WO2003092632A2 - Agents anticancereux de peptides cycliques et leurs procedes d'utilisation - Google Patents

Agents anticancereux de peptides cycliques et leurs procedes d'utilisation Download PDF

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Publication number
WO2003092632A2
WO2003092632A2 PCT/US2003/014373 US0314373W WO03092632A2 WO 2003092632 A2 WO2003092632 A2 WO 2003092632A2 US 0314373 W US0314373 W US 0314373W WO 03092632 A2 WO03092632 A2 WO 03092632A2
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Prior art keywords
peptide
amino acid
cancer
amino acids
cyclic
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PCT/US2003/014373
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English (en)
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WO2003092632A3 (fr
Inventor
M. Reza Ghadiri
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The Scripps Research Institute
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Priority to AU2003232077A priority Critical patent/AU2003232077A1/en
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Publication of WO2003092632A3 publication Critical patent/WO2003092632A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the field of the invention includes cancers, as well as the treatment and/or prophylaxis of cancer and/or tumors and compositions therefor.
  • Cancer is a major disease that continues to be a leading cause of death at any age. In the United States alone, about half a million Americans die of cancer each year, totaling over 1500 people a day. One in four deaths in the U.S. are from cancer and it is the second leading cause of death in the U.S., behind heart disease. The exact causes of all cancers are not known, but links between certain activities such as smoking or exposure to carcinogens and the incidence of certain types of cancers and tumors have been shown by a number of researchers.
  • chemotherapeutic and biologic agents have been shown to be effective against cancers and tumor cells, but not all types of cancers and tumors respond to these agents, and cancers can often reoccur. Unfortunately, many of these agents also destroy normal cells. Despite advances in the field of cancer treatment the development of cytotoxic agents that have specificity for cancer and tumor cells while not affecting normal cells would be extremely desirable.
  • the present invention provides new, fast-acting cyclic peptide anti- cancer agents for treating and/or preventing cancer in a human or other animal.
  • the present cyclic peptides are highly effective for many cancers. Cyclic peptides are fast acting, proteolytically stable and easy to synthesize. Other cyclic peptides of the invention do not have undesired toxicity against normal mammalian cells, for example, as measured by hemolysis of erythrocytes.
  • cancer includes solid mammalian tumors as well as hematological malignancies. "Solid mammalian tumors" include cancers
  • hematological malignancies includes childhood leukemia and lymphomas, Hodgkin's disease, lymphomas of lymphocytic and cutaneous origin, acute and chronic leukemia, plasma cell neoplasm and cancers associated with AIDS.
  • a cancer at any stage of progression can be treated, such as primary, metastatic, and recurrent cancers.
  • Information regarding numerous types of cancer can be found, e.g., from the American Cancer Society (www.cancer.org), or from, e.g., Wilson et al. (1991) Harrison's Principles of Internal Medicine, 12th Edition, McGraw-Hill, Inc. Both human and veterinary uses are contemplated.
  • normal mammalian cell and “normal animal cell” are defined as a cell that is growing under normal growth control mechanisms (e.g., genetic control) and displays normal cellular differentiation. Cancer cells differ from normal cells in their growth patterns and in the nature of their cell surfaces. For example cancer cells tend to grow continuously and chaotically, without regard for their neighbors, among other characteristics well known in the art.
  • the invention provides anti-cancer cyclic peptides and pharmaceutical compositions thereof wherein the cyclic peptides comprise a sequence of from four to about sixteen alternating D- and L- -amino acids, or from three to ten ⁇ - amino acids, wherein the cyclic peptide does not have undesired activity against normal mammalian cells.
  • Activity against cancer cells can be evaluated using assays and techniques known in the art.
  • Activity against normal mammalian cells can also be measured using assays and techniques known in the art, for example, by the ability of peptides to cause hemo lysis of mammalian red blood cells in vitro.
  • Such cyclic peptides and pharmaceutical compositions can be used for treating or preventing cancer in a mammal.
  • Client Reference 893.1 PCT Cyclic peptides may have a lethal dose (LD ⁇ 00 ) or a 50% inhibitory dose (ED 50 ) at which substantially no cancer cells grow in vitro that is less than the peptide concentration needed to cause 50% hemolysis of normal mammalian red blood cells.
  • the LD JOO or ED 0 can be less than half the peptide concentration needed to cause 50% hemolysis of normal mammalian red blood cells.
  • the LD ⁇ 00 or ED 50 can be less than one fifth to less than one quarter the peptide concentration needed to cause 50% hemolysis of mammalian red blood cells.
  • the LD 10 o or ED 50 is less than at least one twentieth to less than at least one tenth the peptide concentration needed to cause 50% hemolysis of mammalian red blood cells.
  • cyclic peptides of the invention are believed to self-assemble into supramolecular structures within or by association with cancer cell membranes.
  • Such supramolecular structures can be nanotubes, barrels of associated, axially parallel nanotubes, a carpet of associated nanotubes, or mixtures thereof. These types of supramolecular structures can selectively induce cancer cell membrane depolarization or disruption while not depolarizing or disrupting normal cell membranes to a substantial or undesired degree. Further, the supramolecular structures can selectively induce cancer cell membrane lysis while not lysing normal cell membranes to a substantial or undesired degree.
  • Cyclic peptides of the invention can have a plurality of amino acids having side chains with affinity for biomolecules integral to cancer cell membranes. Such biomolecules can facilitate selective assembly of the cyclic peptides into supramolecular structures within cancer cell membranes. Most normal mammalian or other animal cells do not have the same biomolecules within their cellular membranes. While not intending to be bound by any particular theory or mechanism of action, cyclic peptides of the invention are believed to selectively associate with cancer cell membranes over normal mammalian or other animal cell membranes.
  • the cyclic peptides can have a half-life in the bloodstream of the mammal of about six hours or less, at an amount that is effective against cancer.
  • Such an effective amount is an amount of the cyclic peptide that is sufficient to induce death or lysis of cancer cells without inducing an undesired amount of death or lysis of normal mammalian cells, for example.
  • the cyclic peptides of the invention preferably induce substantially no hemolysis of normal red blood cells at anti-cancer effective doses.
  • the cyclic peptides of the invention can be administered in multiple doses over a period of one to seven days.
  • An effective amount of the present cyclic peptides is about 0.1 mg/kg to about 100 mg/kg of body weight, alternatively about 0.5 mg/kg to about 50 mg/kg of body weight, about 1.0 mg/kg to about 30 mg/kg of body weight, and other amounts set forth herein.
  • the cyclic peptides of the invention generally have about 25% to about 88% D- and/or L-polar amino acids.
  • the percentage of polar amino acids can be from about 33% or 50% to about 65% or 88% of the total number of D- and L-amino acids.
  • an eight residue cyclic peptide of the invention can have at least one, alternatively, two to seven polar D- and L-amino acids.
  • Other eight residue cyclic peptides will have three to five polar D- and L-amino acids for example.
  • six residue cyclic peptides of the invention can have two to five polar D- and L- amino acids.
  • Other six residue cyclic peptides may have three to four polar D- and L-amino acids. At least one of these polar D- or L-amino acids may be adjacent to at least one other polar D- or L-amino acid. Alternatively, at least one polar D- or L-amino acid may be adjacent only to nonpolar D- or L-amino acids.
  • polar amino acids are available to one of skill in the art.
  • Examples of polar D- and L- ⁇ -amino acids that can be utilized in the peptides of the invention include the D- or L-enantiomers of serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, histidine, arginine, lysine, hydroxylysine or ornithine.
  • the cyclic peptides of the invention generally have about 25% to about 88%) D- and/or L-ionizable amino acids.
  • the percentage of ionizable amino acids can be from about 33% or 50%) to about 65% or 88% of
  • a six or eight residue cyclic peptide can have at least one, or alternatively two or three or more ionizable D- and/or L-amino acids.
  • the cyclic peptides of the invention can have four to six ionizable D- and/or L-amino acids.
  • Such an ionizable D- or L-amino acid can be adjacent to at least one other polar or ionizable D- or L-amino acid.
  • the cyclic peptides of the invention can have at least one ionizable D- or L-amino acid that is adjacent only to nonpolar D- or L-amino acids.
  • ionizable amino acids are available to one of skill in the art and the invention contemplates all such ionizable amino acids.
  • ionizable D- and/or L-amino acids include the D- or L-enantiomers of arginine, aspartic acid, glutamic acid, histidine, lysine, hydroxylysine or ornithine.
  • the cyclic peptides of the invention can have nonpolar D- and/or L-amino acid residues.
  • the cyclic peptides of the invention generally have about 12% to about 75% D- and/or L-nonpolar amino acids.
  • the percentage of nonpolar amino acids can be from about 50% to about 67% or 75%) of the total number of D- and/or L-amino acids.
  • an eight residue cyclic peptide of the invention can have at least one, alternatively, two to seven nonpolar D- and/or L-amino acids.
  • Other eight residue cyclic peptides may have three to five nonpolar D- and/or L-amino acids.
  • six residue cyclic peptides of the invention have two to five nonpolar D- and/or L-amino acids.
  • Other six residue cyclic peptides may have three to four nonpolar D- and/or L-amino acids. At least one of these nonpolar D- and/or L-amino acids may be adjacent to at least one other nonpolar D- and/or L-amino acid. Alternatively, at least one nonpolar D- or L-amino acid may be adjacent only to polar D- or L-amino acids.
  • nonpolar amino acids are available to one of skill in the art and the invention contemplates all such nonpolar amino acids.
  • nonpolar amino acids include the D- and L-enantiomers of alanine, valine, isoleucine, leucine, methionine, norleucine, phenylalanine, tyrosine or tryptophan.
  • the cyclic peptides of the invention can have an amino acid sequence having formula I:
  • m is an integer ranging from 1 to 7; each p is separately an integer ranging from 0 to 7; each X], X 2 , X 3 , X4, X 5 , X 6 , X 7 , X 8 , X 9 , and X 10 is separately a polar D- or L- ⁇ -amino acid; and each Yi, Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 , Y 8 , Y 9 , and Y ]0 is separately nonpolar D- or L- ⁇ -amino acid; and wherein the cyclic peptide has an even number of from four to about sixteen alternating D- and
  • m is an integer ranging from 1 to 7; each p is separately an integer ranging from 0 to 7; each X], X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and X 8 is separately a polar D- or
  • each Yi, Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 , and Y 8 is separately nonpolar D- or L- ⁇ -amino acid; and wherein the cyclic peptide has an even number of from four to about sixteen alternating D- and L- ⁇ amino acids.
  • the cyclic peptides of the invention can have an amino acid sequence having formula III:
  • the cyclic peptide has an amino acid sequence of formula INa or INb:
  • Client Reference 893.1 PCT Yi, Y 2 and Y are each separately nonpolar amino acid; and wherein the cyclic peptide has an even number of from four to about sixteen alternating D- and L- ⁇ amino acids.
  • the cyclic peptide has an amino acid sequence of formula Na or Vb:
  • Va Vb wherein: q is an integer ranging from 2 to 7;
  • X] and X 2 are separately polar amino acids
  • Yi and Y 2 are separately nonpolar amino acids.
  • Cyclic peptides often have, for example, from four to about sixteen D- and L- ⁇ -amino acids. In other embodiments, the cyclic peptides have about six to about ten or twelve D- and L- ⁇ -amino acids. In still other embodiments, cyclic peptides of about six or eight D- and L- ⁇ -amino acids are employed.
  • compositions of the invention can include an effective amount of at least one of the cyclic peptides of the invention, or two or more different cyclic peptides of the invention. These compositions also include a pharmaceutically effective carrier.
  • the cyclic peptides need not be made from D- or L- ⁇ -amino acids and can alternatively have a sequence of from three to about ten homochiral ⁇ -amino acids.
  • Such ⁇ -amino acids are available to one of skill in the art.
  • Beta amino acids can be substituted at the ⁇ - or ⁇ -carbons by one to two substituents.
  • Mono-substituted beta amino acids of either S or R chirality can be employed for the construction of cyclic ⁇ -peptides, provided that the cyclic beta peptide is homochiral.
  • Client Reference 893.1 PCT homochiral ⁇ -peptides of the present invention have the relative R,R or S,S diastereomeric configuration.
  • Cyclic peptides having ⁇ -amino acids generally have at least one ⁇ -amino acid with at least one polar side chain. Preferred ⁇ - peptides cause substantially no undesirable lysis of mammalian cells.
  • the cyclic ⁇ -peptides of the invention can have an amino acid sequence of formula VI:
  • each p is separately an integer ranging from 0 to 7; each Z], Z 3 , Z 5 , Z 7 , Z 9 , Z ⁇ ⁇ , Z ⁇ 3 , Z 15 , Zj 7 , and Z ⁇ g is separately a monosubstituted ⁇ -amino acid; each Z 2 , Z 4 , Z 6 , Z 8 , Z ⁇ 0 , Z 12 , Z ]4 , Z ]6 , Z ] 8 , and Z 20 is separately a disubstituted ⁇ -amino acid; and wherein the cyclic ⁇ -peptide has a sequence of from three to about ten homochiral ⁇ -amino acids.
  • the invention therefore also contemplates pharmaceutical compositions that include an effective amount of at least one cyclic peptide, or two or more different cyclic peptides, wherein these cyclic peptide(s) have a sequence of from three to about ten homochiral ⁇ -amino acids, for example, as provided by formula VI.
  • the invention also provides a method of treating or preventing a cancer in a mammal and other animals, which comprises contacting a cancer cell with a cyclic peptide comprising a sequence of from four to about sixteen amino acids, wherein the sequence has alternating D- and L- ⁇ -amino acids, in an amount sufficient to induce cancer cell death without inducing an undesirable amount of normal cell death.
  • Such cyclic peptides can alternatively have a sequence of from three to about ten ⁇ -amino acids.
  • the invention further provides a method of identifying cyclic peptides selectively cytotoxic to a target cell-type comprising screening one or more cyclic peptides for induction of cell death in target cells without induction of substantial or undesired cell death in a second cell type.
  • cyclic peptides may comprise, for example, an alternating D- and L- ⁇ amino acid sequence of between four and about sixteen amino acids. Altematively, such cyclic peptides may have a sequence of from three to about ten homochiral ⁇ -amino acids.
  • the invention also provides a method of identifying cyclic peptides selectively cytotoxic to a target cell-type comprising: (a) making a combinatorial library of cyclic peptides, wherein each cyclic peptide in the combinatorial library comprises an alternating D- and L- ⁇ amino acid sequence of between four and about sixteen amino acids; and (b) screening cyclic peptides from the combinatorial library for induction of cell death in target cells without induction of substantial or undesired cell death in a second cell type.
  • cyclic peptides may have a sequence of from three to about ten homochiral ⁇ -amino acids.
  • the library can be used to generate single cyclic peptides or mixtures of cyclic peptides. Mixtures of cyclic peptides that show anti-cancer activity can then be further screened to identify one or more anti- cancer cyclic peptides in one or more of the mixtures, which can then be isolated or synthesized and re-tested for induction of cell death in target cancer cells without induction of substantial or undesired cell death in a second cell type.
  • the invention further provides a method of identifying cyclic peptides selectively cytotoxic to a target cell-type. This method involves the step of rationally designing at least one cyclic peptide comprising an alternating D- and L- ⁇ amino acid sequence of between four and sixteen amino acids.
  • such cyclic peptides may have a sequence of from three to about ten homochiral ⁇ -amino acids.
  • the method further involves screening such rationally designed cyclic peptides for induction of cell death in target cells without induction of substantial or undesired cell death in a second cell type.
  • Client Reference 893.1 PCT Rationally designing a cyclic peptide can involve identifying at least one effective cyclic peptide from a combinatorial library that can induce cell death in target cells without inducing substantial or undesired cell death in a second cell type and exchanging at least one amino acid for a different amino acid in the alternating D- and L- ⁇ amino acid sequence of the effective cyclic peptide to generate a rationally designed cyclic peptide.
  • the rationally designed cyclic peptide can then be screened for induction of cell death in target cells without inducing substantial or undesired cell death in a second cell type.
  • such cyclic peptides may have a sequence of from three to about ten homochiral ⁇ -amino acids.
  • the target cell-type can be a cancer cell and the second cell type can be a normal mammalian cell, for example, a mammalian red blood cell. Cell death of the second cell type can be detected by detecting hemolysis, for example.
  • the method can further include screening a third cell type, for example, by determining whether a peptide induces substantial cell death in the third cell type.
  • the method can also include determining the minimum inhibitory dose at which a peptide can kill substantially all, or inhibit the growth, of the target cells.
  • Assays can be performed in vitro by separately contacting a peptide with the target cell type and with other cell types (e.g., a second or third or other cell type).
  • assays can be performed in vivo by administering at least one peptide to a test animal comprising the target cell type and another cell type or types and determining whether the peptide is toxic to the target cell type but does not have substantial or undesired toxicity to another cell type or types.
  • Such a method can also include determining whether the peptide adversely affects the health of the animal. Determining whether the peptide adversely affects the health of the animal can include examining the bodily fluids or the anatomy of the animal by pathological or histological methods.
  • the invention further provides a method of identifying cyclic peptides capable of selective association with one or more target biomolecules on a selected cell surface, comprising contacting a solution of cyclic peptides, each peptide comprising between four to about sixteen amino acids in an alternating
  • the target biomolecule can be displayed, for example, on the surface of a living cell or on the surface of a liposome. Altematively, the peptide can be contacted with the target biomolecule(s).
  • the method can further include determining the structure of the peptides that spontaneously assemble into the supramolecular structure that selectively associates with the biomolecule(s).
  • the present invention also provides methods of evaluating or confirming therapeutically effective dosages for treating a cancer with a cyclic peptide having an amino acid sequence of alternating D- and L- ⁇ -amino acids, or a cyclic peptide comprising from three to about ten ⁇ -amino acids, that includes determining the LDioo or ED 50 of the cyclic peptide at which substantially no cancer cells grow or survive in vitro.
  • the present invention also provides a composition comprising one or more of the present cyclic peptides with one or more other anti-cancer agents.
  • Mammals and other animals including birds may be treated by the methods and compositions described and claimed herein.
  • Such mammals and birds include humans, dogs, cats, and livestock, for example, horses, cattle, sheep, goats, chickens, turkeys and the like.
  • the invention therefore provides a pharmaceutical composition for treating, inhibiting or preventing growth of a cancer cell in an animal comprising a cyclic peptide in an amount effective to treat or prevent a target cancer in the animal, and a pharmaceutically acceptable carrier, wherein the cyclic peptide comprises a sequence of from 4 to about 16 alternating D- and L- ⁇ -amino acids or a sequence of three to about ten ⁇ -amino acids.
  • the cyclic peptides provided herein for example, cause substantially no hemolysis of non-cancerous mammalian red blood cells.
  • the invention also provides a method for treating, inhibiting or preventing growth of a cancer cell in an animal comprising contacting a target
  • the invention further provides a method for treating, inhibiting or preventing growth of a cancer cell in an animal comprising contacting target cancer cell with a cyclic peptide comprising a sequence of from three to about ten ⁇ -amino acids, in an amount sufficient to induce target cancer cell death without inducing an undesirable amount of non-cancerous mammalian cell death.
  • the invention further provides a method of identifying a cyclic peptide selectively cytotoxic to a target cancer cell-type comprising: (a) contacting said target cancer cell-type with a test cyclic peptide comprising a sequence of from four to about sixteen alternating D- and L- a amino acids, or a sequence of three to about ten ⁇ -amino acids; and (b) determining whether said test cyclic peptide induces cell death of said target cancer cell-type without inducing substantial or undesired cell death in a second cell type.
  • FIGURES Figure 1 provides a schematic drawing of the self-assembly of the present cyclic peptides into nanotubules.
  • An eight-residue cyclic D, L- ⁇ -peptide with alternating D- and L-amino acids is depicted to the left, with emphasis on its flat ring-shaped conformation.
  • Side chains (R) decorate the outside surface of the cyclic peptide.
  • a series of cyclic peptides align and undergo inter-peptide hydrogen bonding to form a tubular stmcture referred to herein as a nanotube (center).
  • Self-assembly is directed by inter- subunit backbone-backbone hydrogen bonding resulting in a /3-sheet-like open- ended hollow tubular supramolecular stmcture. This /3-sheet like hydrogen bonding pattern is shown to the right. For clarity most side chains are omitted.
  • Figure 2 illustrates the modes of permeation that are accessible to peptide supramolecular structures. Depending on the composition and sequence
  • Client Reference 893.1 PCT of amino acids employed in the cyclic peptides, supramolecular stmctures can interact with the membranes of cells through (a) pores, (b) barrel stave strictures, (c) carpet-like stmctures, or (d) alternate modes of action. Cyclic peptides are depicted as ring stmctures. Note that in Figure 2b, the hydrophobic tails of the membrane lipids interact with hydrophobic portions of the nanotubes, whereas in Figure 2d the hydrophilic heads of the membrane lipids interact with hydrophilic portions of the nanotubes.
  • Figure 3 provides a plot of the apparent proton transport (Figure 3 a) and the carboxyfluoroscein release ( Figure 3b) mediated by peptide SEQ ID NO: 11 (cyclo-[Gln-D-Lys-(Trp-D-Leu) 2 -Trp-D-Lys-]) 5 as expressed in fractional fluorescence changes as a function time.
  • the peptide was added at about 100 seconds, and the detergent triton X-100 was added at about 200 seconds.
  • Figure 4 provides an attenuated total reflectance (ATR) infrared (IR) spectra for peptide SEQ ID NO: 11 (cyclo-[-Gln-D-Lys-(Trp-D-Leu) 2 -Trp-D- Lys-] in DMPC multibilayers.
  • the solid trace indicates absorbance of parallel- polarized light; the dashed trace provides absorbance of perpendicular-polarized light.
  • Figure 5 provides a structural comparison of supramolecular stmctures composed of: (a) cyclic jS-tetrapeptides, and (b) cyclic D, L- ⁇ -octapeptides.
  • This figure illustrates that, due to the unidirectional arrangement of the polar backbone amide groups, cyclic /3-tetrapeptide supramolecular stmctures may possess a macrodipole moment reminiscent of an ⁇ -helix, while cyclic D, L- ⁇ - octapeptide supramolecular stmctures will, under most circumstances, not have such a net dipole moment.
  • most side chains are omitted from the nanotube stmctures depicted in Figure 5a and 5b.
  • Figure 6 provides a thin section electron microscopy image of untreated S. aureus (ATCC 25923) displaying a normal intact membrane.
  • Figure 7 provides a thin section electron microscopy image of S aureus (ATCC 25923) after exposure to 2XMIC concentration of cyclo[KSKWLWLW] for 2 hours.
  • the image provides direct visualization of the membrane mode of
  • Figure 8 provides a thin section electron microscopy image of S. aureus (ATCC 25923) after exposure to 2xMIC concentration of cyclo[KSKWLWLW] for 2 hours.
  • the image provides direct visualization of the membrane mode of action. Arrows denote abnormal membrane structures caused by the peptide action.
  • Figure 9 illustrates the toxicity of peptides administered via intraperitoneal route.
  • the mice were injected with varying concentrations of cyclo[RRKWLWLW]HCl.
  • the mice were injected with varying concentrations of cyclo[KQRWLWLW]HCl. Mice were monitored over a period of 14 days for activity and mortality. Four mice per dose were used in each experiment.
  • Figure 10 illustrates the toxicity of peptides administered via intraperitoneal route.
  • the mice were injected with varying concentrations of cyclo[KSKWLWLW]HCl.
  • the mice were injected with varying concentrations of cyclo[KKLWLW]HCl. Mice were monitored over a period of 14 days for activity and mortality. Four mice per dose were used in each experiment.
  • Figure 11 provides a graph indicating the toxicity of peptide SEQ ID
  • Figure 12 provides the in vitro testing results for the SEQ ID NO: 12 peptide in a variety of cancel lines.
  • Figures 13a - i provide dose response curves for the SEQ ID NO: 12 peptide in a variety of cancel lines.
  • Figure 14 provides graphs of mean Logio GI30, TGI and LC50 concentrations for the SEQ ID NO: 12 peptide in a variety of cancel lines.
  • Figure 15 provides additional dose response curves for the SEQ ID NO: 12 peptide in a variety of cancel lines.
  • Client Reference 893.1 PCT Figure 16A and B provides a chart illustrating the concentration (moles/liter) of the indicated cyclic peptide that inhibits 50% (GI50) of cancer cell growth. As illustrated, a large variety of cancer cell lines were tested. Cyclic peptides [KSKKLWLW] (SEQ ID NO: 149), [RHKHRWLW] (SEQ ID NO:151), [KRKWLW] (SEQ ID NO:125), and [KSKWLW] (SEQ ID NO:126) were effective against many cancer cell lines when used at micromolar concentrations.
  • the present invention provides small cyclic peptides and compositions that can quickly and selectively kill or inhibit growth of cancer cells without substantial toxicity toward normal cells.
  • the present invention includes cyclic peptides, and pharmaceutical compositions comprising cyclic peptides, with either a sequence of alternating D-, and L- ⁇ -amino acids, or a sequence of ⁇ - amino acids, that can sample flat, ring-shaped conformations.
  • Such ring-shaped conformations project the amino acid side chains of the cyclic peptides away from the center of the ring and orient the amide backbone approximately perpendicular to the plane of the ring stmcture.
  • the cyclic peptides can self-assemble via intermolecular hydrogen bonding to form supramolecular stmctures.
  • Cyclic peptides that simply contain one or more D- amino acids do not adopt a flat ring-shaped conformation and do not have the backbone conformation needed for self-assembly of the cyclic peptide into supramolecular stmctures.
  • Another feature of the present self-assembling peptide supramolecular stmctures is believed to be the potential for a given cyclic peptide to form a number of diastereomeric nanotube assemblies. This property stems from the fact that backbone-backbone hydrogen bonding are believed primarily to direct the self-assembly of the nanotube stmcture. Differently stacked subunits can give rise to topoisomeric supramolecular stmctures that share the same or nearly the same tubular /5-sheet- like hydrogen bonded backbone stmcture.
  • amino acid includes the residues of the natural ⁇ -amino acids
  • amino acid residues are useful in the cyclic peptides and the invention is not limited to natural, genetically-encoded amino acids. Examples of amino acids that can be utilized in the cyclic peptide described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein. Another source of a wide variety of amino acid residues is provided by the website of RSP Amino Acids Analogues, Inc. (www.amino-acids.com).
  • mammal refers to an animal, such as a warmblooded animal, which is susceptible to or has a cancer. Mammals include cattle, buffalo, sheep, goats, pigs, horses, dogs, cats, rats, rabbits, mice, and humans. Also included are other livestock, domesticated animals and captive animals.
  • farm animals includes chickens, turkeys, fish, and other farmed animals.
  • a "nanotube” or “nanotubule” is a small tubule that may spontaneously form from the cyclic peptides of the present invention.
  • the present cyclic peptides are believed to stack to form supramolecular stmctures composed of nanotubes. Hydrogen bonding between cyclic peptides helps to drive the self-assembly of the supramolecular stmctures from the cyclic peptide and, after formation, acts to stabilize the stmcture.
  • Each nanotube has a pore in the center of the tube that is surrounded by the series of peptide backbones of the stacked cyclic peptides that form the nanotubes.
  • the size of the pore depends upon the number of amino acids in the cyclic peptides that form the nanotube.
  • ions, sugars, and other small molecules can travel through the pores of the present nanotubes. Larger molecules can also flow the pores of nanotubes formed from larger cyclic peptides and supramolecular stmctures formed of aggregates of nanotubes.
  • the supramolecular stmcture is thought to be a barrel-like stmcture composed of clusters of nanotubes.
  • the supramolecular stmcture is thought to be a "carpet” or “carpet- like” arrangement of nanotubes.
  • a "carpet” or “carpet-like” arrangement of one or more nanotubes is where the nanotube(s) adopt orientations that can be approximately or somewhat parallel to the plane of the membrane stmcture.
  • the nanotubes can assume other orientations relative to the plane of the membrane.
  • the nanotubes may be situated on the surface of the membrane, or may be partially or fully contained within the interior of the membrane.
  • the carpet-like nanotubes can be oriented with a tilt angle of up to
  • These carpet or carpet-like arrangements may be formed because the contemplated cyclic peptides can possess clusters of hydrophilic and hydrophobic residues, that upon aggregation can form supramolecular stmctures with, for example, a hydrophilic side or face and a hydrophobic side or face.
  • a hydrophilic face of a nanotube can, for example, be positioned so that it is in contact with the hydrophilic portions of the membrane or with the aqueous environment.
  • a hydrophobic face of a nanotube can, for example, be in contact with the hydrophobic portions of the membrane. See Figure 2A-D.
  • peptide as used herein includes a sequence of from four to sixteen amino acids residues in which the ⁇ -carboxyl group of one amino acid is joined by an amide bond to the main chain ( ⁇ - or ⁇ -) amino group of the adjacent amino acid.
  • the peptides provided herein for use in the described and claimed methods and compositions are cyclic. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right. However, where the peptides are shown in cyclic form, the first amino acid in the sequence is arbitrarily chosen. Moreover, for formulae of cyclic peptides where the sequence extends onto two lines, the sequence on the second line extends from the N-terminal side on the right to the C-terminal side on the left.
  • supramolecular stmctures are multi-subunit stmctures, e.g. nanotubes, barrels and carpets of nanotubules, which are believed to be formed through "noncovalent” assembly of cyclic peptides.
  • Supramolecular stmctures may be contrasted with molecular or polymeric systems that are the product of covalent bond formation between reactants or monomers.
  • the proposed peptide supramolecular stmctures are thermodynamically controlled assemblies that can undergo reversible stmctural assembly and disassembly. Such assembly-disassembly will depend, for example, on the environment, subunit stmcture, side group selection, side group interaction, and the nature and combination of noncovalent forces operating on
  • compositions containing peptides that can form supramolecular stmctures are their ability to select amongst various cell membrane types. Such selection is driven by favorable thermodynamic forces determined by the composition of the cyclic peptide relative to the cell membrane environment and the molecular and/or supramolecular constituents of the cell membrane.
  • substantially no with reference to self-assembly, hemolysis, toxicity or cellular lysis, or the like, means that little or no self-assembly, hemolysis, toxicity, cellular lysis or the like is present at the tested or desired peptide dosage or concentration.
  • substantially no hemolysis can mean that less than about 20%, alternatively less than 15% or less than 10%), or no detectable, hemolysis at the tested or desired peptide dosage or concentration has occurred.
  • substantially no toxicity or lysis can mean that less than about 20%, alternatively less than 15% or less than 10%), or no detectable, toxicity or lysis at the tested or desired peptide dosage or concentration has occurred.
  • a therapeutically effective amount is that amount sufficient to control cancer growth.
  • a therapeutically effective amount reduces the size of the cancer in the affected mammal by at least about 20%, by at least about 40%>, by at least about 60%, or by at least about 80% relative to untreated subjects.
  • a therapeutically effective amount reduces the size of the cancer in the affected mammal by at least about 90% or more. These percentages refer to a decrease in the size of the cancer found in the mammal relative to untreated subjects. While not intending to be bound by any particular theory or mechanism of action, a "therapeutically effective amount” may also be that amount of cyclic peptide needed to permeabilize or depolarize the cellular
  • the term "therapeutically effective amount” is that amount needed to control the growth of, or kill, a cancer cell.
  • An effective amount of the therapeutic agent used to control the cancer cell can vary according to factors such as the type of cancer, the amount of cancer already present in the animal, the age, sex, and weight of the mammal, and the ability of the cyclic peptides of the present invention to control cancer growth in the mammal.
  • Therapeutically effective amounts of the peptide and peptide compositions can also be used to prevent cancer, including preventing a recurrence of cancer.
  • the present invention provides cyclic peptides and compositions including cyclic peptides that have an amino acid sequence of alternating D- and L-amino acids that is between four to about sixteen, alternatively about six to about sixteen amino acids in length.
  • the cyclic peptides of the present invention can have between three to about ten ⁇ -amino acids.
  • the cyclic D, L- ⁇ -peptides do not include the amino acids proline and glycine.
  • ⁇ -amino acids can be substituted at the ⁇ - or ⁇ - carbons, or both.
  • Mono-substituted ⁇ -amino acids of either S or R chirality can be employed for the constmction of cyclic ⁇ -peptides, provided that the cyclic beta peptide is homochiral.
  • Disubstituted ⁇ -amino acids employed in the present invention must have the relative R,R or S,S diastereomeric configuration, provided that the ⁇ -amino acid residues in a cyclic peptide stmcture are homochiral.
  • Cyclic peptides having ⁇ -amino acids generally have at least one ⁇ - amino acid with at least one polar side chain.
  • the cyclic peptides of the present invention are believed to undergo self-assembly to form supramolecular stmctures that, upon assembly in or on a cancer cell membrane, can cause depolarization and/or permeablization and/or destabilization of the cancer cell membrane.
  • the cyclic peptides of the present invention are believed to undergo self-assembly to form supramolecular stmctures that, upon assembly in or on a cancer cell membrane, can cause depolarization and/or permeablization and/or destabilization of the cancer cell membrane.
  • the cyclic peptides of the present invention are believed to undergo self-assembly to form supramolecular stmctures that, upon assembly in or on a cancer cell membrane, can cause depolarization and/or permeablization and/or destabilization of the cancer cell membrane.
  • the cyclic peptides of the present invention are believed to undergo self-assembly to form supramolecular st
  • Client Reference 893.1 PCT cause lysis of the cancer cell. While not intending to be bound by any particular theory or mechanism of action, self-assembly into supramolecular stmctures is thought to occur by stacking of the cyclic peptides in an anti-parallel fashion or a parallel fashion with formation of ⁇ -sheet hydrogen bonds between adjacent cyclic peptides. However, it is believed that the preferred cyclic peptides do not readily or undesirably self-assemble into supramolecular stmctures in undesired mammalian cellular membranes as measured, for example, in an assay for toxicity in mammalian cells or hemolysis of mammalian red blood cells at tested or therapeutically effective doses.
  • Cyclic peptides of the present invention can be made from ⁇ -amino acids or ⁇ -amino acids.
  • the amino acid sequence of the present cyclic peptides includes at least one polar amino acid in the case of D,L ⁇ -amino acid cyclic peptides, or at least one polar side chain in the case of cyclic ⁇ -peptides.
  • the percentage of polar amino acids can range, for example, from about 25% or 33% to about 65% or 88%). However, in some embodiments a majority of the amino acids are polar. For example, the percentage of polar amino acids can be from about 50% to about 88% of the total number of amino acids.
  • the sizes for the present cyclic peptides are about six to about ten D,L ⁇ -amino acids or about three to about ten ⁇ -amino acids. In other embodiments, the size for the present cyclic peptides is about six to about eight D,L ⁇ -amino acids or four to about six ⁇ -amino acids.
  • an eight residue cyclic peptide of the invention can have at least one, alternatively, two to seven polar D- and/or L- ⁇ -amino acids.
  • Other eight residue cyclic peptides will have three to five polar D- and/or L- ⁇ -amino acids for example.
  • Preferred eight residue cyclic peptides have three, four or five polar amino acids.
  • six residue cyclic peptides of the invention can have two to five polar D- and/or L- ⁇ -amino acids.
  • Other six residue cyclic peptides may have three to four polar D- and/or L- ⁇ - amino acids. At least one of these polar D- or L- ⁇ -amino acids may be adjacent
  • Client Reference 893.1 PCT to at least one other polar D- or L- ⁇ -amino acid.
  • at least one polar D- or L- ⁇ -amino acid may be adjacent only to nonpolar D- or L- ⁇ -amino acids.
  • Beta peptides having about four to about eight ⁇ -amino acids may have, for example, about two to twelve polar side chains, depending on the level of ⁇ and ⁇ backbone substitution.
  • the cyclic D-L- ⁇ -peptides of the invention generally have about 25% to about 88% ionizable amino acid residues.
  • the percentage of ionizable amino acids can be from about 33% or 50% to about 65% or 88% of the total number of D- and/or L-amino acids.
  • a six or eight residue cyclic peptide can have at least one, or alternatively two or three or more ionizable D- and/or L-amino acids.
  • the cyclic peptides of the invention can have four to six ionizable D- and/or L-amino acids.
  • Such an ionizable D- or L-amino acid can be adjacent to at least one other polar or ionizable D- or L-amino acid.
  • the cyclic peptides of the invention can have at least one ionizable D- or L-amino acid that is adjacent only to nonpolar D- or L-amino acids.
  • the cyclic ⁇ -peptides of the invention generally have about 25% to about 88% ionizable amino acid side chains. In some embodiments, the percentage of ionizable amino acid side chains can be from about 33% or 50% to about 65% or 88%o of the total number of amino acid side chains.
  • a four to six residue cyclic ⁇ -peptide can have at least one, or alternatively two or three or more ionizable amino acid side chains.
  • the cyclic ⁇ -peptides of the invention can have four to six ionizable amino acid side chains.
  • the cyclic peptides of the invention can have nonpolar D- and/or L-amino acid residues.
  • the number of non-polar amino acids chosen can vary as the size of the peptide varies and as the selected cancer cell membrane environment varies.
  • the cyclic peptides of the invention generally have about 12% to about 75%> D- and L-nonpolar amino acids.
  • the percentage of nonpolar amino acids can be from about 50% to about 67% or 75% of the total number of D- and L-amino acids.
  • Client Reference 893.1 PCT peptide of the invention can have at least one, alternatively, two to seven nonpolar D- and or L-amino acids.
  • Other eight residue cyclic peptides may have three to five nonpolar D- and/or L-amino acids.
  • six residue cyclic peptides of the invention have two to five nonpolar D- and/or L-amino acids.
  • Other six residue cyclic peptides may have three to four nonpolar D- and/or L-amino acids. At least one of these nonpolar D- or L- amino acids maybe adjacent to at least one other nonpolar D- or L-amino acid.
  • At least one nonpolar D- or L-amino acid may be adjacent only to polar D- or L-amino acids.
  • the cyclic peptides do not include the amino acid proline or glycine, but certain cyclic peptides may have good activity even though proline or glycine is included.
  • ⁇ -amino acids can have non-polar side chains at the ⁇ - or ⁇ -carbons, or both.
  • the number of non-polar amino acid side chains chosen can vary as the size of the peptide varies and as the selected cancer cell membrane environment varies.
  • the cyclic ⁇ -peptides of the invention generally have about 12%) to about 75% nonpolar amino acid side chains.
  • the percentage of nonpolar amino acid side chains can be from about 50%) to about 67% or 75% of the total number of amino acid side chains.
  • an eight residue cyclic ⁇ -peptide of the invention can have at least one, alternatively, two to seven nonpolar amino acid side chains.
  • cyclic ⁇ -peptides may have three to five nonpolar amino acid side chains.
  • six residue cyclic ⁇ -peptides of the invention have two to five nonpolar amino acid side chains.
  • Other six residue cyclic ⁇ -peptides may have three to four nonpolar amino acid side chains.
  • Amino acids used in the cyclic peptides can be genetically encoded amino acids, naturally occurring non-genetically encoded amino acids, or synthetic amino acids. Both L- and D-enantiomers of any of the above are utilized in the cyclic peptides.
  • the amino acid notations used herein for the twenty genetically encoded L-amino acids and some examples of non-encoded amino acids are provided in Table 1.
  • Certain commonly encountered amino acids that are not genetically encoded and that can be present in the cyclic peptides of the invention include,
  • Client Reference 893.1 PCT but are not limited to, ⁇ -alanine (b-Ala) and other omega-amino acids such as 3- aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; ⁇ -aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); ⁇ - aminovaleric acid (Ava); methylglycine (MeGly); omithine (Om); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 2- naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2- fluor
  • Additional amino acid analogs contemplated include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statine, ⁇ -methyl-alanine, para-benzoyl-phenylalanine, propargylglycine, and sarcosine.
  • Peptides that are encompassed within the scope of the invention can have any of foregoing amino acids in the L- or D- configuration, or any other amino acid known to one of skill in the art.
  • amino acids that are substitutable for each other generally reside within similar classes or subclasses. As known to one of skill in the art, amino acids can be placed into different classes depending primarily upon the chemical and physical properties of the amino acid side chain. For example, some amino acids are generally considered to be hydrophilic or polar amino acids and others are considered to be hydrophobic or nonpolar amino acids.
  • Polar amino acids include amino acids having acidic, basic or hydrophilic side chains and nonpolar amino acids include amino acids having aromatic or hydrophobic side chains.
  • Nonpolar amino acids may be further subdivided to include, among others, aliphatic amino acids.
  • the definitions of the classes of amino acids as used herein are as follows:
  • Nonpolar Amino Acid refers to an amino acid having a side chain that is uncharged at physiological pH, that is not polar and that is generally repelled by aqueous solution.
  • Examples of genetically encoded hydrophobic amino acids include Ala, He, Leu, Met, T ⁇ , Tyr and Val.
  • non-genetically encoded nonpolar amino acids include t-BuA, Cha and Nle.
  • Aromatic Amino Acid refers to a nonpolar amino acid having a side chain containing at least one ring having a conjugated 7T-electron system (aromatic group).
  • aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others.
  • substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others.
  • Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan.
  • Non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, ⁇ -2-thienylalanine, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2- fiuorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.
  • “Aliphatic Amino Acid” refers to a nonpolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids include Ala, Leu, Val and He. Examples of non-encoded aliphatic amino acids include Nle.
  • Polar Amino Acid refers to a hydrophilic amino acid having a side chain that is charged or uncharged at physiological pH and that has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • Polar amino acids are generally hydrophilic, meaning that they have an amino acid having a side chain that is attracted by aqueous solution.
  • genetically encoded polar amino acids include asparagine, glutamine, lysine and serine.
  • non-genetically encoded polar amino acids include citrulline, homocysteine, N-acetyl lysine and methionine sulfoxide.
  • Acidic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples
  • Client Reference 893.1 PCT of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
  • Basic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of greater than 7.
  • Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion.
  • genetically encoded basic amino acids include arginine, lysine and histidine.
  • non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.
  • Ionizable Amino Acid refers to an amino acid that can be charged at a physiological pH.
  • Such ionizable amino acids include acidic and basic amino acids, for example, D-aspartic acid, D-glutamic acid, D-histidine, D-arginine, D- lysine, D-hydroxylysine, D-ornithine, L-aspartic acid, L-glutamic acid, L- histidine, L-arginine, L-lysine, L-hydroxylysine or L-ornithine.
  • the above classifications are not absolute.
  • Several amino acids exhibit more than one characteristic property, and can therefore be included in more than one category.
  • tyrosine has both a nonpolar aromatic ring and a polar hydroxyl group.
  • tyrosine has several characteristics that could be described as nonpolar, aromatic and polar. However, the nonpolar ring is dominant and so tyrosine is generally considered to be nonpolar. Similarly, in addition to being able to form disulfide linkages, cysteine also has nonpolar character. Thus, while not strictly classified as a hydrophobic or nonpolar amino acid, in many instances cysteine can be used to confer hydrophobicity or nonpolarity to a peptide.
  • polar amino acids contemplated by the present invention include, for example, arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, hydroxylysine, omithine, serine, threonine, the corresponding ⁇ -amino acids, and structurally related amino acids.
  • the polar amino is an ionizable amino acid such as arginine, aspartic acid, glutamic acid, histidine, hydroxylysine, lysine, or omithine.
  • nonpolar or nonpolar amino acid residues that can be utilized include, for example, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tryptophan, tyrosine and the like.
  • amino acid sequence of a peptide can be modified so as to result in a peptide variant that includes the substitution of at least one amino acid residue in the peptide for another amino acid residue, including substitutions that utilize the D rather than L form.
  • One or more of the residues of the peptide can be exchanged for another, to alter, enhance or preserve the biological activity of the peptide.
  • Such a variant can have, for example, at least about 10% of the biological activity of the corresponding non- variant peptide.
  • Conservative amino acid substitutions are often utilized, i.e., substitutions of amino acids with similar chemical and physical properties, as described above.
  • conservative amino acids substitutions involve exchanging aspartic acid for glutamic acid; exchanging lysine for arginine or histidine; exchanging one nonpolar amino acid (alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine,) for another; and exchanging one polar amino acid (aspartic acid, asparagine, glutamic acid, glutamine, glycine, serine, threonine, etc.) for another.
  • the substitutions are introduced, the variants are screened for biological activity.
  • the cyclic peptides of the invention can have an amino acid sequence having formula I:
  • m is an integer ranging from 1 to 7; each p is separately an integer ranging from 0 to 7; each Xi, X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , and X] 0 is separately a polar D- or L- ⁇ -amino acid; and
  • each Y 1 ; Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 , Y 8 , Y 9 , and Y 10 is separately nonpolar D- or L- ⁇ -amino acid; and wherein the cyclic peptide has an even number of from four to about sixteen alternating D- and L- ⁇ amino acids.
  • the cyclic peptides of the invention can have an amino acid sequence having formula II:
  • m is an integer ranging from 1 to 7; each p is separately an integer ranging from 0 to 7; each X ⁇ , X , X 3 , X 4 , X 5 , X 6 , X 7 , and X 8 is separately a polar D- or L- ⁇ -amino acid; each Yi, Y 2 , Y , Y 4 , Y 5 , Y 6 , Y 7 , and Y 8 is separately nonpolar D- or L- ⁇ -amino acid; and wherein the cyclic peptide has an even number of from four to about sixteen alternating D- and L- ⁇ amino acids.
  • the cyclic peptides of the invention can have an amino acid sequence having formula III:
  • m is an integer ranging from 1 to 7; each p is separately an integer ranging from 0 to 7; each X], X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , and X ⁇ 0 is separately a polar D- or L- ⁇ -amino acid;
  • each Y Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y7, Yg, Y9, and Y] 0 is separately nonpolar D- or L- ⁇ -amino acid; and wherein the cyclic peptide has an even number of from four to about sixteen alternating D- and L- ⁇ amino acids.
  • the cyclic peptide has an amino acid sequence of formula IVa or IVb:
  • the cyclic peptide has an amino acid sequence of formula Va or Va:
  • Va Vb wherein: q is an integer ranging from 2 to 7;
  • Xi and X 2 are separately polar amino acids
  • Y, and Y are separately nonpolar amino acids.
  • the X amino acids in the above formulae can be D-serine, D-threonine, D-asparagine, D-glutamine, D-aspartic acid, D-glutamic acid, D-
  • one or more of the X amino acids are ionizable amino acids.
  • Such ionizable amino acids include, for example, D-aspartic acid, D-glutamic acid, D-histidine, D-arginine, D-lysine, D-hydroxylysine, D-omithine, L-aspartic acid, L-glutamic acid, L-histidine, L-arginine, L-lysine, L- hydroxylysine or L-omithine.
  • the Y amino acids in the above formulae can be, for example, L-alanine,
  • the Y amino acids may be L-tryptophan, D-tryptophan, L-leucine or D-leucine, provided that the cyclic peptide has a sequence of alternating D- and L-amino acids.
  • the cyclic peptides of the present invention include any of SEQ ID NO:5, 7-22, 26-29, 40, 41, 43-55, 57, 58, 61-67, 72-77, 79-89, 91-93, 97-102, 107, 109-112, 114-117, 119-122, 125, 126, 128, 129, 133, 139, 140 or 141.
  • the cyclic peptides employed are those with SEQ ID NO:8, 9, 12, 17, 18, 26, 29, 47-52, 61, 63, 67, 68, 72-77, 84, 85, 87-89, 91-93, 100, 102, 107, 111, 112, 119, 125 and 139.
  • Formulations or compositions containing the present cyclic peptides can include a mixture of two or more cyclic peptides.
  • the present isolated, purified peptides or variants thereof can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by enzyme catalyzed peptide synthesis or with the aid of recombinant DNA technology.
  • Solid phase peptide synthetic method is an established and widely used method, which is described in references such as the following: Stewart et al., Solid Phase Peptide Synthesis. W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc. 85 2149 (1963); Meienhofer in "Hormonal
  • peptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion- exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; ligand affinity chromatography; or crystallization or precipitation from non-polar solvent or nonpolar/polar solvent mixtures. Purification by crystallization or precipitation is preferred.
  • libraries of peptides can be made using a one-bead-one-compound strategy provided by Lam et al. (97 Chem. Rev. 411-448 (1997) or synthesized on macrobeads by a split and pool method of Furka, et al. (37 Int. J. Pept. Prot. Res. 487-493(1991)).
  • Mass spectrometric sequence analysis techniques enable rapid identification of every peptide within a given library. See, Biemann, K. 193 Methods Enzymol. 455 (1990).
  • synthetic operations including peptide cyclization, are performed on solid support to avoid laborious and difficult to automate solution-phase operations.
  • the final product of the synthesis regimen is generally sufficiently pure for biological assays without laborious purification procedures. Peptide yields from each synthesis can be sufficient for performing 50 to 100 assays. Rapid, automatic mass-spectrometry-based peptide sequence analysis can be performed to identify peptide sequences that have high activity and to discard peptide sequences with low activity.
  • Client Reference 893.1 PCT of peptides can also be generated for testing.
  • Impure preparations of peptides can be used for quick screening of combinations of sequences. When a mixture of peptides shows activity, the peptides in the mixture can either be individually isolated and tested or pure peptides having sequences known to be present in the impure mixture can be individually prepared and tested.
  • Salts of carboxyl groups of a peptide or peptide variant of the invention may be prepared in the usual manner by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide
  • a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate
  • an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • N-acyl derivatives of an amino group of the peptide or peptide variants may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide.
  • O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- acylation and O-acylation may be carried out together, if desired.
  • Acid addition salts of the peptide or variant peptide, or of amino residues of the peptide or variant peptide may be prepared by contacting the peptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid.
  • Esters of carboxyl groups of the peptides may also be prepared by any of the usual methods known in the art.
  • the invention also contemplates cyclic peptides composed of one or more ⁇ amino acids. Such ⁇ -amino acids can be substituted along their peptide backbones by one to two substituents.
  • Such substituents can include cycloalkyl, cycloalkenyl, and heterocyclic rings that encompass the ⁇ and ⁇ carbons of the ⁇ -peptide backbone. These rings can be, for example, C 3 -C 8 cycloalkyl, cycloalkenyl or heterocyclic rings having one or more nitrogen atoms as the sole heteroatom, and can be substituted or unsubstituted.
  • the substituents on the ring or on the ⁇ and ⁇ carbons of the ⁇ -peptide can be, for example, hydroxy, linear
  • the cyclic ⁇ -peptides of the invention can have an amino acid sequence of formula VI:
  • each p is separately an integer ranging from 0 to 7; each Z,, Z 3 , Z 5 , Z , Z 9 , Z , Z 1 , Z 1 , Z, 7 , and Z 19 is separately a monosubstituted ⁇ -amino acid; each Z 2 , Z , Z 6 , Z 8 , Z ⁇ 0 , Z, 2 , Z ⁇ , Z 16 , Z ⁇ 8 , and Z 20 is separately a disubstituted ⁇ -amino acid; and wherein the cyclic ⁇ -peptide has a sequence of from three to about ten homochiral ⁇ -amino acids.
  • the cyclic peptides provided herein are believed self-assemble into supramolecular stmctures.
  • Self-assembly means that a collection of cyclic peptides can associate to form a supramolecular stmcture on or within a cellular membrane without the assistance of anything other than the components of the cellular membrane.
  • the physical and chemical properties of the cellular membrane may facilitate self-assembly of the cyclic peptides and the interaction between the components of cancer cellular
  • Client Reference 893.1 PCT membranes and the cyclic peptides may determine whether the cyclic peptides are selective for those cellular membranes.
  • supramolecular stmctures by the peptides of the invention is supported by high-resolution imaging using cryo-electron microscopy, electron diffraction, Fourier-transform infrared spectroscopy, and molecular modeling. Supramolecular stmctures have been further characterized by IR spectroscopy, low-dose electron microscopy, and the analysis of electron diffraction patterns.
  • cyclic peptide stmctures that are made up of an even number of alternating D- and L-amino acid residues are believed to adopt or sample a flat ring-shaped conformation in which all backbone amide functionalities lie approximately perpendicular to the plane of the ring stmcture.
  • cyclic peptides made up of ⁇ -amino acids can also adopt a flat-ring stmcture.
  • the peptide subunits can stack, under favorable conditions, to furnish a contiguous hydrogen bonded hollow tubular stmcture that is referred to herein as a nanotube (see Figures 1 and 5).
  • Electron diffraction patterns display axial spacing of 4.80 A that is in agreement with the peptide stacking and the formation of tight network of hydrogen bonded b-sheet type stmcture.
  • the meridonial spacing in the electron diffraction patterns display spacing of 12.67 ⁇ 0.06 A and 21.94 ⁇ 0.05 A characteristic of a hexagonal body centered packing of nanotubes. Hexagonal lattice resulting from the close packing of
  • the periodicity in this packing produces diffraction spots at 1/r, 2/r, and so on, and at 1/r, and 2/r, and so on.
  • the observed electron diffraction patterns on the meridional axes extend to third order reflections (4.1 A) signifying the ordered and crystalline state of nanotube stmctures.
  • the diffraction patterns also showed a unit cell with an angle of 99° and no other symmetry than the center of symmetry pursuant to Friedel's law.
  • the model shows stmcture factors similar to the patterns observed in the electron diffraction thus supporting the proposed three- dimensional model.
  • Involvement of intermolecular hydrogen bonding network in a tube-like assembly is also supported by FT-IR spectroscopic analysis according to the method of S. Krimm et al. (Advances in Protein Chemistry; Anfinsen, C.
  • Nanotubes display characteristic IR features of a ⁇ -sheet stmcture signified not only by the amide /bands at 1626 cm “1 and 1674 cm “1 and an amide //band at 1526 cm “1 , but also by the observed NH stretching frequency at 3291 cm "1 supporting formation of a tight network of hydrogen bonds.
  • the IR spectmm is similar to tubular stmctures that have been discovered in nature.
  • nanotubular stmctures for some of the present peptides can be conceptually related to the stmcture of crystalline linear Gramicidin A that is known to form dimeric ⁇ -helical stmctures.
  • Gramicidin A has amide I bands at 1630, 1685 cm “1 , an amide //band at 1539 cm "1 , and an NH stretching frequency at 3285 cm "1 .
  • the observed frequency of NH stretching mode correlates to an average intersubunit distance of 4.76 A that is in close agreement with the value of 4.80 A obtained independently from the electron diffraction patterns.
  • the pore size, or internal diameter, of self-assembled nanotubes can be adjusted by the ring size of the peptide subunit employed.
  • a twelve-residue cyclic peptide stmcture for example cyclo[-(Gln-D-Ala-Glu-D-Ala) 3 -] (SEQ ED NO:l), has a diameter of about 13 A.
  • the eight residue cyclic peptide cyclo[- (Trp-D-Leu) 3 -Gln-D-Leu-] (SEQ ID NO:2) has a diameter of approximately 7.5 A in diameter.
  • a cyclic peptide having SEQ ID NO:2 in synthetic phosphatidylcholine liposomes displays an FTIR amide-I band at 1624 cm “1 and an observed N-H stretching frequency at 3272 cm “1 that support formation of a tight network of ⁇ -sheet-like hydrogen bonds with an average intersubunit distance of 4.7 A to 4.8 A.
  • the flat, ring-shaped cyclic peptides of the present invention are not only structurally predisposed toward intermolecular interaction, but are also energetically favored to self-assemble on selected cancer cell membranes and permeabilize cancer cells through formation of pores or other membrane destabilizing stmctures.
  • Unilamellar vesicles were prepared by the reverse-phase evaporation using DPPC, OPPC, cholesterol in the ratio of 1 : 1 :2 in a solution containing 5(6)-carboxyfluorescein (20 mM in phosphate/saline buffer: 137 mM NaCl, 2.6 mM KC1, 6.4 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , pH 6.5) according to the method of F. Szoka et al.
  • the ring stmcture of this peptide is N- methylated on one face. Such N-methylation does not adversely affect the ability of the peptide to interact with liposomal membranes but predisposes peptides toward a dimeric cylindrical stmcture that cannot span a normal liposomal membrane. Thus, although the peptide has been shown to partition effectively into liposomes, it does not promote proton transport activity in the
  • the present invention is directed to methods of treating cancer in an animal, for example, for human and veterinary uses, which include administering to a subject animal (e.g., a human), a therapeutically effective amount of a cyclic peptide of the present invention. While not intending to be bound by any particular theory or mechanism of action, according to the present invention, it is believed that the cyclic peptide undergoes self-assembly to form a supramolecular stmcture that causes cancer cellular membrane permeation, destabilization or depolarization.
  • Treatment of, or treating, cancer is intended to include the alleviation of or diminishment of at least one symptom typically associated with the disease. The treatment also includes alleviation or diminishment of more than one symptom.
  • the treatment may cure the cancer, e.g., it may substantially kill the cancer cells and/or it may arrest or inhibit the growth of the cancerous tumor.
  • Cancers that can be treated by the present cyclic peptides include solid mammalian tumors as well as hematological malignancies.
  • Solid mammalian tumors include cancers of the head and neck, lung, mesothelioma, mediastinum, esophagus, stomach, pancreas, hepatobiliary system, small intestine, colon, colorectal, rectum, anus, kidney, urethra, bladder, prostate, urethra, penis, testis, gynecological organs, ovaries, breast, endocrine system, skin central nervous system; sarcomas of the soft tissue and bone; and melanoma of cutaneous and intraocular origin.
  • Hematological malignancies include childhood leukemia and lymphomas, Hodgkin's disease, lymphomas of lymphocytic and cutaneous origin, acute and chronic leukemia, plasma cell neoplasm and cancers associated with AIDS.
  • a cancer at any stage of progression can be treated, such as primary, metastatic, and recurrent cancers.
  • Anti-cancer activity can be evaluated against varieties of cancers using methods available to one of skill in the art. Anti-cancer activity, for example, is determined by identifying the lethal dose (LD 10 o) or the 50% effective dose (ED 50 ) or the dose that inhibits growth at 50% (GI 50 ) of a cyclic peptide of the present invention that prevents the growth of a cancer. In one aspect, anti- cancer activity is the amount of the peptide that kills 50% or 100% of the cancer cells, for example, when measured using standard dose response methods.
  • the present invention also provides a method of evaluating a therapeutically effective dosage for treating a cancer with a cyclic peptide having an amino acid sequence of alternating D- and L-amino acids, or of three to about ten homochiral ⁇ -amino acids, that includes determining the LD 100 or ED 50 of the cyclic peptide in vitro.
  • a method permits calculation of the approximate amount of cyclic peptide needed per volume to inhibit cancer cell growth or to kill 50%> to 100% of the cancer cells. Such amounts can be determined, for example, by standard microdilution methods.
  • the cyclic peptides provided herein do not have substantial or undesired toxicity against normal mammalian or other animal cells.
  • hemolysis is one way to measure whether a cyclic peptide can self-assemble within normal mammalian or other animal cell membranes. If a cyclic peptide can self-assemble within a membrane, the membrane will tend to become depolarized and permeabilized. Red blood cells are conveniently used to test for membrane depolarization and permeabilization, because they undergo hemolysis, which can be detected as the release of hemoglobin from the cell. Hemolysis can be observed by methods available to one of skill in the art. For example, after exposure to the present cyclic peptides, the release of hemoglobin can be observed spectrophotometrically by observing the
  • Control samples can be used, for example, the medium in which the cells are tested or maintained can serve as a zero blank.
  • a second control can be used to determine the absorbance value for 100% hemolysis.
  • Such a second control can be a sample that is identical to the test animal cell sample but which has been sonicated to completely dismpt the cells. Screening Methods
  • Assays may be used to identify cyclic peptides that can selectively interact with a cancer cell of interest.
  • a wide variety of assays may be used for this purpose. See, for example, the assays carried out within the National Cancer Institute's "In Vitro Cell Line Screening Project.”
  • such an assay can involve contacting a cancer cell of interest with at least one cyclic peptide and observing whether the cyclic peptide kills the cancer cell and/or has other deleterious effects upon that cell.
  • Methods available in the art can also be used for determining whether the cyclic peptides of the invention interact with the membrane of a cancer cell of interest.
  • cyclic peptides can be labeled with a reporter molecule that permits detection of the peptide.
  • the cyclic peptides can be contacted with the cancer cell of interest for a time and under conditions that permit binding or association of the peptide to cellular membranes.
  • the cells can be washed with physiological solutions to remove unbound or unassociated cyclic peptides, and the cells can then be observed to ascertain whether the reporter molecule is bound or associated with the cells or the cellular membranes.
  • one of skill in the art can test whether the cyclic peptide(s) can selectively penetrate the membranes of selected cancer cells. This may be done by examining whether the reporter molecule remains associated with the cellular membranes of the cancer cell or whether the reporter molecule becomes associated with the interior of the cell.
  • Reporter molecules that can be employed include any detectable compound or molecule available to one of skill in the art that is conjugated directly or indirectly to a cyclic peptide of the invention.
  • the label may itself be
  • Client Reference 893.1 PCT detectable e.g., radioisotope labels or fluorescent labels
  • PCT detectable e.g., radioisotope labels or fluorescent labels
  • an enzymatic label may catalyze chemical alteration of a substrate compound or composition that is detectable.
  • Deleterious effects upon the cancer cell of interest can also be detected as an indication of an interaction between a cyclic peptide of the invention and the cell. Such deleterious effects can involve any evidence that the cyclic peptide has had an adverse or cytotoxic effect upon the cell. For example, one of skill in the art can test whether the cyclic peptide(s) kill the cancer cell, cause membrane depolarization, cause permeabilization of the membranes of the cell, or tend to lyse the cancer cells.
  • cyclic peptides that have little interaction with, and low toxicity for, normal human or other animal cells but that have good anti-cancer properties (depolarizing or permeabilizing cancer cell membranes, lysing or otherwise killing the cancer cell).
  • pluralities of assays are performed in parallel with different cyclic peptides at different concentrations to obtain a differential response to the various concentrations.
  • at least one control assay is included in the testing.
  • Such a control can be a negative control involving exposure of the cancer cells of interest to a physiologic solution containing no cyclic peptide.
  • Another control can involve exposure of the cancer cell of interest to a cyclic peptide that has already been observed to adversely affect the cancer cell of interest, or a second cell that is related to the cell of interest.
  • Another control can involve exposing a cell of interest to a known therapeutic agent that has a desired affect on the cancer cell of interest, for example, an anti-cancer agent with known efficacy at a particular concentration or dosage.
  • a known therapeutic agent that has a desired affect on the cancer cell of interest, for example, an anti-cancer agent with known efficacy at a particular concentration or dosage.
  • One of skill in the art can readily select control compounds and conditions that facilitate screening and analysis of the effects of the cyclic peptides on a cancer cell of interest.
  • cyclic peptides are obtained from a wide variety of sources including libraries of cyclic peptides generated as described herein. Cyclic peptides can also be rationally designed and synthesized to have specific structural features selected by one of skill in the art.
  • any cell type available to one of skill in the art can be assayed by these methods.
  • any mammalian or other animal cancer cell type can be screened to assess whether the cyclic peptides of the invention can selectively interact therewith.
  • Mammalian or other animal cells can also be screened to ascertain whether the peptides of the invention selectively interact therewith and/or to determine whether the peptides of the invention do not interact, bind, lyse, kill or otherwise adversely affect the viability of the mammalian or other animal cell.
  • mammalian or other animal red blood cells are screened with the cyclic peptides to ascertain whether the cyclic peptides have an adverse effect on the red blood cells.
  • the membrane of red blood cells tends to be more susceptible to lysis than many other mammalian or other animal cell types.
  • red blood cells are a useful cell type for quickly screening whether a cyclic peptide would be expected to have any adverse effects on other mammalian or other animal cell types and/or in vivo after therapeutic administration. Methods of assaying for cell lysis are available in the art.
  • red blood cells can be tested to ascertain whether hemolysis has occurred upon exposure to at least one cyclic peptide of the invention.
  • the peptide is tested against other mammalian or other animal cell types or used for in vivo testing in standard animal models.
  • Conditions for screening cyclic peptides include conditions that are used by one of skill in the art to grow, maintain or otherwise culture cell types of interest. Cancer cell types of interest should be assayed under conditions where they would be healthy but for the presence of the cyclic peptide(s). Controls can be performed where the cell types are maintained under the selected culture conditions and not exposed to a cyclic peptide, to assess whether the culture conditions influenced the viability of the cells.
  • One of skill in the art can also perform the assay on cells that have been washed in simple physiological
  • Client Reference 893.1 PCT solutions such as buffered saline, to eliminate, or test for, any interaction between the cyclic peptides or cells and the components in the culture media.
  • culture conditions for the assays generally include providing the cells with the appropriate concentration of nutrients, physiological salts, buffers and other components typically used to culture or maintain cells of the selected type.
  • a variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, albumin, and serum (e.g. fetal calf serum) that are used to mimic the physiologic state of the cell types of interest. Conditions and media for culturing, growing and maintaining cells are available to one of skill in the art.
  • the selected reagents and components are added to the assay in the order selected by one of skill in the art.
  • the cyclic peptides are added last to start the assay.
  • Assays are performed at any suitable temperature, typically between 4 °C and 40° C.
  • the temperature may generally range from about room temperature (about 20 °C) to about 37°C.
  • Incubation periods are selected to ascertain the optimal range of activity or to insure that the cyclic peptides do not adversely affect the cell type of interest.
  • incubation times can be optimized to facilitate rapid high-throughput screening. Typically incubation times are between about one minute and about five days, such as a range from about 30 minutes to about 3 days.
  • Cyclic peptides having the desired selectivity and activity in vitro may be tested for activity and/or lack of toxicity in vivo, in an appropriate animal model.
  • animal models include primates as well as mice, rats, rabbits, cats, dogs, pigs, goats, cattle or horses.
  • the mouse is a convenient animal model for testing whether cyclic peptides of the invention have toxic effects and/or to determine whether the cyclic peptides can inhibit the growth of a cancer cell.
  • cyclic peptides of the invention can readily perform in vivo evaluation of the cyclic peptides of the invention. For toxicity testing, a series of cyclic peptides at different test dosages can be separately administered to different animals. A single dose or, a series of dosages can be administered to the animal. A test
  • Client Reference 893.1 PCT period is selected that permits assessment of the effects of the peptide(s) on the animal.
  • Such a test period can mn from about one day to about several weeks or months.
  • the effect of a cyclic peptide(s) on an animal can be determined by observing whether the peptide adversely affects the behavior (e.g., lethargy, hyperactivity) and physiological state of the animal over the course of test period.
  • the physiological state of the animal can be assessed by standard procedures. For example, during the test period one of skill in the art can draw blood and collect other bodily fluids to test, for example, for various enzymes, proteins, metabolites, and the like.
  • One of skill in the art can also observe whether the animal has bloating, loss of appetite, diarrhea, vomiting, blood in the urine, loss of consciousness, and a variety of other physiological problems.
  • the animal can be sacrificed and anatomical, pathological, histological and other studies can be performed on the tissues or organs of the animal.
  • mice are infected with the selected cancer and a selected test dosage of one or more cyclic peptides is administered shortly thereafter. Mice are observed over the course of several days to several weeks to ascertain whether the cyclic peptide protects the mice from the cancer. At the end of the test period, mice can be sacrificed and examined to ascertain whether the cyclic peptide has optimally protected the mice from cancer and/or to determine whether any adverse side effects have occurred.
  • Controls are used to establish the effects of the cancer when the cyclic peptide is not administered. Other controls can also be performed, for example, the safety and efficacy of the present cyclic peptides can be compared to that of known anti-cancer agents.
  • the invention further provides a method of discovering a cyclic peptide capable of selective association with a target biomolecule on a selected cell surface. This method can involve contacting a solution of cyclic peptides with the target biomolecule and determining whether
  • the target biomolecule can be displayed, for example, on the surface of a living cell or on the surface of a liposome.
  • the peptide can be contacted with the target biomolecule in a hydrogen bond-promoting solution, such as an aqueous solution at a pH of about 7.0 to about 7.6 where simple salts may be present in physiological concentrations.
  • the method can further include determining the stmcture of the peptides that spontaneously assemble into the supramolecular structure that selectively associates with the biomolecule.
  • Cyclic peptides having good anti-cancer properties in vitro and/or in vivo that also have substantially no toxicity are good candidates for therapeutic development into appropriate dosage forms, as described in more detail below.
  • Dosages, Formulations and Routes of Administration for the Peptides are administered so as to achieve a reduction in at least one symptom associated with a cancer, tumor, indication or disease, or a decrease in the amount of antibody associated with the cancer, tumor, indication or disease.
  • the peptide, a variant thereof or a combination thereof may be administered as single or divided dosages, for example, of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages may provide beneficial results.
  • the amount administered will vary depending on various factors including, but not limited to, the cyclic peptide chosen, the disease, the weight, the physical condition, the health, the age of the mammal, whether prevention or treatment is to be achieved, and if the peptide is chemically modified. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.
  • Administration of the therapeutic agents in accordance with the present invention may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the peptides of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • peptides are synthesized or otherwise obtained, purified as necessary or desired and then lyophilized and stabilized. The peptide can then be adjusted to the appropriate concentration, and optionally combined with other agents.
  • the absolute weight of a given peptide included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least one peptide of the invention, or a plurality of peptides specific for a particular cell type can be administered.
  • the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.
  • Daily doses of the cyclic peptides of the invention can vary as well.
  • Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day.
  • one or more suitable unit dosage forms comprising the therapeutic peptides of the invention can be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes.
  • the therapeutic peptides may also be formulated for sustained release (for example, using microencapsulation, see WO 94/ 07529, and U.S. Patent No.4,962,091).
  • the formulations may, where appropriate, be
  • Client Reference 893.1 PCT conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • the therapeutic peptides of the invention are prepared for oral administration, they are generally combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • a pharmaceutically acceptable carrier diluent or excipient
  • the peptides may be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum.
  • the active peptides may also be presented as a bolus, electuary or paste.
  • Orally administered therapeutic peptides of the invention can also be formulated for sustained release, e.g., the peptides can be coated, micro-encapsulated, or otherwise placed within a sustained delivery device.
  • the total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.
  • pharmaceutically acceptable it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious or unsuitably harmful to the recipient thereof.
  • compositions containing the therapeutic peptides of the invention can be prepared by procedures known in the art using well-known and readily available ingredients.
  • the peptide can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like.
  • excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives.
  • Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone.
  • Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium
  • Client Reference 893.1 PCT carbonate and sodium bicarbonate can also be included such as paraffin.
  • Resorption accelerators such as quaternary ammonium compounds can also be included.
  • Surface active agents such as cetyl alcohol and glycerol monostearate can be included.
  • Adsorptive carriers such as kaolin and bentonite can be added.
  • Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added.
  • the compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives.
  • tablets or caplets containing the cyclic peptides of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate.
  • Caplets and tablets can also include inactive ingredients such as cellulose, pre-gelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, com starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like.
  • Hard or soft gelatin capsules containing at least one cyclic peptide of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil.
  • enteric-coated caplets or tablets containing one or more peptides of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.
  • the therapeutic peptides of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes.
  • the pharmaceutical formulations of the therapeutic peptides of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.
  • the therapeutic peptides may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form.
  • the active peptides and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active peptides and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water
  • formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol,” polyglycols and polyethylene glycols, C1-C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol,” isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.
  • organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol,” polygly
  • antioxidants chosen from antioxidants, surfactants, other preservatives, film- forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings.
  • Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and ⁇ -tocopherol and its derivatives can be added.
  • combination products that include one or more cyclic peptides of the present invention and one or more other anti-cancer agents.
  • antibiotics can be included in the pharmaceutical compositions of the invention, such as aminoglycosides (e.g., streptomycin, gentamicin, sisomicin, tobramycin and amicacin), ansamycins (e.g. rifamycin), antimycotics (e.g. polyenes and benzofuran derivatives), ⁇ -lactams (e.g.
  • PCT penicillins and cephalosporins
  • chloramphenical including thiamphenol and azidamphenicol
  • linosamides lincomycin, clindamycin
  • macrolides erythromycin, oleandomycin, spiramycin
  • polymyxins bacitracins
  • tyrothycin capreomycin
  • vancomycin vancomycin
  • tetracyclines including oxytetracycline, minocycline, doxycycline
  • phosphomycin and fusidic acid including oxytetracycline, minocycline, doxycycline
  • the peptides are well suited to formulation as sustained release dosage forms and the like.
  • the formulations can be so constituted that they release the active peptide, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time.
  • Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, draining devices and the like.
  • the therapeutic agents may be formulated as is known in the art for direct application to a target area.
  • Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap.
  • Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols.
  • the therapeutic peptides of the invention can be delivered via patches or bandages for dermal administration.
  • the peptide can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer.
  • an adhesive polymer such as polyacrylate or acrylate/vinyl acetate copolymer.
  • the backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns.
  • Client Reference 893.1 PCT Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • the active peptides can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051,842.
  • the percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1 -85% by weight.
  • Drops such as eye drops or nose drops, may be formulated with one or more of the therapeutic peptides in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents.
  • Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
  • the therapeutic peptide may further be formulated for topical administration in the mouth or throat.
  • the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier.
  • the pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art.
  • pharmaceutically acceptable carriers such as physiologically buffered saline solutions and water.
  • diluents such as phosphate buffered saline solutions pH 7.0-8.0.
  • the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention.
  • dosage forms comprise an amount of at least one of the agents of the invention effective to treat or prevent the clinical symptoms of a specific infection, indication or disease. Any statistically significant attenuation of one or more symptoms of an infection, indication or disease that has been treated pursuant to the method of the present invention is considered to be a treatment of such infection, indication or disease within the scope of the invention.
  • the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator, or a metered-dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung. Clarke, S. W. and Davia, D. eds., pp.
  • MDI pressurized metered dose inhaler
  • the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung. Clarke, S. W. and Davia, D. eds., pp.
  • Therapeutic peptides of the present invention can also be administered in an aqueous solution when administered in an aerosol or inhaled form.
  • other aerosol pharmaceutical formulations may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/ml and about 100 mg/ml of one or more of the peptides of the present invention specific for the indication or disease to be treated.
  • Dry aerosol in the form of finely divided solid peptide or nucleic acid particles that are not dissolved or suspended in a liquid are also useful in the practice of the present invention.
  • Peptides of the present invention may be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 ⁇ m, alternatively between 2 and 3 ⁇ m. Finely divided particles may be prepared by pulverization and screen filtration using techniques
  • the particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder.
  • a predetermined quantity of the finely divided material which can be in the form of a powder.
  • the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units.
  • the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.
  • the therapeutic peptides of the invention are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Nebulizers include, but are not limited to, those described in U.S. Patent Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp.
  • the therapeutic agent may also be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).
  • the active ingredients may also be used in combination with other therapeutic agents, for example, pain relievers, anti-inflammatory agents, antihistamines, bronchodilators and the like, whether for the conditions described or some other condition.
  • the present invention further pertains to a packaged pharmaceutical composition for controlling cancer such as a kit or other container.
  • a packaged pharmaceutical composition for controlling cancer such as a kit or other container.
  • the kit or container holds a therapeutically effective amount of a pharmaceutical
  • Client Reference 893.1 PCT composition for controlling cancer and instmctions for using the pharmaceutical composition for control of a given cancer type.
  • the pharmaceutical composition includes at least one cyclic peptide of the present invention, in a therapeutically effective amount such that cancer is controlled.
  • Solvents and reagents Acetonitrile (ACN, optima grade), dichloromethane (DCM, ACS grade), N,N-dimethylformamide (DMF, sequencing grade), diethyl ether (Et 2 O, ACS grade), N,N-diisopropylethylamine (DIEA, peptide synthesis grade) were purchased from Fisher and used without further purification.
  • ACN Acetonitrile
  • DCM dichloromethane
  • DMF N,N-dimethylformamide
  • Et 2 O diethyl ether
  • DIEA N,N-diisopropylethylamine
  • Trifluoroacetic acid (TFA, New Jersey Halocarbon), 2-(lH- benzotriazol- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU, Richelieu Biotechnologies), benzotriazole- 1 -yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP, Novabiochem) were used as obtained.
  • Commercially available amino acids and resins were used as obtained from Bachem, Novabiochem or Advanced Chemtech.
  • the side-chain protections were as follows. For Fmoc synthesis: Arg (Pbf), His (Boc), Lys (Boc), Ser (t-Bu) and Thr (t-Bu). All other chemicals were used as obtained from Aldrich, Acros, Sigma or Fluka.
  • Cyclization was performed in DMF at a peptide concentration of 1-5 mM using a mixture of PyBOP (5 eq. with respect to cmde peptide) and DIEA (40 eq.). The amount of DIEA was adjusted to achieve an apparent pH 9-10, which was assessed by applying a drop of reaction mixture to a wet pH paper. Reaction was followed by MALDI-MS and HPLC and in most cases it was complete in less than 2 hours. Then DMF was removed by evaporation under vacuum (1 mm Hg) at temperatures less that 30 °C and the residue was dried under vacuum (0.1 mm Hg) overnight.
  • the dried cmde peptide was dissolved in a mixture of TFA/PhOH/H 2 O/thioanisole/EDT/TIS (81.5:5:5:2.5:1) (about 100 mL/g of peptide) at room temperature for 1-3 hours. The completion of the reaction was followed by HPLC and MALDI-MS. The TFA solution was concentrated by 5 times by evaporation under vacuum (1 mm Hg), from which the peptide was precipitated by adding it to an ice-cold Et 2 O. The purity of dried cmde peptides was assessed by HPLC and MALDI-MS.
  • the cmde peptides can be partially purified by dissolving in boiling ACN/water/HCl mixture (30/70/0.1) and cooling the turbid solution in a fridge. In case of peptide high solubility in this mixture, the precipitate can be obtained by adding
  • N-Boc- ⁇ -Fmoc-glutamic acid was loaded onto methyl- benzhydrylamine (MBHA) resin through its side chain carboxylate, then the resin was split into four equimolar fractions of 0.25 mmol each for the rest of the synthesis.
  • MBHA methyl- benzhydrylamine
  • the specified amino acids of the peptide library sequences showing the greatest biological activity were retained for the generation of the next set of libraries.
  • Subsequent generations of peptide libraries were synthesized in a similar fashion, with the splitting of the resin after the coupling of the specified amino acids determined from the previous generation.
  • the peptides of the combinatorial libraries were identified by electrospray-mass spectrometry (ES- MS) or MALDI-TOF mass spectrometry. When a peptide library, peptide pool or cmde preparation of a peptide showed activity, individual peptides were re-synthesized, HPLC-purified and tested again for activity.
  • a cmde preparation of a peptide having a low minimum inhibitory concentration value cyc/ ⁇ [/)-Arg-J-Gln-Z -Arg-E-Trp- E>-Trp-J-Leu-Trp-J-Trp] (SEQ ID NO: 10)
  • SEQ ID NO: 10 a cmde preparation of a peptide having a low minimum inhibitory concentration value, cyc/ ⁇ [/)-Arg-J-Gln-Z -Arg-E-Trp- E>-Trp-J-Leu-Trp-J-Trp]
  • Cyclic D, J- ⁇ -peptide combinatorial libraries were prepared using a one- bead-one-compound strategy on macrobeads by a split and pool method. See, K.S. Lam, M. Lebl, V. Krchnak, "The One-bead-one-compound' combinatorial library method," Chem. Rev. 1997, 97, 411-448. Each bead contained a single sequence and was dispersed into microtiter plates using a density of one bead per well. Cleavage of the peptide from a single bead provided about 70-80 ⁇ g of peptide per well. This amount of peptide could be used for approximately 100 in vitro anti-microbial assays. Mass spectrometric peptide sequencing strategies
  • Client Reference 893.1 PCT were used for rapid identification of selected peptide species within a given library.
  • Solid-phase peptide synthesis was performed on polystyrene macrobeads functionalized with a TFA-labile trityl linker, which considerably facilitated the synthesis, handling, solid-phase cyclization, and final side chain deprotection and peptide isolation.
  • the growing peptide chain was linked through the first amino acid side chain (for example, lysine or histidine) to the trityl moiety allowing for selective "head-to-tail" cyclization of the completed peptide sequence on solid support.
  • the ⁇ -carboxyl group of the first N- ⁇ -Fmoc amino acid was protected as an allyl ester.
  • Resin loading and peptide chain elongation was performed under standard Fmoc solid phase peptide synthesis conditions using chlorotrityl polystyrene macrobead resin (500-560 urn, Peptides International) as the solid support, with HBTU as a coupling reagent and 20% piperidine in DMF for Fmoc deprotection. After completion of the final amino acid coupling, the resin was exposed to palladium tetrakis(triphenylphosphine) and N-methyl morpholine to remove the C-terminal allyl protecting group.
  • N-Fmoc amino acids for solid-phase peptide synthesis and trityl chloride PS 1% DVB, substitution 0.5 - 1.05 mmol g _1 ) resin were used as obtained from Novabiochem or Bachem.
  • Trityl chloride macrobead resin was obtained from Peptides International.
  • Fmoc-Lysine(Boc)-Oallyl Fmoc-Lysine(Boc)-OAllyl was made according to the protocol of Kates, S. A.; Sole, N. A.; Johnson, C. R.; Hudson, D.; Barany, G.; Albericio, F. Tetrahedron Lett. 1993, 34, 1549-1552.
  • Fmoc-Lys-(Boc)-OH (5 g, 10.6 mmol) was added to allyl bromide (25 mL, 0.29 mol), followed by DIPEA (3.73 mL). This mixture was heated at 90 °C for 1 h.
  • Trityl chloride resin was swollen in dry deacidified (Na 2 CO 3 ) dichloromethane for 20 min. A solution of Fmoc-Lys-O Allyl in dichloromethane was added to the resin, immediately followed by 4 eq. of DIPEA. After shaking for 2 hours the resin was washed resin with dichloromethane, then shaken with 10% MeOH: 10% DIPEA: 80% dichloromethane for 10 min. After washing with dichloromethane and drying in
  • Peptide Synthesis Peptides were synthesized using standard solid-phase Fmoc protocols (see Wellings, D. A.; Atherton, E. Methods Enzymol 1997, 289, 44-67) on the Fmoc-Lys-OAllyl loaded trityl resin. Following synthesis of the linear peptide the resin was swollen in dry dichloromethane for 20 min. To the resin was added a degassed solution of 0.5 eq. Pd(PPh 3 ) 4 in 90% CHC1 3 : 10% 4- mefhylmorpholine.
  • Client Reference 893.1 PCT well After the determined exposure time to peptide, media was removed and 90 ⁇ l of HBS buffer was added. 10 ⁇ l of trypan blue was then added to each well. After 10 min, the cells were counted under a light microscope and the percent viable cells in each well was recorded. LC 50 values were determined from this percentage.
  • a peptide having SEQ ID NO: 12 showed toxicity against these HeLa cells at concentrations above 1.5 ⁇ g/ml. This peptide is currently in animal testing phase for antimicrobial activity and shows very low in vivo toxicity.
  • MTT was added to the cell media, and the cells were incubated for four hours. They were then washed and the insoluble dye resulting from any MTT reduced by the cells was dissolved in DMSO. The optical density of the cells was then measured (560 nm). This optical density was proportional to the population of living cells in a sample. The levels of toxicity against the HeLa cell line using the MTT assay was similar to the trypan blue exclusion assay (1.5-3 ⁇ g/ml).
  • Peptide SEQ ID NO: 12 was then screened against a panel of 60 human tumor cell lines. Cells were exposed to peptide for a 48 hour period and cell viability was estimated using a sulforhodamine B assay. For additional details concerning this screen see the following Examples and the website at http://dtp.nci.hih.gov. The peptide was tested at five 10-fold dilutions and percent growth was calculated relative to control cells that received no peptide (growth of control cells for 48 hours was taken as 100% growth). Results of this assay indicated that peptide SEQ ID NO: 12 showed strong activity against two leukemia cell lines (HL-60 and MOLT-4). The data are summarized in Figures 9-15 and Table 7 below.
  • This in vitro anti-tumor screen consisted of 60 human tumor cell lines against which peptide SEQ ID NO: 12 was tested at a minimum of five concentrations using 10-fold dilutions. A 48 hour continuous dmg exposure protocol was used, and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The data are summarized in Figures 9-15.
  • Cyclic peptides were evaluated for anti-cancer activity against breast cancer, lung cancer, and CNS tumor cell lines maintained by The Developmental Therapeutics Program of the National Cancer Institute (NCI). Cyclic peptides were tested at 20 ug/mL against three cell lines as described in the website at dtp.nci.nih.gov/branches/btb/ivclsp.html. Cell lines tested were MCF7 (tumor type: breast), NCI-H460 (tumor type: non-small cell lung), and SF-268 (tumor type: CNS).
  • the cyclic peptides tested are set forth in Table 8, including 22 synthesized cyclic D,L-alpha peptides, 11 peptides having from four to six beta amino acids, 160 peptides from library Hexal (cmde), and 160 peptides from library Octal (cmde).
  • the Hexal cyclic peptides included peptides having the sequence cyclic[KYZZYX] wherein X represents amino acids Lys, His, Arg, Ser, Asn, Glu; Y represents amino acids Lys, His, Arg, Ser, Asn, Glu, Trp, Leu; Z represents amino acids Trp, Leu; K is Lys; and underlined amino acids are D- amino acids while non-underlined amino acids are L-amino acids.
  • the Octal cyclic peptides included peptides having the sequence cyclic[KXYZZZYX] wherein X represents amino acids Lys, His, Arg, Ser, Asn, Glu; Y represents amino acids Lys, His, Arg, Ser, Asn, Glu, T ⁇ , Leu; Z represents amino acids T ⁇ , Leu; and K is Lys.
  • Cmde Hexal and Octal library materials were used for testing. Cyclic peptides from the Hexal library having the least target activity in this assay ([KHKWWW] and [NKHLLL] or [HKNLLL]) were not re- synthesized or purified for additional activity testing.
  • Client Reference 893.1 PCT Table 9 shows the sequences of submitted cyclic peptides having cancer cell cytotoxicity in the above-described assay for at least one of three cell lines at 20 ug/mL.
  • a shorthand for peptide sequences was employed: the one-letter amino acid symbol was used but underlining indicates that the amino acid is a D- amino acid; non-underlining indicates the amino acid is a L-amino acid.
  • jS-amino acids are present a lower case "h" is used before the one letter amino acid symbol for the related ⁇ -amino acid.
  • the MCF-7 cell line was most sensitive to the tested peptides (12 peptides having target activity in this assay).
  • the NCI-H460 and SF-268 cell lines were less sensitive (4 and 5 peptides having target activity in this assay against these lines, respectively).
  • Of the 13 cyclic peptides in only one case was there target activity with the NCI-H460 cell line without corresponding target activity in the MCF-7 cell line. In no cases was there target activity against SF- 268 without corresponding activity in the MCF-7 cell line.
  • One cyclic peptide was included in the list of hits that showed a growth of 35% for MCF7, just over the target 32% toxicity for this experiment. Of the cyclic non-D,L-alpha peptides tested, two were active below 32% growth for at least one cell line in this experiment.
  • the peptides tested were differentially active against the MCF7 (tumor type: breast), NCI-H460 (tumor type: non-small cell lung), and SF-268 (tumor type: CNS) cell lines.
  • MCF7 tumor type: breast
  • NCI-H460 tumor type: non-small cell lung
  • SF-268 tumor type: CNS
  • PCT h represents the beta analog of the parent amino acid
  • the counts of individual hydrophilic residues were normalized relative to the total count of all the hydrophilic residues in the cyclic peptides that showed target activity in this assay and the total submitted cyclic peptides, respectively. Differences in percent representation in the cyclic peptides having target activity in this assay and the total cyclic peptides submitted highlight differences in cytotoxicity selection. Serine residues were represented more heavily in the cyclic peptides having cancer cell target activity in this assay (20%) than in the total tested cyclic peptides (13%). Lysine residues were slightly more
  • Client Reference 893.1 PCT represented in the cyclic peptides having target activity in this assay than in the initial distribution (54%> v 52%). Conversely, His and Arg were less represented in the cyclic peptides having target activity in this assay (His: 15% v 19%, Arg: 7% v 11%) (Table 12).
  • Table 15 provides the percentage growth of breast, non-small cell lung and CNS cancer lines in the presence of 20 ug/mL cyclic D,L-beta peptides.
  • the "h” indicates that the amino acid is a beta amino acid.
  • beta-peptides c-[hK-hK-hW-hL-hW-hL-] and c-[hR-hK-hK-hL-hL-hW-] (sequences cyclic [/3-Lys- /3-Lys- /3-T ⁇ - /3-Leu- /3-T ⁇ - /3-Leu] or cyclic [/3-Arg- /3-Lys- /3-Lys- /3-Leu- /3-Leu -/3-T ⁇ ], respectively) are highly effective at suppressing cancer cell growth.
  • the peptides included the following eleven cyclic peptides and one linear peptide: [KSKKLWLW] (SEQ ID NO: 149), [KSSSKWLW] (SEQ ID NO:91), [KHKHFLWL] (SEQ ID NO:72), [KHKLFLAL] (SEQ ID NO:47), [RHKHRWLW] (SEQ ID NO: 151), [KWKWSWLW] (SEQ ID NO: 107), [SEKHKLWW] (SEQ ID NO: 157), [KKKRHLWL] (SEQ ID NO: 158), -KSKWLWLW-(linear) (SEQ ID NO:159), [KRKWLW] (SEQ ID NO: 125), [KSKWLW] (SEQ ID NO: 126), and [KSKWLWLW] (SEQ ID NO: 12).
  • Human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5%> fetal bovine semm and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated into 96 well microtiter
  • the plates were incubated for an additional 48 h at 37°C, 5 % CO 2 , 95 % air, and 100 % relative humidity.
  • the assay was terminated by the addition of cold TCA.
  • Cells were fixed in situ by the gentle addition of 50 ⁇ l of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with tap water and air dried.
  • Sulforhodamine B (SRB) solution 100 ⁇ l
  • 0.4 % (w/v) in 1 % acetic acid was added to each well, and plates are incubated for 10 minutes at room temperature.
  • the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 ⁇ l of 80 % TCA (final concentration, 16 % TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth
  • GI50 Growth inhibition of 50 %
  • GI50 dmg concentration resulting in a 50%> reduction in the net protein increase (as measured by SRB staining) in control cells during the dmg incubation.
  • Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested. Further procedures can be found at the website at dtp.nci.nih.gov/branches/btb/ivclsp.html.
  • Cell membrane selectivity has also been assessed for various peptides of the invention to ascertain whether the peptides would show antimicrobial activity against prokaryotic cellular membranes as opposed to eukaryotic cellular membranes.
  • PCT Antimicrobial activity can be determined using a broth dilution assay essentially as descried in the guidelines of the National Committee for the Control of Laboratory Standards (NCCLS) [National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Fourth edition. Approved Standard (1997). Document M7-A4. (NCCLS, Nillanova, Pennsylvania, 1997].
  • Test tubes macrodilution method
  • microtiter plates microdilution method containing two-fold serial dilution of peptides were inoculated with various bacterial cultures. Controls included non-inoculated medium (sterility), vehicle control, and various commercially available antibiotics for which minimal inhibitory concentrations against tested organisms were known.
  • Table 16 Several of the strains of bacteria tested are described in Table 16.
  • VanB Gram Vancomycin resistant
  • VanA Vancomycin resistant
  • Escherichia coli EL744 K12 Gram Lacks key efflux pump component (tolC::kan) negative
  • MBC Minimum bactericidal concentrations
  • NCCLS National Committee for Clinical Laboratory Standards. Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved Standard (1999). Document M26-A. (NCCLS, Villanova, Pennsylvania, 1999). Fifty ⁇ l aliquots from MIC, 2xMIC and 4xMIC assay wells were removed and plated in antibiotic-free agar plates by using the lawning technique. Growth and sterility controls were sampled in the same manner. The lawned plates were incubated for 24-48 hours and the MBC was determined as the lowest concentration at which 99.9% killing was achieved.
  • Red blood cells were then treated with serial dilutions of test peptides in a 96 well plate at 37 °C for 30 minutes.
  • Control samples included a saline solution and 1 %> Triton X-100 as 0 and 100 % hemolysis, respectively.
  • mellitin a linear peptide that is hemolytic against mammalian red blood cells in vitro at a concentration of about 10 ⁇ g/ml was used as a further control.
  • Client Reference 893.1 PCT were centrifuged at 1000 g for 10 minutes. Aliquots of the supernatant were diluted 2 times with saline solution and the absorbance was measured at 560 nm.
  • Stock peptide solutions for in vivo experiments were prepared in aqueous sucrose (9%). To facilitate dissolution of the peptide, the initial suspension was sonicated for 15-20 min. The obtained solution was sterilized by passage through a sterile 0.45 ⁇ m filter (COSTAR, ⁇ Star, Coming Inc.).
  • concentrations of various peptide stock solutions were determined by quantitative HPLC analysis using known concentrations of internal standards. Further the peptide solutions were appropriately diluted with sterile aq. sucrose (9%>).
  • S. aureus MRS A bacteria (ATCC 33591) were grown at 37 °C in 5 mL of Antibiotic Medium-3 (AM-3, Difco Laboratories) with agitation for 12 hours to a stationary phase. Cells were collected by centrifugation, washed twice with saline solution and resuspended in about 10 mL of saline to an O.D. 650 of 1.2. This suspension was diluted ten times in a sterilized 5% mucin (Difco) in saline to a concentration of 2-
  • Vancomycin resistant Enterococcus faecium (VREF) bacteria ATCC 515705 were grown at 37 °C in 60 mL of Brain Heart Infusion medium (BHI, Difco Laboratories) with agitation for 16 hours to a stationary phase. Cells were collected by centrifugation, washed twice with saline solution and resuspended in 6 mL of saline to a theoretical O.D. 650 of 9-10 (determined by taking OD. 650 of 10-20 fold diluted samples and correlating it to the original concentration).
  • BHI Brain Heart Infusion medium
  • This suspension was diluted 15 times in a sterilized 5%> mucin (Difco) in saline to a concentration of 5-7x10 8 cfu/ml (actual inoculum size was verified by colony counts on agar plates).
  • the selectivity of different cyclic peptides for different cell membranes is illustrated in this Example by in vitro antibacterial assays against a variety of bacteria.
  • the first cyclic peptide tested had sequence Lys-D-Gln-Arg-D-T ⁇ -Leu- D-T ⁇ -Leu-D-T ⁇ (SEQ ID NO:9).
  • This cyclic peptide displayed potent activity against gram positive Bacillus subtilis and Staphylococcus aureus as well as against gram negative Streptococcus pneumonieae and vancomycin-resistant Enterococcus faecalis (Table 17).
  • Cyclic peptides with SEQ ID NO: 5 and 6 each bear one basic residue between two serine residues, or between a serine and a threonine residue, respectively.
  • Peptide SEQ ID NO: 5 displayed good activity against S. aureus MRS A, while substitution of lysine by histidine in peptide SEQ ID NO:6 decreases the activity.
  • Cyclic peptides with SEQ ID NO: 8- 15 each possess two basic amino acids and one neutral, polar amino acid. These cyclic peptides varied in activity and red blood cell hemolysis. A single glutamic acid substitution affected activity as indicated by the difference in activity against S.
  • aureus MRSA bacteria between peptide SEQ ID NOs: 14 and 16.
  • Increasing the number of basic residues from two to three in peptide SEQ ID NOs: 17-22 provided significant activity against S. aureus MRSA.
  • Peptides with SEQ ED NO: 18 and SEQ ID NO:21 also exhibited activities against E. coli.
  • the in vitro antibacterial activities of hexameric peptides with SEQ ID NOs:26-29 also indicate that use of basic amino acids in the cyclic peptides may increase activity and improve
  • Peptide SEQ ID NO:26 with two consecutive lysine residues, exhibits broad-spectrum activity and has low hemolytic properties.
  • peptide SEQ ID NO:27 which has a histidine instead of the lysine in peptide SEQ ID NO:26, retains high activity against S. aureus MRSA but not against E. coli.
  • Substituting a lysine in peptide SEQ ID NO:26 with serine yields a less active peptide SEQ ID NO:28.
  • peptide SEQ ID NO:29 which possesses two arginine residues, displays potent and selective activity against E. coli with low levels of hemolysis.
  • the spectrum of activity and membrane selectivity observed with the above peptides indicates that single amino acid substitutions can be utilized to influence activity and target cell selectivity.
  • Results for further testing of gram-negative and gram-positive bacteria are shown in Tables 20 and 21 respectively, along with control assays using FDA approved antibiotics. Underlining indicates that amino acid is a D-amino acid residue and brackets indicate that the peptides are circular.
  • cyclic peptides of the invention The selectivity of different types of cyclic peptides of the invention is further illustrated in Table 22, where underlining indicates which amino acids have D-chirality, brackets identify cyclic peptides and a number in parentheses indicates the SEQ ID NO:. These peptides were tested against methicillin- resistant Staphylococcus aureus (MRSA)(ATCC 33591) and vancomycin- resistant Enterococcus faecalis (VRE)(ATCC 51575) and for hemolytic activity against mammalian red blood cells.
  • MRSA methicillin- resistant Staphylococcus aureus
  • VRE vancomycin- resistant Enterococcus faecalis
  • VRE Enterococcus faecalis
  • Cyclic peptides having SEQ ID NO: 13, 18, 26, and 29 were also assayed for proteolytic susceptibility.
  • the peptides have an abiotic structure and conformational preferences, and were stable in the presence of trypsin, - chymotrypsin, subtilisin, and blood plasma. No significant peptide degradation was observed in chromatograms obtained from RP-HPLC over a 24 h time period, whereas control linear L- ⁇ -amino acid peptides were degraded in less than 10 min under similar reaction conditions and within four hours when placed in murine blood plasma.
  • cyclic peptides may kill or affect target cells.
  • Another mechanism by which the cyclic peptides may kill or affect cells is through a receptor that recognizes the cyclic peptides as ligands. See Friederich et al., Antimicrob. Agents Chemother. 44, 2086-2092(2000); Amsterdam, D. in Antibiotics in Laboratory Medicine, 3rd ed. (ed. Lorian, V.) 53-105 (Baltimore, Maryland, USA, 1991), although this mode of action for the cyclic peptides in membranes is less likely for several reasons.
  • cyclic peptides with activity is illustrated by enantiomeric peptides having SEQ ID NO:8 (cyclo-[D-Lys-Gln-D-Arg-Trp-D- Leu-Trp-D-Leu-T ]) and SEQ ID NO:9 (cyclo-[Lys-D-Gln-Arg-D-Trp-Leu-D- Trp-Leu-D-Trp]). These two cyclic peptides have similar in vitro activities despite the differences in chirality of these peptides at each position.
  • Biophysical analyses performed in synthetic lipid membranes also support a membrane permeation mode of action.
  • the peptide SEQ ED NO:2 (cyclo[- Gln-D-Leu-(Trp-D-Leu) ]) facilitates only the transport of analytes that are smaller than its internal diameter across liposome membranes and according to ATR-FTIR analysis in DMPC multibilayes, assembles into a tube-like structure that is oriented perpendicular to the membrane plane.
  • the single nanotube through-pore mechanism it is a likely mode of function for this peptide (see Figure 2a).
  • the homologous peptide SEQ ID NO:3 (cyclo[-Lys-D-Leu-(Trp-D-Leu) j), having a polar charged side chain likely forms a different supramolecular structure with an opening that is larger than the internal tube diameter because it facilitates transport of larger molecules of up to approximately 10,000 MW across membranes.
  • the supramolecular structure formed from peptides having SEQ ID NO:3 (cyclo[-Lys-D-Leu-(Trp-
  • Lipid and peptide nanotube tilt angles are calculated according to methods detailed in H.-S. Kim et al., 120 J. Am. Chem. Soc, 4417-24 (1998).
  • DMPC dimyristoyl phosphatidylcholine. Data are average of two samples with errors ⁇ 2°.
  • FIG. 1 provides a thin section electron microscopy image of untreated S. aureus (ATCC 25923) displaying a normal intact membrane.
  • Figures 7 and 8 provide thin section electron microscopy images of S. aureus (ATCC 25923) after exposure to 2XMIC concentrations of cyclo[KSKWLWLW]. These images provide direct visualization of a membrane mode of action. Arrows denote abnormal membrane structures caused by the peptide action.
  • cytotoxicity is based at least in part on membrane permeation, depolarization, and/or lysis.
  • Such evidence includes the observation that the cyclic peptides act very quickly to kill microbes, that diverse cyclic peptide structures described herein show anti-cancer and antimicrobial activity, that the cyclic peptides can depolarize microbial membranes, that attenuated total reflectance (ATR) FT-IR spectroscopy studies are consistent with a membrane permeation mechanism of action rather than a receptor/ligand- mediated binding/inhibition mechanism, and that electron microscopy reveals an effect of the cyclic peptides of the invention on membrane structure.
  • ATR attenuated total reflectance
  • mice Initial toxicology studies in mice were conducted to evaluate various routes of drug administration, maximum tolerable dose, and blood and tissue toxicity.
  • mice Male Balb-C in groups of 4-8 were given via IV, D? or SQ single bolus doses of peptides and monitored for 14 days. Toxicity of sub-lethal doses was assessed based on the behavior and appearance of the mice after peptide administration compared to control mice receiving only vehicle. Signs of acute toxicity included lack of activity, red feet and tail, faster breathing. Death was defined as the end point for lethal doses of peptides.
  • Peptides of the invention were tested in vivo in mice to evaluate protection against bacterial infection using procedures similar to those described in N. Frimodt-M Her et al., The Mouse Peritonitis/Sepsis Model, in Zak et al. (eds.) HANDBOOK OF ANIMAL MODELS OF INFECTION 127-37 (Academic Press 1999). Male Balb-C mice (6 weeks old, approximately 20 g) were used in the study. Staphylococcus aureus MRSA bacteria (ATCC 33591) were grown at 37 °C with agitation for 12 hours to a stationary phase. Cells were collected by centrifugation, washed twice with saline and resuspended to an O.D.
  • Plasma from each blood sample was separated immediately after collection by spinning down red blood cells over a period of 5 min at 4000 rev./min.
  • the plasma was diluted with an equal volume of saline and refrigerated until analysis. Storage of samples under these conditions over a period of 1 month did not change the concentration of peptide.
  • solutions of peptide in 9% sucrose were injected IP into Balb/C mice at a dose of 100 mg/kg. Blood was collected immediately from one group of mice (3 mice per group) by bleeding the tail, separately from each mouse (50-100 uL per mouse).
  • a solution of peptide in 9% sucrose (2 mg/mL) was injected IN into the tail vein of Balb/C mice at a dose of 5 mg/kg.
  • Blood was then collected immediately from one group of mice (3 mice per group) by bleeding the tail, separately from each mouse (50-100 uL per mouse).
  • Blood was also collected from other groups of mice at times 30, 60, 120, 230, and 300 min after injection.
  • Plasma from each blood sample was separated immediately after collection by spinning down red blood cells over a period of 5 min at 4000 rev./min. Plasma was then diluted with an equal volume of saline and refrigerated until analysis.
  • Plasma was then diluted with an equal volume of saline and refrigerated until analysis. Storage at these conditions over a period of 1 month did not change the concentration of peptide.
  • Detection of c(KSKWLWLW) HCl in plasma by HPLC was accomplished as follows. Samples were diluted with saline plasma (50-100 uL), added to an equal volume of eluent A (0.1 % TFA in 96.5% H 2 O/0.9% AC ⁇ /2.4% MeOH (v/v)), vortexed, and partial precipitation was removed by centrifugation.
  • the clear solution was injected into HPLC and the peptide was detected at 280 nm, in the 18-20 min interval, using a gradient of eluent A (0.1 % TFA in 96.5% H 2 O/0.9% AC ⁇ /2.4% MeOH (v/v)) and eluent B (0.05 % TFA in 8% H 2 O/72% AC ⁇ /20% MeOH (v/v)) using a flow rate of 1.5
  • mice in each dose group were injected with a bolus intravenous (IV), intraperitoneal (IP), or subcutaneous (SQ) doses of peptide SEQ ID NO:8, 18, and 26.
  • IV intravenous
  • IP intraperitoneal
  • SQL subcutaneous
  • Client Reference 893.1 PCT NO:8 indicated that bolus IV injections of 12 mg/kg caused signs of temporary ( ⁇ 10 min in duration) discomfort. In contrast, no signs of toxicity were observed at the maximum tested dose (12 mg/kg) with the IP route of administration. Peptide SEQ ID NO:26 was tolerated up to the maximum tested IP dose (50 mg/kg), with the highest dose causing signs of acute toxicity that were not observable after one hour. Signs of acute toxicity included any one of the following: lack of activity, red feet and tail, or faster breathing. Peptide SEQ ID NO: 18 was tested up to 17.5 mg/kg IP and SQ doses without any apparent signs of toxicity.
  • a regimen of 17.5 mg per day of peptide SEQ ID NO:18 for three consecutive days was administered to two mice IP and two mice SQ. Over four days, no apparent changes in the physical, social, and feeding activities were observed in these mice relative to control mice injected with vehicle without peptide. Moreover, hematology, necropsy and detailed microscopic examination of various tissue and organ samples performed on the fourth day after the initial injection showed normal blood and morpho logical profiles except for the sites of IP and SQ injections (K.G. Osborn, DVM, Ph.D., the Scripps Research Institute). These sites exhibited moderate subacute inflammation typical of IP and SQ drug administration.
  • Peptide SEQ ID NO: 18 was tested for antibacterial activity in vivo by observing whether this peptide could protect mice from bacterial infection.
  • Two groups of mice (4 mice per dose in each group) were infected intraperitoneally (left side) with a lethal dose of MRSA (ATCC 33591) (2-5xl0 7 cfu mouse).
  • peptide bolus doses of 0 (vehicle only), 10, 20, and 40 mg/kg were administered subcutaneously (SQ) in the upper neck compartment soon after MRSA injection.
  • mice To the second group of mice, five bolus peptide doses each 0 (vehicle only), 2.5, and 5 mg/kg were administered intravenously (IV) in 10 hour intervals with the first series of doses administered soon after MRSA injection. All mice in the control groups that received vehicle without peptide (0 mg/kg doses) died within 48 hours. However, at 40 mg/kg bolus SQ dose and 2.5 x 5 mg/kg IV dosage regimen, 75% and 50% of mice survived, respectively, during the course of a fourteen-day study. There was some
  • a larger study with peptides having SEQ ID NO: 8, 12, 17, 18, and 26 was also performed using groups of mice infected with lethal doses of MRSA (ATCC 33591) (IP left side). Each group of mice was treated with a bolus IP (right side) dose of peptide SEQ ID NO: 8, 12, 17, 18, or 26 at 45-60 min after initial infection and observed for up to 14 days. All mice in the control group that received vehicle without peptide died within 48 hours.
  • mice were also infected with lethal doses of VREF (ATCC 51575) (LP left side). Each group of mice was treated with a bolus IP (right side) dose of peptide SEQ ID NO: 12, 17, or 18 at 45-60 min after initial infection and observed for 14 days. All mice in the control group that received vehicle without peptide died within 48 hours. In each case, a single dose of the appropriate amount of peptide was sufficient to completely protect various groups of mice from VFEF infections (Table 24).

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Abstract

L'invention se rapporte à de nouveaux types d'agents anticancéreux et à leur procédés d'utilisation.
PCT/US2003/014373 2002-05-06 2003-05-06 Agents anticancereux de peptides cycliques et leurs procedes d'utilisation WO2003092632A2 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
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US7425535B2 (en) 2000-05-09 2008-09-16 Ghc Research Development Corporation Therapeutic pore-forming peptides
US7456146B2 (en) * 2001-05-09 2008-11-25 Ghc Research Development Corporation Lytic peptide prodrugs
WO2010051667A1 (fr) * 2008-11-10 2010-05-14 复旦大学 Compositions pharmaceutiques comprenant des nanotubes de peptide cyclique et utilisations de celles-ci
ES2342874A1 (es) * 2010-02-19 2010-07-15 Fundacio Privada Institut D'investigacio Biomedica De Girona Dr. Josep Trueta Peptidos ciclicos inhibidores del crecimiento celular.
WO2013025099A1 (fr) * 2011-08-17 2013-02-21 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg Substrats pour la détection de périopathogènes de la cavité buccale
WO2014035345A1 (fr) * 2012-08-29 2014-03-06 Agency For Science, Technology And Research Peptides et utilisations associées
JP2016516752A (ja) * 2013-04-02 2016-06-09 ザ スクリプス リサーチ インスティテュート アテローム性動脈硬化症の治療および予防のための環状ペプチドの使用
CN106699841A (zh) * 2017-01-05 2017-05-24 北京大学深圳研究生院 一种自组装的多肽纳米棒及其制备方法
US10456443B2 (en) 2014-08-27 2019-10-29 Ohio State Innovation Foundation Peptidyl calcineurin inhibitors
US10626147B2 (en) 2014-05-21 2020-04-21 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
US10815276B2 (en) 2014-05-21 2020-10-27 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
US11168310B2 (en) 2018-02-22 2021-11-09 Entrada Therapeutics, Inc. Compositions and methods for treating mitochondrial neurogastrointestinal encephalopathy
US11576946B2 (en) 2018-01-29 2023-02-14 Ohio State Innovation Foundation Peptidyl inhibitors of calcineurin-NFAT interaction
US11987647B2 (en) 2018-05-09 2024-05-21 Ohio State Innovation Foundation Cyclic cell-penetrating peptides with one or more hydrophobic residues

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US6096707A (en) * 1997-07-11 2000-08-01 Biotie Therapies Ltd. Integrin binding peptide and use thereof
US6277818B1 (en) * 1998-10-29 2001-08-21 Angstrom Pharmaceuticals, Inc. Cyclic peptide ligands that target urokinase plasminogen activator receptor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6096707A (en) * 1997-07-11 2000-08-01 Biotie Therapies Ltd. Integrin binding peptide and use thereof
US6277818B1 (en) * 1998-10-29 2001-08-21 Angstrom Pharmaceuticals, Inc. Cyclic peptide ligands that target urokinase plasminogen activator receptor

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7425535B2 (en) 2000-05-09 2008-09-16 Ghc Research Development Corporation Therapeutic pore-forming peptides
US7456146B2 (en) * 2001-05-09 2008-11-25 Ghc Research Development Corporation Lytic peptide prodrugs
WO2010051667A1 (fr) * 2008-11-10 2010-05-14 复旦大学 Compositions pharmaceutiques comprenant des nanotubes de peptide cyclique et utilisations de celles-ci
ES2342874A1 (es) * 2010-02-19 2010-07-15 Fundacio Privada Institut D'investigacio Biomedica De Girona Dr. Josep Trueta Peptidos ciclicos inhibidores del crecimiento celular.
WO2011101510A1 (fr) * 2010-02-19 2011-08-25 Universitat De Girona Peptides cycliques inhibiteurs de la croissance cellulaire
WO2013025099A1 (fr) * 2011-08-17 2013-02-21 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg Substrats pour la détection de périopathogènes de la cavité buccale
WO2014035345A1 (fr) * 2012-08-29 2014-03-06 Agency For Science, Technology And Research Peptides et utilisations associées
CN104583229A (zh) * 2012-08-29 2015-04-29 新加坡科技研究局 肽及其应用
US9221875B2 (en) 2012-08-29 2015-12-29 Agency For Science, Technology And Research Amphiphilic peptides for treatment of keratitis
US9273096B2 (en) 2012-08-29 2016-03-01 Agency For Science, Technology And Research Amphiphilic peptides comprising the formula I: (X1Y1X2Y2)n, and uses thereof
JP2016516752A (ja) * 2013-04-02 2016-06-09 ザ スクリプス リサーチ インスティテュート アテローム性動脈硬化症の治療および予防のための環状ペプチドの使用
EP2981277A4 (fr) * 2013-04-02 2017-03-08 The Scripps Research Institute Utilisations de peptides cycliques pour traiter et prévenir l'athérosclérose
US10626147B2 (en) 2014-05-21 2020-04-21 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
US10815276B2 (en) 2014-05-21 2020-10-27 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
US11225506B2 (en) 2014-05-21 2022-01-18 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
US10456443B2 (en) 2014-08-27 2019-10-29 Ohio State Innovation Foundation Peptidyl calcineurin inhibitors
CN106699841A (zh) * 2017-01-05 2017-05-24 北京大学深圳研究生院 一种自组装的多肽纳米棒及其制备方法
US11576946B2 (en) 2018-01-29 2023-02-14 Ohio State Innovation Foundation Peptidyl inhibitors of calcineurin-NFAT interaction
US11168310B2 (en) 2018-02-22 2021-11-09 Entrada Therapeutics, Inc. Compositions and methods for treating mitochondrial neurogastrointestinal encephalopathy
US11987821B2 (en) 2018-02-22 2024-05-21 Entrada Therapeutics, Inc. Compositions and methods for treating mitochondrial neurogastrointestinal encephalopathy
US11987647B2 (en) 2018-05-09 2024-05-21 Ohio State Innovation Foundation Cyclic cell-penetrating peptides with one or more hydrophobic residues

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