hematopoietic progenitor cells (HPCs) by granulocyte colony-stimulating factor (GCSF) or cyclophosphamide is due to the disruption of the CXCR4/CXCL12 chemotactic pathway. The mobilization of HPCs coincided in vivo with the cleavage of the N terminus of the chemokine receptor CXCR4 found on HPCs. This resulted in the loss of chemotactic response of the HPCs to the CXCR4 ligand, CXCL12. The concentration of CXCL12 was also decreased in vivo in the bone marrow of mobilized mice, and this decrease coincided with the accumulation of serine proteases capable of direct cleavage and inactivation of CXCL12. As both CXCL12 and CXCR4 are essential for the homing and retention of HPCs in the bone marrow, the proteolytic degradation of CXCL12 and CXCR4 may represent a critical step in the mobilization of HPCs into the peripheral blood by GCSF or cyclophosphamide.
The von Hippel-Lindau tumor suppressor protein (VHL) negatively regulates CXCR4 expression owing to its capacity to target hypoxia-inducible factor (HIF 1-alpha; 603348) for degradation under normoxic conditions. This process is suppressed under hypoxic conditions, resulting in HIF-dependent CXCR4 activation. An analysis of clear cell renal carcinoma that manifests mutations in the VHL gene in most cases revealed an association of strong CXCR4 expression with poor tumor-specific survival. These results suggest a mechanism for CXCR4 activation during tumor cell evolution and imply that VHL inactivation acquired by incipient tumor cells early in tumorigenesis confers not only a selective survival advantage but also the tendency to home to selected organs.
Vascularization of organs generally occurs by remodeling of the preexisting vascular system during their differentiation and growth to enable them to perform their specific functions during development. The molecules required for early vascular systems, many of which are receptor tyrosine kinases and their ligands, are revealed by analysis of mutant mice. As most of these mice die during early gestation before many of their organs have developed, the molecules responsible for vascularization during organogenesis are not identified by this approach. CXCR4 is responsible for B-cell lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septum formation.
It has been shown that CXCR4 is expressed in developing vascular endothelial cells. It has been found that mice lacking either CXCR4 or PBSF/SDF1 have defective formation of the large vessels supplying the gastrointestinal tract. In addition, mice lacking CXCR4 die in utero and are defective in vascular development, hematopoiesis and cardiogenesis, like mice lacking PBSF/SDF1, indicating that CXCR4 is a primary physiologic receptor for PBSF/SDF1. It haws been concluded that PBSF/SDF1 and CXCR4 define a new signaling system for organ vascularization.
Some chemokine receptors also serve as co-receptors for HIV, such that they interact with HIV and with the cellular CD4 receptor to facilitate viral entry into cells. In particular, CXCR4 and CCR5 have been found to be the major co-receptors, although many other chemokine and orphan receptors have also been identified as potential co-receptors for HIV-1. Therapeutic approaches based on antagonist of these receptors have been developed, some of which are currently in clinical trials. In addition, expression of CXCR4 has been associated with osteosarcoma, pancreatic cancer, brain, breast, and colon cancer. Some chemokines have been linked to metastasis of cancer from specific organs, including lymph node, bone marrow, and skin; or from carcinomas of breast, head and neck, melanoma or prostate origin.
It has been shown that human epithelial cell derived beta defensins (hBD)-2 and -3 block HIV-1 replication via a direct interaction with virions and through modulation of the CXCR4 co-receptor on immunocompetent cells.
Accordingly, there is a need for agents that induce or increase beta-defensin production and agents that modulate activities of a beta-defensin.
SUMMARY OF THE INVENTIONIt is an aspect of the disclosure to provide agents that affect SDF-1 and CXCR4-mediated processes.
The present invention provides that hBD-3 promotes directly the internalization of CXCR4 yet does not induce calcium flux, extracellular signal-regulated kinase (ERK-1/2) phosphorylation or chemotaxis. HBD-3 competes with stromal derived factor 1 (SDF-1), the natural ligand for CXCR4, for cellular binding and blocks SDF-1-induced calcium flux, ERK-1/2 phosphorylation and chemotaxis, without effects on other G protein coupled receptors. It is envisioned that the novel activity of this endogenous CXCR4 antagonist may provide a new strategy for HIV therapies or immunomodulation.
In certain aspects, the invention relates to the discovery that beta-defensin polypeptides act as antagonists of the CXCR4 receptor. The CXCR4 receptor participates in a range of physiological and pathological processes, including angiogenesis, hematopoiesis, tumor angiogenesis, growth and metastasis, and cardiogenesis. Additionally, as demonstrated herein, beta-defensins modulate certain immune cells, promoting the expression of CD40, CD80 and/or CD46. This immunomodulatory effect may be mediated by CCR6 and/or CXCR4.
An aspect of the invention provides a method for inhibiting the SDF1-CXCR4 signaling pathway in a subject, comprising administering to the subject an effective amount of a BD-inducing agent.
An aspect of the invention provides a method for inhibiting a CXCR4-related process in a subject, comprising administering to the subject an effective amount of a BD-inducing agent.
An aspect of the invention provides a method for treating a subject, comprising administering to the subject an effective amount of a BD-inducing agent.
An aspect of the invention provides a method for mobilizing hematopoietic stem cells in a subject, comprising administering to the subject an effective amount of a BD-inducing agent.
An aspect of the invention provides a method for promoting cardiogenesis or the formation of cardiomyocytes in a subject, comprising administering to the subject an effective amount of a BD-inducing agent.
A further aspect of the invention provides a method for inducing CD40, CD80 and/or CD46 expression in immune cells, particularly myeloid dendritic cells and monocytes, comprising administering to the subject an effective amount of a BD-inducing agent.
In certain embodiments, the methods and compositions disclosed herein may employ, as a BD-inducing agent, a Fusobacterium associated defensin-inducer (FAD-I) polypeptide. In certain embodiments, a FAD-I polypeptide is a polypeptide that comprises an amino acid sequence that is at least 90%, 95%, 97%, 99% or 100% identical to the amino acid sequence of SEQ ID Nos.: 1, 3, 5, and 7. In certain embodiments, a FAD-I polypeptide is a polypeptide comprising a portion of an amino acid sequence that is at least 90%, 95%, 97%, 99% or 100% identical to SEQ ID Nos.: 1, 3, 5, and 7, wherein said portion is sufficient to induce beta-defensin-2 (BD-2) production, beta-defensin-3 (BD-3) production, or both, and preferably induction of human beta-defensin-2 (hBD-2) production, human beta-defensin-3 (hBD-3) production, or both. In certain embodiments a FAD-I polypeptide is a polypeptide obtained when a nucleic acid comprising a nucleic acid sequence at least 90%, 95%, 97%, 99% or 100% identical to a nucleic acid sequence of SEQ ID NOs.: 2, 4, 6, and 8 is expressed in a cell, preferably a bacterial cell, such as F. nucleatum or E. coli. In certain embodiments, a FAD-I polypeptide is a polypeptide derived from a F. nucleatum cell wall, having a monomeric molecular weight range of about 12-14 kDa and which polypeptide induces BD-2 production, BD-3 production, or both. In certain embodiments, the FAD-I polypeptide additionally has a pI of between 4.0 and 5.5. In certain embodiments a FAD-I polypeptide is purified or partially purified. In preferred embodiments, the FAD-I polypeptide and/or a composition comprising the FAD-I polypeptide induces beta-defensin production in at least one epithelial cell type, such as an oral epithelial cell, a corneal epithelial cell, a skin cell. In preferred embodiments, the defensin induced is a BD-2, a BD-3, or both, and in humans, an hBD2, and hBD3, or both. In certain embodiments, the FAD-I polypeptide and/or composition comprising the FAD-I polypeptide induces beta-defensin production in one or more cells of a mucosal epithelium, such as the vagina, rectum, urethra, intestines, nasal epithelium, oral epithelium or corneal epithelium.
In certain embodiments, the BD-inducing agent is a synthetic analog of double stranded RNA. In one particular embodiment, the BD-inducing agent is polyinosine-polycytidylic acid (poly I:C).
In one embodiment, an agent of the invention has a 50% effectiveness at a concentration of about 10 μM or less.
In certain embodiments, the invention provides systemic administration to a subject of an agent of the invention. Systemic administration may include direct administration to the bloodstream. The invention also provides methods and compositions suitable for such systemic administration. In certain embodiments, the invention also provides local administration to a subject of an agent of the invention.
In certain embodiments, the defensin-stimulating composition is formulated for local delivery, such as to a particular epithelium, optionally a mucosal epithelium. For example, a composition may be formulated for delivery to the mouth, the eye, the skin, the vagina, the rectum, the intestines and the nose or other airways. In certain embodiments, the application provides methods for making a medicament comprising a FAD-I and an excipient for the administration by one of the above-described modes.
In certain embodiments, the invention further provides administering an agent of the invention in combination with an additional anticancer agent.
BRIEF DESCRIPTION OF THE DRAWINGS
For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
A “chimeric polypeptide” or “fusion polypeptide” is a fusion of a first amino acid sequence with a second amino acid sequence where the first and second amino acid sequences are not naturally present in a single polypeptide chain.
The term “compound” used herein is meant to include, but not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, natural product extract libraries, and any other molecules (including, but not limited to, chemicals, metals and organometalic compounds).
The term “ED50” means the dose of a drug which produces 50% of its maximum response or effect.
An “effective amount” of, e.g., an agent of the invention, with respect to the subject method of treatment, refers to an amount of the activator in a pharmaceutical preparation which, when applied as part of a desired dosage regimen brings about a meaningful mitigation of a disease state, according to clinically acceptable standards.
“Expression” of a polypeptide refers to the amount of mature polypeptide produced by a cell. Accordingly, the level of expression can be modulated at different stages such as transcription, translation, and posttranslational processing.
An “expression construct” is any recombinant nucleic acid that includes an expressible nucleic acid and regulatory elements sufficient to mediate expression in a suitable host cell. For example, an expression construct may contain a promoter or other RNA polymerase contact site, a transcription start site or a transcription termination sequence. An expression construct for production of a protein may contain a translation start site, such as an ATG codon, a ribosome binding site, such as a Shine-Dalgarno sequence, or a translation stop codon.
The term “heterologous” as used in describing a nucleic acid with respect to another nucleic acid means that the two nucleic acids are not normally operably linked to each other or do not naturally occur in adjacent positions.
The term “LD50” means the dose of a drug which is lethal in 50% of test subjects.
The term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. Nucleic acids may include unconventional modifications to any portion, including, for example, the sugar phosphate backbone or the base portion.
The term “or” is used herein to mean, and is used interchangeably with, the term “and/or”, unless context clearly indicates otherwise.
The term “including” is used herein to mean, and is used interchangeably with, the phrase, “including but not limited to.”
The term “percent identical” refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Various alignment algorithms or programs may be used, including FASTA, BLAST or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
The term “purified protein” refers to a preparation of a protein or proteins which are preferably isolated from, or otherwise substantially free of, other proteins normally associated with the protein(s) in a cell or cell lysate. The term “substantially free of other cellular proteins” (also referred to herein as “substantially free of other contaminating proteins”) is defined as encompassing individual preparations of each of the component proteins comprising less than 20% (by dry weight) contaminating protein, and preferably comprises less than 5% contaminating protein. Functional forms of each of the component proteins can be prepared as purified preparations by using a cloned gene as described in the attached examples. By “purified,” it is meant, when referring to component protein preparations used to generate a reconstituted protein mixture, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture). The term “purified” as used herein preferably means at least 80% by dry weight, more preferably in the range of 85% by weight, more preferably 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term “pure” as used herein preferably has the same numerical limits as “purified” immediately above.
The term “recombinant nucleic acid construct” includes any nucleic acid comprising at least two sequences which are not adjacent on a nucleic acid strand in nature. A recombinant nucleic acid may be generated in vitro, for example by using the methods of molecular biology, or in vivo, for example by insertion of a nucleic acid at a novel chromosomal location by homologous or non-homologous recombination.
The terms “protein” and “polypeptide” are used interchangeably.
A “patient” or “subject” to be treated by the subject method can mean either a human or non-human animal.
“Small molecule” as used herein, is meant to refer to a compound that has a molecular weight of less than about 5 kDa and most preferably less than about 2.5 kDa. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical or biological mixtures comprising arrays of small molecules, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention.
The term “therapeutic index” refers to the therapeutic index of a drug defined as LD50/ED50.
The term “treat,” “treating,” or “treatment” as used herein means to counteract a medical condition to the extent that the medical condition is improved according to a clinically acceptable standard for measurement.
A molecule, or complex of the invention, or the agent used in the method of the invention may be rendered insoluble. For example, a molecule, or agent may be bound to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insoluble molecule or agent may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
In certain aspects, the invention provides methods and compositions relating to BD-inducing agents.
By “BD-inducing agent” is meant an agent that induces or increases expression of a BD-polypeptide in a cell. Preferably, a BD-inducing agent induces or increases expression of an HBD-2 polypeptide or HBD-3 polypeptide in a human cell; such a BD-inducing agent may also be referred to as HBD-inducing agent. Expression of a BD polypeptide can be determined by known methods such as measuring mRNA level or protein level, as described in greater detail below. By “induces or increases” is meant that (a) a BD-polypeptide is expressed in the presence of a BD-inducing agent whereas the BD-polypeptide is not detectably expressed in the absence of the BD-inducing agent or (b) expression of BD-polypeptide in the presence of a BD-inducing agent is greater (e.g., by 1, 2, 5, 10, 20, 100, 1000, 10000 fold) than that in the absence of the BD-inducing agent.
A BD-inducing agent may be essentially any composition, including, for example, one or more polypeptides (including portions thereof or a fusion protein), small molecules, peptidomimetics, nucleic acids, or mixtures thereof.
Certain small molecules were known to induce epithelial beta defensin expression. For example, isoleucine-1 and its analogs can activate transcription of epithelial beta defensin genes and the transcriptional activation appears to involve the NF-kappaB/rel family of trans-activating factors. High calcium concentration (1.7 mM) alone applied in culture medium also appears sufficient to induce HBD-2 and HBD-3 mRNA expression in epidermal cells. As shown in Example 2 and
In certain embodiments, the BD-inducing agent induces a HBD-2 or HBD-3.
In certain embodiments, the methods and compositions disclosed herein may employ, as a BD-inducing agent, a FAD-I polypeptide. In certain embodiments, an FAD-I polypeptide is a polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, or 98-99% identical to an amino acid sequence of SEQ ID NO: 1, 3, 5, or 7. In certain embodiments, an FAD-I polypeptide is a polypeptide obtained by expressing a nucleic acid that is at least 80%, 85%, 90%, 95%, 97%, or 98-99% identical to a nucleic acid of SEQ ID NO: 2, 4, 6, or 8 in a cell. In certain embodiments, a FAD-I polypeptide is a polypeptide encoded by a nucleic acid that is at least 80%, 85%, 90%, 95%, 97%, or 98-99% identical to a nucleic acid of SEQ ID NO: 2, 4, 6, or 8.
In certain embodiments, a FAD-I polypeptide is a polypeptide comprising a portion of an amino acid sequence that is at least 90%, 95%, 97%, 99% or 100% identical to SEQ ID NOs.: 1, 3, 5, or 7, wherein said portion is sufficient to induce BD-2 production, BD-3 production, or both, and preferably induction of hBD-2 production, hBD-3 production, or both. In certain embodiments a FAD-I polypeptide is a polypeptide obtained by expressing a nucleic acid comprising a nucleic acid sequence at least 90%, 95%, 97%, 99% or 100% identical to a nucleic acid sequence of SEQ ID NOs.: 2, 4, 6, or 8 in cell, preferably a bacterial cell, such as F. nucleatum or E. coli. In certain embodiments, a FAD-I polypeptide is a polypeptide derived from an F. nucleatum cell wall, having a monomeric molecular weight range of about 12-14 kDa and which polypeptide induces BD-2 production, BD-3 production, or both. In certain embodiments, the FAD-I polypeptide additionally has a pI of between 4.0 and 5.5. In certain embodiments a FAD-I polypeptide is purified or partially purified. In preferred embodiments, the FAD-I polypeptide and/or a composition comprising the FAD-I polypeptide induces beta-defensin production in at least one epithelial cell type, such as an oral epithelial cell, a corneal epithelial cell, a skin cell. In preferred embodiments, the defensin induced is a BD-2, a BD-3, or both, and in humans an hBD2, an hBD3, or both. In certain embodiments the FAD-I polypeptide and/or composition comprising the FAD-I polypeptide induces beta-defensin production in one or more cells of a mucosal epithelium, such as the vagina, rectum, urethra, intestines, nasal epithelium, oral epithelium or corneal epithelium.
1Gene name; taken from the F. nucleatum ATCC 25586 genome;
2Preprotein; designation of entire protein, containing the signal peptide;
3Mature protein; designation of the protein without the signal peptide;
4% identity; % of matched peptide sequence of entire protein;
5—; no signal peptide found.
Table 1. Predicted Physical Properties of FAD-I Polypeptides. 1: Gene name; taken from the F. nucleatum ATCC 25586 genome; 2: Preprotein; designation of entire protein, containing the signal peptide; 3. Mature protein; designation of the protein without the signal peptide; 4. % identity; % of matched peptide sequence of entire protein; 5 -; no signal peptide found.
Another aspect of the disclosure relates to polypeptides derived from a full-length FAD-I polypeptide. Isolated peptidyl portions of the subject proteins can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such polypeptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, any one of the subject proteins can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function in a cellular assay for BD-2 induction, BD-3 induction, or both.
Various cell lines can be utilized for screening of the candidate BD-inducing agents, e.g., normal human oral epithelial cells. Candidate agents can be screened for their ability to induce in the cells expression of a BD gene such as for example a BD-2 or BD-3. Expression of a BD gene can be determined and measured by level of mRNA transcribed from the BD gene, level of protein translated from the mRNA, or level of mature protein processed from the translated protein. Thus, a BD-inducing agent may induce or increase expression of a BP polypeptide by activating transcription of the BD gene encoding the BD-polypeptide. Alternatively, a BD-inducing agent may facilitate translation from a BD mRNA. Yet another alternative BD-inducing agent may enhance processing of a translated product of a BD gene and thereby increase production of the mature BD-polypeptide.
Conventional methods may be employed or modified to determine mRNA level or protein level. To illustrate, an Example below describes a method of real-time RT-PCR assay to quantify HBD mRNA. Antibodies to a BD-polypeptide may be used to determine protein level. The BD expressed maybe endogenous to the cells utilized in the assays, for example the normal human oral epithelial cells. As is known in the art, cell lines can also be created via transfections with nucleic acids encoding the proteins (e.g., an HBD-2 or HBD-3 polypeptide) desired to be present for a subject assay.
Alternatively, expression of a BD gene may also be measured by determining the activity of a promoter responsible for expression of a native BD gene, which may also be referred to as a BD-promoter. A “promoter” is a control or regulatory sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. A BD-promoter is a control or regulatory sequence that is responsible for expression of a BD gene in a cell. A BD-promoter may comprise a nucleic acid sequence that can interaction with transcriptional activators. For example, the BD-promoter is speculated to be responsive to NF-kappaB/rel family of trans-activating factors. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
Promoter activity can also be determined using conventional methods such as reporter gene assays in which reporter genes are operably linked to a promoter of interest, The phrase “operably linked” means that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
Various reporter gene constructs may be used in accord with the methods of the invention and include, for example, reporter genes which produce such detectable signals as selected from the group consisting of an enzymatic signal, a fluorescent signal, a phosphorescent signal and drug resistance. To illustrate, cells may be transfected with a BD-2 or BD-3 reporter gene construct, wherein a BD-promoter or potential BD-inducing agent responsive regulatory element of a BD-2 or BD-3 gene is operably linked to a reporter gene, and preferably a reporter gene that produces a fluorescent protein (e.g. green fluorescent protein) or an enzyme that can generate a fluorescent substrate or other detectable signal. The cells are then contacted with a candidate BD-inducing agent and reporter gene expression is assessed. In certain embodiments, an assay may comprise employing a cell that naturally has inducible BD-2 or BD-3 expression, such as a normal human oral epithelial cell. Alternatively, the cells maybe transfected with a reporter gene construct.
In certain aspects, the invention provides isolated and/or recombinant nucleic acids encoding FAD-I polypeptides, such as, for example, SEQ ID NOs.: 2, 4, 6, or 8. Nucleic acids of the invention are further understood to include nucleic acids that comprise variants of SEQ ID NOs.: 2, 4, 6, or 8. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include coding sequences that differ from the nucleotide sequence of the coding sequence designated in SEQ ID NOs.: 2, 4, 6, or 8, e.g. due to the degeneracy of the genetic code. For example, nucleic acids encoding FAD-I polypeptides may be nucleic acids comprising a sequence that is at least 90%, 95%, 99% or 100% identical to the sequence of SEQ ID NOs.: 2, 4, 6, or 8, or a sequence that encodes the polypeptide of SEQ ID NOs.: 1, 3, 5, 6, or 7. In other embodiments, variants will also include sequences that will hybridize under highly stringent conditions to a coding sequence of a nucleic acid sequence designated in SEQ ID NOs.: 2, 4, 6, or 8.
One of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed. In one embodiment, the invention provides nucleic acids which hybridize under low stringency conditions of 6×SSC at room temperature followed by a wash at 2×SSC at room temperature.
Isolated nucleic acids which differ from SEQ ID NOs.: 2, 4, 6, or 8 due to degeneracy in the genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among Fusobacterium cultivars. One skilled in the art will appreciate that these variations in one or more nucleotides of the nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.
Optionally, a FAD-I nucleic acid of the invention will genetically complement a partial or complete FAD-I loss of function phenotype in an F. nucleatum cell. For example, a FAD-I nucleic acid of the invention may be expressed in a cell in which endogenous FAD-I has been knocked out, and the introduced FAD-I nucleic acid will mitigate a phenotype resulting from the knockout. An exemplary FAD-I loss of function phenotype is a decrease in the stimulation of hBD-2 expression, hBD-3 expression, or both in NHOECs or similarly sensitive cell types.
In certain aspects, nucleic acids encoding FAD-I polypeptides may be used to increase FAD-I expression in an organism or cell by direct delivery of the nucleic acid. A nucleic acid therapy construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which encodes a FAD-I polypeptide.
In another aspect of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a subject FAD-I polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the FAD-I polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers and “Other expression control elements. For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding a FAD-I polypeptide. Such useful expression control sequences, include, for example, the early and late promoters of SV 40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, the lac system, the tip system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
As will be apparent, the subject gene constructs can be used to cause expression of the subject FAD-I polypeptides in cells propagated in culture, e.g. to produce proteins or polypeptides, including fusion proteins or polypeptides, for purification.
This invention also pertains to a host cell transfected with a recombinant gene including a coding sequence for one or more of the subject FAD-I polypeptides. The host cell may be any prokaryotic or eukaryotic cell. For example, a polypeptide of the present invention may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
Accordingly, the present invention further pertains to methods of producing the subject FAD-I polypeptides. For example, a host cell transfected with an expression vector encoding a FAD-I polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the polypeptide. In a preferred embodiment, the FAD-I polypeptide is a fusion protein containing a domain, which facilitates its purification, such as a FAD-I-GST fusion protein, FAD-I-intein fusion protein, FAD-I-cellulose binding domain fusion protein, FAD-polyhistidine fusion protein etc.
Methods for purifying FAD-I from F. nucleatum cell wall extracts are also disclosed herein.
A nucleotide sequence encoding a FAD-I polypeptide can be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial) cells, are standard procedures.
A recombinant FAD-I nucleic acid can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Expression vehicles for production of a recombinant FAD-I polypeptides include plasmids and other vectors. For instance, suitable vectors for the expression of a FAD-I polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae. These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used.
The preferred mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells are known in the art. In some instances, it may be desirable to express the recombinant FAD-I polypeptide by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the E-gal containing pBlueBac III).
It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins. Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing such recombinant polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP.
In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+ metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified FAD-I polypeptide.
Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence.
A variety of polypeptide agents are described herein, including naturally occurring beta-defensin and beta-defensin-inducing polypeptides. Variants of such polypeptides may be readily prepared and tested for activity. An example of variant agents of the invention may be variant polypeptides, e.g., a FAD-I polypeptide having a modification that enhances the modified polypeptide's stability or other properties. Another example of variant agents of the invention may be created through making peptidomimetics based on, e.g., a BD-polypeptide or a FAD-I polypeptide. Yet another example of variant agents of the invention may be variant nucleic acids, e.g., a nucleic acid having a modification or mutation that enhances the modified or mutated nucleic acid's uses in, e.g., making recombinant proteins or gene therapy.
Variant agents of the invention may be evaluated by the methods described herein. For example, variant BD agents may be evaluated for their CXCR4 binding properties and/or SDF-1 competition. Variant BD-inducing agents may also be evaluated for their ability to induce expression of a BD-polypeptide or a reporter gene operably linked to a promoter responsible for expression of a BD gene. Other variant agents may also be assayed for their effect on the interaction between a BD-polypeptide and a chemokine receptor.
In certain embodiments, small molecules are candidate agents to be screened to identify an agent of the invention. In certain preferred embodiments, small molecules are generated by combinatorial synthesis.
The candidate agents used in the invention may be pharmacologic agents already known in the art or may be agents previously unknown to have any pharmacological activity. The agents may be naturally arising or designed or prepared in the laboratory. They may be isolated from microorganisms, animals, or plants, or may be produced recombinantly, or synthesized by chemical methods known in the art. In some embodiments, candidate agents are identified from small chemical libraries, peptide libraries, or collections of natural products using the methods of the present invention. A library with over two million synthetic compounds that is compatible with miniaturized cell-based assays has been described. It is within the scope of the present invention that such a library may be used to screen for agents that are BD agents, BD-inducing agents, or other agents of the invention. There are numerous commercially available compound libraries, such as the Chembridge DIVERSet. Libraries are also available from academic investigators, such as the Diversity set from the NCI developmental therapeutics program.
One basic approach to search for a subject agent is screening of compound libraries. One may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to identify useful compounds by “brute force.” Screening of such libraries, including combinatorially generated libraries, is a rapid and efficient way to screen a large number of related (and unrelated) compounds for activity.
Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third, and fourth generation variant compounds modeled on active but otherwise undesirable compounds. It will be understood that undesirable compounds include compounds that are typically toxic, but have been modified to reduce the toxicity or compounds that typically have little effect with minimal toxicity and are used in combination with another compound to produce the desired effect.
The invention also provides mimetics, e.g., peptide or non-peptide agents, which are able to mimic action of the authentic protein, e.g., an HBD-2, an HBD-3, or a FAD-I, in a host. Such mutagenic techniques as described herein, as well as the thioredoxin system, are also particularly useful for mapping the determinants which participate in protein-protein interactions of interest. For example, amino acid residues of a BD-polypeptide may be mapped to determine which ones affect the BD-polypeptide's activity, e.g., antimicrobial or antiviral effectiveness, interaction with a chemokine receptor. To illustrate, the critical residues of a BD-polypeptide such as for example an HBD-2 or HBD-3 polypeptide can be determined and used to generate its derived peptidomimetics which can affect the binding between the HBD-2 or HBD-3 polypeptide with another molecule such as the CXCR4 receptor protein. By employing, for example, scanning mutagenesis to map the amino acid residues of a BD-polypeptide which are involved in interacting to another molecule such as CXCR4, peptidomimetic compounds can be generated which mimic those residues involved in the interactions of interest. In other aspects, the critical residues of a BD-inducing polypeptide can be determined with which can induce expression of a BD-polypeptide. Non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine, azepine, substituted gamma lactam rings, keto-methylene pseudopeptides, b-turn dipeptide cores, and b-aminoalcohols.
Many useful pharmacological compounds are compounds structurally related to compounds that interact naturally with the target, e.g., a BD-polypeptide, a CXCR4, a promoter responsible for driving expression of a beta defensin in cells, the binding interface between a BD-polypeptide and a CXCR4. Creating and examining the action of such molecules is known as “rational drug design,” and include making predictions relating to the structure of the targets. Thus, it is understood that a subject agent identified by the present invention may be a small molecule or any other compound (e.g., polypeptide or polynucleotide) that may be designed through rational drug design starting from known binders of the targets.
The goal of rational drug design is to produce structural analogs of biologically active target compounds. By creating such analogs, it is possible to fashion drugs that are more active or stable than the natural or earlier generation molecules, have different susceptibility to alteration or may affect the function of various other molecules. In one approach, one can generate a three-dimensional structure for molecules like the targets, and then design a molecule for its ability to interact with the targets. This could be accomplished by X-ray crystallography, computer modeling, or by a combination of both approaches.
Certain embodiments of the invention employ polypeptides, e.g., a BD-inducing agent comprising a FAD-I polypeptide. Variant polypeptides may be derived from a polypeptide of the invention, e.g., a full-length FAD-I polypeptide. Isolated peptidyl portions of the subject polypeptides can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acids encoding such polypeptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, any one of the subject polypeptides can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as an agent of the invention.
It is also possible to modify the structure of a subject polypeptide for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified polypeptides, when designed to retain at least one activity of the naturally-occurring form of the protein, are considered functional equivalents of the subject polypeptide. Such modified polypeptides can be produced, for instance, by amino acid substitution, deletion, or addition.
For instance, it is reasonable to expect, for example, that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and methionine. Whether a change in the amino acid sequence of a polypeptide results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type protein. For instance, such variant forms of BD-inducing, FAD-I polypeptide can be assessed, e.g., for their ability induce BD-2 or BD-3 production in a cell. Such variant forms of a BD-polypeptide can be assessed for any of the various biochemical and biological activities described herein. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
This invention further contemplates a method of generating sets of combinatorial mutants of the subject polypeptides (e.g., a BD-inducing polypeptide such as FAD-I) as well as truncation mutants. The purpose of screening such combinatorial libraries is to generate, for example, variants or homologs which can act as either agonists or antagonist, or alternatively, which possess novel activities all together. Combinatorially-derived variants or homologs can be generated which have a selective potency relative to a naturally occurring subject polypeptide. Such proteins, when expressed from recombinant DNA constructs, can be used in, e.g., gene therapy protocols.
Likewise, mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding wild-type protein, For example, the variant protein can be rendered either more stable or less stable to proteolytic degradation or other cellular process which result in destruction of, or otherwise inactivation of the polypeptide of interest.
In similar fashion, variants or homologs can be generated by the present combinatorial approach to act as antagonists, in that they are able to interfere with the ability of the corresponding wild-type protein to function.
In a representative embodiment, the amino acid sequences for a population of BD-inducing polypeptides are aligned, preferably to promote the highest homology possible. Such a population of variants can include, for example, homologs from one or more species of Fusobacterium or homologs from the same species but which differ due to mutation. Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences. In a preferred embodiment, the combinatorial library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential sequence that has function similar to a naturally occurring subject polypeptide (e.g., a FAD-I). For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential beta defensin nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display).
There are many ways by which the library of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential sequences. The synthesis of degenerate oligonucleotides is well known in the art. Such techniques have been employed in the directed evolution of other proteins.
Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, BD-inducing variant agents can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like by linker scanning mutagenesis; by saturation mutagenesis; by PCR mutagenesis; or by random mutagenesis, including chemical mutagenesis, etc. Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying truncated and bioactive variants of BD-inducing polypeptides.
A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.
Polypeptides of the invention may further comprise post-translational or non-amino acid elements, such as hydrophobic modifications (e.g. polyethylene glycols or lipids), poly- or mono-saccharide modifications, phosphates, acetylations, etc. Effects of such elements on the functionality of a polypeptide may be tested as described herein.
Polypeptides of the invention may also comprise fusion or chimeric polypeptides. Fusion polypeptides may be useful for making recombinant polypeptides of the invention. Alternatively, fusion polypeptides may have enhanced stability or functionality which can be tested as described herein. An exemplary fusion polypeptide may be a dimeric BD agent which includes a dimerization domain.
In certain embodiments, the application provides methods for treating a variety of diseases by administering a defensin-inducing composition, such as a FAD-I. Examples of diseases to be treated include infectious diseases of the various epithelial tissues, including conjunctivitis, gingivitis, tooth decay, sinusitis, urinary tract infections, gastroenteritis and dermatitis, any of which may be bacterial, fungal or viral in origin. Diseases to be treated include systemic infectious diseases as well. In certain embodiments, compositions comprising an FAD-I may be used to treat infections that are resistant to one or more other antimicrobial agents, such as vancomycin resistant Enterococcus or methicillin resistant Staphylococcus aureus, penicillin or cephalosporin resistant Pneumococcus, multi-drug resistant Pseudomonas, to name only a few. Cancers may also be treated using compositions disclosed herein, including squamous cell carcinomas, such as oral squamous cell carcinomas, and other tumor types. In addition, compositions disclosed herein may be used to bolster the immune system of immunocompromised patients.
In certain aspects the application provides methods for stimulating BD-2 production, BD-3 production, or both, comprising contacting a cell with a composition comprising a FAD-I. In certain preferred embodiments, the cell is an epithelial cell, optionally an epithelial cell located in a vertebrate such as a human. In certain exemplary embodiments, the epithelial cell is an oral epithelial cell, a corneal epithelial cell or a keratinocyte. In certain embodiments, the epithelial cell is a mucosal epithelial cell.
BD-2 polypeptides are known promote maturation and/or production of immune cells, such as dendritic cells. These cells are important in many aspects of immunity, including recognition and destruction of a wide variety of cancers. Accordingly, compositions disclosed herein are envisioned as being suitable for treatment of cancers, as well for bolstering the immune response of immunocompromised patients, such as, for example, patients that have received irradiation therapy or patients that suffer from an immunodeficiency syndrome, such as that caused by HIV.
BD-2s and BD-3s exhibit broad-spectrum antimicrobial activity and are active against a range of bacteria, including gram negative and gram positive bacteria. Accordingly, FAD-I may be used to treat systemic and local infections of any of these bacteria. For example, FAD-I may be used against bacteria that are responsible for periodontal disease, such Porphyromonas gingivalis.
BD-2s and BD-3s are also active against a range of pathogenic fungi. Accordingly, it is envisioned that BD-inducing agents may be used to treat systemic and local fungal infections. For example, FAD-I may be used to treat Candida albicans infections. C. albicans is the causative agent for many yeast infections in women, as well as for monocutaneous fungal disease in HIV patients.
BD-2s and BD-3s are active against viral agents, and particularly enveloped viruses, such as many retroviruses and RNA viruses, including lentiviruses such as HIV and SIV.
Unlike most antimicrobial agents, resistance to beta-defensins is rare in pathogenic organisms. Accordingly, a BD inducing agent may be used in situations where use of a traditional antimicrobial agent would be ill-advised because of the risk of resistance development. For example, FAD-I may be administered to patients that are at risk for an infection, as in the case of immunocompromised patients, as well as people who expect to encounter infectious agents.
Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. In certain aspects, the application provides compositions comprising an agent of the invention and an excipient that is a pharmaceutically acceptable carrier. Such compositions may be designed for delivery systemically or locally, and may be formulated for administration in any convenient way for use in human or veterinary medicine.
Thus, another aspect of the present invention provides compositions, optionally pharmaceutically acceptable compositions, comprising an amount, optionally a therapeutically-effective amount, of one or more of the agents or compositions described above, formulated together with one or more excipients, including additives and/or diluents. As described in detail below, the compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) systemic or local oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue, toothpastes or mouthwashes, films or strips (e.g., Listerine PocketPaks® Strip, which is a micro-thin starch-based film impregnated with ingredients); (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin or mucous membrane; or (4) intravaginally or intrarectally, for example, as a pessary, cream, foam, or film that dissolves (e.g., the type of film used in vaginal contraceptive Films). However, in certain embodiments an agent or composition of the invention maybe simply dissolved or suspended in sterile water.
The phrase “therapeutically-effective amount” as used herein means that amount of a compound, material, or composition comprising an agent or composition of the present invention which is effective for producing some desired therapeutic effect.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “excipient” as used herein means a material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, optionally pharmaceutically-acceptable, involved in administering the subject BD or BD-inducing polypeptide. Each excipient should be compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable excipients include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mamiitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
Compositions may also include excipients that are salts, preferably relatively non-toxic, inorganic and organic acid salts. These salts can be prepared in situ during the final isolation and purification of the agents or compositions of the disclosure, or by separately reacting a purified agent or composition with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the chloride, hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. Other salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
In other cases, the agents or compositions of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like: (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal anchor parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
Methods of preparing these formulations or compositions include the step of bringing into association an agent or composition of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent or composition of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as tooth pastes or mouth washes and the like, each containing a predetermined amount of an agent or composition of the present invention as an active ingredient. An agent or composition of the present invention may also be administered as a bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets maybe made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration of the agents or compositions of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds (e.g., agents or compositions of the invention), may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more agents or compositions of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the FAD-I polypeptide.
Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal (systemic) or dermal (local) administration of an agent or composition of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to an agent or composition of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Ophthalmic formulations, eye drops, ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This maybe accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
When the agents or compositions of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
An agent of the invention may be incorporated into contraceptives, such as condoms, female condoms, spermicidal ointment, contraceptive films or sponges and the like.
In yet another embodiment, the BD-inducing agent can be administered as part of a combinatorial therapy with other agents. For example, the combinatorial therapy can include a BD-inducing agent with at least one antibacterial, antiviral or antifungal agent. In a preferred embodiment, BD-inducing agent is administered with one or more additional anticancer agents. Others will be, in view of this disclosure, known to those of skill in the art.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, in addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the agents of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
In certain aspects, the invention provides transdermal patches to deliver an agent to a subject. By “transdermal patch” is meant a system capable of delivery of a drug to a subject via the skin, or any suitable external surface, including mucosal membranes, such as those found inside the mouth. Such delivery systems generally comprise a flexible backing, an adhesive and a drug retaining matrix, the backing protecting the adhesive and matrix and the adhesive holding the whole on the skin of the subject patient. On contact with the skin, the drug-retaining matrix delivers drug to the skin, the drug then passing through the skin into the patient's system.
A nucleic acid encoding a BD-inducing polypeptide may be administered to a subject so as to stimulate production of the active polypeptide in vivo. For this purpose, various techniques have been developed for modification of target tissue and cells in vivo. A number of viral vectors have been developed which allow for transfection and, in some cases, integration of the virus into the host. The vector maybe administered by injection, e.g. intravascularly or intramuscularly, inhalation, or other parenteral mode. Non-viral delivery methods such as administration of the DNA via complexes with liposomes or by injection; catheter or biolistics may also be used.
In general, the manner of introducing the nucleic acid will depend on the nature of the tissue, the efficiency of cellular modification required, the number of opportunities to modify the particular cells, the accessibility of the tissue to the nucleic acid composition to be introduced, and the like. The DNA introduction need not result in integration. In fact, non-integration often results in transient expression of the introduced DNA, and transient expression is often sufficient or even preferred. It will typically be desirable to achieve expression of BD-inducing polypeptides in various epithelial cell types, particularly those that do not normally express BD polypeptides at high levels. It may also be desirable to achieve expression of such polypeptides in immume cells, e.g. T cells and cells found in proximity to such cells, e.g. cells of the bone marrow, thymus and blood stream generally.
Any means for the introduction of polynucleotides into mammals, human or nonhuman, may be adapted to the practice of this invention for the delivery of the various constructs of the invention into the intended recipient.
As disclosed herein, BD inducers may be used to treat a variety of disorders and to modulate a variety of processes.
In certain embodiments, the present invention provides compositions and methods for inhibiting angiogenesis and for treating angiogenesis-associated diseases (or disorders). In other embodiments, the present invention provides methods of inhibiting or reducing tumor growth and methods of treating an individual suffering from cancer. These methods involve administering to the individual a therapeutically effective amount of one or more BD inducers as described above. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.
As described herein, angiogenesis-associated diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; inflammatory disorders such as immune and non-immune inflammation; chronic articular rheumatism and psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation and wound healing; telangiectasia psoriasis scleroderma, pyogenic granuloma, cororany collaterals, ischemic limb angiogenesis, corneal diseases, rubeosis, arthritis, diabetic neovascularization, fractures, vasculogenesis, hematopoiesis.
It is understood that methods and compositions of the invention are also useful for treating any angiogenesis-independent cancers (tumors). As used herein, the term “angiogenesis-independent cancer” refers to a cancer (tumor) where there is no or little neovascularization in the tumor tissue.
In particular, BD inducers of the present invention are useful for treating or preventing a cancer (tumor), including, but not limited to, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and leukemia.
In certain embodiments of such methods, one or more BD inducers can be administered, together (simultaneously) or at different times (sequentially). In addition, such agents can be administered with another agent for treating cancer or for inhibiting angiogenesis.
In certain embodiments, the subject BD inducers can be used alone. Alternatively, the subject BD inducers may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor). For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present invention recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of one or more BD inducers of the invention.
The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
We have identified in F. nucleatum, a heat stable, cell surface associated factor that induces hBD-2, hBD-3, or both in NHOECs, and consequently protects them from P. gingivalis invasion. We refer to this agent as FAD-I for Fusobacterium associated defensin inducer. KB cells from an oral cell line that originated from a human oral squamous cell carcinoma (ATCC CCL-17), did not express hBD-2 after FAD-I challenge; and were not protected from P. gingivalis invasion. A 40 kDa cell wall associated component bound NHOEC surface proteins but failed to bind an equivalent fraction from KB cells. Organic extraction of the cell wall fraction of F. nucleatum generated a water soluble top layer that induces hBD-2. Proteinase K digestion of the top layer abolished both the hBD-2 inducibility and the 40 kDa component. Further biochemical analyses, along with bioactivity studies of FAD-I identified FAD-I as the polypeptide of SEQ ID NO.:1.
The top layer fraction increased hBD-2 expression in NHOECs with minimal induction of IL-8. This correlates with in vivo findings showing that while hBD-2 is expressed only in the presence of infection or inflammation in skin, trachea and gut epithelium, in oral tissues it is expressed in normal uninflamed conditions. Our results also showed that hBD-3 expression in normal human skin keratinocytes was induced by F. nucleatum cell wall. Our results, therefore demonstrate that F. nucleatum has the ability to activate epithelial cells in a discrete and limited manner; i.e., induction of hBD-2 without IL8. Further, NF-kB, is neither necessary nor sufficient for F. nucleatum induction of hBD-2. Instead, MAP kinase pathways, p38 and JNK are utilized. These results are consistent with the hypothesis that F. nucleatum has evolved to generate a heightened state of readiness of the epithelium it inhabits without fully unleashing other innate immune responses. This does not mean that F. nucleatum doesn't have the ability to upregulate IL-8, nor does it ll1ean that this organism maintains the same symbiotic relationship with the host in all body sites. Other components of F. nucleatum, such as LPS, can activate expression of IL-8 in NHOECs, and this organism may use its inherent resistance to defensins as a virulence strategy in its ability to invade epithelial cells, and possibly in association with systemic complications such as amniotic fluid infections that lead to preterm births.
Untreated cell wall supernatant from F. nucleatum (ATCC 25586) was charged onto a C4 HPLC column and fractions were eluted with an acetonitrile gradient and tested for hBD-2 mRNA induction of normal human oral epithelial cell (NHOEC) monolayers by RT-PCR. The shoulder that eluted at 47-50% acetonitrile, and is designated with an asterisk/arrow in
An aliquot of the active fraction was subjected to SDS-PAGE electrophoresis, and three bands were excised for trypsin digest and amino acid sequencing (
In summary, we have now confirmed, through repeated experiments and multiple MALDI-MS and CID analyses, that the hBD-2 inducing HPLC fraction from soluble F. nucleatum cell wall, elutes at 30-35 minutes and contains a hydrophobic 12.5 kDa peptide that is in the pH range of 3.8-4.2. This is the polypeptide of SEQ ID NO.: 1.
We challenged human corneal epithelial cells and skin keratinocytes with F. nucleatum cell wall (under identical conditions described in Preliminary Studies for NHOEC's) followed by RT-PCR analysis.
We obtained SV40 transformed human corneal epithelial cells (HCE-T) and grew them in monolayers as described by Maldonado and Furcht, 1995. HCE-T cells have been shown to express properties similar to normal corneal epithelial cells and don't produce free viral particles, nor have been shown to revert to a viral producing cell line. Upon challenge with increasing concentrations of the F. nucleatum cell wall fraction, there was a concomitant increase in hBD-2 transcript expression (
Primary normal human skin keratinocyes (NHSKs) were obtained from a keratome biopsy through the Department of Dermatology (University Hospitals, Cleveland, Ohio). They were isolated and cultured as described previously, followed by challenge with F. nucleatum cell wall. RT-PCR analysis revealed that both hBD-2 and -3 mRNA were induced (
We have conducted extensive studies comparing beta defensin regulation by F. nucleatum, P. gingivalis, A. actionmycetemcomitans, and different isolates of C. albicans in NHOEC's and the immortalized human oral cell line OKF/Tert cells (terts). We have concluded that in all our recorded cases, terts behave like NHOEC's. We therefore decided to use terts as our cell source for the generation of a transient reporter gene construct system.
Tert cells were transfected with pGL3-HBD-2p (gift from Jürgen Harder and Jens Schröder, Kiel University, Germany). This construct involved PCR amplifying the hBD-2 promoter (1284 bp upstream of the reading frame) and using Hind III and Xho I to ligate the promoter to the firefly luciferase gene in the pGL3 vector. Transfection was conducted with LipofectAMINE reagent (Invitrogen, Carlsbad, Calif.), following the manufacturer's instruction. Briefly, cells were loaded into 24 well plates and grown to near confluence. Serum free DMEM was used as the transfection medium. The transfection cocktail was prepared in the following way (per well): plasmid DNA (1 μg) was added to 25 μg DMEM, along with 4 μl PLUS reagent mix, followed by incubation at room temperature for 15 min. LipofectAMINE reagent (1 μl) was added to 25 μl DMEM. The plasmid mix and the LipofectAMINE mix were combined and incubated at room temperature for 15 min. After removal of culture medium, the combined DNA-PLUS-LipofectAMINE mixture was added to each well along with 0.2 ml DMEM, followed by incubation at 37 C, 5% CO2, 3 hr. The transfection medium was then replaced with fresh DMEM, and incubated for an additional 24 hrs. Cells were then challenged with either PBS, PMA, or F. nucleatum cell wall (10 μg/ml). We used the luciferase reporter assay system (Promega, Madison, Wis.) to detect the expression of luciferase.
We compared hBD-2 induced normal human oral epithelial cells (NHOECs), after F. nucleatum challenge, with uninduced, quiescent NHOECs to determine protection against P. gingivalis invasion. Semi-confluent monolayers were challenged with F. nucleatum overnight, to induce hBD-2 expression. Unstimulated and F. nucleatum stimulated NHOEC monolayers were challenged with syto 62 labeled P. gingivalis at an MOI of 10:1 and 100:1. After a 90 minute incubation, followed by one hour antibiotic treatment to kill all extracellular bacteria (64, 65), cells were analyzed by flow cytometry. The F. nucleatum prestimulated cells were more than 50% protected when compared to the unstimulated cultures at an MOI of 100:1, and more than 67% protected at an MOI of 10:1 (
These bioassays demonstrate the efficacy of protection elicited from physiologically relevant concentrations of cell associated hBD-2.
F. nucleatum is resistant to recombinant hBD-1 and -2. After generating recombinant forms of hBD-1 and hBD-2, using a baculovirus expression system with Sf21 insect cells, we found that F. nucleatum was resistant to these agents, while P. gingivalis was sensitive to them in low micromolar concentrations (10 μg/ml hBD-1=2.55 μM; 10 μg/ml hBD-2=2.31 μM) (
To demonstrate the internalization of CXCR4 in response to hBD-3, CEM X4/R5 cells (a human T cell line expressing CXCR4 and CCR5) and THP-1 cells (a human monocytic leukemia cell line) were cultured and maintained as described previously (Quinones-Mateu et al., 2003. Human epithelial beta-defensins 2 and 3 inhibit HIV-1 replication. Aids 17:F39-48). To induce fMLP receptor expression, THP-1 cells were differentiated in RPMI 1640 medium supplemented with 300 μM dibutyryl cyclic AMP (Sigma, St. Louis, Mo.) for 2 days. Recombinant hBD-3 was produced by cloning hBD-3 cDNA into pET-30c (a gift from J. Harder and J. Schröder, Kiel University, Germany) (Novagen, Madison, Wis., USA) as previously described (Harder, et al., 2001. Isolation and characterization of human beta-defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 276:5707-5713.). The identity, purity and biological activity of recombinant hBD-3 (rhBD-3) were confirmed by Western analysis run alongside the native peptide, N-terminal amino acid sequencing, matrix assisted laser desorption ionization time of flight mass spectrometry (MALDITOF-MS), and killing of E. coli ML35p, respectively. CEM X4/R5 cells grown in RPMI-1640 medium containing 5% FBS and supplemented with 400 μg/ml G418 (Invitrogen, Carlsbad, Calif.), were collected, washed twice with PBS, resuspended in medium containing 0.5% FBS, with or without 20 μg/ml rhBD-3, followed by incubation at 37° C. in 5% CO2 for 2 h. In both conditions, cells were treated with FACS/Perm (PharMingen, San Jose, Calif.) at room temperature, 10 min, and then washed three times with PBS. Cells were stained with PE-labeled anti-CXCR4 monoclonal antibody (clone 12G5, PharMingen) at room temperature for 90 min, followed by washing three times with PBS. Samples were observed using a dual scanning confocal microscope system (Zeiss LSM 510, Oberkochem, Germany) and analyzed with the Zeiss LSM 5 Image Browser. HBD-3 treatment of CEM cells results in CXCR4 internalization as detected by confocal microscopic analysis of permeabilized cells stained with anti-CXCR4 antibodies (
A competition assay was conducted which demonstrates the ability of hBD-3 to inhibit the binding of the CXCR4 ligand SDF-1 to CEM cells expressing both CXCR4 and CCR5. CEM X4/R5 cells were washed and resuspended in balanced salt solution (BSS) containing 140 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, 1 mg/ml BSA, and 20 mM NaHEPES (pH 7.5). One million cells in a final volume of 300′ L were placed in each tube along with 0.1 nM 125I labeled SDF-1 or RANTES (PerkinElmer NEN, Boston, Mass.). Unlabeled SDF-1 or RANTES (1 μg/ml for each) or various concentrations of hBD-3 were added simultaneously. Only 125I labeled SDF-1 or 125I labeled RANTES was added to control tubes. Cells were incubated at 37° C. for 30 min, or on ice for 4 h, and then washed three times with BSS. The cellular binding of 125I labeled SDF-1 or 125I labeled RANTES was determined by γ-counter readings. All experiments were performed in triplicate. HBD-3 inhibited the binding of 125I labeled SDF-1 (
It has been surprisingly found that, at concentrations ranging from 2.5 to 20 μg/ml (0.48-3.9 μM), hBD-3 does not induce a calcium mobilization response in CEM cells but rather blocks the calcium flux induced by SDF-1 in a dose dependent manner, as illustrated in
In addition to mobilizing calcium, signal transduction through SDF-1/CXCR4 binding coordinates activation of mitogen activated protein kinase (MAPK) pathways, such as extracellular signal-regulated kinase (ERK-1/2). To define further the antagonism of SDF-1/CXCR4 interactions by hBD-3, the ability of hBD-3 to inhibit SDF-1 induced phosphorylation of the ERK-1/2 MAPK was assessed by Western blot analysis. PBMCs were isolated from heparinized whole blood using Ficoll-Paque (Amersham Biosciences, Piscataway, N.J.) density sedimentation. After washing twice with RPMI-1640, the cells were used for subsequent CD4+ cell isolation by negative selection using magnetic cell sorting (MACS) and the CD4+ T cell isolation kit II (Miltenyi Biotec, Auburn, Calif.). Briefly, PBMCs were resuspended in running buffer and incubated with a biotin antibody cocktail containing antibodies against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR gamma/delta, and Glycophorin A. Anti-biotin Microbeads were then added and the cell suspension was passed through the AutoMACS isolation device (Miltenyi Biotec). The resulting purity of CD4+ cells was greater than 90%, as determined by flow cytometry. CEM X4/R5 cells or CD4+ PBMC were washed several times with PBS and resuspended at a concentration of 107 cells/ml in BSS. Aliquots of cell suspension (500 μL) were pre-treated with either 10 nM SDF-1, hBD-3 (20 μg/ml; 3.9 μM), hBD-3 prior to SDF-1, carbachol (10 μM) or phorbol myristate acetate (PMA; 100 nM; Sigma) for 3 min. Reactions were terminated by transfer to an ice bath and rapid centrifugation to pellet the cells. Following aspiration of the test medium and washing with ice-cold PBS, the pelleted cells were routinely disrupted in 200 μL of lysis buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM β-glycerol phosphate, 2 mM Na-Pyrophosphate, 5 mM EDTA, 5 mM EGTA, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonylfluoride, 0.1% aprotinin, and 10 μg/ml leupeptin. After 20 minutes on ice, the lysate was centrifuged at 10,000×g for 10 minutes at 4° C.; 150 μl of the supernatant was denatured by addition of 4×-Laemmli sample buffer and boiling for 5 min. The extracted proteins were separated by SDS-PAGE on 12% gels (Bio-Rad Laboratories, Hercules, Calif.) and transferred to 0.45 μm Immobilon-P polyvinylidene difluoride membranes (Millipore Corporation, Bedford, Mass.). The blots were serially probed with anti-phospho-ERK1/2 and then anti-total ERK1/2. Mouse monoclonal anti-phospho-ERK1/ERK2 (Thr202/Tyr204) and rabbit polyclonal anti-total ERK1/ERK2 were obtained from Cell Signaling Technology (Beverly, Mass.). Horseradish peroxidase conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). Bound primary antibodies were visualized with appropriate peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) using Enhanced Luminol Chemiluminescence Reagent (Amersham Piscataway, N.J.). HBD-3 did not induce ERK-1 or ERK-2 phosphorylation but abrogated SDF-1-dependent ERK1/2 activation in both CEM X4/R5 cells and in freshly isolated CD4+ peripheral blood mononuclear cells as shown in
Because SDF-1/CXCR4 dependent lymphocyte locomotion is well established, and hBD-3 acts antagonistically towards CXCR4, chemotaxis assays were conducted to determine whether hBD-3 would block SDF-1-induced T cell chemotaxis. Briefly, chemotaxis assays were performed as as follows. Jurkat cells (clone E6-1; human T cell line; ATCC#TIB-152) or activated primary T lymphoblasts {kindly provided by Dr. Alan Levine, Dept. Gastroenterology, Case Western Reserve University, Cleveland, Ohio;}, were washed twice with serum-free RPMI-1640, and resuspended in RPMI-1640 containing 0.5% FBS at a concentration of 1×107 cells/ml. A 100 μl aliquot of each cell type was added into respective transwell inserts of chemotaxis chambers (Transwell Permeable Supports, Corning, Acton, Mass.), where the lower compartment of each chamber contained 600 μl of RPMI-1640 with 0.5% FBS. SDF-1 (100 ng/ml) was added into each lower compartment except in control chambers, followed by addition of hBD-3 (0-40 μg/ml). After incubation at 37° C. for 3 hours, the inserts were removed, and cells that migrated into the lower chamber were counted. As shown in
Expression of hBDs may also be induced by agents other than polypeptides as shown in
The experiments reported here demonstrate that recombinant hBD-3 induces internalization of CXCR4 without promoting calcium mobilization or activation of ERK-1/2 MAPK. There is at least some specificity for CXCR4 since hBD-3 does not inhibit calcium mobilization as a result of RANTES, fMLP, ATP, or carbachol binding to their receptors. Althought the applicants do not wish to condition patentability on any particular theory, because these ligands induce calcium flux via either a Gi (SDF-1, RANTES, fMLP) or Gq (carbachol, ATP)-coupled receptor pathway and since hBD-3 also blocks SDF-1 binding, the data suggest that hBD-3-mediated inhibition of calcium flux occurs at the level of the CXCR4 receptor and not at the G protein level. The interaction of agonistic ligands with G protein-coupled receptors (GPCR) induces conformational changes that coordinately elicit activation of the downstream G protein-dependent signaling cascades and also promote desensitization/internalization of the activated receptor. The canonical model for GPCR internalization involves phosphorylation of the receptors, association of arrestin-family adapters with the phosphorylated receptors, targeting of the GPCR-arrestin complexes to clathrin-coated pits, and, finally, dynamin-dependent internalization of the complexes. Previous studies have indicated that SDF-1 induces internalization of CXCR4 via these classical arrestin and dynamin-dependent mechanisms. Although antagonistic ligands for most GPCR do not elicit either coupling to G proteins (by definition) or desensitization/internalization, a small but increasing number of GPCRs have been shown to internalize upon binding antagonists. These results indicate that hBD-3 also induces CXCR4 internalization despite its actions as an antagonist of SDF-1 binding to, and activation of CXCR4. CXCR4 may conceivably assume multiple conformations that can independently regulate G protein coupling versus receptor internalization. SDF-1/CXCR4 interactions have been implicated in a number of vital biological functions, including but not limited to hematopoeisis, neurogenesis, angiogenesis, cardiogenesis and immune cell trafficking. Our findings indicate that hBD-3 may regulate some of these activities at selected sites such as those proximate to mucosal surfaces where the concentration of hBDs is high. These findings also indicate that calcium influx and ERK1/2 phosphorylation are not necessary to induce CXCR4 internalization and thus implicate the likely importance of other as yet undescribed mechanisms in trafficking of CXCR4. This novel finding also may lead to a better understanding of how to inhibit the SDF-1/CXCR4 axis by use of endogenous agents such as hBD-3 or its derivatives and also may provide a new direction for drug design.
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
Claims
1. A method for inhibiting the SDF1-CXCR4 signaling pathway in a subject, comprising administering to the subject an effective amount of a BD-inducing agent.
2. The method of claim 1, wherein the BD-inducing agent is a Fusobacterium associated defensin inducer (FAD-I) polypeptide.
3. The method of claim 1, wherein the BD-inducing agent is a synthetic analog of double stranded RNA.
4. The method of claim 3, wherein the BD-inducing agent is polyinosine-polycytidylic acid
5. The method of claim 1, wherein the selected agent is administered systemically.
6. The method of claim 1, wherein the agent is administered locally.
7. The method of claim 2, wherein the FAD-I polypeptide comprises an amino acid sequence at least 90 percent identical to an amino acid sequence selected from the group consisting of SEQ. ID. NOs.: 1, 3, 5, and 7.
8. The method of claim 7, wherein the FAD-I polypeptide comprises an amino acid sequence at least 95 percent identical to an amino acid sequence selected from the group consisting of SEQ. ID. NOs.: 1, 3, 5, and 7.
9. The method of claim 2, wherein the FAD-I polypeptide in encoded by a nucleic acid comprising a sequence at least 90 percent identical to a nucleic acid sequence selected from the group consisting of SEQ. ID. NOs.: 2, 4, 6, and 8.
10. The method of claim 9, wherein the FAD-I polypeptide in encoded by a nucleic acid comprising a sequence at least 95 percent identical to a nucleic acid sequence selected from the group consisting of SEQ. ID. NOs.: 2, 4, 6, and 8.
11. The method of claim 1, wherein the method inhibits a CXCR4-related process in a subject.
12. The method of claim 1, wherein the method stimulates the mobilization of hematopoietic stem cells in the subject.
13. The method of claim 1, wherein the method inhibits angiogenesis in a subject.
14. The method of claim 12, wherein the angiogenesis is tumor angiogenesis.
15. The method of claim 1, wherein the method stimulates an immune response in the subject.
16. The method of claim 15, wherein the agent is administered systemically.
17. The method of claim 15, wherein the agent is administered locally.
18. The method of claim 1, wherein the method promotes cardiogenesis in the subject.
19. The method of claim 1, wherein the agent is administered systemically.
20. The method of claim 15, wherein the agent is administered locally.
Type: Application
Filed: Apr 28, 2006
Publication Date: Dec 7, 2006
Applicant: Case Western Reserve University (Cleveland, OH)
Inventor: Aaron Weinberg (Shaker Heights, OH)
Application Number: 11/380,836
International Classification: A61K 39/02 (20060101); A61K 48/00 (20060101);
