APOE PEPTIDE DIMERS AND USES THEREOF
The present invention provides novel pharmaceutical compositions comprising ApoE-derived peptide dimers. In particular, the ApoE peptide dimers of the invention comprise at least two ApoE mimetic domains and can comprise one or more protein transduction domains. Methods of treating various conditions, such as cancer, inflammatory conditions, and neurodegenerative diseases, by administering the pharmaceutical compositions of the invention are also disclosed.
This application claims the benefit of U.S. Provisional Application No. 61/292,668, filed Jan. 6, 2010, which is herein incorporated by reference in its entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLYThe contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: COGO—022—01WO_SeqList_ST25.txt, date recorded: Jan. 6, 2011, file size 40 kilobytes).
FIELD OF THE INVENTIONThe present invention relates to pharmaceutical compositions comprising peptide dimers derived from apolipoprotein E (ApoE). The present invention also relates to methods of treating various disease states, such as cancer and neurodegenerative diseases, with the novel compositions.
BACKGROUND OF THE INVENTIONCancer is a class of diseases in which a group of cells exhibit uncontrolled growth, invasion and destruction of adjacent tissues, and metastasis (spread of aberrant cells spread to other locations in the body), or in which cells fail to undergo programmed cell death (e.g. apoptosis) at the appropriate time. Cancer causes about 13% of all deaths worldwide and according to the American Cancer Society, 7.6 million people died from cancer in the world during 2007. Current treatment for cancer depends upon the specific type of cancer and tissue involved, but includes surgery, chemotherapy, radiation therapy, immunotherapy, and monoclonal antibody therapy among other methods. Although these treatment methods have been successful in some cases, they are hindered by adverse side effects or limited efficacy. For example, the efficacy of eliminating cancerous tissue by surgical removal of tumors is often limited by the tendency of cancers to invade adjacent tissue and metastasize to other sites in the body. Chemotherapy, as well as radiation treatment, is often limited by toxicity or damage to other tissues in the body. Thus, cancer remains a major health concern and there is a need for improved methods of treating cancer.
Inflammation is strongly correlated to cancer initiation, progression and metastasis (Mantovani et al. (2008) Nature, Vol. 454: 436-444). Pro-inflammatory mediators such as prostaglandins, cytokines, reactive oxygen/nitrogen species, and growth factors, activate PI3K/Akt signaling that increases pro-survival, proliferative, and metastatic processes (Dillon et al. (2007) Oncogene, Vol. 26: 1338-1345; Qiao et al. (2008) Cell Cycle, Vol. 7: 2991-2996; Prueitt et al. (2007) International Journal of Cancer, Vol. 120: 796-805; Wang and DuBois (2006) Gut, Vol. 55: 115-122). Mutations in the PI3K/Akt pathway are common in human tumors, which result in unregulated PI3K/Akt signaling (Carnero et al. (2008) Curr Cancer Drug Targets, Vol. 8: 187-98; Dillon et al., 2007; Yuan and Cantley (2008) Oncogene, Vol. 27: 5497-5510). Thus, pharmacological control of the PI3K/Akt signaling axis is an aim for cancer therapeutics.
Akt kinase activity is directly regulated by the tumor suppressor protein phosphatase 2A (PP2A), which functions to dephosphorylate Akt at threonine 308 and serine 473 (Andjelkovic et al. (1996) Proc. Natl. Acad. Sci., Vol. 93: 5699-5704; Resjö et al. (2002) Cellular Signalling, Vol. 14: 231-238). However, PP2A activity is commonly decreased in human cancers (Chen et al. (2004) Cancer Cell, Vol. 5: 127-136). One mechanism by which PP2A activity is suppressed in cancer is by the formation of complexes with endogenous protein inhibitors such as CIP2A and I2PP2A (Junttila et al. (2007) Cell, Vol. 130: 51-62; Li et al. (1996) J. Biol. Chem., Vol. 271: 11059-11062). I2PP2A, which is also known as SET, is a potent inhibitor of PP2A and has been implicated in AML and blast crisis CML (Li et al., 1996; Neviani et al. (2005) Cancer Cell, Vol. 8: 355-368). Despite the endogenous inhibition in many human cancers, PP2A activity can be pharmacologically increased and is a potential molecular target for cancer therapeutics (Guichard et al. (2006) Carcinogenesis, Vol. 27: 1812-1827; Perrotti and Neviani (2008) Cancer and Metastasis Reviews, Vol. 27: 159-168; Switzer et al. (2009) Oncogene, Vol. 28: 3837-3846).
ApoE-derived peptides have shown promising effects in abating injury in inflammation-associated neuropathologies, such as Alzheimer's disease, multiple sclerosis and traumatic brain injury (Hoane et al. (2009) Journal of Neurotrauma, Vol. 26: 121-129; Li et al. (2006) J Pharmacol Exp Ther, Vol. 318: 956-965; Wang et al. (2007) Neuroscience, Vol. 144: 1324-33; WO 2006/029028; WO 2003/026479). Inflammation is a common feature of both neurological diseases and cancer, and PI3K/Akt signaling is also unregulated in neurodegenerative diseases such as Alzheimer's (Griffin et al. (2005) J Neurochem, Vol. 93: 105-17; Pei et al. (2003) Acta Neuropathol, Vol. 105: 381-92). Also, the expression of PP2A subunits is decreased in Alzheimer's patients, which is consistent with increased tau hyper-phosphorylation observed in this pathology (Vogelsberg-Ragaglia et al. (2001) Experimental Neurology, Vol. 168: 402-412). ApoE peptides have also been reported to increase PP2A activity by relieving inhibition by SET (see, e.g., WO 2008/080082). Thus, ApoE peptides represent a viable therapeutic approach for treating various conditions, including cancer, inflammatory conditions, and neurodegenerative diseases.
Although several ApoE peptides have proven to be effective in treating specific conditions, there is a need in the art to develop new ApoE-derived peptides with increased potency and greater safety windows. In particular, it is desirable to develop new ApoE-based peptide therapeutics that can effectively treat multiple conditions.
SUMMARY OF THE INVENTIONThe present invention is based, in part, on the discovery that dimerization of ApoE peptides increases their biological activity. Thus, the present invention provides novel ApoE peptide therapeutics with increased potency as compared to monomeric ApoE peptides. For instance, in one embodiment, a peptide dimer of the invention comprises a first ApoE peptide and a second ApoE peptide, wherein said first and second ApoE peptides are covalently linked by a linking moiety. In certain embodiments, the first and second ApoE peptides contain a sequence derived from the LDL receptor binding region of the native ApoE holoprotein. The first and second ApoE peptides may be identical or may be different.
In some embodiments, at least one of the ApoE peptides in the dimer is conjugated to a protein transduction domain through, optionally, one or more linking residues. In other embodiments, both the first and second ApoE peptides in the dimer are each conjugated to a protein transduction domain through, optionally, one or more linking residues. The one or more linking residues can include cysteine residues, or modified amino acids, such as azidohomoalanine or propargylglycine. The protein transduction domain can be a peptide derived from antennapedia, TAT, SynB1, SynB3, SynB5, and polyarginine.
The first and second ApoE peptides in the peptide dimers of the invention may be covalently linked by a linking moiety. The linking moiety can include a disulfide bridge, a bismaleimide (e.g., bismaleimido-ethane or bismaleimido-hexane), a 1,4-disubstituted triazole, and N,N-dipropargylamine.
The present invention also includes pharmaceutical compositions of the ApoE peptide dimers of the invention. In one embodiment, the pharmaceutical composition comprises an effective amount of an ApoE peptide dimer as described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions may further comprise additional therapeutic compounds depending on the particular condition to be treated.
The present invention also provides methods of treating, preventing, or ameliorating various conditions or diseases, including cancer, neurodegenerative disorders (e.g., ALS, Alzheimer's disease, Parkinson's disease, and Huntington's disease), and inflammatory conditions (e.g., multiple sclerosis, inflammatory bowel disease, Crohn's disease, and rheumatoid arthritis) by administering an effective amount of at least one ApoE peptide dimer as described herein.
The present invention also includes a method for predicting or evaluating the efficacy of a therapeutic intervention for treating cancer in a patient. In one embodiment, the method comprises measuring the expression level of SET protein in a biological sample from a patient, and comparing the measured level to the expression level of SET protein in a control sample, wherein the measured expression level of SET protein is indicative of the therapeutic efficacy of the therapeutic intervention. In certain embodiments, the therapeutic intervention is an ApoE peptide or peptide dimer described herein. The biological sample can be, for example, a tumor biopsy from a solid tumor or mononuclear cells isolated from a blood sample. In one embodiment, the biological sample is CD19+/CD5+ leukemia cells.
The present invention also encompasses a kit for predicting the therapeutic efficacy of ApoE peptides or peptide dimers for treating cancer in a patient. In one embodiment, the kit comprises a reagent for measuring SET protein expression in a biological sample and instructions for measuring SET protein expression for predicting or evaluating the efficacy of an ApoE peptide or peptide dimer for treating cancer in a patient.
The inventors previously discovered that ApoE synthetic peptides were useful in treating various types of cancer. See WO 2010/002982, filed Jul. 1, 2009, which is herein incorporated by reference in its entirety. Here, the inventors have expanded upon their earlier work finding surprisingly that dimers of the synthetic ApoE peptides exhibit increased biological activity as compared to their monomeric counterparts. The inventors discovered that one particular lot of ApoE peptide, which was particularly potent in activity assays, had been oxidized to form peptide dimers. Additional experiments demonstrated that ApoE peptide dimers formed through irreversible linkages were even more potent than the reversibly-linked dimers. Accordingly, the present invention provides novel peptide dimers derived from the receptor binding region of ApoE. In one embodiment, the peptide dimer comprises a first ApoE peptide and a second ApoE peptide, wherein said first and second ApoE peptides are covalently linked by a linking moiety.
ApoE peptides, also referred to as COG peptides, are peptides derived from the native ApoE holoprotein. The peptide dimers of the present invention comprise at least two ApoE peptides or ApoE mimetic domains. The ApoE peptides or mimetic domains may be derived from the LDL receptor binding region of the ApoE holoprotein, namely amino acids 130-150 of full-length ApoE protein. In certain embodiments, the ApoE peptides or mimetic domains of the invention may be derived from at least amino acids 133-140 of ApoE. In one embodiment of the invention, the ApoE peptide is derived from amino acids 130-149 of ApoE. In another embodiment, the ApoE peptide is derived from amino acids 133-149 of ApoE. In still another embodiment, the ApoE peptide is derived from amino acids 138-149 of ApoE. As used herein, the phrase “derived from” refers to a peptide that contains at least 80% identity to a particular amino acid sequence from the ApoE protein or a peptide that has at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acid residues from a receptor binding region of the ApoE protein (e.g. amino acids 130-150). By way of example, a peptide having a sequence corresponding to amino acids 133-149 with one, two, or three point mutations or amino acid modifications would be considered to be derived from amino acids 133-149 of the ApoE protein. ApoE peptides or mimetic domains can be derivatives of a peptide containing five or more, ten or more residues, or 15 or more residues from amino acids 133-149 of native ApoE protein, including derivatives having non-natural amino acid substitutions, such as amino isobutyric acid and acetyl lysine, and other modifications that enhance the alpha-helical content of the peptide.
In one embodiment of the invention, the first and/or second ApoE peptide of the peptide dimer has a sequence of LRVRLASHLRKLRKRLL (SEQ ID NO: 3 (COG133)). The COG133 monomer has previously proven useful in treating or reducing cerebral ischemia or cerebral inflammation. See U.S. Application Publication No. 2003/0077641 A1, filed Sep. 23, 2002, incorporated herein by reference in its entirety. In another embodiment, the first and/or second ApoE peptide is an analog or derivative of COG133. For instance, the first and/or second ApoE peptide has a sequence selected from the group consisting of AS(Aib)LRKL(Aib)KRLL (SEQ ID NO: 5 (COG1410)), LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL (SEQ ID NO: 4 (COG248)), and LRVRLAS(Aib)LRKLR(K-Ac)RLL (SEQ ID NO: 35 (COG345)). Other ApoE analogs or derivatives can be incorporated into the ApoE peptide dimers of the invention. For instance, a large number of analogs of the ApoE 130-150 peptide were previously created and their activity tested in a cell-based assay for suppression of release of inflammatory cytokines and free radicals and in receptor binding assays. Lynch et al. (2003) J. Biol. Chem., Vol. 278(4): 48529-33 and U.S. Application Publication Serial No. 2003/0077641 A1, filed Sep. 23, 2002; U.S. Pat. No. 7,205,280, issued Apr. 17, 2007; and U.S. application Ser. No. 09/260,430, filed Mar. 1, 1999, the contents of each of which are incorporated herein by reference in their entireties. In particular, the improved ApoE analogs described in WO 2006/029028, which is herein incorporated by reference in its entirety, are suitable first and/or second ApoE peptides or mimetic domains of the peptide dimers of the invention. For instance, ApoE peptides or mimetic domains can include, but are not limited to:
wherein (NMe)-L is an N-methylated Leucine, Aib is amino iso-butyric acid, (orn) is ornithine, (narg) is nitroarginine, (NLe) is norleucine, (harg) is homoarginine, (dmarg) is dimethyl arginine, (aclys) is acetyl lysine, (azlys) is azalysine and Ac is an acetylated amino terminus. Other ApoE peptides or mimetic domains derived from the receptor binding region of ApoE protein are also contemplated. For instance, ApoE peptides or mimetic domains that comprise a sequence corresponding to amino acids 133-149 of ApoE protein and retain (i.e. not substituted) one or more key residues selected from the group consisting of S139, R142, K143, L144, K146, R147 and L149, but have one or more amino acid substitutions at other positions are suitable first and/or second ApoE peptides or mimetic domains of the peptide dimers of the invention.
In certain embodiments of the invention, the first and second ApoE peptides of the peptide dimer are the same. For example, in one embodiment, the peptide dimer comprises a first ApoE peptide and a second ApoE peptide, wherein the first and second peptide have a sequence of SEQ ID NO: 3 (COG133). In other embodiments, the first and second ApoE peptides of the peptide dimer are different. By way of example, the peptide dimer can comprise a first ApoE peptide and a second ApoE peptide, wherein the first ApoE peptide has a sequence of SEQ ID NO: 3 (COG133) and the second ApoE peptide has a sequence of SEQ ID NO: 5 (COG1410). Peptide dimers including all possible permutations of the different ApoE peptides described herein are encompassed by the present invention.
In another embodiment of the invention, the first and/or second ApoE peptide of the peptide dimer is conjugated to a protein transduction domain (PTD). PTDs are short basic peptides that enhance the intracellular delivery of cargo. Some non-limiting examples of PTDs that may be conjugated to the ApoE peptides include peptides derived from antennapedia, SynB1, SynB3, SynB5, TAT, and polyarginine. For instance, exemplary PTD sequences that can be conjugated to the first and/or second ApoE peptides include:
In certain embodiments, the first and/or second ApoE peptide is conjugated to a PTD having a sequence of RQIKIWFQNRRMKWKK (SEQ ID NO: 8); YGRKKRRQRRR (SEQ ID NO: 9), or WKK. In one embodiment, the first ApoE peptide is conjugated to a first PTD through one or more first linking residues. Thus, the peptide dimers of the invention can comprise a first ApoE peptide conjugated to a first PTD and a second ApoE peptide that is not conjugated to a PTD. In such embodiments, the peptide dimers comprise two ApoE peptides or mimetic domains and a single PTD. In another embodiment, the first ApoE peptide is conjugated to a first PTD through one or more first linking residues and the second ApoE peptide is conjugated to a second PTD through one or more second linking residues. In such embodiments, the peptide dimers comprise two ApoE peptides or mimetic domains and two PTDs. Thus, the peptide dimers of the invention can comprise two ApoE peptide domains with zero, one, or two PTDs. See Example 3 and
The ApoE peptides and PTDs of the dimers can be any combination of the ApoE peptides and PTDs described herein. In particular embodiments, the PTDs are selected from peptides derived from antennapedia or TAT (e.g., SEQ ID NO: 8, SEQ ID NO: 9, or the WKK sequence) and the ApoE peptides are selected from COG133 (SEQ ID NO: 3), COG248 (SEQ ID NO: 4), COG1410 (SEQ ID NO: 5), or COG345 (SEQ ID NO: 35) as described in Tables II and IV of Example 3. In a certain embodiment, the ApoE peptide is COG133 (SEQ ID NO: 3) and the PTD has a sequence of SEQ ID NO: 8. In another embodiment, the ApoE peptide is COG133 (SEQ ID NO: 3) and the PTD has a sequence of WKK. In another embodiment, the peptide dimer comprises a first peptide and a second peptide, wherein said first peptide and said second peptide are covalently linked by a bismaleimido-ethane, and wherein the first and second peptide have a sequence of SEQ ID NO: 1, SEQ ID NO: 15, or SEQ ID NO: 90. For example, in one particular embodiment, the peptide dimer comprises a first peptide and a second peptide, wherein the first and second peptide have a sequence of SEQ ID NO: 1, and wherein said first peptide and said second peptide are covalently linked by a bismaleimido-ethane linker between the cysteine residues at position 17 in SEQ ID NO: 1 (e.g., the peptide dimer is COG449; see Table I). In another particular embodiment, the peptide dimer comprises a first peptide and a second peptide, wherein the first and second peptide have a sequence of SEQ ID NO: 15, and wherein said first peptide and said second peptide are covalently linked by a bismaleimido-ethane linker between the cysteine residues at the amino terminus of each peptide (i.e. at position 1 in SEQ ID NO: 15) (e.g., the peptide dimer is COG492; see Table I). In still another particular embodiment, the peptide dimer comprises a first peptide and a second peptide, wherein the first and second peptide have a sequence of SEQ ID NO: 90, and wherein said first peptide and said second peptide are covalently linked by a bismaleimido-ethane linker between the cysteine residues at position 4 in SEQ ID NO: 90 (e.g., the peptide dimer is COG493; see Table I).
The first and/or second ApoE peptide may optionally be conjugated to the PTD through one or more linking residues. As used herein, a “linking residue” refers to at least one amino acid or modified amino acid that is capable of undergoing a reaction to cross-link ApoE peptide monomers to form stable dimers. In some embodiments, the linking residues are amenable to cross-linking using maleimide groups, such as those described in
The peptide dimers of the invention comprise a first ApoE peptide and a second ApoE peptide, wherein said first and second ApoE peptides are covalently linked by a linking moiety. As used herein, a “linking moiety” may be a compound or molecule that cross-links peptide monomers such that the peptide chains are separated by at least four atoms. The linking moiety can be selected to create various lengths of the linker between the peptide monomers. For instance, the linking moiety may be selected such that the peptide chains are separated by at least 6 atoms, at least 8 atoms, at least 10 atoms, or at least 12 atoms. Linking moieties can be heterologous amino acids not found in the native ApoE sequence, such as additional cysteine residues or modified amino acids, such as azidohomoalanine or propargylglycine. In some embodiments, linking moieties can include molecules or compounds that are produced from a cross-linking reaction with amino acids in the peptide chains. For instance, in certain embodiments, the linking moiety is selected from the group consisting of a disulfide bridge, a bismaleimide, a 1,4-disubstituted triazole, and N,N-dipropargylamine. The bismaleimide can include, but is not limited to, bismaleimido-ethane or bismaleimido-hexane. In some embodiments, the linking moiety is not a peptide bond.
In embodiments in which the peptide dimer comprises ApoE peptides that are not conjugated to PTDs, the two ApoE peptides can be linked such that the carboxy terminus of the first ApoE peptide is linked to the amino terminus of the second ApoE peptide (e.g., direct linkage). Alternatively, the two ApoE peptides can be linked such that the two ApoE peptides are in reverse orientation relative to each other. For example, the carboxy terminus of the first ApoE peptide can be linked to the carboxy terminus of the second ApoE peptide or the amino terminus of the first ApoE peptide can be linked to the amino terminus of the second ApoE peptide. Such linkages may be accomplished by adding one or more amino acid residues capable of undergoing cross-linking reactions (e.g. cysteine, azidohomoalanine, or propargylglycine residues) to the appropriate terminus of the first and second ApoE peptides. By way of example, cysteine residues added to the amino terminus of both the first and second ApoE peptides will generate a dimer in which the first and second ApoE peptides are linked at their amino termini (see, e.g., COG492 in Example 2).
In embodiments, in which the peptide dimer comprises at least one ApoE peptide conjugated to a PTD, the dimer can be formed by cross-linking at least one of the linking residues in the ApoE-PTD conjugate and an amino acid at either the carboxy or amino terminus of the second, unconjugated ApoE peptide. If both ApoE peptides are conjugated to PTDs, the dimer is preferably formed by cross-linking at least one of the linking residues in each ApoE-PTD conjugate such that the two peptide chains are linked through internal amino acid residues.
ApoE peptides or mimetic domains may be incorporated into multimers such that an ApoE multimer contains three or more ApoE peptides or mimetic domains. One or more of the ApoE peptides in the multimer can be conjugated to a PTD as described herein. In one embodiment, the present invention provides an ApoE trimer comprising a first ApoE peptide, a second ApoE peptide, and a third ApoE peptide, wherein the first, second, and third ApoE peptides are covalently linked by a linking moiety. Other ApoE multimers are contemplated within the scope of the invention.
Peptides of the present invention can be produced by standard techniques as are known in the art. The peptides of the invention may have attached various label moieties such as radioactive labels, heavy atom labels and fluorescent labels for detection and tracing. Fluorescent labels include, but are not limited to, luciferin, fluorescein, eosin, Alexa Fluor, Oregon Green, rhodamine Green, tetramethylrhodamine, rhodamine Red, Texas Red, coumarin and NBD fluorophores, the QSY 7, dabcyl and dabsyl chromophores, BODIPY, Cy5, etc.
Modification of the peptides disclosed herein to enhance the functional activities associated with these peptides could be readily accomplished by those of skill in the art. For instance, the peptide dimers of the present invention can be chemically modified or conjugated to other molecules in order to enhance parameters such as solubility, serum stability, etc., while retaining functional activity. In particular, the first and/or second ApoE peptide of the dimer may be acetylated at its N-terminus and/or amidated at its C-terminus, or the dimers can be further conjugated, complexed or fused to molecules that enhance serum stability, including but not limited to albumin, immunoglobulins and fragments thereof, transferrin, lipoproteins, liposomes, α-2-macroglobulin and α-1-glycoprotein, PEG and dextran. Such molecules are described in detail in U.S. Pat. No. 6,762,169, which is herein incorporated by reference in its entirety.
The ApoE peptides of the inventive peptide dimers can further include conservative variants of the peptides described herein. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the peptide. A substitution, insertion or deletion is said to adversely affect the peptide when the altered sequence prevents or disrupts a biological function associated with the peptide. For example, the overall charge, structure or hydrophobic/hydrophilic properties of the peptide may be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the peptide. Ordinarily, the conservative substitution variants, analogs, and derivatives of the peptides, will have an amino acid sequence identity to the disclosed sequences, SEQ ID NOs: 3, 4, 5, and 35, of at least about 55%, at least about 65%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% to 99%. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.
Thus, the first and/or second ApoE peptides of the peptide dimers of the present invention include molecules having the amino acid sequence disclosed in SEQ ID NOs: 3, 4, 5, and 35; fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, or more amino acid residues of the therapeutic peptide; amino acid sequence variants of such peptides wherein an amino acid residue has been inserted N- or C-terminal to, or within, the disclosed sequence; and amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by another residue. Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding peptides of other animal species, including but not limited to rabbit, rat, porcine, bovine, ovine, equine and non-human primate species, and derivatives wherein the peptide has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example, a detectable moiety such as an enzyme or radioisotope).
Methods of cross-linking peptides to form peptide dimers are known to those in the art and can include, but are not limited to, coupling via maleimide groups and coupling using “click chemistry” (see Example 3 and
The ApoE peptide dimers of the invention can be in free form or the form of a salt, where the salt is pharmaceutically acceptable. These include inorganic salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and the like. Various organic salts of the peptide may also be made with, including, but not limited to, acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benozic acid, cinnamic acid, salicylic acid, etc.
In one embodiment, the peptide dimers of the present invention are used in combination with a pharmaceutically acceptable carrier. Thus, the present invention also provides pharmaceutical compositions suitable for administration to a subject. Such compositions comprise an effective amount of an ApoE peptide dimer of the present invention in combination with a pharmaceutically acceptable carrier. The carrier can be a liquid, so that the composition is adapted for parenteral administration, or can be solid, i.e., a tablet or pill formulated for oral administration. Further, the carrier can be in the form of a nebulizable liquid or solid so that the composition is adapted for inhalation. When administered parenterally, the composition should be pyrogen free and in an acceptable parenteral carrier. Active agents can alternatively be formulated encapsulated in liposomes, using known methods. Preparation of a peptide dimer of the present invention for intranasal administration can be carried out using techniques as are known in the art. The inventive peptide dimers may also be formulated for topical administration, for example in the form of creams or gels. Topical formulations are particularly useful for treating skin cancers or inflammatory skin conditions. In other embodiments, the ApoE peptide dimers may be formulated for rectal administration, such as in the form of suppositories. In some embodiments, rectal administration of the ApoE peptide dimers may be preferred for treatment of colorectal cancer, inflammatory bowel disease, or Crohn's disease.
Pharmaceutical preparations of the peptide dimers of the present invention can optionally include a pharmaceutically acceptable diluent or excipient.
The ApoE peptide dimers of the invention may contain further modifications or be formulated to specifically target specific tissues, such as inflamed tissues or cancerous tumors. For instance, the ApoE peptide dimers may be conjugated to other peptides that localize to tumor cells, such as those described in U.S. Pat. No. 6,380,161, U.S. Publication No. 2003/0232013, WO 2009/155556, and WO 2009/143023. Additionally or alternatively, the ApoE peptide dimers may be encapsulated into liposomes. The liposomes may contain a targeting ligand to localize the liposomes to particular tissues or tumor sites.
An effective amount of an ApoE peptide dimer of the present invention is an amount that decreases at least one symptom or pathology associated with cancer, such as tumor size, tumor growth, spread of cancer cells, number of cancer cells, and survival, compared to that which would occur in the absence of the peptide. An effective amount of an ApoE peptide dimer can also be an amount that decreases microglial activation (i.e., an amount that decreases the production of neurotoxic and neuromodulatory compounds by microglia) as compared to that which would occur in the absence of the compound. The effective amount (and the manner of administration) will be determined on an individual basis and will be based on the specific composition of the peptide dimer being used and a consideration of the subject (size, age, general health), the specific condition being treated (e.g. cancer, neurodegenerative disorder, inflammatory condition), the severity of the symptoms to be treated, the result sought, the specific carrier or pharmaceutical formulation being used, the route of administration, and other factors as would be apparent to those skilled in the art. The effective amount can be determined by one of ordinary skill in the art using techniques as are known in the art. Therapeutically effective amounts of the peptide dimers described herein can be determined using in vitro tests, animal models or other dose-response studies, as are known in the art.
The peptide dimers of the present invention can be administered acutely (i.e., during the onset or shortly after events leading to a diagnosis of a particular condition), or can be administered prophylactically (e.g., before scheduled surgery, or before the appearance of signs or symptoms of a particular condition), or administered during the course of a particular disease or condition to reduce or ameliorate the progression of symptoms that would otherwise occur. The timing and interval of administration is varied according to the subject's symptoms, and can be administered at an interval of several hours to several days, over a time course of hours, days, weeks or longer, as would be determined by one skilled in the art.
The typical daily regime can be from about 0.01 μg/kg body weight per day, from about 1 mg/kg body weight per day, from about 10 mg/kg body weight per day, from about 100 mg/kg body weight per day, from about 1,000 mg/kg body weight per day. Depending on the particular ApoE peptide dimer to be administered, dosages can be between about 1 mg/kg and about 500 mg/kg body weight per day, preferably between about 25 mg/kg and about 400 mg/kg body weight per day, or more preferably between about 50 mg/kg and about 250 mg/kg body weight per day.
The present invention provides methods of treating cancer in a subject in need thereof by administering an effective amount at least one ApoE peptide dimer as described herein. In certain embodiments, said at least one ApoE peptide dimer comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 35, 90, and effective fragments and variants thereof. ApoE peptide dimers can reduce one or more symptoms associated with cancer, including but not limited to tumor formation, tumor growth, number of cancerous cells, spread of cancerous cells to healthy tissue, and decreased survival. Cancers that may be treated with the peptide dimers and methods of the invention include, but are not limited to, various forms of leukemia (CLL, CML, ALL, AML), breast cancer, ovarian cancer, cervical cancer, prostate cancer, colorectal cancer, lung cancer, pancreatic cancer, brain cancer (e.g., gliomas), skin cancer (melanoma and nonmelanoma), head and neck cancers, bladder cancer, endometrial cancer, renal cell cancer, thyroid cancer, stomach cancer, esophageal cancer, gall bladder cancer, liver cancer, lymphoma (e.g. non-Hodgkin's lymphoma), and sarcoma. In one embodiment, the ApoE peptide dimers can reduce activation of signaling pathways, such as the Akt pathway, that are aberrantly activated in various forms of cancer (see Example 5). ApoE peptides can also activate PP2A (Examples 2, 5, and 10). PP2A has been reported to negatively regulate endothelial cell motility, which is required for angiogenesis and tumor metastasis in cancers (Gabel et al., 1999, Otolaryngol Head Neck Surg. 121: 463-468; Young, MR., 1997, Adv Exp Med Biol. 407: 311-318). Inhibition of PP2A by okadaic acid increased cell motility by disrupting the cytoskeletal network thereby enhancing the invasive properties of the tumor cells. Thus, peptide dimers of the present invention would reduce tumor cell metastasis and cancer-associated angiogenesis by activating PP2A. In one embodiment of the invention, administration of the ApoE peptide dimer increases PP2A activity in a cancer cell of the subject. In another embodiment, administration of the ApoE peptide dimer decreases Akt kinase activity in a cancer cell of the subject. In yet another embodiment, administration of the ApoE peptide dimer induces cytotoxicity in cancer cells in the subject.
The present invention also provides a method for the treatment of leukemia comprising administering at least one ApoE peptide dimer in an amount that would reduce symptoms of the disease as compared to that which would occur in the absence of the peptide dimer. In one embodiment, the leukemia is chronic myelogenous leukemia (CML). SET (i.e., I2PP2A), an endogenous negative regulator of PP2A, is overexpressed in CML and inhibits PP2A, thus maintaining activation of the oncogenic BCR/ABL kinase pathway (Neviani et al. (2005) Cancer Cell. 8: 355-368). Therefore, administration of an ApoE peptide dimer of the invention would activate PP2A, which would then be free to dephosphorylate regulators of cell proliferation and survival as well as suppress the oncogenic activity of the BCR/ABL kinase thus reducing leukemogenesis. In another embodiment, the leukemia is chronic lymphocytic leukemia (CLL). In preferred embodiments, administration of the ApoE peptide dimer decreases the number of CD5+ B cells in the subject. In another embodiment, the leukemia is acute lymphocytic leukemia (ALL).
The present invention also encompasses methods of treating breast cancer in a subject by administering an effective amount of at least one ApoE peptide dimer to the subject. In one embodiment, the breast cancer is characterized by Her2 expression. In another embodiment, the breast cancer is characterized by estrogen receptor expression. In another embodiment, the breast cancer is characterized by progesterone receptor expression. The ApoE peptide dimers of the invention can be used to treat any of the three main subtypes of breast cancer: luminal tumors (ER+/HER2−), HER2 amplified tumors (HER2+), and triple negative breast cancer (TNBC, ER−/PR−/HER2−). In certain embodiments, the breast cancer to be treated with an ApoE peptide dimer of the invention is triple negative breast cancer characterized by lack of expression of the estrogen receptor, progesterone receptor, and HER2 receptor. Administration of ApoE peptide dimers preferably reduce tumor growth following their administration.
The ApoE peptide dimers of the present invention may be used alone to treat cancer or in combination with other therapeutic agents commonly used to treat cancer, such as, e.g. chemotherapy agents (chlorambucil, cyclophosphamide), corticosteroids (prednisone, prednisolone), fludarabine, pentostatin, cladribine, imatinib (Gleevec), dasatinib (Sprycel), hormonal therapy (tamoxifen, aromatase inhibitors), sorafenib, gefitinib, and radiation. In some embodiments, the ApoE peptide dimers are administered in combination with sorafenib or gefitinib to treat cancer. As used herein, “in combination” means that the ApoE peptide dimer and other therapeutic agents are administered such that their effects overlap in time. Thus, the ApoE peptide dimer can be administered simultaneously with the other therapeutic agent or before or after the other therapeutic agent.
The present invention provides a method for predicting or evaluating the efficacy of a therapeutic intervention for treating cancer in a patient. In one embodiment, the method comprises measuring the expression level of SET protein in a biological sample from a patient, and comparing the measured level to the expression level of SET protein in a control sample, wherein the measured expression level of SET protein is indicative of the therapeutic efficacy of the therapeutic intervention. In certain embodiments, the therapeutic intervention is an ApoE peptide or peptide dimer of the invention. The present inventors have discovered that ApoE mimetic peptides and peptide dimers bind to SET (i.e., I2PP2A) and relieve its inhibition of endogenous PP2A, thereby increasing PP2A activity in the cell. Without being bound by any particular theory, it is believed that this increase in PP2A activity induced by ApoE peptides or peptide dimers triggers apoptosis leading to cytotoxicity of cancer cells. Therefore, cancer cells that overexpress SET protein are particularly susceptible to ApoE peptide-induced cytotoxicity. Accordingly, the present invention includes a method for predicting therapeutic efficacy of an ApoE peptide or peptide dimer for treating cancer in a patient by measuring the expression level of SET protein in a biological sample from the patient and comparing the measured level to the expression level of SET protein in a control sample, wherein the measured expression level of SET protein is predictive of the therapeutic efficacy of the ApoE peptide or peptide dimer.
In one embodiment, a measured SET expression level of at least 2-fold relative to the control sample is predictive of therapeutic efficacy of an ApoE peptide or peptide dimer for treating cancer in the patient. In some embodiments, a measured SET expression level of at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold relative to the control sample is predictive of therapeutic efficacy of an ApoE peptide or peptide dimer for treating cancer in the patient. In one embodiment, the method is predictive of therapeutic efficacy of an ApoE peptide or peptide dimer for treating breast cancer in the patient. In another embodiment, the method is predictive of therapeutic efficacy of an ApoE peptide or peptide dimer for treating triple negative breast cancer (estrogen receptor negative, progesterone receptor negative, and HER2 receptor negative) in the patient. In another embodiment, the method is predictive of therapeutic efficacy of an ApoE peptide or peptide dimer for treating B-cell lymphoma (e.g. non-Hodgkin's lymphoma) in the patient. In still another embodiment, the method is predictive of therapeutic efficacy of an ApoE peptide or peptide dimer for treating leukemia (e.g. CML or CLL) in the patient.
SET expression can be measured by assessing the level of SET protein or SET transcript. SET expression can be measured by methods known in the art including, but not limited to, Northern Blot, PCR, RT-PCR, Western Blot, immunoassay (e.g. ELISA or multiplexed assays), 2D gel electrophoresis, and hybridization. In one embodiment, SET protein expression is measured.
In certain embodiments, the method for predicting the efficacy of ApoE peptide therapy for treating cancer in a patient further comprises administering at least one ApoE peptide or peptide dimer to the patient following assessment of SET expression in the patient's biological sample. Any ApoE peptide or dimer thereof described herein is suitable for use in the method. For instance, in some embodiments, an ApoE peptide or dimer thereof having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 35, 90 and effective fragments and variants thereof is administered to the patient. In another embodiment, the method further comprises adjusting the particular type of ApoE peptide or peptide dimer or dosage of the ApoE peptide or peptide dimer based on the expression level of the SET protein in the patient's biological sample. SET expression levels can be measured multiple times over a particular period of time or treatment period in a patient.
The biological sample can be any tissue sample that contains cancerous cells. For instance, the biological sample can include, but is not limited to, a biopsy from a solid tumor (e.g. breast cancer, lymphoma, sarcoma, etc.) or peripheral blood mononuclear cells (PBMCs) isolated from blood. In one embodiment, the biological sample is CD19+/CD5+ leukemia cells. The control sample can be any tissue sample that contains normal or non-cancerous cells such as PBMCs isolated from a normal, age-matched patient or non-cancerous tissue (e.g. breast, lymph, skin, etc.) isolated from the patient to be treated or from a normal, age-matched control patient.
The present invention also encompasses a kit for predicting or evaluating the efficacy of an ApoE peptide or peptide dimer for treating cancer in a patient. In one embodiment, the kit comprises a reagent for measuring SET protein expression in a biological sample and instructions for measuring SET protein expression for predicting or evaluating the efficacy of an ApoE peptide or peptide dimer for treating cancer in a patient. In some embodiments, the reagent for measuring SET expression can include SET-specific antibodies, ELISA reagents, and primers and probes for amplifying and detecting SET mRNA. In other embodiments, the kit may further comprise one or more normalization controls. For example, the normalization control may be an exogenously added RNA or protein that is not naturally present in the sample or it may be a protein or RNA known to be expressed constitutively in a particular biological sample or cell, such as beta-actin. In such embodiments, the kit may further provide reagents (antibodies, primers, probes, etc.) for detecting and quantitating the normalization control. In some embodiments, the kit can further comprise a set of reference values to which the measured SET expression levels can be compared.
The present invention provides a method of reducing glial activation or microglial activation in a subject in need thereof by administering to the subject at least one of the ApoE peptide dimers of the invention. In one embodiment, the microglial activation is associated with central nervous system (CNS) inflammation, traumatic brain injury, cerebral ischemia or cerebral edema. Thus, the present methods and compositions are useful in preventing, suppressing or reducing the activation of glia in the CNS that occurs as a part of acute or chronic CNS disease. The effect of the present methods and peptide dimers can be assessed at the cellular or tissue level (e.g., histologically or morphometrically), or by assessing a subject's neurological status. The suppression or reduction of glial activation can be assessed by various methods as would be apparent to those in the art; one such method is to measure the production or presence of compounds that are known to be produced by activated glia, and compare such measurements to levels of the same compounds in control situations. Alternatively, the effects of the present methods and peptide dimers in suppressing, reducing or preventing microglial activation can be assessed by comparing the signs and/or symptoms of CNS disease in treated and control subjects, where such signs and/or symptoms are associated with or secondary to activation of microglia.
The present methods and peptide dimers are useful in preventing, treating, or ameliorating neurological signs and symptoms associated with acute CNS injury. As used herein, acute CNS injury includes but is not limited to stroke (caused by thrombosis, embolism or vasoconstriction), closed head injury, global cerebral ischemia (e.g., ischemia due to systemic hypotension of any cause, including cardiac infarction, cardiac arrhythmia, hemorrhagic shock, and post coronary artery bypass graft brain injury), focal ischemia and intracranial hemorrhage. Ischemic damage to the central nervous system can result from either global or focal ischemic conditions. Global ischemia occurs where blood flow to the entire brain ceases for a period of time, such as during cardiac arrest. Focal ischemia occurs when a portion of the brain is deprived of normal blood flow, such as during thromboembolytic occlusion of a cerebral vessel, traumatic head injury, edema and brain tumors. Much of the CNS damage due to cerebral ischemia occurs during the hours or even days following the ischemic condition, and is secondary to the release of cytotoxic products by damaged tissue.
The present methods and peptide dimers are also useful in preventing, treating, or ameliorating the neurological signs and symptoms associated with inflammatory conditions affecting the nervous system including the CNS, including but not limited to multiple sclerosis, vasculitis, acute disseminated encephalomyelitis and Guillain-Barre syndrome. In this regard, the ApoE peptide dimers of the invention can be used alone or in combination with other known anti-inflammatory drugs or cytokines to formulate pharmaceutical compositions for the treatment of CNS inflammatory conditions.
In another embodiment, the present invention provides a method of reducing neuronal cell death in a subject in need thereof comprising administering to the subject an effective amount of at least one ApoE peptide dimer described herein. In some embodiments, the neuronal cell death is associated with glutamate excitotoxicity. It was previously found that the COG 133 monomer peptide significantly suppressed neuronal cell death and calcium influx associated with N-methyl-D-aspartate exposure (see, e.g., U.S. Application Publication No. 2003/0077641 A1, herein incorporated by reference in its entirety). Thus, the peptide dimers of the present invention provide the basis for improved therapeutic compositions for treating diseases associated with glutamate excitotoxicity mediated by overstimulation of the NMDA receptor. For instance, glutamate excitotoxicity has been associated with neurolathyrism, amyotrophic lateral sclerosis (ALS) (Doble (1999) Pharmacol. Ther., Vol. 81:163-221), schizophrenia (Nguimfack (2002) Encephale, Vol. 28: 147-153), Huntington's chorea, Parkinson's (Nguimfack, 2002; Mytilineou et al. (1997) J. Neurochem., Vol. 68: 33-39; Klopman and Sedykh (2002) BMC Pharmacol., Vol. 2: 8; Le and Lipton (2001) Drugs Aging, Vol. 18: 717-724), bipolar disorder (Farber et al. (2002) Mol. Psychiatry, Vol. 7: 726-733), multiple sclerosis in humans and experimental autoimmune encephalitis (EAE) in animals (Paul and Bolton (2002) J. Pharmacol. Exp. Ther., Vol. 302: 50-57), depression, stroke (Le and Lipton, 2001), epilepsy and the inherited neurometabolic disease d-2-hydroxyglutaric aciduria (Kolker et al. (2002) Eur. J. Neurosci., Vol. 16: 21-28), in addition to Alzheimer's Disease (Bi et al. (2002) Neuroscience, Vol. 112: 827-840; Bi et al. (2002) J. Neurol. Sci., Vol. 200: 11-18) and traumatic brain injury (Rao et al. (2001) Brain Res., Vol. 911: 96-100; Regner et al. (2001) J. Neurotrauma, Vol. 18: 783-792; Xu and Luo (2001) Chin. J. Traumatol., Vol. 4: 135-138).
Thus, the present invention includes the use of the disclosed peptide dimers in methods and pharmaceutical formulations for the treatment of any of the above diseases or disorders, and in combined therapeutic compositions containing other known compounds useful for treating the various disorders. For instance, in some embodiments, the peptide dimers of the invention can be used to treat neurolathyrism, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, or schizophrenia in a subject in need thereof.
Riluzole (RILUTEK®, Rhone-Poulenc) is a substance with glutamate antagonistic properties that is used for neuroprotective treatment in amyotrophic lateral sclerosis and which has been tested in clinical trials for treatment of Huntington's disease and Parkinson's disease (Schiefer et al. (2002) Mov. Disord., Vol. 17: 748-757; Doble, 1999). Schiefer and colleagues recently demonstrated that riluzole prolongs survival time and alters nuclear inclusion formation in a transgenic mouse model of Huntington's disease. Thus, given the probable NMDA antagonistic role of the peptide dimers of the invention, these peptide dimers could be used in pharmaceutical formulations for the treatment of ALS, Huntington's and Parkinson's, alone or in combination with other glutamate antagonists, such as riluzole.
L-deprenyl is an inhibitor of monoamine oxidase (MAO)-B that delays the emergence of disability and the progression of signs and symptoms of Parkinson's disease, and is predicted to exert a protective effect from events occurring downstream from activation of glutamate receptors (Mytilineou et al., 1997). MAO-B inhibitors, dopamine receptor antagonists, such as levodopa, and NMDA receptor antagonists have all been shown to have an antiparkinson effect, and multidrug combinations have been shown to synergistically enhance the antiparkinson effects of the drugs (Klopman and Sedykh, 2002). Thus, given the probable NMDA antagonistic role of the peptide dimers of the invention, these peptide dimers could be used in pharmaceutical formulations for the treatment of Parkinson's, alone or in combination with other NMDA receptor antagonists, MAO-B inhibitors, such as L-deprenyl, and dopamine receptor agonists, such as levodopa.
The production of free radicals as a result of glutamate excitotoxicity has been implicated in the pathogenesis of schizophrenia (Nguimfack, 2002). Thus, researchers have begun to examine treatment of schizophrenia with antioxidizing substances used in other neurological diseases such as ALS, Parkinson's and Huntington's disease. Given that the peptide dimers of the invention likely have NMDA receptor antagonistic properties and can be used to inhibit the production of free radicals as a result of glutamate excitotoxicity, these peptide dimers can be used in pharmaceutical formulations for the treatment of schizophrenia, alone or in combination with other antioxidizing substances.
The present invention also includes a method of treating, preventing or ameliorating the symptoms of multiple sclerosis in a subject in need thereof by administering to the subject an effective amount of at least one ApoE peptide dimer of the invention. Multiple sclerosis (MS) is an immunologically mediated disease, as determined by observation of the response to immunotherapy and the existence of an animal model, experimental autoimmune encephalitis (EAE). See, for example, Mix et al. (2004) J. Neuroimmunol., Vol. 151(1-2): 158-70, Anderson, et al. (2004), Ann. Neurol., Vol. 55(5):654-9, and Ni et al. (2004) Mult. Scler., Vol. 10(2): 158-64. Interferon (IFN) beta-1b, IFN beta-1a, and glatiramer acetate (COPAXONE®, Teva), current therapies used for relapsing or remitting MS, have mechanisms of action that address the immunologic pathophysiology of MS (Dhib-Jalbut (2002) Neurology, Vol. 58: S3-S9). For instance, the interferons bind to cell surface-specific receptors, initiating a cascade of signaling pathways that end with the secretion of antiviral, antiproliferative, and immunomodulatory gene products. Glatiramer acetate, a synthetic molecule, inhibits the activation of myelin basic protein-reactive T cells and induces a T-cell repertoire characterized by anti-inflammatory effects. Several currently marketed treatments, including IV immunoglobulin (GAMAGARD®, Baxter), methotrexate (RHEUMATREX©, American Cyanamid), and azathioprine (IMURAN®, GlaxoSmithKline), have been evaluated as treatments for relapsing-remitting multiple sclerosis in combination with the approved therapies (Calabresi (2002) Neurology, Vol. 58: S10-S22). Given that the NMDA receptor antagonist memantine (NAMENDA®, Merz) has been shown to prevent the breakdown of and restore the blood-brain barrier and reduce symptoms associated with pathogenesis of EAE in vivo (Paul and Bolton, 2002), the peptide dimers of the present invention can be used alone or in combination with other NMDA receptor antagonists or in addition to interferons or glatiramer acetate for the treatment of MS in humans.
The present invention encompasses a method of treating, preventing or ameliorating the symptoms of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or polyarticular-course juvenile rheumatoid arthritis in a subject in need thereof by administering to the subject at least one ApoE peptide dimer as described herein. Current therapies for arthritis include peptides and proteins that bind with tumor necrosis factor. Etanercept (ENBREL®, Amgen) is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human 75 kd tumor necrosis factor receptor linked to the Fc portion of human IgG1. Adalimumab (HUMIRA®, Abbott) is a recombinant human IgG1 monoclonal antibody. Tumor necrosis factor binding proteins have shown outstanding results in slowing the progression and lessening the symptoms of rheumatoid arthritis and other rheumatic diseases. Thus, the ApoE peptide dimers of the present invention can be used alone or in combination with other drug for the treatment of rheumatic diseases, including for example, rheumatoid arthritis, ankylosing spondylitis, polyarticular-course juvenile rheumatoid arthritis, and psoriatic arthritis.
The present methods and ApoE peptide dimers are also useful in treating, preventing, or ameliorating neurological signs and symptoms associated with chronic neurological disease, including but not limited to Alzheimer's disease (AD) and HIV-associated encephalopathy. The finding by the present inventors that ApoE peptide dimers are particularly potent in suppressing microglial activation provides a role for the peptide dimers of the invention in the treatment of any neurological disease involving microglial activation. For example, microglia express markers of activation in AD, suggesting that crucial inflammatory events in AD involve microglia. Such activated microglia cluster near amyloid plaques (Griffin et al. (1995) J. Neuropath. Exp. Neurol., Vol. 54: 276). Microglia are also activated in epilepsy (Sheng et al. (1994) J. Neurochem, Vol. 63: 1872).
It has been shown that uptake and pathogenic effects of amyloid beta peptide are blocked by NMDA receptor antagonists (Bi et al., 2002). Other studies indicate that anti-inflammatory drugs can delay the onset or progression of AD (Breitner et al. (1995) Neurobiol. Aging, Vol. 16: 523; Rogers et al. (1993) Neurology, Vol. 43: 1609). Thus, the peptide dimers of the present invention can be used alone or in combination with other NMDA receptor antagonists or other known pharmaceuticals and especially anti-inflammatory drugs used for the treatment of AD in compositions and methods for the treatment of AD in humans.
The present invention includes a method of treating, preventing or ameliorating the symptoms of bacterial sepsis in a subject in need thereof by administering to the subject an effective amount of an ApoE peptide dimer of the invention. Monomeric ApoE receptor binding peptides have been shown to protect against LPS-induced production of cytokines in the periphery in an in vivo animal model of sepsis. See U.S. Application Publication No. 2003/0077641 A1, which is herein incorporated by reference in its entirety. Thus, the peptide dimers of the present invention can be used alone or in combination with other known anti-inflammatory cytokines and antibodies in compositions and methods for the treatment of sepsis.
It is known that the inflammatory process mediates an aspect of the atherosclerotic process. See, e.g., Hansson (1994) Basic Res. Cardiol., Vol. 89: 41; Berliner et al. (1995) Circulation, Vol. 91: 2488; Watanabe et al. (1997) Int. J. Cardiol., Vol. 54: 551. ApoE is known to be secreted by macrophages locally at blood vessel walls (although the amount secreted by macrophages in an individual is trivial compared to the amount of ApoE produced by the liver). In the classic model of atherosclerosis, ApoE functions to remove cholesterol from the blood stream and deliver it to macrophages or to the liver. However, it has become apparent that ApoE secreted by macrophages at the blood vessel wall decreases atherosclerotic plaque formation, independent of any lipid metabolism effects. For instance, ApoE-deficient mice are accepted as a model of hypercholesteremia and atherosclerotic disease. Providing ApoE-secreting macrophages to such mice dramatically decreases atherosclerotic plaque formation. Linton et al. (1995) Science, Vol. 267: 1034. Conversely, replacing a wild-type mouse's macrophages with ApoE-deficient macrophages accelerates atherosclerotic changes, even though the animal continues to produce ApoE by the liver. Fazio et al. (1997) Proc. Natl. Acad. Sci., Vol. 94: 4647.
In atherosclerosis, it is hypothesized that ApoE, via a receptor-mediated event, downregulates macrophage activation in the vicinity of blood vessel walls. Such down-regulation of macrophage activation interrupts or interferes with the cascade of events associated with atherosclerotic plaque formation, to thereby reduce or slow the formation of atherosclerotic lesions. The cascade of events known to be associated with atherosclerosis includes smooth muscle cell and endothelial cell proliferation, and foam cell formation. Evidence exists that ApoE downregulates each of these processes. ApoE thus affects the presence and progression of atherosclerosis in vivo, independent of its effects on lipids. The progression of atherosclerosis can be assessed by measuring the amount or size of atherosclerotic plaques, or the percentage of the blood vessel blocked by an atherosclerotic lesion, or the rate of growth of such plaques.
Atherosclerosis refers to the thickening of the arterial intima and accumulation of lipid in artherosclerotic plaques. The present invention provides a method of treating atherosclerosis or of reducing the formation of atherosclerotic plaques in a subject in need thereof by administering one or more peptide dimers of the present invention. Conditions that can be treated by the present method include atherosclerosis of the coronary arteries; arteries supplying the CNS, such as carotid arteries; arteries of the peripheral circulation or the splanchnic circulation; and renal artery disease. Administration, such as parenteral administration, can be site-specific or into the general blood stream. In some embodiments, the peptide dimers can be combined with an additional anti-atherosclerotic drug, including HMG-CoA reductase inhibitors, also termed statins. Suitable statins for use in the methods of the invention include, for example, lovastatin (MEV ACOR®, Merck), simvastatin (ZOCOR®, Merck), pravastatin (PRAVACHOL®, Bristol Myers Squibb), rosuvastatin (CRESTOR®, AstraZeneca), fluvastatin (LESCOL®, Novartis) and atorvastatin (LIPITOR®, Warner-Lambert).
The present invention further provides a method of treating, preventing or ameliorating the symptoms of inflammatory bowel disease (IBD), Crohn's disease, or ulcerative colitis in a subject in need thereof by administering an effective amount of at least one ApoE peptide dimer of the invention. In practicing the methods of this invention, the therapeutic peptides and/or derivatives thereof may be used alone or in combination with other active ingredients. If desired, one or more agents typically used to treat inflammatory bowel disease may be used as a substitute for or in addition to the therapeutic peptides in the methods and compositions of the invention. Such agents include biologics (e.g., inflixamab, adelimumab, and CDP-870), small molecule immunomodulators (e.g., VX 702, SCIO 469, doramapimod, RO 30201 195, SCIO 323, DPC 333, pranalcasan, mycophenolate, and merimepodib), non-steroidal immunophilin-dependent immunosuppressants (e.g., cyclosporine, tacrolimus, pimecrolimus, and ISAtx247), 5-amino salicylic acid (e.g., mesalamine, sulfasalazine, balsalazide disodium, and olsalazine sodium), DMARDs (e.g., methotrexate and azathioprine) and alosetron.
Suitable subjects benefiting from the compositions and methods of the present invention include male and female mammalian subjects, including humans, non-human primates, and non-primate mammals. Subjects include veterinary (companion animal) subjects, as well as livestock and exotic species.
The examples which follow are set forth to illustrate the present invention, and are not to be construed as limiting thereof.
EXAMPLES Example 1 The Cytotoxic Activity of ApoE Peptides is Enhanced by Formation of Disulfide DimersWe have previously shown that the addition of a protein transduction domain (PTD), such as an antennapedia peptide, to the apoE-mimetic COG133 peptide (LRVRLASHLRKLRKRLL (SEQ ID NO: 3)) enhances its anti-inflammatory activity. A series of fusion peptides with COG133 conjugated to a PTD were prepared by chemical synthesis. Notably, we found that COG112 with the sequence RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL (SEQ ID NO: 1), was effective in suppressing production of NO, TNFα and IL-6 with IC50s of 21 nM, 58 nM, and 12 nM, respectively, in BV2 cells following stimulation with LPS (
During the course of testing various compounds for cytotoxicity against CLL cells, we found that COG112 had an ED50 of 220 nM. These data were generated using lot #313 of the COG112 peptide. Upon depletion of the stock of lot #313, we began using a new synthesis of COG112 (lot #411) and discovered that the ED50 was reduced to 1.2 μM. While still being more potent than the apoE-mimetic COG133 lacking the antennapedia PTD, this lot was less active than lot #313.
To determine any possible structural differences in the two lots of COG112, we assayed COG112 from the two different lots using liquid chromatography/mass spectrometry (LC/MS) techniques with electrospray ionization in the positive detection mode. For COG112 from lot #313, a predominant peak with a mass to charge ratio (M/Z) of 1126.3 and peaks at M/Z=1800.9, 1286.6, and 1001.5 were observed (red arrows in
To confirm this finding, we prepared the reduced COG112 by treatment of COG112 from lot #313 with dithiothreitol to reduce the disulfides to the free thiol and repeated the LC/MS analysis (
Having confirmed the dimer structure of COG445, we then evaluated this peptide in both the BV2 cell assay for NO release and the CLL cytotoxicity cell assay. In the BV2 assay, we confirmed an IC50 of 20 nM for NO release and an ED50 of 110 nM for cytotoxicity of CLL cells. In the case of COG445, it is important to note that the previous ED50 values (e.g., 220 nM) were reported using the molecular weight of the monomer of 4502 rather than the actual molecular weight of the dimer of 9004. Adjusting for the correct molecular weight of COG445, the ED50 value for CLL cytotoxicity is reduced to 110 nM.
Example 2 Non-Reducible COG112 Dimer Peptides Activate PP2A and are Cytotoxic to Cancer CellsAfter discovery that COG112 was active as a disulfide-linked dimer, we sought a method to stabilize the dimer state of COG112. We initially treated the reduced COG112 peptide with a 5-fold molar excess of bismaleimidoethane (BMOE) in dilute solution. The peptide was precipitated by addition of ether, collected by filtration, and the unreacted BMOE removed by washing prior to drying under vacuum. The BMOE-linked peptide was dissolved in buffer and mixed with a 1.5-2.0 molar excess of freshly reduced COG112. Coupling was monitored by HPLC until the reaction was complete and the resultant peptide-BMOE-linker-peptide dimer was precipitated with ether, collected, washed, and purified by reverse phase HPLC to a purity of 98%. The identity of this peptide (known as COG449) was confirmed by MS and was assayed in the BV2 NO release assay. As shown in
To further evaluate the effect of COG449, we measured the ability of the stable dimerized COG449 compound to activate PP2A in 32D:p210BCR/Abl chronic myelogenous leukemia cells. Treatment with either COG445 or COG449 resulted in increased phosphate release due to activation of PP2A relative to untreated control cells (
Based on the activation of PP2A in serum containing media and potent suppression of NO in the BV2 assay, we tested COG 445 and COG449 for cytotoxicity against patient-derived CLL cells and normal B cells (
In order to more fully understand the role that the PTD domain and the apoE domain of the COG peptides play in anti-CLL cytotoxic activity, we tested additional compounds with an HIV-TAT PTD (COG226) and peptides with altered apoE sequences (COG1410, and COG248) as shown in Table I. It is notable that either the antennapedia (ANTP) or TAT PTD increases potency of COG1410 from 5.7 μM to 1.0 μM and 1.4 μM, respectively. It is also interesting that COG1410 attached to ANTP (COG 120) is more potent as a monomer than the COG112 monomer, with EC50 values of 1.0 μM and 1.4 μM for COG120 and COG112, respectively. This result suggests that creation of dimeric peptides containing altered apoE domains plus the ANTP or TAT PTD domains may further improve the potency of the peptides. These data demonstrate that apoE-mimetic compounds display potent and selective cytotoxic activity for freshly isolated human B-CLL cells with a wide safety margin.
We also evaluated the various peptides for efficacy in suppressing nitric oxide production induced by LPS stimulation of BV2 microglia cells as a measure of anti-inflammatory activity as well as the maximum dose tolerated in mice (Table I and
Next, we examined the potency of an ApoE peptide dimer versus the monomeric form on proliferation of the MDA-MB-231 breast cancer cell line. MDA-MB-231 cells were treated with various concentrations of either COG435 (monomer; SEQ ID NO: 90) or COG493 (a BMOE-linked dimer of COG 435) peptides for 48 hours. Following peptide treatment, cells were quantified using a MTT assay. The results, shown in
To determine whether ApoE peptide dimers were cytotoxic to other types of cancer, we evaluated the effect of three different ApoE BMOE-linked peptide dimers (COG449, COG492, COG493; see Table I) on the growth characteristics of U87MG glioblastoma cells. Various concentrations of COG449, COG492, COG493 or Sorafenib were used to treat U87MG glioblastoma cells and viable cells were quantitated using MTT. Sorafenib, which has previously been reported to be cytotoxic to glioblastoma cells (Yang et al. (2010) Mol Cancer Ther., Vol. 9(4):953-962), was used as a positive control. The dose response curve shown in
This series of experiments demonstrate that ApoE peptides are cytotoxic to three different types of cancer cells. Interestingly, the dimeric form of the ApoE peptides is considerably more potent in inducing cytotoxicity of cancer cells than the monomeric form.
Based on the results demonstrated in Examples 1 and 2 that dimers of ApoE peptides are more potent in inducing cytotoxicity of cancer cells and activating PP2A, twenty eight different monomer ApoE peptides are synthesized that can be coupled with two different coupling chemistries to create a dimer library of sixty four unique compounds. The goal of this Example is to establish a library of chemically stable peptide dimers designed to explore the structure activity relationship between apoE-mimetic peptides and cytotoxicity for CLL cells. Our initial screens with COG peptides were limited to a single dimer peptide, COG112, which has the sequence Ac-RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL-amide (SEQ ID NO: 1) that contained a disulfide bridge through the cysteine at position 17. This peptide has an antennapedia-derived PTD domain at the N-terminal end and an apoE-mimetic domain in the C-terminal portion such that the dimerized peptide contained two PTD domains and two apoE-mimetic domains. While this dimer of COG112 demonstrated superior potency, it is not possible to determine whether the PTD domain is essential for improved potency or whether a dimer composed of two COG peptides will be sufficient for high potency in cytotoxicity assays. It appears that a PTD does improve cytotoxic potency based on the observation that monomeric COG112 had an ED50 of 1.4±0.7 μM while COG133 that lacks a PTD was three fold less potent with an ED50 of 4.4±1.5 μM (Table I). Based on these results, our strategy for making dimer peptides relies on the combinatorial mixing of peptides that contain a PTD and an apoE-mimetic domain.
The first method used to create apoE-mimetic dimer peptides utilizes the reaction of a maleimide group with the sulfhydryl group of cysteine. Monomer peptides are created with a single cysteine residue in the peptide monomers and coupled to form a dimer using a bismaleimide linker to create the stable dimer (
Following synthesis of the peptides listed in Table II, the peptides are coupled to form dimers using either BMOE or BMH as listed in Table III. In Table III, an X indicates which monomer peptides are coupled together, thereby creating a focused library containing 40 unique members. The initial library is not strictly combinatorial in that only matched apoE sequences are coupled, resulting in dimer peptides that contain two COG133, two COG1410, two COG248, or two COG345 apoE domains. Final compounds that contain two PTD domains and two apoE domains are highlighted in the blue area of the Table, compounds that contain one PTD domain and two apoE domains are highlighted in the yellow area of the Table, and compounds lacking a PTD domain but containing two apoE domains are found in the green area of the Table.
The second method for coupling of the peptides utilizes “Click” chemistry for coupling the monomers. This coupling method relies on the copper catalyzed 3+2 Huisgen cycloaddition of an azide and a primary alkyne to form a stable 1,4-disubstituted triazole as shown in
Following synthesis of the peptides listed in Table IV, the peptides are coupled to form dimers using either the direct Click coupling (
Bismaleimide Coupling.
Coupling of the cysteine containing peptides with bismaleimide compounds is performed as a two step process where a 3-5 fold molar excess BMOE or BMH are initially reacted with the reduced monomer peptide in dilute solution. The peptide is precipitated by addition of ether, collected by filtration, and unreacted BMOE/BMH is removed by washing prior to drying under vacuum. The BMOE/BMH-linked peptide monomer peptide is dissolved in buffer and mixed with a 1.5-2.0 molar excess of the second monomer peptide. Coupling is monitored by HPLC until the reaction is complete. The resultant peptide is precipitated with ether, collected, washed, purified by reverse phase HPLC to a purity of >90%, and analyzed by LC/MS to determine the molecular mass of the product. Any peptides that do not match the expected mass are rejected and the coupling is repeated.
Click Coupling.
Coupling of the azidohomoalanine and propargylglycine containing peptides is performed using previously described protocols (Chan et al. (2004) Org Lett, Vol. 6(17): 2853-2855). Briefly, equimolar amounts of each monomer peptide are mixed together with copper iodide and ascorbic acid or tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA). Initially coupling is accomplished with ascorbic acid (Rostovtsev et al. (2002) Angew Chem Int Ed Engl, Vol. 41(14): 2596-2599), but TBTA is used if the ascorbic acid coupling results in poor yield or if damage to the peptide scaffolds is observed. Following coupling, peptides are precipitated with ether, collected and purified by reverse phase HPLC to a purity of >90%. Peptides are analyzed by LC/MS to determine the molecular mass of the product and any peptides that do not match the expected mass are rejected and the coupling is repeated.
Example 4 Evaluation of ApoE Peptide DimersThis example outlines experiments designed to evaluate the potency of the peptide dimers from the library described in Example 3. A two step screening cascade is employed. It has previously been shown that high cytotoxicity and leukemia cell apoptosis occur when CLL cells are cultured with nitric oxide synthase (NOS) inhibitors, thereby reducing the concentration of nitric oxide (NO)(Thomas et al. (2008) Free Radic Biol Med, Vol. 45: 18-31). This phenomenon occurs because low to moderate levels of NO are required for maintenance of the anti-apoptotic state of CLL cells (Zhao et al. (1998) Blood, Vol. 92(3): 1031-1043; Levesque et al. (2008) Leuk Res, Vol. 32(7): 1061-70; Kolb et al. (2003) Cardiovasc Haematol Disord, Vol. 3(4): 261-86). Within cells, NO is produced from L-arginine by three NOS isoforms in humans that are encoded by separate genes. NOS1 (“neuronal” NOS) and NOS3 (“endothelial” NOS) generally produce low levels of NO and are constitutively expressed, while inducible NOS (NOS2) is induced by cytokines and microbial factors through activation of NFκB. Like BV2 cells, human cells express NOS2 and produce NO in response to several stimuli including IFN-α, IFN-γ, IL-1, TNFα, IL-6, and LPS (Weinberg (1998) Molecular Med, Vol. 4: 577-591). In human CLL cells, it has been reported that high levels of NOS2 mRNA and protein are constitutively expressed and the cells have high NOS enzyme activity (Zhao et al. (1998) Blood, Vol. 92(3): 1031-1043). Based on these data and the limitations on use of human CLL cells, compounds are initially screened for suppression of NO production in lipopolysaccharide stimulated BV2 microglial cells. This assay is used for the initial screen because BV2 cells grow rapidly and readily express inducible NO synthase (NOS) in response to LPS treatment, leading to measurable amounts of NO production. Therefore, the first screening assay entails treating BV2 cells in an 8 point dose titration curve followed by stimulation with LPS and measuring the ability of the peptide dimers to suppress NO production.
Following determination of the IC50 for NO production in BV2 cells, the twenty peptide dimers with the greatest potency in the NO suppression assay are screened for cytotoxic activity against purified CD19+/CD5+ leukemia cells from CLL patients. Whole blood from CLL patients is obtained and the CD19+/CD5+ CLL cells are isolated by using RosetteSep™ Human B Cell Enrichment Cocktail as described in the methods section below. Unlike homogeneous BV2 cells from culture, it is difficult to obtain enough cells from one patient to screen many peptide dimers with cells from a single patient. Furthermore, due to the diverse nature of CLL with many documented chromosomal abnormalities and phenotypes, it is necessary to screen a single peptide dimer against CLL cells from multiple patients. Therefore, to ensure that pharmacogenomic effects are minimized in this screening step, ED50 values for cytotoxicity of each peptide dimer is determined for CLL cells from not less than six randomly selected patients and the ED50 curves from each individual patient are averaged. Following completion of this screening step, the safety window of each of the 10 most potent peptide dimers from the CLL cell cytotoxicity assays is determined by isolating CD19+ B-cells from normal, age-matched volunteers and determining the ED50 for cytotoxicity of normal B-cells. Similar to the analysis with the CLL cells, the ED50 in B-cell samples is determined from not less than 4 volunteers. A safety window for each peptide dimer is calculated by dividing the ED50 value for cytotoxicity on CLL cells by the ED50 value for cytotoxicity on normal B-cells. The five peptide dimers with the greatest safety window is selected for use in pharmacokinetic profiling.
Pharmacokinetic Profiling of Peptide DimersMale C57Bl/6 mice (20-24 g, Charles River, Raleigh, N.C.) are injected with a peptide dimer subcutaneously at a volume of 5 mL/kg in the scruff of the neck. Ten minutes before the desired timepoint, mice are anesthetized and at the desired timepoint blood is drawn by cardiac puncture. Blood from at least 4 mice is used for each timepoint with the samples collected at 5, 10, 15, 30, 45, 60, 90, 120, 180 and 240 minutes for the initial analysis. The samples are processed and the pharmacokinetic analysis is completed. These timepoints were selected based on previous experience with subcutaneously injected COG1410 that shows a dose dependent Tmax of 20-30 minutes and a half life for clearance of 60-90 minutes. In the event that the half life has not been reached, a repeat of the experiment is performed that uses one timepoint before the observed Tmax, a timepoint at the observed Tmax and timepoints extending long enough to adequately determine the half life based on extrapolation of the previous observations of clearance from the plasma. Analysis of three concentrations of the peptide dimer is also performed to determine if there is a dose dependent effect on the pharmacokinetic parameters.
Noncompartmental model analysis is used to estimate pharmacokinetic parameters (Gibaldi and Perrier, 1982) including area under the plasma-concentration time curve from time 0 to time infinity (AUC, 0-∞), peak plasma concentration (Cmax), systemic clearance (CL, which is calculated based on the ratio between the dose and AUC, 0-∞), volume of distribution at steady state (Vss), terminal half-life (t½), which is calculated using a minimum of the last three concentration-time data, and mean residence time in the body (MRT). Data analysis is conducted using Win-Nonlin professional version 3.1 (Pharsight Corporation, Cary, N.C., USA).
Peptide Dimer Treatment of Eμ-TCL1 Transgenic MiceFollowing pharmacokinetic profiling, select peptide dimers are tested in a transgenic mouse model of CLL. Blood is drawn by retro-orbital bleeding from transgenic Eμ-TCL1 mice aged to 9 months for initial analysis to total white cell counts and leukemia cell burden. Animals that show leukemia signs are randomly assigned to treatment groups. At the initiation of treatment, blood is drawn to determine baseline CD5+/CD19+ CLL cell counts and groups of animals (n=20) are subcutaneously injected with a vehicle control (lactated Ringer's solution) or one of the selected peptide dimers at doses and a dose frequency schedule determined by the pharmacokinetic profile data for a total treatment time of 35 days.
Blood is collected from each mouse on a weekly basis by retro-orbital bleeds. This blood is used for determination of total blood leukocyte and lymphocyte counts as well as CD19+/CD5+ cell counts to determine the leukemia burden. After 35 days of treatment, mice are euthanized and the post treatment leukemia burden is measured by cell counting, spleen weight, and histological analysis of bone marrow, spleen, liver, and lymph nodes. All mice dying before 35 days are analyzed in a comparable fashion.
MethodsBV2 Cell Growth, LPS Stimulation and NO Quantitation.
Low passage BV2 microglial cell cultures are maintained in 10% HI (Heat Inactivated) FBS DMEM media (supplemented with MEM NEAA (Non-Essential Amino Acids), sodium pyruvate and Pen-Strep) and continuously cultured until needed. To determine the IC50 values for NO production, peptides are added to BV2 cultures in 1% HI FBS DMEM media (supplemented with MEM NEAA and sodium pyruvate) at a range of final assay concentrations from 1.0 μM in 2 fold dilutions to 7.8 nM followed immediately by addition of LPS (100 ng/mL final concentration) as previously described (Laskowitz et al. (2001) Exp Neurol, Vol. 167(1): 74-85). After incubating for 18±1 hours, conditioned media is removed for analysis of nitrite (the stable oxidation product of released nitric oxide) by the Griess colorimetric assay (Promega). Remaining cells are assayed for viability in an MTT assay (Promega) and the nitrite assay values are normalized for each concentration using the percent viability of cells in the MTT assay. IC50 values for NO inhibition are calculated under the assumption that LPS-only (no peptide added) cultures exhibit a 100% response and no-LPS (no peptide added) cultures exhibit a 0% response. Typically, the absence of LPS exposure to BV2 cells results in levels below the limit of detection in our assays. Similarly, addition of any of the peptides up to 25 μM without LPS results in undetectable NO levels.
CLL Cell Isolation.
Normal B-cells from volunteers and B-CLL cells from patients are isolated using the RosetteSep™ Human B Cell Enrichment Cocktail according to the manufacturer's instructions. This method depletes whole blood of T cells, monocytes, and NK cells using a proprietary antibody cocktail that cross-links unwanted cells in human whole blood to multiple red blood cells (RBCs) forming immunorosettes. This increases the density of the unwanted (rosetted) cells, such that they pellet along with the free RBCs when centrifuged over a buoyant density medium such as Ficoll-Paque®. This leaves the highly enriched B-cell or B-CLL cells at the interface between Ficoll and the plasma. The antibodies in the cocktail contain anti-CD14, anti-CD2 and anti-CD16 antibodies to remove T cells, monocytes, and NK cells, respectively. The purity of these B-cell and B-CLL preparations is then determined by flow cytometry. Preparations typically average less than 2% CD3+ T-cells and less than 0.5% CD14+ monocytes. In the case of B-CLL cell isolation, we routinely obtain preparations that contain less than 1.5% normal CD19+/CD5− B-cells using this method.
CLL Cell Culture/Cytotoxicity Assays.
For cytotoxicity assays, 3×106 CLL cells/well are cultured in 24 well tissue culture plates in 1.5 mL of Hybridoma SFM™ (Gibco, Long Island, N.Y.) as described by Levesque et al. (2001, 2003). All cultures are incubated at 37° C., 5% CO2 in air. Peptide dimers are applied to B-CLL cells (0.25×106 cells/well in a 96 well plate) and after 72 hours, viable cells are assessed using the MTS assay (Pharmacia) to determine the concentration of peptide dimer that is effective in killing 50% of the input CLL cells (ED50) (Levesque et al., 2003).
Example 5 An ApoE Peptide Dimer Inhibits Akt Signaling in Cancer CellsAkt signaling is often dysregulated in cancers thereby promoting cellular survival and proliferation. ApoE-based peptides have shown anti-inflammatory effects in various neuropathologies associated with increased Akt signaling. Given the effect of ApoE peptides on other inflammatory pathologies, we investigated the effect of an ApoE peptide dimer on Akt signaling in breast and brain cancer cells. A disulfide dimer of COG112 (SEQ ID NO: 1) was prepared as described in Example 1 (COG445). Adherent breast cancer cells, MDA-MB-231 (MB231), or glioblastoma cells, U87-MG (U87), were serum starved overnight and then exposed to COG445 for 2 hours before stimulating with epidermal growth factor (EGF) for 5 minutes. Western blot analysis showed that COG445 at concentrations up to 1 μM did not alter EGFR activation as determined by EGFR tyrosine 1045 phosphorylation in both U87 (
Inhibition of Akt phosphorylation by COG445 appears to be downstream of EGFR/PI3K signaling as EGFR and PDK1 phosphorylation were not altered (
To further characterize the effect of COG445 on PP2A activity, MB231 cells were treated as above and the phosphatase activity of immunoprecipitated PP2A was measured. EGF stimulation caused a significant decrease in total PP2A activity compared to untreated serum starved cells (
To examine the mechanism of COG445 on PP2A activity, recombinant human PP2A catalytic subunit was incubated with COG445 and activity was measured. COG445 did not have any effect on the rate of phosphatase activity up to 10 μM (data not shown). These data indicate that COG445 affects PP2A activity on a biochemical level. To further explore the changes of PP2A protein complexes, co-immunoprecipitation experiments were performed on MB231 cells. The potent endogenous inhibitor of PP2A, I2PP2A (also known as SET), was strongly associated to PP2A in EGF stimulated cells, corresponding to the low PP2A activity, while the association was significantly decreased in unstimulated cells (
Akt exerts its proliferative signal by phosphorylating protein substrates such as mTOR and GSK-3β. To examine the effects of COG445 on downstream Akt signaling in MB231 cells, mTOR activation and GSK-3β inhibition was measured by western blot analysis. COG445 caused a dose-dependent decrease in mTOR and GSK-3β phosphorylation upon EGF stimulation (
Because COG445 inhibited Akt activation and downstream signaling, we examined the effects of COG445 on cellular proliferation. Adherent MB231 and U87 cells were grown for ˜24 hours in the presence of COG445 and cellular proliferation was measured by MTT reduction. COG445 caused a dose-dependent decrease in proliferation for both cancer cell lines tested (
The results of these experiments described in this example suggest that ApoE-based peptides may have beneficial effects in cancer chemotherapy by activating the tumor suppressor PP2A.
Example 6 An ApoE Peptide Dimer Reduces c-myc Phosphorylation in a Burkitt's Lymphoma Cell LineAberrant c-myc expression has been implicated in various forms of cancer. It has been reported that phosphorylation at serine 62 of c-myc stabilizes the c-myc protein, while dephosphorylation at this serine residue by PP2A directs ubiquitin-mediated degradation of c-myc (see, e.g., Sears et al. (2004) Cell Cycle, Vol. 3: 1133-1137). Because an ApoE peptide dimer increases PP2A activity and promotes c-myc degradation in cancer cell lines (see Examples 2 and 5), we further examined the effect of a stabilized ApoE peptide dimer on c-myc phosphorylation in a c-myc dependent human Raji cell line of Burkitt's lymphoma. We treated Raji cells with COG449, a BMOE-linked dimer of COG112 (see Example 2), or a vehicle control for 20 hr and probed the extracts by Western blotting with an antibody for P-S62 and a total c-myc antibody, which showed a significant reduction of phosphorylation at S62 (
These results are consistent with those obtained in the experiments described in Examples 2 and 5 and suggest that ApoE peptide dimers can modulate c-myc protein levels in cancer cells perhaps by antagonizing SET and relieving the inhibition of PP2A. Thus, ApoE peptide dimers may represent a new approach to cancer treatment, especially in cancers where SET is overexpressed.
Example 7 SET is Overexpressed in CLL and B-Cell Lymphoma CellsIn seeking to study the dysregulation of PP2A in chronic lymphocytic leukemia (CLL), we chose to focus on the endogenous physiological inhibitory proteins of PP2A. Recently, Neviani et al. (Cancer Cell, Vol. 8: 355-68, 2005) reported that the SET oncoprotein (also known as Inhibitor-2 of PP2A, I2PP2A) was overexpressed in patient-derived chronic myelocytic leukemia (CML) cells and that the SET concentration increased as the patient developed blast crisis. This reference also demonstrated that PP2A activity decreased during blast crisis, resulting in reduced ability of cells to regulate the Akt signaling pathway following BCR/Abl stimulated Akt phosphorylation. Given the convergence of dysregulated Akt signaling in both CML and CLL, we sought to determine whether SET might also be overexpressed in fresh, patient-derived CLL cells. Using primary B-CLL samples from 16 patients and normal B-cells from volunteers, we prepared cell lysates and 40 μg of each lysate was loaded on to a SDS PAGE, transferred to nitrocellulose, and immunoblotting was performed. The bands detected using an anti-SET antibody were quantitated and normalized using β-Actin as a loading control on a LiCor Odyssey fluorescence scanner. We discovered that SET was significantly overexpressed (p<0.05) in the CLL patient samples relative to normal B-cells from volunteers (
To expand this work, we also evaluated SET overexpression in the Raji and Ramos cell lines of Burkitt's lymphoma, a B-cell Non-Hodgkin's lymphoma (NHL), which unlike CLL cells, is proliferating and can be genetically manipulated. We grew these cells using conditions recommended by ATCC. Cell lysis for Western blotting and isolation of mRNA was performed using standard protocols from 3 separate cultures. Following synthesis of cDNA from the isolated mRNA, qPCR was performed analyzing SET mRNA and 18S primers and the fold change of the SET expression in Raji and Ramos cells were normalized to that of normal B-cell cDNA expression level (standardized to 1). SET expression levels were 10.5±0.7 fold higher than normal B-cells in Raji cells and 8.2±0.4 for the Ramos cells (p<0.001) (
To analyze the effect of reducing the SET activity in cancerous B-cells, we used lentivirus to introduce a shRNA construct to silence SET production in the Raji cell line. When we transduced cells with a SET-specific shRNA construct, the growth rate measured using the MTT assay was significantly decreased relative to a noncoding control shRNA construct (
To determine if SET levels are indicative of more rapid CLL disease progression, we used Western blotting to quantify SET levels in cell extracts from 226 of the 435 patients in our repository. We created a receiver operator curve to determine a cutoff for high and not-high SET levels. Analysis of the time to first treatment for each of these two groups showed a significant difference—the group with highest SET levels had a reduced time to first treatment relative to patients with lower SET levels (
Overall, our data demonstrate that SET is overexpressed in CLL and NHL relative to normal B-cells and that antagonism of SET function by reducing its level in cells inhibits growth. Furthermore, our results indicate that measurement of the overexpression of SET from CLL cells or biopsied NHL tissue might be a useful biomarker to predict which patients may require therapy sooner and which patients may benefit from anti-SET therapy, such as ApoE peptide dimers described herein.
Example 8 ApoE Peptide Dimers Reduce Cellular Concentrations of the Anti-Apoptotic Mcl-1 ProteinThe Myeloid Cell Leukemia-1 (Mcl-1) protein is a member of the Bcl-2 family that regulates apoptosis. Members of this family include the anti-apoptotic members Bcl-2, Bcl-XL and Mcl-1 while pro-apoptotic members include BAD, BID, and BAX (Buggins and Pepper (2010) Leuk Res, Vol. 34: 837-842). The anti-apoptotic Bcl-2 family members associate with pro-apoptotic family members to inhibit mitochondrial outer membrane permeabilization that releases cytochrome-C and initiates the intrinsic apoptotic pathways. CLL cells have been shown to overexpress both Bcl-2 and Mcl-1 (Buggins and Pepper (2010) Leuk Res, Vol. 34: 837-842) and high levels of Bcl-2 and Mcl-1 correlate with poor response to fludaribine therapy in patients (Kitada et al. (1998) Blood, Vol. 91: 3379-8947). Mcl-1 overexpression was demonstrated to arise from B-cell receptor (BCR) engagement and that stimulation of BCR may promote selection of neoplastic B-cell clones (Stevenson and Caligaris-Cappio (2004) Blood, Vol. 103: 4389-9548).
Recently, Peppers et al. measured Mcl-1, Bcl-2, and BAX levels from 185 CLL patients and found that patients with high Mcl-1 levels and low BAX levels, giving rise to a high Mcl-1/BAX ratio had significantly shorter time-to-first-treatment and lower overall survival than patients with lower Mcl-1 levels and ratios between Mcl-1 and BAX (Pepper et al. (2008) Blood, Vol. 112: 3807-3817). In addition to CLL, Mcl-1 overexpression had been reported in B-cell non-Hodgkin's lymphoma (NHL) patients and the expression level correlated with tumor grade where higher expression levels were found in high grade lymphomas (Cho-Vega et al. (2004) Hum Pathol, Vol. 35: 1095-1100). Taken together, these data have been used to suggest that Mcl-1 is the most significant anti-apoptotic protein associated with B-cell malignancies (Gandhi et al. (2008) Blood, Vol. 112: 3538-4051).
Given our results with destabilization of c-myc (see Examples 5 and 6), we began our evaluation of the Mcl-1 protein by analyzing the sequence of c-myc near the T58 and S62 sites that regulates the ubiquitination and proteosomal degradation process and compared this sequence to the Mcl-1 sequence. We observed that there is distinct homology between the c-myc and Mcl-1 motifs with S/T residues at 159 and 163 in Mcl-1 that correspond to the T58 and S62 residues of c-myc as indicated by red arrows in
To test this hypothesis, we performed immunoprecipitation of Mcl-1 from human CLL cells and checked for co-immunoprecipitation of Pin1, PP2A, Axin, and SET (
We next sought to determine whether SET antagonism would destabilize Mcl-1. Destabilization of Mcl-1 by treatment with SET antagonists was evaluated by treating primary human CLL cells for 24 hr with the ApoE peptide dimer COG449 (a BMOE-linked dimer of COG112; see Example 2) and evaluating the level of Mcl-1 in the cells. We observed a significant dose-dependent decrease in the Mcl-1 concentration relative to β-Actin as a loading control (
To evaluate the effect of COG449, an ApoE peptide dimer (see Example 2), on cancer cell growth in vivo, we analyzed the effects of COG449 treatment of the Ramos cell line of Burkitt's lymphoma, a B-cell non-Hodgkin's lymphoma. Ramos cells are B-cells that overexpress c-myc and form tumors throughout the body. After determining that COG449 inhibited growth with an EC50 of 125 nM in culture, 107 Ramos cells were subcutaneously injected into the left flank of female SCID mice (Schliemann et al. (2009) Blood, Vol. 113: 2275-2283). Tumor growth was monitored daily by palpitation and caliper measurement until tumors reached approximately 150 mm3. At day 11, mice were assigned to two groups so that initial tumor size was approximately equal between groups.
Tumor measurements and treatment with COG449 (5 mg/kg, subcutaneous injection into the right shoulder area) or a vehicle control were performed by a technician who was blinded to the treatment solutions. At day 19, the tumor volume reached the predetermined size for termination of the experiment and animals were photographed (
The measured tumor volumes and final tumor weights are plotted in
Breast cancer, the most common cancer affecting women, is a heterogeneous disease comprised of several molecular subtypes (Tang et al. (2009) Diagn Mol Pathol, Vol. 18(3): 125-132). Three main subgroups have been defined based on the pattern of expression of the hormone receptors estrogen (ER) and/or progesterone (PR), and the HER2 receptor status: luminal tumors (ER+/HER2−), HER2 amplified tumors (HER2+), and triple negative breast cancer (TNBC, ER−/PR−/HER2−). The identification of subgroups of breast cancer based on biologic differences has allowed the development of targeted therapeutic agents (Di Cosimo and Baselga (2010) Nat Rev Clin Oncol, Vol. 7(3): 139-147). For example, hormone therapies are effective in the treatment of hormone-receptor positive breast cancers while HER2-targeted therapies are useful in the treatment of HER2-positive tumors. TNBC, which lack expression of hormone receptors and HER2, is therefore insensitive to such targeted therapies. TNBC, and the molecularly related Basal-type breast cancer accounts for 15-20% of all invasive breast cancer cases and is characterized by its aggressive clinical behavior, high rates of relapse following chemotherapy, and poor patient survival (Di Cosimo and Baselga, 2010; Ray and Polite (2010) Cancer J, Vol. 16(1): 17-22). In addition, TNBC/Basal-like BC disproportionately afflicts African-American women with incidence as high as 39% (Carey et al. (2006) JAMA, Vol. 295(21): 2492-2502).
The most promising approach to treating women with this devastating disease is the use of molecularly targeted drugs that are specific for activated oncogenic pathways in the disease and thus generally present less toxicity. Thus, understanding the unique molecular changes associated with the development of TNBC is necessary in order to develop new targeted therapies that will be effective against this aggressive tumor type. However, little is actually known about the underlying genetic changes associated with the development of TNBC. Recent work has described aberrant activation of various receptor tyrosine kinase signaling pathways in TNBC, including the EGF, HGF, FAK, FGF, VEGF, and IGF-1 pathways, which can upregulate kinase cascades including Raf/MEK/ERK and PI3K/AKT (Di Cosimo and Baselga, 2010; Turner et al. (2009) Oncogene, Vol. 29(14): 2013-2023; Kurebayashi (2009) Breast Cancer, Vol. 16(4): 275-280). Additionally, defects in apoptotic pathways, including p53, Bcl2, and Mcl-1, are also common in TNBC.
Several naturally occurring inhibitors of PP2A have been identified, including SET (also known as I2PP2A) and Cellular Inhibitor of PP2A (CIP2A). CIP2A has recently been described as an important PP2A inhibitor in multiple cancer types (Khanna et al. (2009) Cancer Inst, Vol. 101(11): 793-805). It is overexpressed in 39% of breast cancers and this is associated with clinical aggressiveness (Come et al. (2009) Clin Cancer Res, Vol. 15(16): 5092-5100). CIP2A overexpression cooperates with Ras and c-Myc for cell transformation, while its suppression inhibits tumor growth (Sablina et al. (2008) Cancer Metastasis Rev, Vol. 27(2): 137-146). CIP2A has been shown to interact with c-Myc and PP2A and display c-Myc stabilization activity (Junttila et al. (2007) Cell, Vol. 130(1): 51-62). CIP2A appears to selectively inhibit PP2A targeted to c-Myc (Westermarck and Hahn (2008) Trends Mol Med, Vol. 14(4): 152-160). The phosphoprotein SET is reported to have general PP2A inhibitory activity (Li et al. (1995) Biochemistry, Vol. 34(6): 1988-1996). SET was originally identified as a fusion protein in acute myelogenous leukemia and it is upregulated in multiple cancer types (Westermarck and Hahn, 2008).
To determine whether these endogenous PP2A inhibitors are expressed in triple negative breast cancers as well, we evaluated SET and CIP2A expression by qRT-PCR in 13 cDNA samples from TNBC tumors and found overexpression in 7 of 13 for SET and 12 of 13 for CIP2A (
ApoE mimetic peptides bind to SET and activate PP2A (see Example 5). We previously found that a peptide derived from amino acids 133-149 (known as COG133) inhibited inflammation and the production of cytokines and nitric oxide through reduced activation of the NF-κB pathway (Singh et al. (2008) J Biol Chem, Vol. 283(24):16752-16761). In order to study the underlying mechanism of this effect, COG133 was biotin labeled and used to affinity purify protein binding partners. Interestingly, the primary binding partner was the SET oncoprotein. The identification of SET, a potent PP2A inhibitor (Li et al. (1996) Journal of Biological Chemistry, Vol. 271(19): 11059-11062, as the binding partner of the apoE-mimetic peptide suggested that the peptides would bind SET and prevent it from binding to and inhibiting PP2A. COG449, a dimer derivative of COG133 (see Example 2) with improved potency and bioavailability, was also found to bind SET (data not shown).
Together, these studies emphasize a critical role for PP2A suppression in breast cancer and support an innovative approach for re-activating the PP2A tumor suppressor through therapies antagonizing its inhibitors.
Example 11 SET Antagonism with an ApoE Peptide Dimer Reduces Phosphorylation of a Cancer Related PP2A TargetTo determine if antagonism of SET with COG449, a BMOE-linked dimer of COG112 (see Example 2), reduced the phosphorylation of a known PP2A target that is implicated in breast cancer, we evaluated the status of eIF4E.
We previously demonstrated that antagonism of SET using related COG peptides reduced the phosphorylation of Akt (see Example 5), and the activity of NFκB (Singh et al., 2008). To analyze the effect of PP2A activation on the mTOR pathway, we analyzed the phosphorylation status of eIF4E and found that SET antagonism by COG449 treatment resulted in decreased phosphorylation of eIF4E (
Following demonstration of the activation of PP2A by the BMOE-linked COG112 peptide dimer, COG449 (see Example 2), and the inhibitory effects of COG449 on several PP2A targets, we explored whether this peptide had any anti-tumor activity. To determine whether COG449 might be effective in the treatment of human breast cancers, we treated several breast cancer cell lines with COG449 and found that COG449 was cytotoxic to all cell lines, including several triple negative breast cancer (TNBC) lines (MDA-231, MDA-468, and HCC38) (
To begin assessment of the potential of combination therapies with COG449, we analyzed the effect of treating MDA-231 cells with sub-lethal doses of COG449 and the multi-kinase inhibitor Sorafenib or the EGFR inhibitor Gefitinib at concentrations below their ED50 doses. The combination of COG449 and Sorafenib or Gefitinib produced a robust cytotoxic effect that was greater than the effect of either compound alone (
It is understood that the disclosed invention is not limited to the particular methodology, protocols and reagents described as these may vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods, devices, and materials are as described. All patents, patent applications and other publications cited herein and the materials for which they are cited are specifically incorporated by reference in their entireties.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims
1. A peptide dimer comprising a first ApoE peptide and a second ApoE peptide, wherein said first and second ApoE peptides are covalently linked by a linking moiety, and wherein the first and second ApoE peptides contain a sequence derived from amino acids 133-149 of ApoE protein.
2. The peptide dimer of claim 1, wherein said linking moiety is selected from the group consisting of a disulfide bridge, a bismaleimide, a 1,4-disubstituted triazole, and N,N-dipropargylamine.
3. The peptide dimer of claim 2, wherein said bismaleimide is bismaleimido-ethane or bismaleimido-hexane.
4. The peptide dimer of claim 1, wherein said first and second ApoE peptides are the same.
5. The peptide dimer of claim 4, wherein said first and second ApoE peptides are peptides having a sequence selected from the group consisting of LRVRLASHLRKLRKRLL (SEQ ID NO: 3), AS(Aib)LRKL(Aib)KRLL (SEQ ID NO: 5), LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL (SEQ ID NO: 4), and LRVRLAS(Aib)LRKLR(K-Ac)RLL (SEQ ID NO: 35).
6. The peptide dimer of claim 1, wherein said first and second ApoE peptides are different.
7. The peptide dimer of claim 1, wherein said first ApoE peptide is conjugated to a first protein transduction domain through one or more first linking residues.
8. The peptide dimer of claim 7, wherein said first protein transduction domain is selected from the group consisting of peptides derived from antennapedia, TAT, SynB1, SynB3, SynB5, and polyarginine.
9. The peptide dimer of claim 8, wherein said first protein transduction domain has a sequence of RQIKIWFQNRRMKWKK (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), or WKK.
10. The peptide dimer of claim 7, wherein said one or more first linking residues is cysteine, azidohomoalanine, or propargylglycine.
11. The peptide dimer of claim 7, wherein said first and second ApoE peptides are peptides having a sequence selected from the group consisting of LRVRLASHLRLRKRLL (SEQ ID NO: 3), AS(Aib)LRKL(Aib)KRLL (SEQ ID NO: 5), LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL (SEQ ID NO: 4), and LRVRLAS(Aib)LRKLR(K-Ac)RLL (SEQ ID NO: 35).
12. The peptide dimer of claim 7, wherein said second ApoE peptide is conjugated to a second protein transduction domain through one or more second linking residues.
13. The peptide dimer of claim 12, wherein said second protein transduction domain is selected from the group consisting of peptides derived from antennapedia, TAT, SynB1, SynB3, SynB5, and polyarginine.
14. The peptide dimer of claim 13, wherein said second protein transduction domain has a sequence of RQIKIWFQNRRMKWKK (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), or WKK.
15. The peptide dimer of claim 12, wherein said one or more second linking residues is cysteine, azidohomoalanine, or propargylglycine.
16. The peptide dimer of claim 12, wherein said first and second ApoE peptides are peptides having a sequence selected from the group consisting of LRVRLASHLRKLRKRLL (SEQ ID NO: 3), AS (Aib)LRKL(Aib)KRLL (SEQ ID NO: 5), LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL (SEQ ID NO: 4), and LRVRLAS(Aib)LRKLR(K-Ac)RLL (SEQ ID NO: 35).
17. A pharmaceutical composition comprising an effective amount of a peptide dimer of claim 1 and a pharmaceutically acceptable carrier.
18. A method of treating cancer, CNS inflammation, traumatic brain injury, cerebral ischemia, cerebral edema, neurolathyrism, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, schizophrenia, atherosclerosis, bacterial sepsis, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, polyarticular-course juvenile rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, or Alzheimer's disease in a subject in need thereof comprising administering to the subject a composition of claim 17.
19. The method of claim 18, wherein said cancer is leukemia, lymphoma, or breast cancer.
20. (canceled)
21. The method of claim 19, wherein said leukemia is chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), or acute lymphocytic leukemia (ALL).
22-43. (canceled)
Type: Application
Filed: Jan 6, 2011
Publication Date: Jan 3, 2013
Inventors: Michael P. VITEK (Rescarch Triangle, NC), Dale J. Christensen (Research Triangle, NC)
Application Number: 13/520,623
International Classification: A61K 38/17 (20060101); A61P 35/00 (20060101); A61P 25/00 (20060101); A61P 25/28 (20060101); A61P 9/10 (20060101); A61P 35/02 (20060101); A61P 25/16 (20060101); A61P 25/18 (20060101); A61P 29/00 (20060101); A61P 17/06 (20060101); A61P 1/00 (20060101); A61P 1/04 (20060101); C07K 14/775 (20060101); A61P 7/10 (20060101);
