WO1995027497A1 - Methode permettant de combattre la neoplasie mammalienne et peptides lytiques utilises dans ladite methode - Google Patents

Methode permettant de combattre la neoplasie mammalienne et peptides lytiques utilises dans ladite methode Download PDF

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Publication number
WO1995027497A1
WO1995027497A1 PCT/US1995/004335 US9504335W WO9527497A1 WO 1995027497 A1 WO1995027497 A1 WO 1995027497A1 US 9504335 W US9504335 W US 9504335W WO 9527497 A1 WO9527497 A1 WO 9527497A1
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peptide
lytic peptide
lytic
peptides
naturally occurring
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PCT/US1995/004335
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English (en)
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Jesse M. Jaynes
Gordon R. Julian
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Demeter Biotechnologies, Ltd.
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Priority to AU22815/95A priority Critical patent/AU2281595A/en
Publication of WO1995027497A1 publication Critical patent/WO1995027497A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees

Definitions

  • the present invention relates to a method of combating mammalian neoplasias, including specifically female mammalian neoplasias, and to non-naturally lytic peptides and their use in such method.
  • Naturally occurring amphipathic peptides play an important if not critical role as immunological agents in insects and have some, albeit secondary, defense functions in a range of other animals.
  • the function of these peptides is to destroy prokaryotic and other non-host cells by disrupting the cell membrane and promoting cell lysis.
  • Common features of these naturally occurring amphipathic, lytic peptides include an overall basic charge, a small size (23-39 amino acid residues), and the ability to form amphipathic ⁇ -helices.
  • Several types of amphipathic lytic peptides have been identified: cecropins (described in U.S. Patents 4,355,104 and 4,520,016 to Hultmark et al .
  • lytic peptides disruption of the membrane lipid bilayer by the amphipathic ⁇ -helix portion of the lytic peptide; lytic peptide formation of ion channels, which results in osmotically induced cytolysis; lytic peptide promotion of protein aggregation, which results in ion channel formation; and lytic peptide-induced release of phospholipids.
  • an ordered secondary conformation such as an ⁇ -amphipathic helix and positive charge density are features that appear to participate in the function of the lytic peptides.
  • the specificity of the lytic action depends upon the sequence and structure of the peptide, the concentration of the peptide, and the type of membrane with which it interacts.
  • Jaynes et al . Peptide Research. 2: 157 (1989) discuss the altered cytoskeletal characteristics of transformed or neoplastic mammalian cells that make them susceptible to lysis by the peptides. In these experiments, normal, human non-transformed cells remained unaffected at a given peptide concentration while transformed cells were lysed. However, when normal cells were treated with the cytoskeletal inhibitors cytochalasin D or colchicine, sensitivity to lysis increased. The experiments show that the action of lytic peptides on normal mammalian cells is limited.
  • amphipathic peptide concentration can be manipulated to effect lysis of one cell type but not another at the same locus.
  • Synthetic peptide analogs can also act as agents of eukaryotic cell proliferation. Amphipathic peptides that promote lysis of transformed cells will, at lower concentrations, promote cell proliferation in some cell types.
  • the synthetic amphipathic peptide analogs typically contain as few as 15 and as many as 40 amino acid residues.
  • a phenylalanine residue is often present at the amino terminus of the protein to provide an aromatic moiety analogous to the tryptophan residue located near the amino terminus of natural cecropins, and a UV-absorbing moiety with which to monitor the purification of the synthetic peptide.
  • the basis for the design of these lytic peptide analogs is that an amphipathic peptide of minimal length and containing overall positive charge density effects lytic activity.
  • lytic peptides as a class may include species that are efficacious in destroying neoplastic cells, the concomitant mediative effect characteristic of lytic peptides, of promoting cell proliferation, would render such lytic peptides poor therapeutic agents for the treatment or prophylaxis of neoplastic conditions.
  • kidney and rental carcinoma increased by 15.2%; melanoma of the skin by 21.3%; multiple myeloma by 25.8%; non-Hodgkin's lymphoma by 26.5%; and cancer of the larynx by 39.2%.
  • lung cancer increased 18% in men, while in women, the increase was 118.3%.
  • the present invention relates generally to a method of combating, treating neoplastic conditions, comprising in vivo delivery to a corporeal neoplastic site of an effective amount of a non-naturally occurring, non-oncocytologically proliferative lytic peptide, and to peptides useful for such treatment, as hereinafter more fully described.
  • non-naturally occurring means that the peptide or other species referred to, is a product of human agency and not a wild or otherwise naturally existing peptide.
  • non-oncocytologically proliferative lytic peptide means a peptide which in vivo does not mediate or otherwise effect a net increase of more than 15% of the population of the neoplastic cells against which the peptide is lytically active.
  • the proliferative effect of lytic peptides on the neoplastic cells can be readily determined within the skill of the art by suitable cellular count assays, e.g., utilizing a cytometer and suitable flow cytometry methods, relative to a corresponding control group of neoplastic cells under the same environmental conditions.
  • amphipathic as used herein in reference to peptide species means that the peptides have hydrophobic amino acid side chains which are oriented on one face or conformational region of the peptide, e.g., on one face of an alpha helix in the case of amphipathic ⁇ -helices as the peptide conformational structure, while the hydrophilic amino acid side chains are oriented on the other face or conformational region of the peptide.
  • amphipathic refers to the distribution of hydrophobic and hydrophilic amino acid residues along opposing faces of the ⁇ -helix structure, which results in one face of the ⁇ -helix structure being predominantly hydrophobic and the other face being predominantly hydrophilic.
  • the degree of amphipathy of a peptide can be assessed by plotting the sequential amino acid residues on an Edmunson helical wheel.
  • the present invention relates to a method of treating neoplasias, comprising in vivo delivery of an effective amount of a non-naturally occurring, non-oncocytologically proliferative lytic peptide to a neoplastic locus.
  • the invention relates in a further aspect to an appertaining method of treating mammalian neoplasia disease states such as female cancers, including breast, ovarian, uterine, and cervical cancers, wherein a portion of the cells of the neoplastic locus are oncologically transformed and such peptide has a lytic effect on the transformed cells but not normal (i.e., non-oncologically transformed) cells.
  • mammalian neoplasia disease states such as female cancers, including breast, ovarian, uterine, and cervical cancers, wherein a portion of the cells of the neoplastic locus are oncologically transformed and such peptide has a lytic effect on the transformed cells but not normal (i.e., non-oncologically transformed) cells.
  • peptide as used herein is intended to be broadly construed as inclusive of polypeptides per se having molecular weights of up to 10,000 daltons, as well as proteins having molecular weights of greater that about 10,000 daltons, wherein the molecular weights are number average molecular weights.
  • methylated means that the specified amino groups have been chemically reacted by a method of reductive alkylation or methylation so that the associated hydrogen atoms are replaced by covalently coupled methyl groups.
  • glycolated means that the specified guanido and ⁇ -amino groups have been chemically reacted such that each is covalently coupled to a glyoxal group.
  • the terms “treating” or “combating” in reference to a physiological condition or disease state is intended to be broadly construed as comprehending treatment of an existing condition or disease state, for amelioration thereof, as well as prophylactic treatment for prevention or diminution of the potential severity of such condition or disease state.
  • Figure 1 is a graph of percentage survival of mouse melanoma cells as a function of time (days following tumor induction) for treatment of the melanoma cells with an amphipathic lytic peptide of Sequence ID No. 39 (as hereinafter described), DP-1, an amphipathic lytic peptide of Sequence ID No. 40 (as hereinafter described), DP-2, and a saline control (no lytic peptide treatment).
  • the present invention provides a method of treating neoplasias and other oncocytological disease states, such as breast, ovarian, uterine, cervical, prostatic, dermal, and bronchogenic cancers, kidney and renal carcinomas, multiple myeloma, non-Hodgkins lymphoma, and laryngeal cancer, that avoids many of the problems associated with current treatments and alternative approaches.
  • the method of the present invention involves the use of lytic peptides which are "cytocidal", i.e., killingly effective, against neoplastic (transformed, cancerous) cells, attacking and destroying such cells while being non-cytocidal against normal (untransformed, non-cancerous) cells.
  • the invention contemplates the in vivo treatment of oncological conditions as well as the in vitro usage of lytic peptides for assay or. analytical purposes.
  • the lytic peptides of the present invention may be delivered to the corporeal neoplastic site by any suitable method of delivery efficacious therefor.
  • the peptides delivered to the gastrointestinal locus may be "latently lytic", i.e., non-lytic prior to action at the neoplastic site, but activated in vivo, under local conditions at the neoplastic site, so as not to affect normal gastrointestinal flora or metabolic order.
  • the lytic peptides may be inherently stabilized proteolytically resistant (due to chemical modification) to accommodate oral delivery thereof.
  • Peptides delivered to a neoplastic locus in active lytic form may usefully exhibit broad spectrum lytic activity for lysing pathogenic bacteria and virally infected cells, as well as transformed neoplastic cells, and thereby effect an enhanced therapeutic result.
  • Lytic peptides of the present invention are useful in treating animals, e.g., mammals such as humans, for conditions in which amelioration of cytologically manifested or mediated conditions such as neoplasia is desired, and against which the peptides are lytically effective.
  • a method of producing an antineoplastic response in an animal subject in need of such treatment comprises administering to the animal subject an antineoplasia-inducing amount of a composition including an effective lytic peptide which is non-oncocytologically proliferative in character.
  • Subjects to be treated by the methods of the present invention include both human and non-human animal (e.g., bird, dog, cat, cow, horse) subjects, and are preferably mammalian subjects, and most preferably human subjects.
  • non-human animal e.g., bird, dog, cat, cow, horse
  • animal subjects may be administered therapeutic composition containing lytic peptides of the invention at any suitable therapeutically effective and safe dosage, as may readily be determined within the skill of the art, and without undue experimentation.
  • suitable therapeutic doses of the active lytic peptide agent, as delivered to the targeted corporeal site, and for achievement of therapeutic benefit will generally be in the range of 0.05 to 15 milligrams (mg) per kilogram body weight of the recipient per day, preferably in the range of 0.10 ⁇ g to 5.0 mg per kilogram body weight per day, and most preferably in the range of 0.5 ⁇ g to 2.5 mg per kilogram body weight per day.
  • the desired dose is preferably presented as two, three, four, five, six, or more sub-doses administered at appropriate intervals throughout the day.
  • These sub-doses may be administered in unit dosage forms, for example, containing from 0.01 to 1.5 mg, and preferably from 0.025 to 1.25 mg of active ingredient per unit dosage form.
  • the doses may be administered as a continuous infusion, via iv perfusion, osmotic pump or transdermal delivery techniques, or by direct injection into the tumor (neoplastic) site.
  • the mode of administration and dosage forms will of course affect the therapeutic amounts of the compounds which are desirable and efficacious for the given treatment application.
  • orally administered dosages typically are at least twice, e.g., 2-10 times, the dosage levels used in parenteral administration methods, for the same lytic peptide active ingredient.
  • Intrathecal administration dosage levels generally are on the order of about 10% of the levels characteristic of parenteral administration dosage levels .
  • the lytic peptide therapeutic agents of the invention are to be avoided, to maintain a conservative dosage level below the ALD iv of 40 milligrams per kilogram of body weight per day. It is noted that there is no determinate intramuscular approximate lethal dose (ALD im ).
  • the lytic peptide in a suitable formulation is administered by direct injection into the tumor, as a syringable composition efficacious for treatment of the neoplastic condition at the injection site.
  • other direct modes of administration may be employed.
  • iv perfusion may be used to introduce the lytic peptide to the neoplastic site.
  • as osmotic pump may be placed in the vicinity of, or within, the tumor mass, and arranged to selectively release the lytic peptide, on a continuous, or non-continuous basis, to the neoplastic site.
  • transdermal delivery means and method may be employed, and in the case of cutaneous or subcutaneous tumors such transdermal means as transdermal patches may be utilized to deliver the lytic peptide to the neoplastic site.
  • the present invention also contemplates pharmaceutical formulations, both for veterinary and for human medical use, which comprise as the active agent one or more lytic peptide(s) of the invention, as well as the use of a lytic peptide of the invention in the manufacture of a medicament for the treatment or prophylaxis of the neoplastic conditions and/or other disease states variously described herein.
  • the active agent preferably is utilized together with one or more pharmaceutically acceptable carrier(s) therefor and optionally any other therapeutic ingredients.
  • the carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof.
  • the active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.
  • the formulations include those suitable for parenteral as well as non-parenteral administration, and specific administration modalities include oral, rectal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal, intra-articular, intra-arterial, subarachnoid, bronchial, lymphatic, and intra-uterine administration.
  • Formulations suitable for parenteral administration are preferred.
  • the formulation advantageously may be administered parenterally.
  • the active agent when employed in a liquid suspension formulation or as a powder in a biocompatible carrier formulation, the formulation may be advantageously administered orally, rectally, or bronchially.
  • the active agent When the active agent is utilized directly in the form of a powdered solid, the active agent may advantageously administered orally. Alternatively, it may be administered bronchially, via nebulization of the powder in a carrier gas, to form a gaseous dispersion of the powder which is inspired by the patient from a breathing circuit comprising a suitable nebulizer device .
  • the active agent in a "vectorized” form, such as by encapsulation of the active agent in a liposome or other encapsulant medium, or by fixation of the active agent, e.g., by covalent bonding, chelation, or associative coordination, on a suitable biomolecule, such as those selected from proteins, lipoproteins, glycoproteins, and polysaccharides.
  • the formulations comprising the active agent of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the active compound(s) into association with a carrier which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the active compound(s) into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the active ingredient as a powder or granules; or a suspension in an aqueous liquor or a non-aqueous liquid, such as a syrup, an elixir, an emulsion, or a draught.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, with the active compound being in a free-flowing form such as a powder or granules which optionally is mixed with a binder, disintegrant, lubricant, inert diluent, surface active agent, or discharging agent.
  • Molded tablets comprised of a mixture of the powdered active compound with a suitable carrier may be made by molding in a suitable machine.
  • a syrup may be made by adding the active compound to a concentrated aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredient(s).
  • a sugar for example sucrose
  • Such accessory ingredient(s) may include flavorings, suitable preservative, agents to retard crystallization of the sugar, and agents to increase the solubility of any other ingredient, such as a polyhydroxy alcohol, for example glycerol or sorbitol.
  • Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound, which preferably is isotonic with the blood of the recipient (e.g., physiological saline solution).
  • Such formulations may include suspending agents and thickening agents and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose form.
  • Nasal spray formulations comprise purified aqueous solutions of the active compounds with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes.
  • Formulations for rectal administration may be presented as a suppository with a suitable carrier such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acids.
  • Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye.
  • Topical formulations comprise the active compound dissolved or suspended in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations .
  • Transdermal formulations may be prepared by incorporating the active agent in a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose, with the resulting formulation then being packed in a transdermal device adapted to be secured in dermal contact with the skin of a wearer.
  • a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose
  • the specific mode of delivery and administration of the lytic peptides of the present invention will depend on the specific corporeal site to be treated. In a given application, the optimum delivery route may differ depending on the form and stage of the cancer being treated.
  • a generally useful route is intravenous administration, which is the route by which most chemotherapeutic drugs are administered at present. This administration route may be usefully employed, for example, for treatment of breast or uterine cancer, however intra-arterial administration may afford advantage as well in treatment of such types of cancer.
  • Intra-arterial administration permits the introduction of the therapeutic agent(s) into the blood supply flowing directly to a tumor site, so that the agent(s) are delivered to the site prior to complete dilution in the total blood volume and prior to passage through the liver.
  • intraperitoneal administration may usefully be employed, since the ovaries reside in the peritoneal cavity and are exposed directly to the fluid therein.
  • direction injection into the uterine cervix may be usefully employed for delivery of the therapeutic agent(s), or alternatively a cervical cap used with a gel-form or other type composition containing the therapeutic agent(s) may be utilized.
  • formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavoring agents, binders, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.
  • accessory ingredient(s) selected from diluents, buffers, flavoring agents, binders, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.
  • lytic peptides as cancer drugs depends upon their unique mechanism of action. Normal mammalian cells are resistant to lysis (cell death) due to their highly organized and well-developed cytoskeletal system. Cancerous cells, whether actively growing or dormant, are known to possess an inferior and compromised structure which when acted upon by the lytic peptides causes them to swell and burst. Hence, the killing of cancer cells while leaving normal cells unharmed, in the practice of the present invention, forms the basis for new chemotherapeutic cancer agents against cancers resistant to other forms of treatment.
  • the lysis is achieved by the assemblage of protein structures (membrane attack complexes) responsible for the lytic lesions formed in the lipid bilayers of cells of inferior cytoskeletal structure, such as cancer cells.
  • the attack complex kills the cell by forming a tubule that traverses the membrane.
  • This hollow cylinder approximately 15 nm long and 10 nm in diameter, inserts its end into the lipid bilayer, and projects from it.
  • a structure of this form perturbs the lipid bilayer sufficiently to allow for the free exchange of electrolytes and water across the membrane.
  • the consequence of this action for a living cell is the net influx of sodium ions, calcium ions, and water due to the high internal colloid osmotic pressure. It is believed that one extremely small (10 nm) hole is sufficient to rapidly kill a single cell.
  • Example 1 The features and advantages of the invention are more fully shown by the following illustrative examples and embodiments, which are not to be limitingly construed as regards the broad scope, utility, and applicability of the invention.
  • Example 1 The features and advantages of the invention are more fully shown by the following illustrative examples and embodiments, which are not to be limitingly construed as regards the broad scope, utility, and applicability of the invention.
  • amphipathic peptide analogs of the present invention are the amino acid sequences of a family of related peptide analogs.
  • the peptides may be synthesized according to conventional methods using a MilligenTM solid phase peptide synthesizer.
  • Representative peptides from this group in some instances, are glyoxylated or methylated to stabilize same against proteolytic digestion, and used in subsequent experimental examples.
  • the three letter amino acid symbols are as follows: Ala, alanine; Arg, arginine; Asp, aspartate; Gly, glycine; Ile, isoleucine; Leu, leucine; Lys, lysine; Phe, phenylalanine; and Val, valine.
  • SEQ ID NO. 1-40 amphipathic peptide analogs are designated for ease of reference as SEQ ID NO. 1-40.
  • amphipathic peptide analogs offer certain advantages. If the modifications are made in such a way that the peptides retain all or most of their amphipathic characteristics, then the physiologically active peptides have enhanced stability to proteolysis. With enhanced stability, oral delivery of the peptide is advantageously accommodated without excessive loss of activity due to proteolytic digestion.
  • the stabilized lytic peptides of such type are suitably stabilized so as to remain resistant to proteolysis so that the peptide reaches the neoplastic locus in an active condition.
  • the lytic peptide may be originally synthesized in a stabilized form, or it may be chelated or otherwise coupled with an associated complexing agent so that the complexed peptide is initially non-lytically active in character, but under conditions existing at the neoplastic corporeal locus the complexed composition dissociates or otherwise "unbinds" to provide the lytically active peptide for antineoplastic activity at such locus.
  • Preferred antineoplastically effective lytic peptides within the general practice of the present invention include small (23-39 amino acid units) amphipathic cationic lytic peptides selected from those identified by amino acid sequence above which are non-oncocytologically proliferative in the specific neoplasia treatment application.
  • the antineoplastically effective peptides of the invention are synthetic amphipathic lytic peptide analogs of melittin, cecropin, magainin, and defensin peptides. Most preferred are synthetic amphipathic lytic peptide analogs of melittin and cecropin peptides.
  • the lytic peptides of the present invention achieve their antineoplastic effect by causing severe perturbation of the cellular membranes of neoplastic (malignant) cells.
  • the lytic peptides also exhibit concentration-dependent cell- proliferating, as well as neoplastic effects.
  • the therapeutically useful peptides of the present invention selected for antineoplastic treatment are chosen to be non-oncocytologically proliferative in character, as is readily determinable within the skill of the art for a given neoplasia treatment application. Alterations in peptide charge density, hydrophobicity or length may affect the neoplastic and proliferative character of the peptide.
  • the peptides of the invention have been designed to provide high antineoplastic activity and to minimize concentration-dependent proliferation of neoplastic cells.
  • lytic peptide concentrations on the order of 1 nM to 10 ⁇ M are usefully employed, particularly in the treatment of female mammalian neoplasias.
  • the preferred method for reductive alkylation uses pyridine borane as the reducing agent.
  • This reagent is one of a class of reducing agents known as amine boranes .
  • Pyridine borane exhibits a slightly higher reducing capacity than sodium cyanoborohydride, another reducing agent that can be used for the reductive alkylation.
  • Pyridine borane drives the reductive alkylation reaction to complete dimethylation with no monomethyl products when excess reagents are used, as demonstrated by Wong, W.S.D., et al . Analytical Biochemistry 139: 58 (1984).
  • the alkylation reagent may suitably comprise formaldehyde as a methyl group (methylation) precursor. Shown below are the agents of reductive alkylation, formaldehyde and pyridine borane, the substrate, peptidyl lysine, and the chemical formulae of the reaction scheme species.
  • the reaction residue was reconstituted to approximately 2.0 ml with 0.1 M acetic acid and applied to a 2.4 cm ⁇ 31 cm G-15-120 ⁇ SephadexTM column to purify the reaction product.
  • 0.1 M acetic acid was the elution reagent
  • 20 ml of eluate containing the product was collected and the eluate was lyophilized to dryness.
  • the peptides were stored at -20°C in the presence of a desiccant as their acetate salt.
  • a desiccant as their acetate salt.
  • they are dissolved in a saline buffer, pH 7.0, at a concentration of 0.1 mg/ml to 10 mg/ml.
  • reagents which are capable of modifying the guanido group arginine with glyoxal under mild conditions and do not require an additional reduction reaction are 2,3- butanedione, phenylglyoxal, and glyoxal.
  • the adducts from 2,3- butanedione and phenylglyoxal were judged to be too unstable, and glyoxal was therefore chosen as the preferred reagent for glyoxylation.
  • the agent of glyoxylation, glyoxal, the substrate, peptidyl arginine, and the chemical reaction scheme are described below.
  • the pyridine as a representative heterocyclic amine, was essential to the reaction, in order to maintain the glyoxal/peptide mixture in solution.
  • Other water-soluble dielectric solvents such as the heterocyclic amine piperidine were tested and can be used in the place of pyridine.
  • glacial acetic acid was added drop-wise to bring the pH to 6.0.
  • a two-phase extraction using three parts ether to one part acetone for the organic phase was repeated three times to remove the majority of the glyoxal.
  • the pyridine was not removed to a significant extent.
  • the preparation was dried in a lyophilizer and the crusty residue was rinsed with three parts ether to one part acetone.
  • the residual ether-acetone was removed in vacuo.
  • the cloudy ether-acetone supernatant was centrifuged to recover a precipitate which was pooled with the remaining residue by washing the tube with glacial acetic acid.
  • the peptides were stored at -20°C in the presence of a desiccant as their acetate salt.
  • a desiccant as their acetate salt.
  • they were dissolved in a saline buffer, pH 7.0 at a concentration of 0.1 mg/ml to 10 mg/ml.
  • Hecate-1 homologous to SEQ ID NO. 4
  • SEQ ID NO. 4 The effect of a lytic peptide was tested against antibiotic-resistant pathogenic bacteria in vitro.
  • antibiotic-resistant cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae were obtained from deceased patients.
  • the lytic peptide bioassay was performed as described below.
  • a flask containing 49 ml of nutrient broth was inoculated with 1 ml of an overnight culture of the test bacteria.
  • the culture was allowed to grow to mid-log phase at 37°C with shaking (approximately 4 hours).
  • the cells were transferred to a sterile tube and centrifuged for 10 minutes at 3000 rpm.
  • the pellet was resuspended in 3 ml of phosphate buffer and centrifuged for 10 minutes at 3000 rpm.
  • the pellet was resuspended once again in sufficient (but measured) volume to calculate the absorbence of the suspension at 600 nm. Using the resulting absorbence and a previously constructed growth curve, the required dilution to achieve a concentration of 10 6 cells/ml was determined.
  • test peptide was dissolved in 1.0 ml of 0.01 % acetic acid to make a 1 mM solution and serial dilutions were made to give a range of peptide concentrations from 10 ⁇ M to 1 mM.
  • the test culture tubes for the bioassay contained 800 ⁇ l of phosphate buffer, pH 7.0, 100 ⁇ l of cells at 10 6 cells/ml and 100 ⁇ l of peptide solution (10 ⁇ M to 1 mM). The final concentration of peptide in the assay was from 1 ⁇ M to 100 ⁇ M.
  • a reaction system minus peptide was included as a control. The tubes were incubated at 37°C for one hour.
  • a lytic peptide concentration in the range of 1 ⁇ m to 100 ⁇ M was effective for lysis of antibiotic resistant Pseudomonas aeruginosa and Klebsiella pneumoniae, most preferably in the range of 5 ⁇ M to 50 ⁇ M.
  • Pneumocystis carinii Hemophilus influenzae, Klebsiella pneumoniae, Chlamydia pneumoniae, and Pseudomonas cepacia are tested in the same bioassay for lytic activity.
  • Peptide concentration in the range of 1 ⁇ m to 100 ⁇ M is effective for lysis of the tested pathogenic bacteria, most preferably in the range of 5 ⁇ M to 50 ⁇ M. This concentration of peptide will be compared with the amount required to treat the pulmonary epithelial cells in a non-toxic manner in order to develop an effective combination dose for concurrent treatment of CF and accompanying bronchopulmonary infections, as well as other pulmonary diseases.
  • a lytic peptide and a chemically modified non-lytic peptide selected from the group shown in Table 1 are tested in vitro with normal and cystic fibrosis affected lung and gastrointestinal epithelial cells, and the cells are assayed for survival.
  • Cell culture is performed according to standard protocols (see for example Reed, W.A. et al . Molecular Reproduction and Development 31: 106 [1992]), and the cytotoxicity assay by 51 Cr release is performed as in Jaynes, J.M. et al . Peptide Research 2: 157 (1989).
  • This test shows a range of peptide concentration that is non-toxic for the cells in vitro.
  • the purpose of the experiment is to formulate a range of safe doses of peptide for in vitro and in vivo experiments. Peptide concentration above 100 ⁇ M to 500 ⁇ M is toxic for the epithelial cells.
  • a lytic peptide and a chemically modified non-lytic peptide selected from the group shown in Table 1 are tested in vitro for stimulation of chloride efflux with pulmonary and gastrointestinal epithelial cells, using a range of peptide concentration that is non-toxic to the cells as shown by the experiments in Example 5.
  • the peptides used in this experiment are chemically modified and non-lytic, for the gastrointestinal epithelial cells, and non-modified, lytic peptides for the pulmonary epithelial cells.
  • Peptide concentration in the range of 1 ⁇ M to 50 ⁇ M is effective for stimulating chloride efflux from pulmonary and gastrointestinal epithelial cells. Combining the results of the pathogenic bacterial lysis experiment, the epithelial cell toxicity experiment, and the stimulation of chloride efflux experiment yields the following conclusion: a peptide concentration corresponding to 1 ⁇ M to 50 ⁇ M is the preferred range for treatment of CF affected epithelial cells, microbial infections and other disease states in vitro.
  • mice that have bronchopulmonary infections of Mycobacterium tuberculosis, Pseudomonas aeruginosa, or Pseudomonas cepacia. Mice infected with both antibiotic-resistant and non-resistant bacteria are used, and treatment with antibiotics is compared to treatment with a lytic peptide.
  • a concentration of peptide in the range of 10 ⁇ g to 25 mg per kg body weight for the recipient per day is the preferred range for treatment.
  • the desired dose is preferably presented as two, three, four, five, six, or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example containing from 10 ⁇ g to 1000 mg, preferably from 50 ⁇ g to 500 mg, and most preferably from 50 ⁇ g to 250 mg of active ingredient per unit dosage form.
  • An advantageous modality of in vivo pulmonary delivery of the peptide is via a liquid nebulizer inhaler device or a dry powder nebulizer inhaler device, depending on the physical state, solubility, and dosage of the peptide.
  • Suitable nebulizers are commercially available under the trademarks "ROTAHALER”, “SPINHALER”, and "TURBOHALER”.
  • Another potentially suitable powder nebulizer apparatus and method of nebulization is disclosed in U.S. Patent No. 5,186,166 to Riggs et al.
  • the effect of a representative lytic, non-chemically modified peptide from Table 1 is tested on previously engineered transgenic mice that are homozygous for the CF defect.
  • the peptide is delivered to a pulmonary locus as described in Example 7.
  • a concentration of peptide in the range of 10 ⁇ g to 25 mg per kg body weight for the recipient per day is employed as the preferred range for treatment.
  • the desired dose is preferably presented as two, three, four, five, six, or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example containing from 10 ⁇ g to 1000 mg, preferably from 50 ⁇ g to 500 mg, and most preferably from 50 ⁇ g to 250 mg of active ingredient per unit dosage form.
  • the experiment shows that peptide in the preferred range of 10 ⁇ g to 25 mg per kg body weight for the recipient per day is effective for treatment of mice with bronchopulmonary infections.
  • the effect of a representative non-lytic, chemically modified peptide from Table 1 is tested on previously engineered transgenic mice that are homozygous for the CF defect, as described in Example 8.
  • the peptide is orally delivered to the gastrointestinal locus, and the chemical modification (glyoxylation or methylation) of the peptide confers enhanced proteolytic resistance, as described in Examples 2-3.
  • a concentration of peptide in the range of 10 ⁇ g to 25 mg per kg body weight for the recipient per day is utilized as a preferred range for treatment.
  • the desired dose is preferably presented as two, three, four, five, six, or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example containing from 10 ⁇ g to 1000 mg, preferably from 50 ⁇ g to 500 mg, and most preferably from 50 ⁇ g to 250 mg of active ingredient per unit dosage form.
  • the experiment shows that peptide in the preferred range of 10 ⁇ g to 25 mg per kg body weight for the recipient per day is effective for treatment of mice with gastrointestinal problems due to CF.
  • various compounds of the present invention having appertaining therapeutic ability may be usefully employed in the treatment of other pulmonary disease states including: various neoplasias, bronchogenic cancers, pneumonia, bronchitis, bronchopulmonary viral infections, and bronchopulmonary microbial infections.
  • the lytic peptides of the present invention may be usefully employed in the treatment of neoplasias as well as the concurrent treatment of other conditions for which the peptides are therapeutically useful, i.e., the administered peptide may effect neoplastic activity at the same time it is physiologically and/or pharmaceutically useful for the treatment of other conditions or disease states in the subject receiving treatment.
  • other conditions or disease states include microbial, parasitic and viral infections, dermal and subdermal wounds (as to which the peptide is healingly effective), etc.
  • the lytic peptides of the invention may suitably be utilized in combination with one another, as a mixture of selected different peptides, as well as in single peptide form.
  • the use of multiple lytic peptide species may for example be beneficial when the neoplastic site contains multiple tumors of differing type, as to which different peptides of the invention are therapeutically effective.
  • the peptide denoted DP-1 had the amino acid sequence identified hereinabove as SEQ. ID NO. 39 and the peptide denoted DP-2 had the amino acid sequence identified hereinabove as SEQ. ID NO. 40, and the control was saline.
  • mice Mouse melanoma cells were introduced onto the scapula of the test mice. Seventy two hours later, treatment was begun with ten mice in each treatment group. One group was administered DP-1 peptide, one group was administered DP-2 peptide, and the third group was administered saline control. Each treatment consisted of a 25 ⁇ g intraperitoneal injection of 0.1 ml volume once a day every other day for one week. Each mouse received four injections in total. As shown by the resulting data in Figure 1, while the DP-2 group and the control group both showed death of all animals after 33 days, the DP-1 group maintained a 62% survival rate through the 38 day study.
  • the peptide denoted DP-1 had the amino acid sequence identified hereinabove as SEQ. ID NO. 39
  • the peptide denoted DP-lm had the same amino acid sequence identified hereinabove as SEQ. ID NO. 39 but was fully methylated to stabilize same against proteolysis in accordance with the methylation procedure as described hereinabove
  • the peptide denoted D5-C had the amino acid sequence identified hereinabove as SEQ. ID NO. 9
  • the peptide denoted D5-F had the same amino acid sequence identified hereinabove as SEQ. ID NO. 12.
  • Riv iv administration in rat.
  • Rim intramuscular administration in rat
  • Miv iv administration in mouse
  • Mim intramuscular administration in mouse.
  • mice and rats were two replicates performed with both mice and rats.
  • the animals one animal/dose
  • the animals were exposed to a range of doses, 10.0, 5.0, 2.5,1.0, 0.5, 0.25, 0.1, 0.05, 0.025, and 0.01 ⁇ M.
  • the lethal dose was diluted to produce an intermediate dose between the lethal dose and the subsequent nonlethal dose.
  • mice were dead within four hours after treatment.
  • the lethality of the high dose was verified by dosing one animal with the lethal dose determined by Replicate 1.
  • the rats used for the evaluation were remaining from the original shipment and were naive for treatment.
  • the doses were the following: D5F, 10.0 ⁇ M; DP1, 10.0 ⁇ M; and DP1m, 10.0 ⁇ M.
  • the rats were deal within four hours after treatment.
  • Rats exposed to 2.5 and 5.0 ⁇ M D5F exhibited discoloration of the tail soon after dosing. In particular, the tails appeared to darken from the usual pink coloration to bluish purple. The appearance of the tail resembled a cyanotic condition. Later during the two-week postdosing period, the ends of the tails of the animals exposed to 2.5 and 5.0 ⁇ M of D5F sloughed off and the remaining tail portion exhibited a blue-black coloration.
  • D5F and DP1 two replicates were performed with rats.
  • the animals one animal/dose
  • the dose range used was smaller than that originally planned due to the availability of the compound to prepare an adequate amount of dosing solution.
  • DP-1 had the amino acid sequence identified hereinabove as SEQ. ID NO.
  • the peptide denoted DP-lm had the same amino acid sequence identified hereinabove as SEQ. ID NO. 39 but was fully methylated to stabilize same against proteolysis in accordance with the methylation procedure as described hereinabove
  • the peptide denoted D5-C had the amino acid sequence identified hereinabove as SEQ. ID NO. 9
  • the peptide denoted D5-F had the same amino acid sequence identified hereinabove as SEQ. ID NO. 12.
  • Various peptides having sequences shown in Table 1 were tested for antineoplastic activity.
  • the cells utilized in the testing with one exception were human clinical isolates (obtained at University of North Carolina Memorial Hospital, Chapel Hill, NC) and were derived from the following tumors: MG-63 - chemotherapy resistant osteosarcoma; T47D - hormone sensitive breast tumor; MDA-MB231 - hormone insensitive breast tumor; BT474 - hormone sensitive breast tumor; B16F1 - mouse melanoma; and KBATCC - nasopharyngeal carcinoma.
  • test procedure was a conventional MTT ID 50 assay.
  • Tests were done in quadruplicate and data were tabulated for the average ⁇ M concentration necessary to achieve the ID 50 (ranging from about 2 to about 20 ⁇ g/ml).
  • the present invention also contemplates pharmaceutical formulations for human medical use, which comprise as one of the active agents therapeutic amounts of the peptides of Table 1 above as well as other physiologically active compounds.
  • These formulations may for example include as additional components nebulizable compounds such as Survanta ® TA pulmonary surfactant (Burroughs Wellcome Co.), MucomistTM mucolytic agent (Mead-Johnson), RibavirinTM virazole (TCN Pharmaceuticals), and DNase (Genentech), as well as other physiologically active therapeutic agents such as antibiotics.

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Abstract

Méthode de traitement de néoplasies, dont les néoplasies mammaliennes de la femme telles que les néoplasies du sein, du col de l'utérus, de l'utérus et des ovaires, ainsi que d'autres néoplasies, dont les cancers de la prostate, de la peau et bronchopulmonaires, qui consiste à administrer un peptide lytique efficace, non naturel et non cytologiquement prolifératif sur un site approprié du corps pour traiter efficacement ces maladies. En particulier, les agents peptidiques lytiques préférés comprennent de petits (de 22 à 39 acides aminés) peptides lytiques cationiques amphipathiques de la classe des dérivés d'analogues synthétiques des peptides mélittine, cécropine, magainine et défensine, de préférence des peptides mélittine et défensine de la classe des analogues synthétiques des peptides mélittine, cécropine, magainine et défensine, de préférence encore des analogues synthétiques de peptides mélittine et défensine.
PCT/US1995/004335 1994-04-08 1995-04-06 Methode permettant de combattre la neoplasie mammalienne et peptides lytiques utilises dans ladite methode WO1995027497A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011700A1 (fr) * 1994-10-13 1996-04-25 Enzon, Inc. Utilisation de substances activatrices de la phospholipase a2 pour reduire les neoplasmes chez les mammiferes
WO1998006419A1 (fr) * 1996-08-15 1998-02-19 Southern Illinois University Facilitation de l'activite de peptides antimicrobiens au moyen d'ions metalliques
US6800727B2 (en) * 2001-09-19 2004-10-05 Chosun University Peptides with increased + charge and hydrophobicity by substituting one or more amino acids of CA-MA peptide and pharmaceutical compositions containing thereof
WO2007107748A2 (fr) * 2006-03-21 2007-09-27 Lytix Biopharma As Inhibition de la croissance tumorale
US8283315B2 (en) 1998-08-28 2012-10-09 Lytix Biopharma As Inhibition of tumour growth

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US4810777A (en) * 1987-03-04 1989-03-07 The United States Of America As Represented By The Department Of Health And Human Services Antimicrobial compounds
WO1990012866A1 (fr) * 1989-04-10 1990-11-01 Louisiana State University And Agricultural And Mechanical College Peptides lytiques, leur emploi pour la croissance, l'infection et le cancer

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US4810777A (en) * 1987-03-04 1989-03-07 The United States Of America As Represented By The Department Of Health And Human Services Antimicrobial compounds
WO1990012866A1 (fr) * 1989-04-10 1990-11-01 Louisiana State University And Agricultural And Mechanical College Peptides lytiques, leur emploi pour la croissance, l'infection et le cancer

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Title
BIOCHEMISTRY, Volume 7, Number 6, issued June 1968, MEANS et al., "Reductive Alkylation of Aminio Groups in Proteins", pages 2192-2201. *
DRUG NEWS AND PERSPECTIVES, Volume 3, Number 2, issued March 1990, JAYNES, "Lytic Peptides Portend an Innovative Age in the Management and Treatment of Human Disease", pages 69-78. *
JOURNAL OF PROTOZOOLOGY, Volume 38, Number 6, issued November-December 1991, ARROWOOD et al., "Hemolytic Properties of Lytic Peptides Active Against the Sporozoites of Cryptosporidium Parvum", pages 161S-163S. *
PEPTIDE RESEARCH, Volume 2, Number 2, issued 1989, JAYNES et al., "In Vitro Cytocidal Effect of Lytic Peptides on Several Transformed Mammalian Cell Lines", pages 157-160. *
PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, Volume 35, issued March 1994, MOORE et al., "Preliminary Experimental Anti-Cancer Activity of Cecropin B", page 410, Abstract No. 2444. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 243, Number 23, issued 10 December 1968, TAKAHASHI, "The Reaction of Phenylglyoxal with Arginine Residues in Proteins", pages 6171-6179. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011700A1 (fr) * 1994-10-13 1996-04-25 Enzon, Inc. Utilisation de substances activatrices de la phospholipase a2 pour reduire les neoplasmes chez les mammiferes
WO1998006419A1 (fr) * 1996-08-15 1998-02-19 Southern Illinois University Facilitation de l'activite de peptides antimicrobiens au moyen d'ions metalliques
US6042848A (en) * 1996-08-15 2000-03-28 The Board Of Trustees Of Southern Illinois University Enhancement of antimicrobial peptide activity by metal ions
US9109048B2 (en) 1998-08-21 2015-08-18 Lytix Biopharma As Inhibition of tumor growth
US8283315B2 (en) 1998-08-28 2012-10-09 Lytix Biopharma As Inhibition of tumour growth
US6800727B2 (en) * 2001-09-19 2004-10-05 Chosun University Peptides with increased + charge and hydrophobicity by substituting one or more amino acids of CA-MA peptide and pharmaceutical compositions containing thereof
WO2007107748A2 (fr) * 2006-03-21 2007-09-27 Lytix Biopharma As Inhibition de la croissance tumorale
WO2007107748A3 (fr) * 2006-03-21 2007-12-06 Lytix Biopharma As Inhibition de la croissance tumorale
JP2009530359A (ja) * 2006-03-21 2009-08-27 リティックス バイオファーマ エイエス 腫瘍成長の阻害

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