WO2011066453A2 - Pacap et protéines apparentées destinés à traiter le cancer - Google Patents

Pacap et protéines apparentées destinés à traiter le cancer Download PDF

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WO2011066453A2
WO2011066453A2 PCT/US2010/058093 US2010058093W WO2011066453A2 WO 2011066453 A2 WO2011066453 A2 WO 2011066453A2 US 2010058093 W US2010058093 W US 2010058093W WO 2011066453 A2 WO2011066453 A2 WO 2011066453A2
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polypeptide
pacap
vip
cancer
therapeutically active
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WO2011066453A3 (fr
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Sami I. Said
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Said Sami I
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to compositions and methods for cancer therapy, and more particularly to methods of treating cancer that employ polypeptides in the secretin
  • PACAP38 PACAP38 and therapeutically active variants thereof.
  • Breast cancer gene 1 and 2 predispose women to breast and ovarian cancers and men to an increased risk for prostate cancer. Aside from non- melanoma skin cancer, breast cancer is the most common form of cancer in women and the number one cause of cancer death in Hispanic women. The incidence remains high despite several significant advances in treatment, including targeted therapies such as HerceptinTM.
  • the present invention is based, in part, on our realization that PACAP (pituitary adenylate cyclase activating polypeptide), VIP (vasoactive intestinal protein), and related proteins in the VIP/PACAP family can be used as anti-cancer therapeutic agents.
  • PACAP epidermal growth factor
  • VIP vasoactive intestinal protein
  • PACAP38 in particular is an effective PARP inhibitor.
  • Other, distinct types of PARP inhibitors have recently been shown to increase the sensitivity of certain tumors to cytotoxic therapy and ionizing radiation. Such tumors are those associated with BRAC1 and BRAC2 mutations, and they include breast, ovarian, and prostate cancer (Fong et ah, New England J. Med. 361(2): 123-133 (2009)).
  • the present invention features compositions and methods of treating cancer by administering a therapeutically effective amount of a polypeptide in the
  • VIP/PACAP family e.g. , PACAP38
  • PACAP PACAP38
  • PACAP PACAP (1-38)
  • a variant of that polypeptide that is also therapeutically active can also be used.
  • polypeptide or a combination or mixture of the polypeptides described herein, can be administered alone (i.e., as the sole type of therapeutic agent) to a patient who has a cancer, including a cancer associated with a BRAC1 or BRAC2 mutation, such as a breast, ovarian, or prostate cancer.
  • a cancer including a cancer associated with a BRAC1 or BRAC2 mutation, such as a breast, ovarian, or prostate cancer.
  • a polypeptide is a PARP inhibitor
  • it can be used as a part of a therapeutic regime.
  • a polypeptide in the VIP/PACAP family that inhibits PARP can be used in combination with another
  • any of the methods of the invention can include one or more of the following steps: a step of providing a tissue sample (e.g. , a biopsy sample) and determining whether there is a mutation (e.g., a cancer-related or cancer- indicative mutation) in a BRAC gene (e.g.
  • the patient may have or may be diagnosed or suspected of having breast cancer, ovarian cancer or prostate cancer (as noted above), or pancreatic cancer, melanoma, leukemia, lymphoma, myelodysplasia, or small cell lung carcinoma.
  • Any of the methods of the invention can include a step of diagnosing a patient (e.g., using a molecular assay, diagnostic marker, or an imaging technique to determine whether a patient has cancer or a pre-cancerous condition).
  • the polypeptide administered can be VIP (SEQ ID NO:l) or a therapeutically active variant of VIP (e.g., a polypeptide comprising a sequence that is at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to VIP).
  • the polypeptide administered can be PACAP (1-27) (residues 1 -27 of SEQ ID NO:2) or a therapeutically active variant of PACAP (1-27) (e.g., a polypeptide comprising a sequence that is at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to PACAP (1 -27)).
  • the polypeptide administered can be any therapeutically active variant of PACAP (1-27) (residues 1 -27 of SEQ ID NO:2) or a therapeutically active variant of PACAP (1-27) (e.g., a polypeptide comprising a sequence that is at least or about 60%, 65%,
  • PACAP38 also known as PACAP (1-38) (residues 1-38 of SEQ ID NO:2) or a therapeutically active variant of PACAP (1-38) (e.g., a polypeptide comprising a sequence that is at least or about 60%, 65%, 70%, 75%, 80%, 85%», 90%, 95%, or 98% identical to PACAP (1-38)).
  • PACAP 1-38
  • PACAP a therapeutically active variant of PACAP
  • PACAP e.g., a polypeptide comprising a sequence that is at least or about 60%, 65%, 70%, 75%, 80%, 85%», 90%, 95%, or 98% identical to PACAP (1-38).
  • ViP/PACAP family members include secretin, glucagon, PHI (peptide histidine-isoleucine), PHM (peptide histidine-methionine), and helodermin.
  • PACAP, VIP, and PHI are known to act via common receptors, namely VPAC 1 and VPAC2.
  • Human preprovasoactive intestinal polypeptide contains PHM, which differs by only two amino acid residues from PHI and which is also closely related in sequence to VIP.
  • polypeptides in the PACAP/VIP family inhibit PARP (such that it cannot or cannot effectively repair mitochondrial damage and the affected cell subsequently dies) and that their efficacy can be improved by co-administration of an agent that permeabilizes the cell membrane and thereby improves access to PARP.
  • a polypeptide in the PACAP/VIP family inhibit PARP (such that it cannot or cannot effectively repair mitochondrial damage and the affected cell subsequently dies) and that their efficacy can be improved by co-administration of an agent that permeabilizes the cell membrane and thereby improves access to PARP.
  • compositions of the present invention include compositions comprising one or more VIP/PACAP family polypeptides (e.g. , PACAP38) and an agent that permeabilizes or increases the permeability of the cellular membrane. While the invention is not so limited, compositions including the permeablization agent may include less polypeptide than would otherwise be required or administered (in the absence of the permeabilization agent). Where nucleic acid constructs are administered, their delivery can be facilitated by the transfection methods described in U.S. Patent No. 6,573,101, which is hereby incorporated by reference in its entirety.
  • cytotoxic therapy e.g. , administration of a cytotoxic therapy
  • chemotherapeutic agent other than a polypeptide as described herein they can include a step of administering any of the agents currently known and used to treat cancer, particularly those known and used to treat cancers that are more susceptible to treatment when PARP is inhibited.
  • the agent can be, for example, a ceramide analog. More specifically, the agent can be paclitaxel, docetaxel, Cytoxan, or gemcitabine.
  • a VIP/PACAP polypeptide as described herein can be fused (directly or via a linker) to a heterologous polypeptide.
  • the heterologous polypeptide can be one that, for example, increases the solubility of the VIP/PACAP polypeptide, enhances cell- or receptor- binding or affinity, or increases the circulating half-life of the VIP/PACAP polypeptide.
  • polypeptides are relatively inexpensive to manufacture, the cost should not be prohibitive.
  • the use of the present polypeptides can occur in the context of treating a mammal, including a human, or a domesticated animal or pet, and in the manufacture of a medicament for treating a mammal who is suffering from a cancer as described herein.
  • the patient can be treated with a gene construct that comprises a sequence encoding the present polypeptide(s) (rather than the polypeptide(s) per se).
  • the invention also features methods of treating cancer that comprise administering to a patient a therapeutically effective amount of a gene construct comprising a sequence encoding a polypeptide in the PACAP/VIP family or a therapeutically active variant thereof.
  • the cancer treated can be one associated with a BRAC mutation and/or one in which PARP inhibition increases the sensitivity to cytotoxic therapy or ionizing radiation (e.g.
  • polypeptide encoded can be: (a) VIP; (b) a therapeutically active variant of VIP; (c) PACAP27; (d) a therapeutically active variant of PACAP27;
  • PACAP38 PACAP38;
  • f a therapeutically active variant of PACAP38; or
  • g another member of the PACAP/VIP family that inhibits PARP or a therapeutically active variant thereof.
  • kits comprising (a) a polypeptide in the PACAP/VIP family or a therapeutically active variant thereof;
  • kits for use in the treatment of a cancer, including treatments in which PARP inhibition increases the sensitivity of the cancerous cells to cytotoxic therapy or ionizing radiation.
  • the kit can also include a chemotherapeutic agent. Any of the polypeptides described herein can be included.
  • the kit can include VIP (SEQ ID NO:l) or a therapeutically active variant thereof (e.g. , a polypeptide comprising a sequence that is at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to VIP).
  • the kit can include PACAP27 or a therapeutically active variant thereof (e.g., a polypeptide comprising a sequence that is at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to PACAP27).
  • the polypeptide administered can be PACAP38 or a therapeutically active variant of
  • PACAP38 ⁇ e.g., a polypeptide comprising a sequence that is at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to PACAP38).
  • the kit can include a nucleic acid construct that, encodes a polypeptide as described above.
  • the chemotherapeutic agent included in the kit can be any of those conventionally used or later determined to treat a BRAC1- orBRAC2-related cancer and/or a cancer in which the cancerous cells become more susceptible to cytotoxic treatment when that treatment is administered in conjunction with PARP inhibition.
  • the chemotherapeutic can be a ceramide analog.
  • the chemotherapeutic can also be paclitaxel, docetaxel, Cytoxan, or gemcitabine.
  • the instructions can be conveyed via any medium, including printed materials or audio or video presentations.
  • the invention further encompasses methods of screening polypeptides to identify additional polypeptides (e.g. , polypeptides in the PACAP/VIP family or variants thereof) that inhibit PARP and/or that are effective chemotherapeutic agents. These methods can be carried out by (a) providing a polypeptide; (b) analyzing the ability of the polypeptide to inhibit PARP; and (c) analyzing the ability of the polypeptide to suppress the growth of a tumor or cancerous cells, either alone or in the presence of a chemotherapeutic agent.
  • the invention further encompases the use of the polypeptides, nucleic acids, and compositions including them (as described herein) in the preparation of a medicament and, further, use of the polypeptides, nucleic acids, and compositions including them in the preparation of a medicament for the treatment of cancer.
  • the cancer can be one as described herein (e.g., a cancer associated with a mutation of a BRAC gene),
  • FIG. 1 is an alignment of two polypeptides, VIP (SEQ ID NO:l) and PACAP (1-38) (SEQ ID NO:2). Residues that are identical to one another in each sequence are underlined.
  • PACAP38 is a 38- amino acid peptide that was first isolated from ovine hypothalamic extracts on the basis of its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al. , Biochem.
  • PACAP is believed to play a fundamental role in biological systems because its sequence is highly conserved from protochordates to mammals. Depending upon the cellular
  • PACAP functions as a hypophysiotropic hormone, a neurotransmitter and neuromodulator. In addition, it plays a role in paracrine and autocrine regulation of certain types of cells.
  • the cDNA and amino acid sequences encoding representative PACAP nucleotide and polypeptide sequences include Genbank number NMJ301099733.1, public GI: 153266791 ; Genbank number NP_001093203.1 ,, public GI: 153266792; Genbank number NM_001117.3, public GI: 153266794; and Genbank number NPJXH 108.2, public
  • PACAP sequence can be used, the invention is not so limited. Any PACAP exhibiting PARP inhibition and/or therapeutic efficacy can be used in the present methods.
  • the PACAP can be a mammalian PACAP.
  • a mature version of PACAP is known in the art as PACAP48.
  • VIP is a 28-amino acid polypeptide. It is synthesized in normal lung cells and functions naturally as a bronchodilator (Morice et al., Lancet 1:457, 1984). This action is mediated by cell surface receptors localized to alveoli and smooth muscle cells (Robberecht et al., Regul. Peptides, 4:241, 1982). VIP receptors have also been identified on human B lymphocyte cell lines and cells in the peripheral circulation (O'Dorisio et al., J. Immunol., 15: 142, 1989).
  • a polypeptide of the PACAP/VIP family having PARP inhibitory activity is administered to a patient in a therapeutically effective amount.
  • Determining an appropriate dosage and dosing regimen is generally considered to be within the ability of one of ordinary skill in the art. Regardless of the precise amount delivered, we refer to a therapeutically effective amount as the amount expected to elicit a favorable clinical outcome. Where a patient has a solid tumor, the most favorable outcome is regression of the tumor to a point where it is undetectable. Similarly, for dispersed cancers, the most favorable outcome is a reduction in cancerous cells to below detectable levels. The patient can, however, be considered treated even where the outcome is not as positive. For example, a patient is also treated where the progression of the cancer stops or slows.
  • the polypeptide can be a therapeutically active variant of a naturally occurring polypeptide within the PACAP/VIP family.
  • the variant may be a mutant of the corresponding naturally occurring protein, and the extent of the mutation can be described in terms of the percent of the amino acid residues that are identical between the mutant and the naturally occurring counterpart. A lack of identity may be due to the substitution and/or deletion of one or more amino acid residues.
  • the therapeutically active variant may be described as a fragment of a PACAP/VIP polypeptide.
  • amino acid residues in a biologically active variant may be a non- naturally occurring amino acid residue.
  • Naturally occurring amino acid residues include those naturally encoded by the genetic code as well as non-standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration).
  • the present peptides can also include amino acid residues that are modified versions of standard residues (e.g.
  • Non-naturally occurring amino acid residues are those that have not been found in nature, but that conform to the basic formula of an amino acid and can be incorporated into a peptide. These include D-alloisoleucine(2R,3S)-2-amino-3-methylpentanoic acid and L- cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. For other examples, one can consult textbooks or the worldwide web (a site is currently maintained by the California Institute of Technology and displays structures of non-natural amino acids that have been successfully incorporated into functional proteins).
  • Non-natural amino acid residues and amino acid derivatives listed in U.S. Application No. 20040204561 can also be used.
  • one or more of the amino acid residues in a biologically active variant can be a naturally occurring residue that differs from the naturally occurring residue found in the corresponding position in a wildtype sequence of a
  • biologically active variants can include one or more amino acid substitutions.
  • substitutions can include a substitution, addition, or deletion of amino acid residues as a mutation of the wildtype sequence.
  • the substitution can replace a naturally occurring amino acid residue with a non-naturally occurring residue or just a different naturally occurring residue. Further the substitution can constitute a conservative or non-conservative substitution.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
  • the therapeutically active variant may be at least or about 60% identical to a naturally occurring counterpart polypeptide.
  • the variant may be at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to the naturally occurring counterpart polypeptide.
  • the variant will be one that retains sufficient biological activity to be therapeutically beneficial.
  • the PARP proteins are a multi-gene family of enzymes that catalyze the transfer of ADP-ribose moieties from nicotinamide adenine dinucleotide (NAD + ) to a variety of acceptor proteins resulting in the attachment of linear or branched polymers of ADP-ribose (PAR).
  • exemplary PARP enzymes include PARP-1, PARP-2, PARP-4, PARP-5a or TNKS, and PARP-5b or TNKS2.
  • Cellular targets of PARP include PARP itself, as well as other nuclear proteins, for example, chromatin structural proteins, e.g. , histone proteins and topoisomerases; DNA synthesis and repair enzymes, e.g., DNA ligases, DNA polymerases, terminal transferase; transcription enzymes, e.g., RNA polymerases and HMG proteins; cell cycle proteins, e.g., Fos, p53 and various structural proteins such as nuclear lamins and nucleolin.
  • the ADP-ribose can be added to glutamate, aspartate, and lysine residues.
  • PARP inhibition assays can be carried out in vitro and are well known in the art. Standard detection assays involve, for example, quantification of labeled NAD + , e.g., radiolabled or biotinylated NAD + , or quantification of ADP-ribose, e.g., using an anti-ADP- ribose antibody. Any one of the cellular targets described above may serve as a substrate for such assays, e.g., histones, DNA replication and repair enzymes, transcriptional proteins. In addition, kits and reagents for performing such assays are readily available from commercial sources, for example, Trevigen, Inc., and Perkin-Elmer. By way of illustration, any given polypeptide can be tested in the HT Universal Chemiluminescent PARP Assay kit (Catalogue # 4676-096-K) commercially available from Trevigen®. The latter measures the
  • biotinylated poly(ADP -ribose) incorporation of biotinylated poly(ADP -ribose) on to histone proteins that are coated on to microwell strips.
  • Predetermined amounts of recombinant PARP enzyme and biotinylated NAD + are incubated in the microwells for one hour in the absence and presence of PARP inhibitors, e.g. , polypeptides in the PACAP/VIP family as well as any variant thereof.
  • the amount of PARP used in the assay will be that which is sufficient to produce a signal within the linear range of detection; thus the absolute amount will vary according to a number of factors, including, for example, the sensitivity of the detection system and the specific activity of the particular preparation of PARP.
  • the wells are then washed to remove PARP and unbound biotinylated NAD + ; biotinylated NAD + that has been incorporated on to the histone substrate is detected with streptavidin-horse radish peroxidase and chemiluminescent substrates.
  • the chemiluminescent signal obtained with a particular PARP inhibitor e.g., a polypeptide in the PACAP/VIP family as well as any variant thereof, can be expressed as a percentage of the signal obtained in the absence of the inhibitor.
  • polypeptides of the invention can also include structural modifications that may, for example, enhance the therapeutic efficacy of the polypeptides or assist clinicians in monitoring a course of treatment with the polypeptides. Structural modifications can be made during or after polypeptide translation or chemical synthesis.
  • the polypeptides can be amidated.
  • the polypeptides can include a detectable marker. Both the form and position of the detectable marker can vary, as long as the polypeptides retain sufficient biological activity to remain useful.
  • the marker can be, for example, a photoaffmity ligand, a radioisotope, or a fluorescent or chemiluminescent compound.
  • the bonds between the amino acid residues can be conventional peptide bonds or another covending bond (such as an ester or ether bond), and the polypeptides can be modified, for example, by amidation, phosphorylation or glycosylation.
  • a modification can affect the polypeptide backbone and/or one or more side chains.
  • Chemical modifications can be naturally occurring modifications made in vivo following translation of an mRNA encoding the polypeptide (e.g., glycosylation in a bacterial host) or synthetic modifications made in vitro.
  • a polypeptide in the PACAP/VIP family as well as any variant thereof can include one or more structural modifications resulting from any combination of naturally occurring (i.e., made naturally in vivo) and synthetic modifications (i.e., naturally occurring or non- naturally occurring modifications made in vitro).
  • modifications include, but are not limited to, amidation (e.g. , replacement of the free carboxyl group at the C-terminus by an amino group); biotinylation (e.g., acylation of lysine or other reactive amino acid residues with a biotin molecule); glycosylation (e.g., addition of a glycosyl group to either
  • acetylation e.g., the addition of an acetyl group, typically at the N-terminus of a polypeptide
  • alleviation e.g., the addition of an alkyl group
  • isoprenylation e.g., the addition of an isoprenoid group
  • lipoylation e.g. attachment of a lipoate moiety
  • phosphorylation e.g., addition of a phosphate group to serine, tyrosine, threonine or histidine.
  • polypeptides can be incorporated into fusion proteins.
  • PACAP38 and VIP can be fused, either directly or via a linker sequence.
  • the polypeptides can also be fused to heterologous polypeptides known in the art to increase the circulating half-life of a polypeptide to which they are attached.
  • any polypeptide in the PACAP/VIP family (or any combination thereof) can be fused to albumin or a portion of an
  • immunoglobulin e.g. , the Fc region of an immunoglobulin such as an IgG.
  • polypeptides of the invention can be chemically synthesized, obtained from natural sources (insofar as they constitute fragments of a naturally occurring polypeptide in the PACAP/VIP family as well as any variant thereof), or purified from cells in which they are recombinantly produced.
  • molecular techniques can be used to express polypeptides having a sequence that is identical to a portion of a polypeptide in the
  • PACAP/VIP family as well as any variant thereof; the methods required for polypeptide synthesis, expression and purification are well known in the art.
  • polypeptides can be chemically synthesized using standard f moc chemistry and purified using high pressure liquid chromatography (HPLC). Fragments of a polypeptide in the PACAP/VIP family as well as any variant thereof can be purified by any method known in the art, including without limitation, fractionation, centrifugation, and chromatography (e.g., gel filtration, ion exchange chromatography, reverse-phase HPLC and immunoaffinity purification).
  • HPLC high pressure liquid chromatography
  • polypeptides may be, but are not necessarily, substantially pure.
  • a polypeptide of the invention whether it contains a sequence that is identical to a portion of a polypeptide in the PACAP/VIP family as well as any variant thereof, should be considered substantially pure when it has been separated from a substantial amount of the material with which it was previously associated (e.g. , cellular components where the polypeptide is recombinantly produced or reagents where the polypeptide is chemically synthesized).
  • a polypeptide of the invention is substantially pure when it is present in a composition in which it constitutes at least or about 60% of the composition by weight (e.g. , at least or about 65%, 70%, 80%, 90%, 95%, or 99%).
  • polypeptides may have the sequence of the human homolog, homologs from species other than humans can also be used ⁇ e.g. , a PACAP/VIP polypeptide of murine, porcine, bovine, canine, feline, or ovine origin can also be used).
  • nucleic acids that encode a given polypeptide are well known in the art.
  • the nucleic acids that encode a polypeptide in the PACAP/VIP family as well as any variant thereof, include those that are codon optimized.
  • the nucleic acids can readily be incorporated into a vector (e.g. , a plasmid or viral vector), and such vectors are encompassed by the invention.
  • the nucleic acids can be operably linked to a regulatory region suitable for use in either a prokaryotic or a eukaryotic system, many of which are laiown in the art and can be used to produce the polypeptides described herein.
  • the regulatory region can be, for example, a promoter or enhancer.
  • Useful promoters include cell type-specific promoters, tissue-specific promoters,
  • host cells including vectors that express a polypeptide of the invention are also encompassed by the invention, and these cells can be prokaryotic (e.g. , bacterial) or eukaryotic (e.g., mammalian).
  • nucleic acid sequence encoding the polypeptide can be incorporated into (e.g., ligated into) an expression vector and used to transform a prokaryotic cell (e.g. , a bacterial cell) or transfect a eukaryotic host cell (e.g. , an insect, yeast, or mammalian host cell).
  • a prokaryotic cell e.g. , a bacterial cell
  • a eukaryotic host cell e.g. , an insect, yeast, or mammalian host cell.
  • nucleic acid constructs can include one or more regulatory sequences operably linked to a nucleic acid sequence encoding a polypeptide of the invention.
  • Regulatory sequences do not typically encode a protein/polypeptide, but instead affect the expression of a nucleic acid sequence.
  • Such transformed or transfected cells can then be used, for example, for large or small scale production of the selected fragment of a polypeptide in the PACAP/VIP family as well as any variant thereof by methods known in the art. In essence, such methods involve culturing the cells under conditions suitable for production of the polypeptide and isolating the polypeptide from the cells or from the culture medium.
  • a construct can include a tag sequence designed to facilitate subsequent
  • the tag can facilitate purification or localization.
  • Tag sequences such as green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, ⁇ galactosidase, or FlagTM tag (Kodak) sequences are typically expressed as a fusion with the polypeptide encoded by the nucleic acid sequence.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • c-myc hemagglutinin
  • ⁇ galactosidase or FlagTM tag (Kodak) sequences are typically expressed as a fusion with the polypeptide encoded by the nucleic acid sequence.
  • Such tags can be inserted in a nucleic acid sequence such that they are expressed anywhere along an encoded polypeptide including, for example, at either the carboxyl or amino termini.
  • the type and combination of regulatory and tag sequences can vary with each particular host, cloning or expression system, and desired outcome
  • cloning and expression vectors containing combinations of regulatory and tag sequences are commercially available. Suitable cloning vectors include, without limitation, pUC18, pUC19, and pBR322 and derivatives thereof (New England Biolabs, Beverly, MA), and pGEN (Promega, Madison, WI).
  • representative prokaryotic expression vectors include, without limitation, pBAD (Invitrogen, Carlsbad, CA), the pTYB family of vectors (New England Biolabs), and pGEMEX vectors (Promega); representative mammalian expression vectors include, without limitation, pTet-On/pTet-Off (Clontech, Palo Alto, CA), pIND, pVAXl, pCR3.1 , pcDNA3.1, pcDNA4, or pUni (Invitrogen), and pCI or pSI
  • representative insect expression vectors include, without limitation, pBacPAO or pBacPAK9 (Clontech), and p2Bac (Invitrogen); and representative yeast expression vectors include, without limitation, MATCHMAKER (Clontech) and pPICZ A, B, and C
  • Escherichia coli can be used to express a polypeptide in the PACAP/VIP family as well as any variant thereof.
  • the E.coli strain. DH10B (Invitrogen) can be transformed with the gram negative broad host range vector, pCM66 containing a nucleic acid sequence encoding a polypeptide in the PACAP/VIP family as well as any variant thereof.
  • BL-21 cells can be transformed with a pGEX vector containing a nucleic acid sequence encoding a polypeptide of the invention. The transformed bacteria can be grown exponentially and then stimulated with
  • IPTG isopropylthiogalactopyranoside
  • the polypeptides produced from a pGEX expression vector can be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors can be designed to include thrombin or factor Xa protease cleavage sites so that the expressed polypeptide can be released from the GST moiety.
  • nucleic acid and “polynucleotide” interchangeably to refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs, any of which may encode a polypeptide of the invention and all of which are encompassed by the invention.
  • Polynucleotides can have essentially any three-dimensional structure.
  • a nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand).
  • Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA) and portions thereof, transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
  • nucleic acids can encode a polypeptide in the PACAP/VIP family as well as any variant thereof
  • an "isolated" nucleic acid can be, for example, a naturally-occurring DNA molecule or a fragment thereof, provided that at least one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences (e.g. , a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restriction endonuclease treatment).
  • An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote.
  • an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid.
  • Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein (i.e. a polypeptide in the PACAP/VIP family as well as any variant thereof). PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995.
  • sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid (as one may wish to do, for example, when making a polypeptide in the PACAP/VIP family as well as any variant thereof).
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g. , using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides e.g., >50-100 nucleotides
  • each pair containing a short segment of complementarity e.g., about 15 nucleotides
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
  • Isolated nucleic acids of the invention also can be obtained by mutagenesis of, e.g., a naturally occurring portion of a polypeptide in the PACAP/VIP family-encoding DNA (in accordance with, for example, the formula above).
  • Two nucleic acids or the polypeptides they encode may be described as having a certain degree of identity to one another.
  • a polypeptide in the PACAP/VIP family as well as any variant thereof may be described as exhibiting a certain degree of identity. Alignments may be assembled by locating short sequences in the PACAP/VIP family as well as any variant thereof in the Protein Information Research (PIR) site.
  • PIR Protein Information Research
  • percent sequence identity refers to the degree of identity between any given query sequence and a subject sequence.
  • a naturally occurring polypeptide in the PACAP/VIP family can be the query sequence and a variant thereof can be the subject sequence.
  • PACAP/VIP family can be the query sequence and a biologically active variant thereof can be the subject sequence.
  • a query nucleic acid or amino acid sequence can be aligned to one or more subject nucleic acid or amino acid sequences, respectively, using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). See Chenna et al., Nucleic Acids Res. 31:3497-3500, 2003.
  • ClustalW calculates the best match between a query and one or more subject sequences and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5.
  • gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
  • ClustalW divides the number of identities in the best alignment by the number of residues compared (gap positions are excluded), and multiplies the result by 100.
  • the output is the percent identity of the subject sequence with respect to the query sequence. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • nucleic acids and polypeptides described herein may be referred to as
  • exogenous nucleic acid indicates that the nucleic acid or polypeptide is part of, or encoded by, a recombinant nucleic acid construct, or is not in its natural environment.
  • an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct.
  • An exogenous nucleic acid can also be a sequence that is native to an organism and that has been
  • exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.
  • a recombinant nucleic acid construct comprises a nucleic acid encoding a polypeptide in the PACAP/VIP family as well as any variant thereof as described herein, operably linked to a regulatory region suitable for expressing the polypeptide in the PACAP/VIP family as well as any variant thereof in the cell.
  • a nucleic acid can comprise a coding sequence that encodes any of the fragments of a polypeptide in the PACAP/VIP family as well as any variant thereof as set forth in, for example, SEQ ID NOs: 1 and 2.
  • a recombinant nucleic acid construct can include a nucleic acid comprising a coding sequence, a gene, or a fragment of a coding sequence or gene in an antisense orientation so that the antisense strand of RNA is transcribed.
  • nucleic acids can encode a polypeptide having a particular amino acid sequence.
  • the degeneracy of the genetic code is well known in the art. For many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
  • codons in the coding sequence for a given a polypeptide in the PACAP/VIP family as well as any variant thereof can be modified such that optimal expression in a particular organism is obtained, using appropriate codon bias tables for that organism.
  • Vectors containing nucleic acids such as those described herein also are provided.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs.
  • the term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors.
  • An “expression vector” is a vector that includes a regulatory region.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).
  • the vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers.
  • a marker gene can confer a selectable phenotype on a host cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin).
  • an expression vector can include a tag sequence designed to facilitate, manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c- myc, hemagglutinin, or FlagTM tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c- myc hemagglutinin
  • hemagglutinin or FlagTM tag (Kodak, New Haven, CT) sequences
  • FlagTM tag Kodak, New Haven, CT sequences
  • the vector can also include a regulatory region.
  • regulatory region refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • operably linked refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence.
  • the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site.
  • a promoter typically comprises at least a core (basal) promoter.
  • a promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • control element such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • the choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.
  • polypeptides in the PACAP/VIP family as well as any variant thereof disclosed herein are generally and variously useful for treatment of cancer.
  • a patient is effectively treated whenever a clinically beneficial result ensues. This may mean, for example, a complete resolution of the symptoms of a disease, a decrease in the severity of the symptoms of the disease, or a slowing of the disease's progression.
  • These methods can further include the steps of a) identifying a subject (e.g., a human patient) who has cancer; and b) providing to the subject a composition comprising a polypeptide in the PACAP/VIP family as well as any variant thereof,
  • the present methods may also include a monitoring step to help optimize dosing and scheduling as well as predict outcome.
  • a sample of tissue ⁇ e.g., a biopsy
  • monitoring can be used to detect the onset of drug resistance and to rapidly distinguish responsive patients from nonresponsive patients. Where there are signs of resistance or nonresponsivness, a physician can choose an alternative or adjunctive agent before the tumor develops additional escape mechanisms.
  • the polypeptides in the PACAP/VIP family as well as any variant thereof described herein can be administered directly to a mammal, which we may also refer to as a "subject" or "patient.”
  • the polypeptides in the PACAP/VIP family as well as any variant thereof can be suspended in a pharmaceutically acceptable carrier (e.g. , physiological saline or a buffered saline solution) to facilitate their delivery (e.g., by intravenous administration).
  • a pharmaceutically acceptable carrier e.g. , physiological saline or a buffered saline solution
  • the polypeptides in the PACAP/VIP family of the invention can be formulated in accordance with their use.
  • the polypeptides in the PACAP/VIP family as well as any variant thereof can be formulated within compositions for application to cells in tissue culture or for administration to a patient.
  • any of the present the polypeptides in the PACAP/VIP family as well as any variant thereof can be administered in the form of pharmaceutical compositions.
  • These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral.
  • Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon, catheter or ophthalmic inserts surgically placed in the conjunctival sac.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous,
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions which contain, as the active ingredient, one or more of the polypeptides in the PACAP/VIP family as well as any variant thereof described herein in combination with one or more pharmaceutically acceptable carriers.
  • the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container.
  • the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g. , normal saline), which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10 % by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the type of diluent can vary depending upon the intended route of administration.
  • the resulting compositions can include additional agents, such as preservatives.
  • polypeptides in the PACAP/VIP family as well as any variant thereof may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device.
  • a device e.g., a catheter
  • the polypeptides in the PACAP/VIP family of the invention can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline).
  • a pharmaceutically acceptable excipient or carrier e.g., physiological saline
  • the excipient or carrier is selected on the basis of the mode and route of administration.
  • Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W.
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose,
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • lubricating agents such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying and suspending agents such as methyl- and propylhydroxy-benzoates
  • sweetening agents and flavoring agents.
  • the pharmaceutical compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • the compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 5 mg; from about 1 mg from to about 50 mg, from about 1 mg to about 30 mg s> from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, from about 5 mg to about 10 mg; from about 10 mg to about 100
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein and/or known in the art.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or
  • compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.
  • the compositions administered to a patient can be in the form of one or more of the pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques or may be sterile filtered.
  • Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the compound preparations typically will be between about 3 and 11 , for example, between about 5 to 9, between 6 and 7, between 7 and 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers could result in the formation of pharmaceutical salts.
  • the proportion or concentration of the polypeptides in the PACAP/VIP family of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g. , hydrophobicity), and the route of administration.
  • the polypeptides in the PACAP/VIP family of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral adminstration.
  • a nucleic acid encoding the PACAP/VIP polypeptides may be administered.
  • sequences encoding the polypeptides described herein can readily be incorporated into a vector, such as a plasmid or viral vector, and administered to a patient by methods known in the art.
  • a polynucleotide containing a nucleic acid sequence encoding an immune conjugate of interest can be delivered to an appropriate cell of the animal. This can be achieved, for example, by the use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages.
  • PLGA poly- lacto-co-glycolide microparticles approximately 1-10 ⁇ in diameter
  • the polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide. Once released, the DNA is expressed within the cell.
  • a second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro -particle through biodegradation. These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5 ⁇ and preferably larger than 20 ⁇ ).
  • liposomes prepared by standard methods.
  • the vectors can be incorporated alone into these delivery vehicles or co- incorporated with tissue-specific antibodies.
  • Poly-L-lysine binds to a ligand that can bind to a receptor on target cells.
  • Delivery of "naked DNA" i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site, is another means to achieve in vivo expression.
  • Polynucleotides can be administered in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to a human or other mammalian subject, e.g., physiological saline.
  • a therapeutically effective amount is an amount of the polynucleotide which is capable of producing a medically desirable result (e.g., an anti-tumor response) in a treated mammal.
  • the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be
  • a preferred dosage for administration of polynucleotide is from approximately 10 6 to 10 12 copies of the polynucleotide molecule. This dose can be repeatedly administered, as needed. Routes of administration can be any of those listed below.
  • the polypeptides, nucleic acids, vectors, and host cells of the invention can be formulated as pharmaceutical compositions.
  • these compositions can be formulated as non-toxic preparations for intravenous administration.
  • the pharmaceutical compositions can include polypeptides of more than one sequence.
  • the pharmaceutical compositions can include a first polypeptide having a sequence that is identical to that of a polypeptide in the PACAP/VIP family ,and a second polypeptide that is a biologically active variant thereof.
  • the pharmaceutical formulations can include mixtures of polypeptides.
  • polypeptides of the invention encompass those in which more than one of the polypeptides in the PACAP/VIP family of the invention are included within a longer polypeptide, and such multimers can also be formulated for administration to a patient.
  • Carriers and stabilizing agents may be added to facilitate drug delivery and to insure shelf-life. For example, encapsulation of the
  • polypeptides in a suitable delivery vehicle may increase the efficiency of delivery.
  • the pharmaceutically effective amount of the present polypeptides will vary according to the patient and/or with the severity of the disease or condition. These variables can be ascertained by one skilled in the art by routine experimentation. An appropriate dosage range, as a starting point, can be derived from animal models or with reference to PCT/AU2005/000835.
  • the polypeptides can be delivered by any number of conventional routes of administration. Given the nature of the condition to be treated, we expect local application to the site of a tumor (or the site from which a tumor has been surgically removed) and intravenous administration (e.g. , intravenous infusion) may be most efficacious.
  • the present polypeptides can be formulated for administration by oral, intravenous, intramuscular or subcuticular routes. Particularly preferred are formulations suitable for intravenous infusion over a period of hours ⁇ e.g. , 6-12 hours, inclusive).
  • compositions described herein can be administered to any part of the host's body for subsequent delivery to a target cell.
  • a composition can be delivered to, without limitation, the brain, the cerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, or the peritoneal cavity of a mammal.
  • routes of delivery a composition can be administered by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal administration, or by gradual perfusion over time.
  • an aerosol preparation of a composition can be given to a host by inhalation.
  • the dosage required will depend on the route of administration, the nature of the formulation, the nature of the patient's illness, the patient's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending clinicians. Suitable dosages are in the range of 0.01-1 ,000 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of cellular targets and the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art. Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).
  • Encapsulation of the polypeptides in the PA CAP/VIP family in a suitable delivery vehicle may increase the efficiency of delivery.
  • the duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years).
  • a polypeptide in the PACAP/VIP family as well as any variant thereof can be administered once a week (for, for example, 4 weeks to many months or years); once a month (for, for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer.
  • the frequency of treatment can be variable.
  • the present polypeptides in the PACAP/VIP family as well as any variant thereof can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.
  • an effective amount of any composition provided herein can be administered to an individual in need of treatment.
  • the term "effective" as used herein refers to any amount that induces a desired response while not inducing significant toxicity in the patient. Such an amount can be determined by assessing a patient's response after administration of a known amount of a particular composition.
  • the level of toxicity if any, can be determined by assessing a patient's clinical symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular composition administered to a patient can be adjusted according to a desired outcome as well as the patient's response and level of toxicity. Significant toxicity can vary for each particular patient and depends on multiple factors including, without limitation, the patient's disease state, age, and tolerance to side effects.
  • Any method known to those in the art can be used to determine if a particular response is induced.
  • Clinical methods that can assess the degree of a particular disease state can be used to determine if a response is induced.
  • the particular methods used to evaluate a response will depend upon the nature of the patient's disorder, the patient's age, and sex, other drugs being administered, and the judgment of the attending clinician.
  • polypeptides in the PACAP/VIP family as well as any variant thereof may also be administered with another therapeutic agent, such as a cytotoxic agent, or cancer
  • chemotherapeutic Concurrent administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.
  • the pharmaceutical compositions can also include other therapeutic antibodies, e.g., antibodies that recognize additional cellular targets.
  • exemplary immunoglobulins are listed below. Each immunoglobulin is identified by its proper name and its trade name. Numbers in parenthesis beginning with "DB” refer to the identifiers for each antibody on The DrugBank database available at the University of Alberta. The DrugBank database is described in Wishart D S, Knox C, Guo A C, et al. (2008). "DrugBank: a knowledgebase for drugs, drug actions and drug targets”. Nucleic Acids Res. 36 (Database issue): D901-6 and can be accessed at www.drugbank.ca.
  • Useful immunoglobulins include: Abciximab (ReoProTM) (DB00054), the Fab fragment of the chimeric human-murine monoclonal antibody 7E3, the synthesis of which is described in EP0418316 (Al) and W0891 1538 (Al), which are herein incorporated by reference; Adalimumab (HumiraTM) (DB00051), a fully human monoclonal antibody that binds to Tumor Necrosis Factor alpha (TNF-a) and blocks TNF-a binding to its cognate receptor; alemtuzumab (CampathTM) (DB00087), a humanized monoclonal antibody that targets CD52, a protein present on the surface of mature lymphocytes, used in the treatment of chronic lymphocytic leukemia (CLL), cutaneous T cell lymphoma (CTCL) and T-cell lymphoma; basiliximab (SimulectTM) (DB00074), a chimeric mouse-human monoclon
  • muromonab-CD3 Orthoclone OKT3TM
  • natalizumab TysabriTM
  • DB00108 a humanized monoclonal antibody against the cellular adhesion molecule a- 4 integrin, the sequence of which is described in Leger O J, Yednock T A, Tanner L, Homer H C, Hines D K, Keen S, Saldanha J, Jones S T 5 Fritz L C, Bendig M M. Humanization of a mouse antibody against human a-4 integrin: a potential therapeutic for the treatment of multiple sclerosis.
  • Hum Antibodies Humanization of a mouse antibody against human a-4 integrin: a potential therapeutic for the treatment of multiple sclerosis.
  • omalizuraab XolairTM
  • DB00043 a humanized IgGlk monoclonal antibody that selectively binds to human immunoglobulin E (IgE); palivizumab (SynagisTM) (DB00110), a humanized monoclonal antibody (IgG) directed against an epitope in the A antigenic site of the F protein of the Respiratory
  • Syncytial Virus (RSV), the amino acid sequence of which is described in Johnson S, Oliver C, Prince G A, Hemming V G, Reifen D S ⁇ Wang S C, Dormitzer M, O'Grady J, Koenig S, Tamura J K, Woods R, Bansal G, Couchenour D, Tsao E, Hall W C > Young J F.
  • a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J Infect Dis. 1997 November; 176(5): 1215- 24; panitumumab (Vectibix 1M ), a fully human monoclonal antibody specific to the epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1 and HER1 in humans); ranibizumab (LucentisTM), an affinity matured anti-VEGF-A monoclonal antibody fragment derived from the same parent murine antibody as bevacizumab (Avastin); rituximab
  • the antibodies can include bioequivalents of the approved or marketed antibodies (biosimilars).
  • a biosimilar can be for example, a presently known antibody having the same primary amino acid sequence as a marketed antibody, but may be made in different cell types or by different production, purification or formulation methods. Generally any deposited materials can be used.
  • the pharmaceutical compositions may also include or be administered along with a cytotoxic agent, e.g. , a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • a cytotoxic agent e.g. , a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • cytotoxic agents include radioactive isotopes (e.g., I31 I, l25 I, 90 Y and 186 Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin or synthetic toxins, or fragments thereof.
  • a non- cytotoxic agent refers to a substance that does not inhibit or prevent the function of cells and/or does not cause destruction of cells.
  • a non-cytotoxic agent may include an agent that can be activated to be cytotoxic.
  • a non-cytotoxic agent may include a bead, liposome, matrix or particle (see, e.g. , U.S. Patent Publications 2003/0028071 and 2003/0032995 which are incorporated by reference herein). Such agents may be conjugated, coupled, linked or associated with an antibody disclosed herein.
  • Useful medicaments include anti-angiogenic agents, i.e., agents that block the ability of tumors to stimulate new blood vessel growth necessary for their survival. Any anti-angiogenic agent known to those in the art can be used, including agents such as Bevacizurnab (Avastin®, Genentech, Inc.) that block the function of vascular endothelial growth factor (VEGF).
  • anti-angiogenic agents i.e., agents that block the ability of tumors to stimulate new blood vessel growth necessary for their survival.
  • Any anti-angiogenic agent known to those in the art can be used, including agents such as Bevacizurnab (Avastin®, Genentech, Inc.) that block the function of vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • DNA damage can typically be produced by radiation therapy and/or chemotherapy.
  • radiation therapy include, without limitation, external radiation therapy and internal radiation therapy (also called
  • Energy sources for external radiation therapy include x-rays, gamma rays and particle beams; energy sources used in internal radiation include radioactive iodine (iodine or iodine ), and from strontium , or radioisotopes of phosphorous, palladium, cesium, iridium, phosphate, or cobalt. Methods of administering radiation therapy are well know to those of skill in the art.
  • DNA-damaging chemotherapeutic agents include, without limitation, Busulfan (Myleran), Carboplatin (Paraplatin), Carmustine (BCNU), Chlorambucil
  • alkylating agents such as carboplatin and cisplatin
  • nitrogen mustard alkylating agents such as carmustine (BCNU)
  • antimetabolites such as methotrexate; folic acid
  • purine analog antimetabolites such as mercaptopurine
  • pyrimidine analog antimetabolites such as fiuorouracil (5-FU) and gemcitabine (Gemzar®)
  • hormonal antineoplastics such as goserelin, leuprolide, and tamoxifen
  • natural antineoplastics such as aldesleukin, interleukin- 2, docetaxel, etoposide (VP- 16), interferon alfa, paclitaxel (Taxol®), and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin,
  • doxorubicin daunomycin and mitomycins including mitomycin C; and vinca alkaloid natural antineoplastics, such as vinblastine, vincristine, vindesine; hydroxyurea; aceglatone, adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, antitumor platelet factors, cyclophosphamide (Cytoxin®), Schizophyllan, cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa, tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11 (irinotecan), mitozantrone
  • Patent No. 4,675,187 neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan, honvan, peplomycin, bestatin (Ubenimex®), interferon- ⁇ , mepitiostane, mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur/uracil, estramustine (estrogen/mechlorethamine).
  • Additional agents which may be used as therapy for cancer patients include EPO, G- CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 through 18, including mutants and analogues; interferons or cytokines, such as interferons a, ⁇ , and ⁇ hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor-p (TGF- ⁇ ), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factor-a & ⁇ (TNF-a & ⁇ ); invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7);
  • Useful therapeutic agents include, produgs, e.g., precursors or derivative forms of a pharmaceutically active substance that is less cytotoxic or non-cytotoxic to tumor cells compared to the parent drug and is capable of being converted, either enzymatically or non- enzymatically, into an active or the more active parent form.
  • produgs e.g., precursors or derivative forms of a pharmaceutically active substance that is less cytotoxic or non-cytotoxic to tumor cells compared to the parent drug and is capable of being converted, either enzymatically or non- enzymatically, into an active or the more active parent form.
  • Prodrugs include, but are not limited to, phosphate-containing prodrugs,
  • cytotoxic drugs that can be derivatized into a prodrug form for use herein include, but are not limited to, those chemotherapeutic agents described above.
  • kits can include measured amounts of a pharmaceutically acceptable composition including a polypeptide in the PACAP/VIP family as well as any variant thereof.
  • the instructions for use can be conveyed by any suitable media. For example, they can be printed on a paper insert in one or more languages or supplied audibly or visually (e.g., on a compact disc).
  • the packaging materials can include vials, packets, or intravenous bags, and the kit can also include instruments useful in administration, such as needles, syringes, tubing, catheters, bandages, and tape.
  • the components of the kit are sterile and suitable for immediate use.
  • kits that included concentrated formulations and/or materials that may require sterilization prior to use.
  • the kits may include one or more therapeutic agents, for example, a chemotherapeutic agent such as paclitaxel, docetaxel, Cytoxan, or gemcitabine.

Abstract

La présente invention concerne des compositions comprenant des polypeptides de la famille des PACAP/VIP (par exemple, des polypeptides pratiquement purs de la famille des PACAP/VIP) ou des variants thérapeutiquement actifs de ceux-ci. Les compositions peuvent comprendre un ou plusieurs types de peptides et peuvent comprendre d'autres substances, par exemple, des transporteurs pharmaceutiquement acceptables et des agents chimiothérapiques. L'invention concerne également des procédés d'utilisation des compositions pour traiter le cancer, par exemple, le cancer du sein, de l'ovaire et de la prostate.
PCT/US2010/058093 2009-11-25 2010-11-24 Pacap et protéines apparentées destinés à traiter le cancer WO2011066453A2 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050043237A1 (en) * 2000-09-27 2005-02-24 Bayer Pharmaceuticals Corporation Pituitary adenylate cyclase activating peptide (PACAP) receptor 3 (R3) agonists and their pharmacological methods of use
US20060223748A1 (en) * 2003-07-14 2006-10-05 Dorian Bevec Biologically active substance of a vasoactive intestinal peptide for treating interstitial lung infections
WO2008022716A2 (fr) * 2006-08-22 2008-02-28 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé de criblage pour ligands gpcr
US20080311138A1 (en) * 2004-12-13 2008-12-18 Maria Teresa De Magistris Adjuvant Activity of Gastrointestinal Peptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050043237A1 (en) * 2000-09-27 2005-02-24 Bayer Pharmaceuticals Corporation Pituitary adenylate cyclase activating peptide (PACAP) receptor 3 (R3) agonists and their pharmacological methods of use
US20060223748A1 (en) * 2003-07-14 2006-10-05 Dorian Bevec Biologically active substance of a vasoactive intestinal peptide for treating interstitial lung infections
US20080311138A1 (en) * 2004-12-13 2008-12-18 Maria Teresa De Magistris Adjuvant Activity of Gastrointestinal Peptides
WO2008022716A2 (fr) * 2006-08-22 2008-02-28 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé de criblage pour ligands gpcr

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