WO2016004067A1 - Formulations de peptide aromatique-cationique, compositions et procédés d'utilisation - Google Patents

Formulations de peptide aromatique-cationique, compositions et procédés d'utilisation Download PDF

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Publication number
WO2016004067A1
WO2016004067A1 PCT/US2015/038598 US2015038598W WO2016004067A1 WO 2016004067 A1 WO2016004067 A1 WO 2016004067A1 US 2015038598 W US2015038598 W US 2015038598W WO 2016004067 A1 WO2016004067 A1 WO 2016004067A1
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aromatic
peptide
phe
arg
lys
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PCT/US2015/038598
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English (en)
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D. Travis Wilson
George K. MOONEY
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Stealth Biotherapeutics Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • 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 present technology relates to aromatic-cationic peptide pharmaceuticals where the active compounds include a plurality of amino acids and at least one peptide bond in the molecular structure, and to methods of providing pharmaceuticals of such peptide active compounds that are bioavailable when administered to subjects.
  • Peptide pharmaceuticals are frequently administered by injection or by nasal administration.
  • injection and nasal administration are significantly less convenient, and involve more patient discomfort than, for example, oral administration.
  • Proteolytic enzymes of both the stomach and intestines may degrade peptides, rendering them inactive before they can be absorbed into the bloodstream. Any amount of peptide that survives proteolytic degradation by proteases of the stomach (typically having acidic pH optima) is later confronted with proteases of the small intestine and enzymes secreted by the pancreas (typically having neutral to basic pH optima).
  • both patents describe peptide dosage formulations which target release of the peptide to the intestine and which enhance bioavailability by administering the peptide in an oral dosage formulation which comprises, in addition to the peptide, at least one pharmaceutically acceptable pH-lowering agent and at least one absorption enhancer effective to promote bioavailability of the peptide.
  • the dosage formulation is coated with an enteric coating capable of conducting the peptide, the absorption enhancer and the pH-lowering agent through a subject's stomach, while protecting the peptide from degradation by stomach proteases. Thereafter, the coating dissolves and the peptide, absorption enhancer and pH lowering agent are released together into the intestine of the subject.
  • condition to be treated by the oral peptide would benefit from more rapid remediation than that provided by the relatively slow dissolution of an enteric coating and related release of the active component(s) within the intestine.
  • One particular example of a condition which benefits from such rapid remediation involves the area of pain relief, where the speed with which relief is achieved is obviously an important, if not critical, factor to a patient.
  • aromatic- cationic peptide be transported all of the way through the stomach and into the intestine.
  • the plasma membrane of eukaryotic cells is impermeable to large peptides or proteins.
  • certain hydrophobic amino acid sequences variously called as ferry peptides or membrane translocating sequences, when fused to the N- or C-terminus of functional proteins, can act as membrane translocators, and mediate the transport of these proteins into living cells.
  • This method of protein delivery into cells while potentially very useful, has two main drawbacks. First, the protein cannot be targeted to any specific cell type. Therefore, once it is injected and enters the circulation, it will presumably enter all cell types in a non-specific, non-receptor mediated manner.
  • Nasal delivery is also frequently plagued by low bioavailability of the therapeutic peptide. Even where nasal delivery is possible, manufacturing costs can be undesirably high because of the large concentration of therapeutic peptide required to provide clinical efficacy in view of low bioavailability occasioned by the difficulty of peptides crossing the nasal mucosa.
  • Therapeutic peptides are often poorly absorbed by tissues, and are readily degraded by bodily fluids. For this reason, formulations were developed for the administration of peptide therapeutics via the nasal route.
  • the nasal formulation was designed to be stored in a multi-dose container that was stable for an extended period of time and resisted bacterial contamination.
  • the preservative in the formulation benzalkonium chloride
  • benzalkonium chloride was found to enhance the absorption of the peptide therapeutic.
  • benzalkonium chloride was reported (P. Graf et al , Clin. Exp. Allergy 25 :395-400; 1995) to aggravate rhinitis medicamentosa in healthy volunteers who were given a decongestant nasal spray containing the preservative.
  • the present technology provides a composition including a therapeutically effective amount of an aromatic-cationic peptide and at least one counter ion, wherein the aromatic-cationic peptide and counter ion are encapsulated within a lipid particle.
  • the lipid particle of the composition includes a phospholipid.
  • the composition also includes one or more absorption enhancers.
  • the composition also includes one or more additional composition selected from the group consisting of a membrane translocator (MT), a membrane fluidizing agent, a peptide active ingredient, a second peptide, a lubricant, and combinations thereof.
  • MT membrane translocator
  • a membrane fluidizing agent selected from the group consisting of a peptide active ingredient, a second peptide, a lubricant, and combinations thereof.
  • the aromatic-cationic peptide of the composition is D-Arg- 2'6'-Dmt-Lys-Phe-NH 2 . In some embodiments, the aromatic-cationic peptide of the composition is Phe-D-Arg-Phe-Lys-NH 2 .
  • the composition also includes an enteric coating.
  • the present technology provides a process for producing a
  • composition including combining to form a first composition: a
  • the first composition further comprises a matrix forming polymer.
  • the first composition is in a solid phase.
  • the first composition is a suspension.
  • the first composition is an adsorbate.
  • the first composition also includes one or more absorption enhancers.
  • the process also includes adding an enteric coating.
  • the lipid or the phospholipid of the composition or the process includes a medium chain fatty acid.
  • the aromatic-cationic peptide is D-Arg-2'6'-Dmt-Lys-Phe- ⁇ 3 ⁇ 4. In some embodiments, the aromatic-cationic peptide is Phe-D-Arg-Phe-Lys-NH2.
  • the counter ion is selected from the group consisting of tosylate, napsylate, docusate, sodium laurel sulfate (SLS), caprate, laurate, myristate, palmitate, stearate, and oleate.
  • the present technology provides a method for increasing the bioavailabilty or stability of an aromatic-cationic peptide comprising: (i) linking the aromatic-cationic peptide to at least one counter ion to generate a peptide-counter ion complex; and (ii) encapsulating the peptide-counter ion complex within a lipid particle, thereby increasing the bioavailabilty or stability of the aromatic-cationic peptide.
  • the counter ion is selected from the group consisting of tosylate, napsylate, docusate, sodium laurel sulfate (SLS), caprate, laurate, myristate, palmitate, stearate, and oleate.
  • SLS sodium laurel sulfate
  • the aromatic-cationic peptide is D-Arg-2'6'- Dmt-Lys-Phe-NH 2 . In some embodiments, the aromatic-cationic peptide is Phe-D-Arg-Phe- Lys-NH 2 .
  • the lipid particle comprises a phospholipid.
  • the lipid particle or the phospholipid includes a medium chain fatty acid.
  • FIG. 1 is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 acetate salt in the absence (triangle) and presence (square) of 20 ⁇ trypsin.
  • D-Arg-2'6'- Dmt-Lys-Phe-NH 2 Dose 0.78 mgA/mL.
  • FIG. 2A is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate in the absence (triange) and presence (square and diamond) of 20 ⁇ trypsin.
  • D- Arg-2'6'-Dmt-Lys-Phe-NH 2 Dose 0.70 mgA/mL.
  • FIG 2B is a graph showing the dissolution of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate in the absence (triangle) and presence (square and diamond) of 20 ⁇ trypsin.
  • D- Arg-2'6'-Dmt-Lys-Phe-NH 2 Dose 0.73 mgA/mL.
  • FIG. 3A is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 napsylate in the absence (triangle) and presence (square and diamond) of 20 ⁇ trypsin.
  • D- Arg-2'6'-Dmt-Lys-Phe-NH 2 Dose 0.75 mgA/mL.
  • FIG. 3B is a graph showing the dissolution of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 napsylate in the absence (triangle) and presence (square and diamond) of 20 ⁇ trypsin.
  • D- Arg-2'6'-Dmt-Lys-Phe-NH 2 Dose 0.77 mgA/mL.
  • FIG. 4A is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 oleate in the absence (triangle) and presence (square and diamond) of 20 ⁇ trypsin.
  • D- Arg-2'6'-Dmt-Lys-Phe-NH 2 Dose 0.39 mgA/mL.
  • FIG. 4B is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 stearate in the absence (triangle) and presence (square and diamond) of 20 ⁇ trypsin.
  • D- Arg-2'6'-Dmt-Lys-Phe-NH 2 Dose 0.39 mgA/mL.
  • FIG. 5A is a graph showing the degradation of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complex in the presence of 20 ⁇ trypsin dosed from the Type I (square), Type II (diamond), or Type III (triangle) lipid formulation at 15 mgA/g the lipid formulation.
  • D-Arg- 2'6'-Dmt-Lys-Phe-NH 2 Dose 0.75 mgA/mL.
  • FIG. 5B is a graph showing the degradation of D-Arg-2'6'-Dmt-Lys-Phe-NH 2 acetate (square), 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complex (diamond), and 1 :6 D- Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complex (triangle) in the presence of 20 ⁇ trypsin dosed from the Type II lipid formulation at 15 mgA/g in the lipid formulation.
  • D-Arg-2'6'- Dmt-Lys-Phe-NH 2 Dose 0.75 mgA/mL.
  • FIG. 6A is a graph showing the degradation of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complex in the presence of 20 ⁇ trypsin dosed from the Type II lipid formulation after varying stability times and conditions.
  • the initial (square) samples were pre-emulsified for 2 hours, while the other samples were pre-emulsified for 1 hour.
  • D-Arg-2'6'-Dmt-Lys- Phe-NH 2 Dose 0.75 mgA/mL.
  • FIG. 7 shows some of the known intestinal proteases.
  • FIG. 8 exemplifies amino acid sequences, each of which can be used as a membrane translocator.
  • FIG. 9 shows some plasma proteases as well as their target sequences.
  • a range includes each individual member.
  • a group having 1-3 atoms refers to groups having 1 , 2, or 3 atoms.
  • a group having 1-5 atoms refers to groups having 1 , 2, 3, 4, or 5 atoms, and so forth.
  • the "administration" of an agent, drug, or peptide to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or
  • Administration includes self-administration and the administration by another.
  • amino acid includes naturally-occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids.
  • Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups ⁇ e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally- occurring amino acid.
  • Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the term "antigen” refers to a molecule or a portion of a molecule capable of stimulating an immune response, which is additionally capable of inducing an animal or human to produce antibody capable of binding to an epitope of that antigen.
  • bioactive molecule refers to those compounds that have an effect on or elicit a response from living cells, tissues, or the organism as a whole.
  • a non-limiting example of a bioactive molecule is D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • biological barrier is meant to include biological membranes such as the plasma membrane as well as any biological structures sealed by tight junctions (or occluding junctions) such as the mucosal or vascular epithelia, (including, but not limited to, the gastrointestinal or respiratory epithelia), and the blood brain barrier.
  • tight junctions or occluding junctions
  • translocation may occur across a biological barrier in a tissue containing cells such as epithelial cells or endothelial cells.
  • Coupled is meant to include all such specific interactions that result in two or more molecules showing a preference for one another relative to some third molecule, including any type of interaction enabling a physical association between, e.g., an aromatic-cationic peptide and a penetrating peptide.
  • the term "effective amount" refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or disorder or one or more signs or symptoms associated with a disease or disorder.
  • the amount of a composition administered to the subject will depend on the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder. For example, a
  • therapeutically effective amount of the aromatic-cationic peptide is meant levels in which the physiological effects of a disease or condition are, at a minimum, ameliorated.
  • a therapeutically effective amount can be given in one or more administrations.
  • the amount of a compound which constitutes a therapeutically effective amount will vary depending on the compound, the disorder and its severity, and the general health, age, sex, body weight and tolerance to drugs of the subject to be treated, but can be determined routinely by one of ordinary skill in the art.
  • effective translocation or “efficient translocation” refers to the introduction of the composition to a biological barrier that results in at least 5%, at least 10%, or at least 20% translocation of the composition (e.g., a composition comprising an aromatic- cationic peptide) across the biological barrier.
  • composition e.g., a composition comprising an aromatic- cationic peptide
  • epitope refers to the portion of any molecule capable of being recognized by and bound by a major histocompatibility complex (“MHC") molecule and recognized by a T cell or bound by an antibody.
  • MHC major histocompatibility complex
  • glycosaminoglycan refers to a polysaccharide that contains amino containing sugars.
  • impermeable molecules are molecules that are unable to efficiently cross biological barriers, such as the cell membrane or tight junctions.
  • anionic impermeable molecules are polysaccharides, e.g., glycosaminoglycans, nucleic acids, or net negatively charged proteins.
  • cationic impermeable molecules are polysaccharides, e.g., glycosaminoglycans, nucleic acids, or net negatively charged proteins.
  • cationic impermeable molecules are polysaccharides, e.g., glycosaminoglycans, nucleic acids, or net negatively charged proteins.
  • cationic impermeable molecules are polysaccharides, e.g., glycosaminoglycans, nucleic acids, or net negatively charged proteins.
  • cationic impermeable molecules are polysaccharides, e.g., glycosaminoglycans, nucleic acids, or net negatively charged proteins.
  • impermeable molecules are net positively charged proteins.
  • isolated polypeptide or peptide refers to a polypeptide or peptide that is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the agent is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • an isolated aromatic-cationic peptide would be free of materials that would interfere with diagnostic or therapeutic uses of the agent.
  • interfering materials may include enzymes, hormones and other proteinaceous and nonproteinaceous solutes.
  • laminate shall have its conventional meaning as something which is composed of layers of firmly united material, but which involves little, if any, interaction between the layers.
  • membrane fluidizing agents are defined as medium chain alcohols which have a carbon chain length of from 4 to 15 carbon atoms (e.g., including 5 to 15, 5 to 12, 6, 7, 8, 9, 10, or 1 1 carbon atoms).
  • a membrane fluidizing agent can be a linear (e.g., saturated or unsaturated), branched (e.g., saturated or unsaturated), cyclical (e.g. , saturated or unsaturated), or aromatic alcohol.
  • Suitable linear alcohols include, but are not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, and pentadecanol.
  • branched alcohols include, but are not limited to, geraniol, farnesol, rhodinol, citronellol.
  • An example of a cyclical alcohol includes, but is not limited to, menthol, terpineol, myrtenol, perillyl and alcohol.
  • suitable aromatic alcohols include, but are not limited to, benzyl alcohol, 4-hydroxycinnamic acid, thymol, styrene glycol, and phenolic compounds.
  • phenolic compounds include, but are not limited to, phenol, m-cresol, and m- chlorocresol.
  • a protein's net charge is determined by two factors: 1) the total count of acidic amino acids vs. basic amino acids, and 2) the specific solvent pH surroundings, which expose positive or negative residues.
  • net positively or net negatively charged proteins are proteins that, under non-denaturing pH surroundings, have a net positive or net negative electric charge.
  • parenteral refers to injections given through some other route than the alimentary canal, such as subcutaneously, intramuscularly, intraorbitally (i.e., into the eye socket or behind the eyeball), intracapsularly, intraspinally, intrasternally, or intravenously.
  • a "penetration composition” includes any composition of a water soluble composition immersed in a hydrophobic medium, that facilitates the effective translocation of a substance, e.g., aromatic-cationic peptide, across a biological barrier.
  • penetration compositions utilize at least one membrane fluidizing agent.
  • pharmaceutically acceptable salt refers to a salt prepared from a base or an acid which is acceptable for administration to a patient, such as a mammal (e.g., salts having acceptable mammalian safety for a given dosage regime).
  • pharmaceutically active agent and “therapeutic agent” are used interchangeably to refer to a chemical material or compound, which, when administered to an organism, induces a detectable pharmacologic and/or physiologic effect.
  • polysaccharide refers to a linear or branched polymer composed of covalently linked monosaccharides; glucose is the most common monosaccharide and there are normally at least eight monosaccharide units in a polysaccharide and usually many more.
  • Polysaccharides have a general formula of Cx(I3 ⁇ 40)y where x is usually a large number between 200 and 2500. Considering that the repeating units in the polymer backbone are often six-carbon monosaccharides, the general formula can also be represented as (CgHio0 5 )n where there are normally between 40 and 3000 monosaccharide units in a polysaccharide.
  • polynucleotide refers to any molecule composed of DNA nucleotides, RNA nucleotides or a combination of both types which comprises two or more of the bases guanidine, citosine, timidine, adenine, uracil or inosine, inter alia.
  • polynucleotide may include natural nucleotides, chemically modified nucleotides and synthetic nucleotides, or chemical analogs thereof and may be single-stranded or double- stranded.
  • the term includes "oligonucleotides” and encompasses "nucleic acids.”
  • polypeptide refers to a molecule composed of covalently linked amino acids and the term includes peptides, polypeptides, proteins and peptidomimetics.
  • a peptidomimetic is a compound containing non-peptidic structural elements that is capable of mimicking the biological action(s) of a natural parent peptide. Some of the classical peptide characteristics such as enzymatically scissile peptidic bonds are normally not present in a peptidomimetic.
  • selectively translocating refers to the relative translocation of the aromatic-cationic peptide as compared to the relative impermeability of other non-aromatic- cationic peptides such as bystander molecules (e.g., impermeable molecules other than the aromatic-cationic peptide itself).
  • small molecule refers to a low molecular weight organic compound which may be synthetically produced or obtained from natural sources and typically has a molecular weight of less than 2000 Da, or less than 1000 Da or even less than 600 Da e.g., less than or about 550 Da or less than or about 500 Da or less than or about 400 Da; or about 400 Da to about 2000 Da; or about 400 Da to about 1700 Da.
  • stabilizers of protein structure refer to any compounds that can stabilize protein structure under aqueous or non-aqueous conditions, such as polycationic molecules, polyanionic molecules, and uncharged polymers.
  • a polycationic molecule that can function as a protein stabilizer is a polyamine such as spermine.
  • polyanionic molecule that can function as protein stabilizers include, but are not limited to, phytic acid and sucrose octasulfate.
  • uncharged polymers that can function as protein stabilizers include polyvinylpyrrolidone and polyvinyl alcohol.
  • the terms “treating” or “treatment” or “alleviation” refers to therapeutic treatment, wherein the object is to reduce or slow down (lessen) the targeted pathologic condition or disorder.
  • a subject is successfully “treated” for a disease or condition if, after receiving a therapeutic amount of the aromatic-cationic peptides according to the methods described herein, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of the disease or condition.
  • prevention or “preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • Treating a disease or condition also refers to treating any one or more of the conditions underlying the disease or condition to be treated.
  • a “signal peptide” or “signal sequence” refers to a sequence of amino acids generally but not necessarily of a length of about 10 to about 50 or more amino acid residues, many (typically about 55-60%) residues of which are hydrophobic such that they have a hydrophobic, lipid-soluble portion.
  • water soluble composition refers to compositions which can be solubilized in a hydrophilic or partially hydrophilic solvent.
  • a hydrophilic or partially hydrophilic solvent may include water, or a non-aqueous medium such as mono-alcohols, di- alcohols, or tri-alcohols.
  • suitable mono-alcohols include, but are not limited to, ethanol, propanol, isopropanol and butanol.
  • An example of a di-alcohol includes, but is not limited to, propylene glycol.
  • An example of a tri-alcohol includes, but is not limited to, glycerol.
  • Aromatic-cationic peptides which may benefit from oral delivery in accordance with the present technology include aromatic-cationic peptides that are physiologically active and have a plurality of amino acids and at least one peptide bond in its molecular structure.
  • the present formulations by several mechanisms, suppress the degradation of the active ingredients (e.g., aromatic-cationic peptides) by protease that would otherwise tend to cleave one or more of the peptide bonds of the active ingredient.
  • the molecular structure may further include other constituents or modifications. Both man-made and natural peptides can be orally delivered in accordance with the present technology.
  • the present technology provides an aromatic-cationic peptide or a pharmaceutically acceptable salt thereof such as acetate salt or trifluoroacetate salt.
  • the peptide comprises at least one net positive charge; a minimum of three amino acids; a maximum of about twenty amino acids; a relationship between the minimum number of net positive charges (p m ) and the total number of amino acid residues (r) wherein 3p m is the largest number that is less than or equal to r + 1; and a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p t ) wherein 2a is the largest number that is less than or equal to p t + 1 , except that when a is 1 , p t may also be 1.
  • one or more amino acids (e.g., 1, 2, 3, 4 or all) of the peptides are D amino acids.
  • the peptide comprises the amino acid sequence Phe-D-Arg- Phe-Lys-NH2 or D-Arg-2'6'-Dmt-Lys-Phe-NH2. In some embodiments, the peptide comprises one or more peptides of Table A:
  • the aromatic-cationic peptide is de ined by Formula I.
  • R 1 and R 2 are each independently selected from
  • R 3 and R 4 are each independently selected from
  • halogen encompasses chloro, fluoro, bromo, and iodo
  • R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from
  • halogen encompasses chloro, fluoro, bromo, and iodo; and n is an integer from 1 to 5.
  • R 1 and R 2 are hydrogen; R 3 and R 4 are methyl; R 5 , R ( R 7 , R s , and R 9 are all hydrogen; and n is 4.
  • the aromatic-cationic peptide is defined by Formula II:
  • R 1 and R 2 are each independently selected from
  • R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are each independently selected from
  • halogen encompasses chloro, fluoro, bromo, and iodo; and n is an integer from 1 to 5.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are all hydrogen; and n is 4.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 11 are all hydrogen; R 8 and R 12 are methyl; R 10 is hydroxyl; and n is 4.
  • the aromatic-cationic peptides of the present technology have a core structural motif of alternating aromatic and cationic amino acids.
  • the peptide may be a tetrapeptide defined by any of Formulas III to VIII set forth below:
  • Aromatic is a residue selected from the group consisting of: Phe (F), Tyr (Y), and Trp (W).
  • the Aromatic residue may be substituted with a saturated analog of an aromatic residue, e.g., Cyclohexylalanine (Cha).
  • Cationic is a residue selected from the group consisting of: Arg (R), Lys ( ), and His (H).
  • the peptides disclosed herein may be formulated as pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt means a salt prepared from a base or an acid which is acceptable for administration to a patient, such as a mammal (e.g. , salts having acceptable mammalian safety for a given dosage regime).
  • the salts are not required to be pharmaceutically acceptable salts, such as salts of intermediate compounds that are not intended for administration to a patient.
  • Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
  • salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts, and the like.
  • Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, ⁇ , ⁇ '-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
  • Salts derived from pharmaceutically acceptable inorganic acids include salts of boric, carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts derived from
  • organic acids include salts of aliphatic hydroxyl acids (e.g. , citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g. , citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g. , citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g. , citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g. , citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic hydroxyl acids (e.g. , citric, gluconic, glycolic, lactic, lactobionic
  • monocarboxylic acids e.g. , acetic, butyric, formic, propionic and trifluoroacetic acids
  • amino acids e.g., aspartic and glutamic acids
  • aromatic carboxylic acids e.g., benzoic, p- chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylacetic acids
  • aromatic hydroxyl acids e.g., o-hydroxybenzoic, p-hydroxybenzoic, 1 -hydroxynaphthalene-2- carboxylic and 3-hydroxynaphthalene-2-carboxylic acids
  • ascorbic dicarboxylic acids (e.g., fumaric, maleic, oxalic and succinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids (e.g.
  • the salt is an acetate salt. Additionally or alternatively, in other embodiments, the salt is a trifluoroacetate salt. In some embodiments, the salt is a tartrate salt.
  • the pharmaceutically acceptable salt includes the peptides of Formulas I or II and a pharmaceutically acceptable acid.
  • the pharmaceutically acceptable acid includes l-hydroxy-2 -naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4- aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L),
  • benzenesulfonic acid benzoic acid, camphoric acid (+), camphor- 10-sulfonic acid (+), capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid (- L), malonic acid, mandelic acid
  • the pharmaceutically acceptable acid is tartaric acid, where such embodiments of the pharmaceutically acceptable salt are referred to as a tartrate salt.
  • the peptide comprises the amino acid sequence 2'6'-Dmt-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe-Lys-NH2, or D-Arg- 2'6'-Dmt-Lys-Phe-NH 2 .
  • the eptide comprises one or more peptides of Table A:
  • the pharmaceutically acceptable salt is a tartrate salt and the peptide includes the amino acid sequence 2'6'-Dmt-D- Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe-Lys-NH 2 , or D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • aromatic-cationic peptides of the present technology disclosed herein may be synthesized by any of the methods well known in the art. Suitable methods for chemically synthesizing the protein include, for example, liquid phase and solid phase synthesis, and those methods described by Stuart and Young in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984), and in Methods Enzymol., 289, Academic Press, Inc, New York (1997). Recombinant peptides may be generated using conventional techniques in molecular biology, protein biochemistry, cell biology, and microbiology, such as those described in Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed.
  • Additional peptide active compounds of the present technology include, but are not limited to, polypeptides such as insulin, vasopressin, salmon calcitonin, glucagon-like peptide 1, calcitonin, and analogs thereof.
  • Other examples include, but are not limited to, calcitonin gene-related peptide, parathyroid hormone (full length or truncated, amidated or in the free acid form, further modified or not), luteinizing hormone-releasing factor, erythropoietin, tissue plasminogen activators, human growth hormone, adrenocorticototropin, various interleukins, enkephalin, DALDA derivatives such as dmt-DALDA and the like.
  • the aromatic-cationic peptide includes the sequence 2'6'- Dmt-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe-Lys-NH 2 , D-Arg-2'6'-Dmt-Lys-Phe-NH 2 , or combinations of two or more thereof.
  • the aromatic-cationic peptide is from about 0.02 to 0.2 percent by weight relative to the total weight of the overall pharmaceutical composition.
  • Other aromatic-cationic peptides of the present technology may be present at higher or lower concentrations depending on desired target blood concentrations for the peptide and its bioavailability in the oral delivery system of the present technology.
  • Aromatic-cationic peptide precursors may be made by either chemical (e.g., using solution and solid phase chemical peptide synthesis) or recombinant synthesis methods known in the art.
  • Precursors of e.g. , amidated aromatic-cationic peptides of the present technology may be made in like manner.
  • recombinant production is believed significantly more cost effective.
  • precursors are converted to active peptides by amidation reactions that are also known in the art. For example, enzymatic amidation is described in U.S. Pat. No. 4,708,934 and European Patent Publications 0 308 067 and 0 382 403.
  • Recombinant production can be used for both the precursor and the enzyme that catalyzes the conversion of the precursor to the desired active form of the aromatic-cationic peptide.
  • Such recombinant production is discussed in Biotechnology, Vol. 11 (1993) pp. 64-70, which further describes a conversion of a precursor to an amidated product.
  • a keto-acid such as an alpha-keto acid, or salt or ester thereof, wherein the alpha-keto acid has the molecular structure RC(0)C(0)OH, and wherein R is selected from the group consisting of aryl, a C 1 -C 4 hydrocarbon moiety, a halogenated or hydroxylated Ci-C 4 hydrocarbon moiety, and a C1-C4 carboxylic acid, may be used in place of a catalase co-factor.
  • keto acids include, but are not limited to, ethyl pyruvate, pyruvic acid and salts thereof, methyl pyruvate, benzoyl formic acid and salts thereof, 2-ketobutyric acid and salts thereof, 3-methyl-2-oxobutanoic acid and salts thereof, and 2-keto glutaric acid and salts thereof.
  • the production of the recombinant aromatic-cationic peptide may proceed, for example, by producing glycine-extended precursor in E. coli as a soluble fusion protein with glutathione-S-transferase.
  • An a-amidating enzyme catalyzes conversion of precursors to active aromatic-cationic peptide. That enzyme is recombinantly produced, for example, in Chinese Hamster Ovary (CHO) cells as described in the Biotechnology article cited above.
  • Other precursors to other amidated peptides may be produced in like manner.
  • Peptides that do not require amidation or other additional functionalities may also be produced in like manner.
  • Other peptide active agents are commercially available or may be produced by techniques known in the art.
  • administering the pharmaceutical formulations of this technology without an enteric coating increases the speed of peptide absorption (relative to corresponding enteric-coated formulation) without reducing bioavailability below practical levels. While some reduction in bioavailability does occur, this reduction is not expected to preclude effective medical treatment, or to unduly detract from the advantages of e.g., in the case of pain relief.
  • the present formulations permit more rapid absorption of the active aromatic-cationic peptides of the present technology or pharmaceutically acceptable salts thereof, such as acetate salt or trifluoroacetate salt, due to the reduction in the time necessary for the vehicle (e.g.
  • the formulations also permit such release further upstream in the alimentary canal, e.g. , in the esophagus and/or stomach, instead of awaiting passage of the material into the intestine. See e.g., U.S. Patent Publication No. 2005/0282756 and U.S. Patent Publication No.
  • subjects in need of treatment with aromatic-cationic peptide active ingredients are provided with a finished pharmaceutical product, for example in tablet form of an ordinary size in the pharmaceutical industry, formed of an oral pharmaceutical composition comprising one or more of such peptide active ingredients (at appropriate dosage).
  • the finished pharmaceutical product may additionally be prepared, if desired, in (for example) capsule form.
  • the dosages and frequency of administering the products are discussed in more detail below.
  • Subjects who may benefit are any who suffer from disorders that respond favorably to increased levels of a peptide-containing compound.
  • oral formulations include a peptide and one or more of an absorption enhancer, a pH lowering agent, other optional agents and an enteric coating.
  • oral peptide formulations described herein are useful in the treatment of disorders stemming from or related to mitochondrial permeability transition (MPT) and/or cellular oxidative damage.
  • MPT mitochondrial permeability transition
  • oral peptide formulations of the aromatic-cationic peptides of the present technology Phe-D- Arg-Phe-Lys-NH 2 and D-Arg-2'6'-Dmt-Lys-Phe-NH 2 , or pharmaceutically acceptable salts thereof, may be used to treat subjects suffering from vascular occlusion, kidney ischemia, tissue ischemia-rep erfusion injury, acute myocardial infarction, diseases or disorders of the eye, or neurological disorders such as Alzheimer's and Parkinson's diseases.
  • Pharmaceutically acceptable salts include, but are not limited to, e.g., acetate salt and trifluoroacetate salt.
  • the pharmaceutical formulations described herein are believed to overcome a series of different and unrelated natural barriers to bioavailability.
  • Various components of the pharmaceutical compositions act to overcome different barriers by mechanisms appropriate to each, and in some embodiments, result in synergistic effects on the bioavailability of a peptide active ingredient.
  • inherent physical and chemical properties of peptides make certain absorption enhancers more effective than others in boosting its bioavailability.
  • the aromatic-cationic peptide active compound of the present technology is contained within a formulation adopted for oral administration.
  • proteolytic degradation of the peptide by stomach proteases (most of which are active in the acid pH range) is reduced due to administration of the formulation to the patient on an empty stomach (although this is not required in order to achieve adequate results), while degradation by intestinal or pancreatic proteases (most of which are active in the neutral to basic pH range) is reduced due to the effect of the pH lowering agent in adjusting the pH of the intestinal environment to sub- optimal levels.
  • solubility enhancers are employed to aid passage of the aromatic-cationic peptide through the intestinal epithelial barrier.
  • the pH-lowering agent is believed to lower the local pH (where the active agent has been released) to levels below the optimal range for many intestinal proteases. This decrease in pH reduces the proteolytic activity of the intestinal proteases, thus affording protection to the peptide from potential degradation should the peptide be present within the intestine.
  • the activity of these proteases is diminished by the temporarily acidic environment as discussed herein. For example, sufficient acid should be provided that local intestinal pH is lowered temporarily to 5.5 or below, 4.7 or below, or 3.5 or below.
  • the sodium bicarbonate test described below (in the section captioned "the pH-Lowering Agent") is indicative of the required acid amount.
  • conditions of reduced pH typically persist for a time period sufficient to protect the aromatic-cationic peptide from proteolytic degradation until at least some of the aromatic-cationic peptide has had an opportunity to cross into the bloodstream.
  • conditions of reduced pH typically persist for a time period sufficient to protect the aromatic-cationic peptide from proteolytic degradation until at least some of the aromatic-cationic peptide has had an opportunity to cross into the bloodstream.
  • salmon calcitonin a 32 amino acid peptide
  • experiments have demonstrated T max of 5-15 minutes for blood levels of salmon calcitonin when the active components are injected directly into the duodenum, ilium or colon.
  • the absorption enhancers of the present formulations synergistically promote peptide absorption into the blood while conditions of reduced proteolytic activity prevail.
  • the mechanism by which the present formulations are believed to accomplish the goal of enhanced bioavailability is aided by having active components of the finished pharmaceutical product released together as simultaneously as possible.
  • the absorption enhancer which may be a solubility enhancer and/or transport enhancer (as described in more detail below), aids transport of the aromatic-cationic peptide from the alimentary canal into the blood, and may promote the process so that it better occurs during the time period of reduced intestinal pH and reduced intestinal proteolytic activity.
  • Many surface active agents may act as both solubility enhancers and transport (uptake) enhancers.
  • enhancing solubility provides (1) a more simultaneous release of the active components of the present formulations into the aqueous portion of the alimentary tract, (2) better solubility of the peptide in, and transport through, a mucous layer such as that found along the intestinal walls.
  • an absorption enhancer provides better transport through the brush border membrane of the intestine into the blood, via either transcellular or paracellular transport.
  • an absorption enhancer may provide both functions. In those instances, embodiments utilizing both of these functions may do so by adding only one additional compound to the pharmaceutical composition. In other embodiments, separate absorption enhancers may provide the two functions separately.
  • many charged lipophilic species can interact with an aromatic-cationic peptide via charge and via hydrophobic interactions such that transport may be facilitated not just by the direct effect of the permeation enhancer on the absorbing membrane but also enabling the interaction of the peptide with the membrane either as a complex or single entity that cannot occur in the presence of peptide alone.
  • the total amount of the pH-lowering compound to be administered with each administration of aromatic-cationic peptide is typically an amount which, when released into the intestine for example, is sufficient to lower the local intestinal pH substantially below the pH optima for proteases found there.
  • the quantity required will necessarily vary with several factors including the type of pH-lowering agent used (discussed below) and the equivalents of protons provided by a given pH-lowering agent.
  • the amount required to provide good bioavailability is an amount which, when the pharmaceutical product of the present technology is added to a solution of 10 milliliters of 0.1 M sodium bicarbonate, lowers the pH of that sodium bicarbonate solution to no higher than 5.5, no higher than 4.7, or no higher than 3.5.
  • Enough acid to lower pH, in the foregoing test, to about 2.8 has been used in some embodiments.
  • at least 50, 100, 200, 300 or at least 400 milligrams of the pH-lowering agent are used in the pharmaceutical composition of the present technology.
  • the foregoing values relate to the total combined weight of all pH- lowering agents where two or more of such agents are used in combination.
  • the oral formulation should not include an amount of any base which, when released together with the pH-lowering compound, would prevent the pH of the above- described sodium bicarbonate test from dropping to 5.5 or below.
  • the pH-lowering agent of the present formulations may be any pharmaceutically acceptable compound that is not toxic in the gastrointestinal tract and is capable of either delivering hydrogen ions (a traditional acid) or of inducing higher hydrogen ion content from the local environment. It may also be any combination of such compounds.
  • at least one pH-lowering agent used in the present formulations has a p a no higher than 4.2, or no higher than 3.0.
  • the pH lowering agent has a solubility in water of at least 30 grams per 100 milliliters of water at room temperature.
  • Non-limiting examples of compounds that induce higher hydrogen ion content include aluminum chloride and zinc chloride.
  • Pharmaceutically acceptable traditional acids include, but are not limited to acid salts of amino acids (e.g., amino acid hydrochlorides) or derivatives thereof. Examples of these are acid salts of acetylglutamic acid, alanine, arginine, asparagine, aspartic acid, betaine, carnitine, carnosine, citrulline, creatine, glutamic acid, glycine, histidine, hydroxylysine, hydroxyproline, hypotaurine, isoleucine, leucine, lysine, methylhistidine, norleucine, ornithine, phenylalanine, proline, sarcosine, serine, taurine, threonine, tryptophan, tyrosine and valine.
  • pH-lowering compounds include dicarboxylic and tricarboxylic carboxylic acids. Acids such as acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyceric, glycocolic, glyoxylic, isocitric, isovaleric, lactic, maleic, oxaloacetic, oxalosuccinic, propionic, pyruvic, succinic, tartaric, valeric, adipic, benzoic, phthalic, sorbic, edetic, benzenesulfonic, p-toluenenesulfonic, methansulfonic, boric, saccharinic,, and the like have been found useful.
  • Acids such as acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyceric, glycocolic, glyoxylic
  • pH-lowering agents that might not usually be called “acids” in the art, but which may nonetheless be useful in accordance with the present technology are phosphate esters (e.g., fructose 1 ,6 diphosphate, glucose 1,6 diphosphate, phosphoglyceric acid, and diphosphoglyceric acid). Carbopol® and polymers such as polycarbophil may also be used to lower pH. Additional pH-lowering agents include, but are not limited to, alginic acid and other naturally occurring or synthetic polysaccharide acids, such as the substituted carboyxmethyl celluloses, xanthan gum, polymethacrylic acid and substituted derivatives, cellulose glycollic acid, and pectins. Any combination of pH lowering agents may be used.
  • the pH lowering agent achieves a pH level of between about 1 to about 6. In some embodiments, the pH lowering agent achieves a pH level of between about 1.5 to about 5.5, or between about 2 to about 5, or between about 2.5 to about 4.5, or between about 3 to about 4.
  • One embodiment utilizes, as at least one of the pH-lowering agents in the finished pharmaceutical product, an acid selected from the group consisting of citric acid, tartaric acid and an acid salt of an amino acid.
  • aromatic-cationic peptides of the present technology or a pharmaceutically acceptable salt thereof, such as acetate salt or trifluoroacetate salt are the active agent
  • certain ratios of pH- lowering agent to peptide may prove especially effective.
  • the weight ratio of pH-lowering agent to aromatic-cationic peptide exceed 200: 1 , 800: 1 , or 2000: 1.
  • the weight ratio of pH-lowering agent to aromatic-cationic peptide exceeds 40: 1, 400:1, or 4000: 1.
  • An alternative or a supplement to the use of pH-lowering agents is the use of protease inhibitors, in particular inhibitors of intestinal proteases.
  • FIG. 7 illustrates some of the known intestinal proteases.
  • the protease inhibitors can be proteins or peptides.
  • the protease inhibitor are trypsin inhibitors (TI).
  • TI include, but are not limited to, soy protein (e.g., SBTI, Kunitz inhibitor, or Glycine Max®) of 20.1 kDa, bovine pancreas of 6.5 kDa, aprotonin, chicken or turkey ova-mucoid TI of 28 kDA, partially hydrolysed gelatin fractions, epsilon-aminocaproic acid, and di-Sodium EDTA.
  • absorption enhancers are present in a quantity that constitutes from 0.1 to 20.0 percent by weight, from about 0.5 to about 1 percent by weight, from about 1 to about 10 percent by weight, from about 3 to about 7 percent by weight, or from about 4 to about 6 percent by weight relative to the overall weight of the pharmaceutical composition.
  • absorption enhancers are surface active agents which act both as solubility enhancers and uptake enhancers.
  • solubility enhancers improve the ability of the components of the present formulations to be solubilized in either the aqueous environment into which they are originally released or into, for example, the lipophilic environment of the mucous layer lining the intestinal walls, or both.
  • Transport (uptake) enhancers are those which facilitate the ease by which aromatic-cationic peptides of the present technology cross the intestinal wall.
  • One or more absorption enhancers may perform one function only (e.g. , solubility), or one or more absorption enhancers may perform the other function only (e.g. , uptake), within the scope of the present technology. It is also possible to have a mixture of several compounds some of which provide improved solubility, some of which provide improved uptake and/or some of which perform both. Without intending to be bound by theory, it is believed that uptake enhancers may act by (1) increasing disorder of the hydrophobic region of the membrane exterior of cells, allowing for increased transcellular transport; or (2) leaching membrane proteins resulting in increased transcellular transport; or (3) widening pore radius between cells for increased paracellular transport.
  • detergents are useful in (1) solubilizing all of the active components quickly into the aqueous environment where they are originally released, (2) enhancing lipophilicity of the components of the present formulations, especially the aromatic-cationic peptide, aiding its passage into and through the intestinal mucus, (3) enhancing the ability of the normally polar aromatic-cationic peptide to cross the epithelial barrier of the brush border membrane; and (4) increasing transcellular or paracellular transport as described above.
  • the surface active agents when used as the absorption enhancers, are free flowing powders for facilitating the mixing and loading of capsules during the manufacturing process. Because of inherent characteristics of specific peptides (e.g., their isoelectric point, molecular weight, amino acid composition, etc.) certain surface active agents will likely interact better with certain peptides. For example, some agents may undesirably interact with the charged portions of a peptide and prevent its absorption, thus undesirably resulting in decreased bioavailability.
  • specific peptides e.g., their isoelectric point, molecular weight, amino acid composition, etc.
  • a surface active agent used as an absorption enhancer may include one or more of the following: (i) anionic surface active agents that are cholesterol derivatives (e.g., bile acids), (ii) cationic surface agents (e.g., acyl carnitines, phospholipids and the like), (iii) non-ionic surface active agents, and (iv) mixtures of anionic surface active agents (especially those having linear hydrocarbon regions) together with negative charge neutralizers.
  • anionic surface active agents that are cholesterol derivatives (e.g., bile acids), (ii) cationic surface agents (e.g., acyl carnitines, phospholipids and the like), (iii) non-ionic surface active agents, and (iv) mixtures of anionic surface active agents (especially those having linear hydrocarbon regions) together with negative charge neutralizers.
  • Negative charge neutralizers include but are not limited to acyl carnitines, cetyl pyridinium chloride, and the like.
  • the absorption enhancer is soluble at acid pH, particularly in the 3.0 to 5.0 range.
  • one combination useful with aromatic-cationic peptides of the present technology mixes cationic surface active agents with anionic surface active agents that are cholesterol derivatives, and which are soluble at acid pH.
  • an acid soluble bile acid is combined with a cationic surface active agent.
  • an acyl carnitine and sucrose ester are combined.
  • acyl carnitines e.g., lauroyl L- carnitine
  • phospholipids and bile acids are used as absorption enhancers, especially acyl carnitine.
  • Anionic surfactants that are cholesterol derivatives are also used in some embodiments.
  • biodegradable or reabsorbable detergents e.g., biologically recyclable compounds such as bile acids, phospholipids, and/or acyl carnitines
  • bile acids e.g., biologically recyclable compounds
  • phospholipids e.g., phospholipids
  • acyl carnitines are useful in enhancing paracellular transport.
  • aromatic-cationic peptides of the present technology are better transported both to and through the intestinal wall.
  • Absorption enhancers include but are not limited to, e.g. , (a) salicylates such as sodium salicylate, 3-methoxysalicylate, 5-methoxysalicylate and homovanilate; (b) bile acids such as taurocholic, tauorodeoxycholic, deoxycholic, cholic, glycholic, lithocholate, chenodeoxycholic, ursodeoxycholic, ursocholic, dehydrocholic, fusidic, etc.; (c) non-ionic surfactants such as polyoxyethylene ethers (e.g.
  • Tween-20, Tween-80 etc. anionic surfactants such as dioctyl sodium sulfosuccinate; (e) lyso-phospholipids such as lysolecithin and lysophosphatidylethanolamine; (f) acylcarnitines, acylcholines and acyl amino acids such as lauroyl L-carnitine, myristoylcarnitine, palmitoylcarnitine,
  • lauroylcholine myristoylcholine, palmitoylcholine, hexadecyllysine, N-acylphenylalanine, N-acylglycine etc.
  • water soluble phospholipids such as diheptanoylphosphatidylcholine, dioctylphosphatidylcholine etc.
  • medium-chain glycerides which are mixtures of mono-, di- and triglycerides containing medium-chain-length fatty acids (caprylic, capric and lauric acids); (i) ethylene-diaminetetraacetic acid; (j) cationic surfactants such as cetylpyridinium chloride; (k) fatty acid derivatives of polyethylene glycol such as Labrasol, Labrafac, etc.; and (1) alkylsaccharides such as lauryl maltoside, lauroyl sucrose, myristoyl sucrose, palmitoyl sucrose, etc.
  • cationic ion exchange agents e.g. , detergents
  • cationic ion exchange agents may prevent the binding of aromatic-cationic peptides of the present technology or other therapeutic agents to mucus.
  • exemplary cationic ion exchange agents include protamine chloride or any other polycation.
  • a water-soluble barrier separates the pH-lowering agent from an acid resistant enteric coating.
  • a conventional pharmaceutical capsule may, for example, be used for the purpose of providing this barrier.
  • Many water soluble barriers are known in the art and include, but are not limited to, hydroxypropyl methylcellulose and conventional pharmaceutical gelatins.
  • a second peptide such as albumin, casein, soy protein, other animal or vegetable proteins and the like
  • non-specific adsorption e.g., binding of peptide to the intestinal mucus barrier
  • the second peptide in some embodiments, is between about 0.1 percent to about 10.0 percent by weight relative to the weight of the overall pharmaceutical composition. In some embodiments, the second peptide is between about 1.0 percent to about 10.0 percent, or between about 2.0 percent to about 9.0 percent, or between about 3.0 percent to about 8.0 percent, or between about 4.0 percent to about 7.0 percent, or between about 5.0 percent to about 6.0 percent by weight relative to the weight of the overall pharmaceutical composition.
  • the second peptide is between about 0.1 percent to about 1.0 percent, or between about 0.2 percent to about 0.9 percent, or between about 0.3 percent to about 0.8 percent, or between about 0.4 percent to about 0.7 percent, or between about 0.5 percent to about 0.6 percent by weight relative to the weight of the overall pharmaceutical composition.
  • This second peptide is typically not physiologically active and is typically not a food peptide such as soy bean peptide or the like. Without intending to be bound by theory, this second peptide may also increase
  • the second peptide may also aid the active compound's passage through the liver.
  • compositions of the present technology may optionally also include common pharmaceutical diluents, glycants, lubricants, gelatin capsules,
  • aromatic-cationic peptide formulations include an enteric coating, a carrier or vehicle that protects the formulation from stomach proteases. Any carrier or vehicle that protects the aromatic-cationic peptide from stomach proteases and then dissolves so that the other ingredients of the composition may be released in the intestine is suitable, in some embodiments.
  • enteric coatings are known in the art, and are useful in accordance with the present technology. Examples include cellulose acetate phthalate, hydroxypropyl methylethylcellulose succinate, hydroxypropyl methylcellulose phthalate,
  • aromatic-cationic peptides of the present technology are included in a sufficiently viscous protective syrup to permit protected passage of the components of the composition through the stomach.
  • Suitable enteric coatings for protecting the aromatic-cationic peptide from stomach proteases may be applied, for example, to capsules after the remaining components have been loaded within the capsule.
  • enteric coating is coated on the outside of a tablet or coated on the outer surface of particles of active components which are then pressed into tablet form, or loaded into a capsule, which is itself coated with an enteric coating.
  • the vehicle or carrier releases the active components in the small intestine where uptake enhancers that increase transcellular or paracellular transport are less likely to cause undesirable side effects than if the same uptake enhancers were later released in the colon. It is emphasized, however, that formulations of the present technology are believed effective in the colon as well as in the small intestine. Numerous vehicles or carriers, in addition to those discussed above, are known in the art. In some embodiments, it is desirable keep the amount of enteric coating low.
  • the enteric coating adds no more than 30% to the weight of the remainder of pharmaceutical composition (the "remainder” being the pharmaceutical composition exclusive of enteric coating itself).
  • the formulation includes less than 20%, e.g., from about 12% to about 20% to the weight of the uncoated composition.
  • the enteric coating should be sufficient to prevent breakdown of the pharmaceutical composition of the present technology in 0. IN HC1 for at least two hours, then capable of permitting complete release of all contents of the
  • composition within thirty minutes after pH is increased to 6.3 in a dissolution bath in which the composition is rotating at 100 revolutions per minute.
  • the weight ratio of pH-lowering agent(s) to absorption enhancer(s) is 3: 1 to 20: 1 , 4: 1 to 12: 1 , or 5 : 1 to 10: 1.
  • the total weight of all pH-lowering agents and the total weight of all absorption enhancers in a given pharmaceutical composition is included in the foregoing ratios. For example, if a
  • composition includes two pH-lowering agents and three absorption enhancers, the foregoing ratios will be computed on the total combined weight of both pH- lowering agents and the total combined weight of all three absorption enhancers.
  • the pH-lowering agent, the aromatic-cationic peptide and the absorption enhancer are uniformly dispersed in the finished pharmaceutical product.
  • the finished pharmaceutical product may be produced in the form of a laminate having two or more layers, wherein the aromatic-cationic peptide is contained within a first layer and the pH-lowering agent and absorption enhancer are contained within a second layer laminated with the first layer.
  • the composition of the product comprises granules that include a pharmaceutical binder having the aromatic-cationic peptide, the pH-lowering agent and the absorption enhancer uniformly dispersed within the binder. Granules may also include an acid core, surrounded by a uniform layer of organic acid, a layer of enhancer and a layer of peptide that is surrounded by an outer layer of organic acid.
  • Granules may be prepared from an aqueous mixture including pharmaceutical binders such as polyvinyl pyrrolidone or hydroxypropyl methylcellulose, together with the pH- lowering agents, absorption enhancers and aromatic-cationic peptides of the present technology to be used in the present formulations.
  • pharmaceutical binders such as polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • patients in need of treatment with aromatic-cationic peptides of the present technology are provided with an oral pharmaceutical composition thereof.
  • the composition is in the form of a tablet or capsule form of an ordinary size in the pharmaceutical industry.
  • the dosages and frequency of administering the products are discussed in more detail below.
  • Patients who may benefit are any who suffer from disorders that respond favorably to increased levels of a peptide-containing compound.
  • aromatic-cationic peptides of the present technology display higher bioavailability when administered orally in accordance with the present compositions, formulations and/or methods compared to controls.
  • bioavailability of aromatic-cationic peptides of the present technology when linked to a membrane translocator (MT) according to the methods disclosed herein is significantly increased.
  • compositions of the present disclosure are believed to overcome a series of different and unrelated natural barriers to bioavailability.
  • Various components of the pharmaceutical compositions act to overcome different barriers by mechanisms appropriate to each, and result, in some embodiments, in synergistic effects on the bioavailability of a peptide active ingredient.
  • the aromatic-cationic peptide may be administered orally.
  • the MT link to the active peptide can be cleaved by an enzyme in the blood or the lymphatic system, thereby leaving the active peptide free to reach its target.
  • proteolytic degradation of the peptide and of the membrane translocator by stomach enzymes most of which are active in the acid pH range
  • intestinal or pancreatic proteases most of which are active in the neutral to basic pH range
  • the peptide is transported through the stomach under the protection of an appropriate acid-resistant protective vehicle for substantially preventing contact between the aromatic-cationic peptide or other peptide and any stomach proteases capable of degrading it.
  • an appropriate acid-resistant protective vehicle for substantially preventing contact between the aromatic-cationic peptide or other peptide and any stomach proteases capable of degrading it.
  • the acid is believed to lower the local intestinal pH, where the aromatic-cationic peptide has been released, to levels below the optimal range for many intestinal proteases and other intestinal enzymes. This decrease in pH reduces the proteolytic activity of the intestinal proteases, thus affording protection to the peptide and the membrane translocator from potential degradation. The activity of these proteases is diminished by the temporarily acidic environment provided by the composition. According to embodiments of methods disclosed herien, sufficient acid is provided that local intestinal pH is lowered temporarily to 5.5 or below, 4.7 or below, or 3.5 or below.
  • the sodium bicarbonate test described herein is indicative of the required acid amount.
  • Conditions of reduced intestinal pH persist for a time period sufficient to protect the aromatic-cationic peptide and the membrane translocator from proteolytic degradation until at least some of the aromatic-cationic peptide has had an opportunity to cross the intestinal wall into the bloodstream.
  • Salmon calcitonin experiments have demonstrated a Tmax of 5-15 minutes for blood levels of salmon calcitonin when the active components are injected directly into the duodenum, ileum or colon of rats.
  • protease inhibitors are believed to reduce the proteolytic activity of the intestinal proteases, thus affording protection to the peptide and the membrane translocator from premature potential degradation.
  • compositions of the present technology can optionally contain absorption enhancers.
  • the absorption enhancers of the disclosure in some embodiments, synergistically promote peptide absorption into the blood while conditions of reduced proteolytic activity prevail.
  • the volume of enteric coating is kept as low as possible consistent with providing protection from stomach proteases.
  • enteric coating is less likely to interfere with peptide release, or with the release of other components in close time proximity with the peptide.
  • the enteric coating is less than about 30% to the weight of the remainder of pharmaceutical composition (i.e., the other components of the composition excluding enteric coating). In some embodiments, it is less than 20%. In some embodiments, the enteric coating adds between 10%> and 20% to the weight of the uncoated ingredients.
  • the absorption enhancer which may be a solubility enhancer and/or transport enhancer (as described in more detail below) aids transport of the aromatic- cationic peptide from the intestine to the blood, and may promote the process so that it better occurs during the time period of reduced intestinal pH and reduced intestinal proteolytic activity.
  • Many surface agents may act as both solubility enhancers and transport (uptake) enhancers.
  • enhancing solubility provides (1) a more simultaneous release of the active components of the present methods into the aqueous portion of the intestine, (2) better solubility of the peptide in, and transport through, a mucous layer along the intestinal walls.
  • an uptake enhancer is useful to provide better transport through the brush border membrane of the intestine into the blood, via either transcellular or paracellular transport.
  • some compounds may provide both functions. In those instances, embodiments utilizing both of these functions may do so by adding only one additional compound to the pharmaceutical composition. In other embodiments, separate absorption enhancers may provide the two functions separately.
  • Formulations including combinations of multiple pH-lowering agents, multiple enhancers, multiple MTs and multiple peptide active agents can be used as well as using just a single pH-lowering agent and/or single enhancer.
  • peptide active agent e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH 2
  • MT sequence to facilitate absorption from the intestine.
  • the MT is typically protected from cleavage by proteases in the stomach and intestine before its absorption. However, once absorbed, the MT should be able to be at least partially removed by proteases to free up the active peptide.
  • the MT can comprise an amino acid sequence, such as a signal peptide or signal sequence.
  • the hydrophobic portion is a common, major motif of the signal peptide, and it is often a central part of the signal peptide of protein secreted from cells.
  • a signal peptide is a peptide capable of penetrating through the cell membrane to allow the export of cellular proteins.
  • the signal peptides are also "importation competents," e.g., are capable of penetrating through the cell membrane from outside the cell to the interior of the cell.
  • the amino acid residues can be mutated and/or modified (i.e., to form mimetics) so long as the modifications do not affect the translocation-mediating function of the peptide.
  • the word “peptide” includes mimetics and the word “amino acid” includes modified amino acids, as used herein, unusual amino acids, and D-form amino acids.
  • the importation competent signal peptides have the function of mediating translocation across a cell membrane from outside the cell to the interior of the cell. In some embodiments, they may also retain their ability to allow the export of a protein from the cell into the external milieu. A putative signal peptide can easily be tested for this importation activity following the teachings provided herein, including testing for specificity for any selected cell type.
  • FIG. 8 exemplifies amino acid sequences, each of which can be used as an MT.
  • the MT can also comprise fatty acids and/or bile acids.
  • such molecules when used, are linked to the active peptide by an amino acid bridge which is subject to cleavage by proteases in the plasma.
  • the MT can be linked to the active peptide by a non-peptidyl linkage, in which case the in vivo enzyme that cleaves the linkage may be an enzyme other than protease.
  • the amino acid bridge is a target for cleavage by at least one plasma protease. Plasma proteases as well as their target sequences are well known in the art. FIG. 9 illustrates some of these enzymes as well as their specific targets.
  • the formulations disclosed herein by several mechanisms, suppresses the degradation of the active ingredient linked to an MT by protease that would otherwise tend to cleave one or more of the peptide bonds of the active ingredient.
  • the molecular structure of the active ingredient e.g. , aromatic-cationic peptide
  • aromatic- cationic peptides of the present technology can be amidated at the C-terminus. Both synthetic and natural peptides can be orally delivered in accordance with the method.
  • peptide active compounds or active agents of the present disclosure include, but are not limited to, aromatic-cationic peptides of the present technology, such as, e.g. , D-Arg-2'6'-Dmt-Lys-Phe-NH 2 as well as polypeptides such as insulin, vasopressin, and calcitonin.
  • aromatic-cationic peptides of the present technology such as, e.g. , D-Arg-2'6'-Dmt-Lys-Phe-NH 2
  • polypeptides such as insulin, vasopressin, and calcitonin.
  • Other examples include calcitonin gene-related peptide, parathyroid hormone, luteinizing hormone -releasing factor, erythropoietin, tissue
  • the peptide has the amino acid sequence Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu- Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Xaa-Xaa-Gly-Xaa-Xaa-Thr-Xaa, wherein amino acids 26, 27, 28, 29, and 31 can be any naturally occurring amino acid, and wherein amino acid 31 is optionally amidated.
  • the peptide may comprise from 0.02 to 0.2 percent by weight relative to the total weight of the overall pharmaceutical composition (exclusive of enteric coating).
  • Other peptide peptides may be present at higher or lower concentrations depending on desired target blood concentrations for the active compound and its bioavailability in the oral delivery system of the methods.
  • Aromatic-cationic peptides of the present technology may be made by either chemical or recombinant syntheses known in the art. Precursors of other amidated peptides may be made in like manner. Recombinant production is believed to be significantly more cost effective. For example, enzymatic amidation is described in U.S. Pat. No. 4,708,934 and European Patent Publications 0 308 067 and 0 382 403. Recombinant production may be used for both the precursor and the enzyme that catalyzes the conversion of the precursor to the final product. Such recombinant production is discussed in Biotechnology, Vol. 1 1 (1993) pp. 64-70, which further describes a conversion of a precursor to an amidated product.
  • linking as used herein is meant that the biologically active peptide is associated with the MT in such a manner that when the MT crosses the cell membrane, the active peptide is also imported across the cell membrane.
  • Examples of such means of linking include (A) linking the MT to the active peptide by a peptide bond, i.e., the two peptides (the peptide part of the MT and the active peptide) can be synthesized contiguously; (B) linking the MT to the active peptide by a non-peptide covalent bond (such as conjugating a signal peptide to a protein with a cross-linking reagent); (C) chemical ligation methods can be employed to create a covalent bond between the carboxy- terminal amino acid of an MT such as a signal peptide and the active peptide.
  • a peptide is synthesized, by standard means known in the art, (Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963; and Lin et al., Biochemistry 27:5640-5645, 1988) and contains, in linear order from the amino- terminal end, a signal peptide sequence (the MT), an amino acid sequence that can be cleaved by a plasma protease, and a biologically active amino acid sequence.
  • a signal peptide sequence the MT
  • an amino acid sequence that can be cleaved by a plasma protease an amino acid sequence that can be cleaved by a plasma protease
  • a biologically active amino acid sequence Such a peptide could also be produced through recombinant DNA techniques, expressed from a recombinant construct encoding the above-described amino acids to create the peptide.
  • a peptide bond as above, can be utilized or a non-peptide covalent bond can be used to link the MT with the biologically active peptide, polypeptide or protein.
  • This non-peptide covalent bond can be formed by methods standard in the art, such as by conjugating the MT to the peptide, polypeptide or protein via a cross-linking reagent, for example, glutaraldehyde. Such methods are standard in the art. (Walter et al., Proc. Natl. Acad. Sci. USA 77:5197; 1980).
  • method (C) standard chemical ligation methods, such as using chemical crosslinkers interacting with the carboxy-terminal amino acid of a signal peptide, can be utilized. Such methods are standard in the art (Goodfriend et al. , Science 143: 1344; 1964, which uses water-soluble carbodimide as a ligating reagent) and can readily be performed to link the carboxy terminal end of the signal peptide to any selected biologically active molecule.
  • compositions of the present disclosure e.g. , formulations including one or more aromatic-cationic peptides, and optionally, one or more of the compounds disclosed herein (e.g. , pH lowering agent, absorption enhancer, enteric coating, MT, etc.) are formulated and/or manufactured as follows.
  • Micelles or Lipidic Emulsions are formulated and/or manufactured as follows.
  • one or more aromatic-cationic peptide, and optionally at least one absorption enhancer or other agents are sequestered at a molecular level in solution or colloid state, rather than as a solid, by liposomes or other lipid complexes.
  • the aromatic-cationic peptide and absorption enhancer are encapsulated in a lipid or lipid-polymer particle or nanoparticle (termed lipid emulsion or micelle).
  • the interaction between the heavily positively charged cationic drug and negatively charged phospholipids or lipid anions ⁇ e.g., oleic acid) produce complexes that tend to propel the charge to the exterior in polar environments and the hydrophobic tails into the lipid core or bi-lipid membrane.
  • This structure is able to protect against proteases, e.g., trypsin, from attacking the aromatic-cationic peptide, and optionally another pharmaceutical agent.
  • Other additives can be used to stabilize and balance the dispersion or bi-lipid layer.
  • Micelles and lipidic emulsion formulations can alleviate the need for protease inhibitors.
  • the lipids are selected from the group consisting of caprylic/capric triglyceride ⁇ e.g.
  • glyceryl monocaprate e.g., Capmul MCM CIO, Abitec, Columbus, Ohio
  • non-ionic surfactant e.g., polysorbate 80 or Tween 80
  • the process for making a lipid complex ⁇ e.g., lipid emulsions and micelles) encapsulating one or more aromatic-cationic peptides, and optionally at least one absorption enhancer or other agents, includes dissolving the aromatic-cationic peptide in a polar solvent, ⁇ e.g.
  • Examples of a polar solvent include, but are limited to, methanol, acetone, isopropanol, ME , ethanol, butanol, n-propanol, benzyl alcohol, di-methyl acetamide (DMA), DMF, or a combination thereof.
  • re-suspension of the lipid complexes is in polar solvents that are non-solvents for peptide but solvents for the lipids.
  • polar solvents include, but are not limited to, methanol, acetone-methylene chloride, cyclohexane, ether, chloroform, toluene, ethyl acetate, isopropyl acetate, cyclohexanone, or any combination thereof.
  • one or more absorption enhancers are added in the re- suspension step.
  • the ratio of absorption enhancer to aromatic-cationic peptide is about 10: 1.
  • the ratio of with absorption enhancer to aromatic-cationic peptide is about 1 : 1 , or about 2 : 1 , or about 3 : 1 , or about 4 : 1 , or about 5: 1 , or about 6: 1, or about 7: 1 , or about 8: 1 , or about 9: 1.
  • the re-suspension step includes adding one or more oils.
  • oils include, but are not limited to, oleic acid, stearic acid, ethyl oleate, castor oil, mineral oil, long chain ester oils, such as vegetable oils.
  • the re- suspension step includes adding one or more of: (a) glyceryl monooleate; (b) a sterol, such as cholesterol; (c) bile acids, such as taurocholic, tauorodeoxycholic, deoxycholic, cholic, glycholic, lithocholate, chenodeoxycholic, ursodeoxycholic, ursocholic, dehydrocholic, fusidic; (d) non-ionic surfactants, such as polyoxyethylene ethers (e.g., Brij 36T, Brij 52, Brij 56, Brij 76, Brij 96, Texaphor A6, Texaphor A14, or Texaphor A60 ), p-t-octyl phenol polyoxyethylenes (e.g., Triton X-45, Triton X-100, Triton X-l 14, or Triton X-305), nonylphenoxypoloxyethylenes (e.g.
  • lauroylcholine myristoylcholine, palmitoylcholine, hexadecyllysine, N-acylphenylalanine, or N-acylglycine; or (h) water soluble phospholipids, such as diheptanoylphosphatidylcholine or dioctylphosphatidylcholine.
  • the re-suspension step includes adding suspended or dissolved 1-20% hydroxypropylmethylcellulose acetate succinate (HPMCAS),
  • the inert solid includes, but are not limited to, Avicel, lactose, calcium carbonate, fumed silicon dioxide, di-calcium phosphate (various grades), and mixture of excipients, modified starches (Sta-Rx), and maltodextrins.
  • the 10 mM solution of phospholipids in methanol includes 1) DPG cardiolipin at 0.1275 mg/10 ml; 2) phosphatidyl glycerol-synthetic at 0.0801 mg/10 ml; 3) and phosphatidyl serine-synthetic at 0.081 mg/10 ml.
  • phospholipids include, but are limited to, phosphatidyl inositol, phosphatidic acid, phosphatidic inositol or mixtures of the above with neutral or positively charged lechithins, cyclodextrins (e.g., HPMCD), and sulfobutylether CD beta -CD can also be used to modulate the lipid interactions.
  • the mixing of phospholipid and aromatic-cationic peptide solutions includes the steps of: 1) mixing 2 ml of phospholipid solution and 1 ml of aromatic- cationic peptide into a 20 ml centrifuge or mixing tube; 2) adding 3 ml of methylene chloride solvent; 3) vortexing/mixing the mixture; 4) ultrasonicating for about 1 minute; 5) evaporating to a film on the mixing tube using gentle flow of nitrogen over the solution; 6) mixing 1 ml of phospholipid solution and 1 ml of aromatic-cationic peptide into the mixing tube; 7) repeating steps 2-5; 8) mixing 3 ml of phospholipid solution and 1 ml of aromatic- cationic peptide into the mixing tube; and repeating steps 2-5.
  • the phospholipid -peptide mixtures are micro fluidized using high shear emulsifiers, which can assist in formation of multi-lamellar vesicles (liposomes) in the 100-200 nm range as an alternative to ultrasonification.
  • the mixture is then evaporated in a rotovaporator under vacuum and/or nitrogen flow dry the film out completely.
  • an enteric coat or polymeric coating is applied to the tablet or capsule.
  • the coating is between about 0.5% to about 20%, or between about 1% to about 18%, or between about 3% to about 1 %, or between about 6% to about 12%, or between about 8% to about 10% of total tablet weight.
  • the coating is supplemented with between about 1% to about 15%, or between about 3% to about 12%), or between about 4% to about 10%, or between about 6%> to about 8% by tablet weight with water soluble HPMC or similar polymer.
  • the pharmaceutical compositions described herein include one or more aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH 2 , one or more phospholipid, and optionally at least one absorption enhancer, in contact or association with a substantially hydrophobic (lipophilic) medium.
  • the pharmaceutical composition includes an aromatic-cationic peptide, a phospholipid, and a matrix forming polymer, and optionally an absorption enhancer in contact or association with a substantially hydrophobic (lipophilic) medium.
  • the aromatic-cationic peptide and a medium chain fatty acid or derivative thereof may be coated, suspended, sprayed by or immersed in a substantially hydrophobic medium forming a suspension.
  • the aromatic-cationic peptide and the medium chain fatty acid or derivative thereof are in a solid form within the hydrophobic medium forming a suspension.
  • the compositions of the present technology are not emulsions.
  • the compositions include oily suspensions and the amount of water in the compositions is very low.
  • the compositions incorporate octanoic acid (e.g., about 60-80%), which, in some embodiments, is a suspension at the concentration of solids exemplified, but in some embodiments, at a lower concentration of solids (below the saturation threshold) a solution is obtained.
  • the suspension may be a liquid suspension incorporating solid material, or a semi-solid suspension incorporating solid material (an ointment).
  • the compositions described herein comprise a suspension which comprises an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of an aromatic-cationic peptide and at least one salt of a medium chain fatty acid.
  • the phospholipid is present in the composition at an amount of about 10% or more by weight.
  • the solid form may comprise a particle (e.g., consist essentially of particles, or consist of particles). The solid particle may be produced by lyophilization or by granulation or by spray-drying or by other means.
  • 90% (v/v) of the particles are below 500 microns, and 50% (v/v) of the particles are below 45 microns.
  • about 10% (v/v) of the particles are above about 50-250 microns, and about 50% (v/v) of the particles are above about 40-50 microns.
  • the phospholipid may generally facilitate or enhance permeability and/or absorption of the aromatic-cationic peptide.
  • the phospholipid may generally facilitate or enhance permeability and/or absorption of the aromatic-cationic peptide and an added absorption enhancer.
  • a matrix forming polymer serves to enhance permeability.
  • the phospholipids include derivatives of phospholipids.
  • the aromatic-cationic peptide, the phospholipid, and/or the matrix forming polymer, and optionally the absorption enhancer are in solid form.
  • the solid form may be a solid particle such as a lyophilized particle, a granulated particle, a pellet or a micro-sphere.
  • the aromatic-cationic peptide, the phospholipid, and/or the matrix forming polymer are all in the same solid form, e.g., all in the same particle. In other embodiments, the aromatic-cationic peptide, the phospholipid, and/or the matrix forming polymer may each be in a different solid form, e.g., each in a distinct particle.
  • the compositions described herein are substantially free of any membrane fluidizing agents.
  • the compositions include no membrane fluidizing agents.
  • compositions may include for example less than 1% or less than 0.5% or less than 0.1% by weight of membrane fluidizing agents.
  • a solid form such as a particle containing the aromatic-cationic peptide is provided.
  • the solid form is then associated with the hydrophobic (lipophilic) medium.
  • the amount of water in the compositions is less than about 3% by weight, usually less than about 2% or about 1% or less by weight.
  • the compositions described herein include one or more aromatic-cationic peptide, such as, e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH2, and a phospholipid, wherein the phospholipid is a medium chain fatty acid, and wherein the phospholipid is included in a solid form.
  • the salt of the medium chain fatty acid is in the form of a particle such as a solid particle.
  • the particle may be characterized as a granulated particle.
  • the solid form may generally result from a spray drying or evaporation process.
  • the salt of the medium chain fatty acid is in the same particle as the aromatic- cationic peptide.
  • the aromatic-cationic peptide and the salt of the medium chain fatty acid can be prepared together by first preparing a solution such as an aqueous solution comprising both the aromatic-cationic peptide and the salt of the medium chain fatty acid and co-lyophilizing the solution to provide a solid form or particle that comprises both the aromatic-cationic peptide and the salt of the medium chain fatty acid (and other ingredients).
  • the resulting solid particles are associated with a hydrophobic medium.
  • the solid particles may be suspended or immersed in a hydrophobic medium.
  • the medium chain fatty acid salt and/or a matrix forming polymer may be in the same particle or in a different particle than that of the aromatic-cationic peptide.
  • bioavailability of the aromatic-cationic peptide will be lower if the medium chain fatty acid is in a different particle than the aromatic-cationic peptide e.g., there will be improved bioavailability if the medium chain fatty acid salt and the aromatic-cationic peptide are together ⁇ e.g., dried together) after solubilization in the hydrophilic fraction.
  • the medium chain fatty acid salt, the aromatic-cationic peptide, and/or the matrix forming polymer are dried, after solubilization, together in the hydrophilic fraction then they are all in the same particle in the final powder.
  • Medium chain fatty acid salts include those having a carbon chain length of from about 6 to about 14 carbon atoms.
  • fatty acid salts are sodium hexanoate, sodium heptanoate, sodium octanoate (also termed sodium caprylate), sodium nonanoate, sodium decanoate, sodium undecanoate, sodium dodecanoate, sodium tridecanoate, and sodium tetradecanoate.
  • the medium chain fatty acid salt contains a cation selected from the group consisting of potassium, lithium, ammonium and other monovalent cations e.g., the medium chain fatty acid salt is selected from lithium octanoate or potassium octanoate or arginine octanoate or other monovalent salts of the medium chain fatty acids. It was found that raising the amount of medium chain fatty acid salt increased the rate of medium chain fatty acid salt.
  • the medium chain fatty acid salt for example, sodium octanoate
  • the medium chain fatty acid salt is present at between about 1% to about 5%, or between about 5% to about 10%), or between about 10% to about 15%, or between about 15% to about 20%, or between about 20% to about 25%, or between about 25% to about 30%, or between about 30% to about 35%, or between about 35% to about 40%, or between about 40% to about 45%, or between about 45% to about 50% by weight of the bulk pharmaceutical composition.
  • the amount of medium chain fatty acid salt in the compositions described herein may be from about 0.01% up to about 50% by weight of the bulk pharmaceutical composition.
  • the medium chain fatty acid salt may be present at an amount of between about 10% to about 50%, or at an amount of between about 10%> to about 20% or between about 10 to about 15% or between about 15% to about 20%>, or between about 1 1% to about 40% by weight.
  • the amount of medium chain fatty acid salt in the compositions described herein may be, for example, from between about 1 1% to about 28%), or between about 12% to about 13%, or between about 13% to about 14%, or between about 14% to about 15%>, or between about 15%> to about 16%>, or between about 16% to about 17%, or between about 17% to about 18%, or between about 18% to about 19%, or between about 19% to about 20%, or between about 20% to about 21%, or between about 21 ) to about 22%, or between about 22% to about 23%, or between about 23% to about 24%), or between about 24% to about 25%, or between about 25% to about 26 %, or between about 26% to about 27%), or between about 21% to about 28%) by weight of the bulk pharmaceutical composition.
  • the medium chain fatty acid salt may be present at an amount of at least about 1 1%, at least about 12%, at least about 13%, at least about 14%, at least about 15% at least about 16%, at least about 17%), at least about 18%, at least about 19%, at least about 20%, at least about 21 %, at least about 22%, at least about 23%, at least about 24%o, at least about 25%, at least about 26%, at least about 27% or at least about 28% by weight of the bulk pharmaceutical composition.
  • the medium chain fatty acid salt (sodium, potassium, lithium or ammonium salt or a mixture thereof) is present between about 12% to about 21 % by weight of the bulk pharmaceutical composition, for example, between about 1 1 % to about 18%), or between about 1 1% to about 17%, or between about 12% to about 16%, or between about 12% to about 15%>, or between about 13% to about 16%, or between about 13% to about 15%, or between about 14% to about 16%), or between about 14% to about 15%, or between about 15%) to about 16%), or, for example, about 15% or about 16%).
  • the medium chain fatty acid salt (having a carbon chain length of from between 6 to about 14 carbon atoms; in some embodiments, 8, 9 or 10 carbon atoms) is present at between about 12% to about 21 % by weight of the bulk pharmaceutical composition, for example, between about 1 1 % to about 18%), or between about 1 1% to about 17%), or between about 12% to about 16%, or between about 12% to about 15%, or between about 13% to about 16%>, or between about 13% to about 15%, or between about 14% to about 16%, or between about 14% to about 15%, or between about 15% to about 16%, or, for example, at about 15% or about 16%.
  • the medium chain fatty acid salt (for example salts of octanoic acid, salts of suberic acid, salts of geranic acid) is present at between about 12% to about 21% by weight of the bulk pharmaceutical composition, for example, between about 11 ) to about 18%o, or between about 11% to about 17%, or between about 12% to about 16%), or between about 12% to about 15%, or between about 13% to about 16%, or between about 13% to about 15%>, or between about or 14% to about 16%, or between about 14% to about 15%, or , or between about 15% to about 16%> or most for example about ⁇ 5% or about 16%).
  • the medium chain fatty acid salt is present in the solid powder at an amount between about 50% to about 90%, for example, at an amount between about 70% to about 80%.
  • Some embodiments of the present technology comprise a composition comprising a suspension which consists essentially of an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of the aromatic- cationic peptide and at least one salt of a medium chain fatty acid and/or a matrix forming polymer, and wherein the medium chain fatty acid salt is not a sodium salt.
  • the salt may be the salt of another cation, e.g., lithium, potassium or ammonium; an ammonium salt.
  • the salt of the fatty acid is sodium octanoate and the hydrophobic medium is glyceryl tricaprylate or castor oil; in some embodiments the composition further comprises glyceryl monooleate and sorbitan monopalmitate or glyceryl monocaprylate and glyceryl tricaprylate and
  • composition further comprises glyceryl tributyrate or lecithin or ethylisovalerate or a combination thereof and at least one stabilizer. In some embodiments the composition includes an absorption enhancer.
  • the absorption enhancer is octreotide, insulin, growth hormone, parathyroid hormone, or analogs thereof (e.g., parathyroid hormone amino acids 1-34 termed teriparatide, interferon-alfa (IFN-a)), a low molecular weight heparin, leuprolide, fondaparinux, somatostatin and analogs (agonists) thereof including peptidomimetics, exenatide, terlipressin, vancomycin or gentamicin inter alia, cholecytokinin or analogs thereof, cholecytokinin-8 (CCK-8) or analogs thereof, calcitonin or aliskiren or salts of these the absorption enhancers.
  • teriparatide interferon-alfa (IFN-a)
  • IFN-a interferon-alfa
  • composition further comprises a bile salt.
  • bile salts are sodium taurocholate, sodium deoxycholate, sodium glycocholate, sodium chenodeoxycolate, sodium cholate, sodium lithocholate, in some sodium taurocholate. Hydrophilic Fraction
  • the above compounds including the aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH2, the phospholipid, optional an absorption enhancer, and/or the matrix forming polymer (or substitute) are solubilized in an aqueous medium and then dried to produce a powder.
  • the drying process may be achieved, for example, by lyophilization or granulation or by spray-drying or by other means.
  • the powder obtained is termed the "hydrophilic fraction".
  • water in the hydrophilic fraction, water is normally present at an amount of less than 6% drying, and the water in the final bulk composition comprises residual water from the hydrophilic fraction.
  • the amount of solid form in the hydrophilic fraction of the formulations of the present technology is normally from about 0.5% to about 50% of the formulation (w/w). In certain aspects of the present technology, the amount of solid form is from about 17% to about 40%.
  • Lyophilization may be carried out as described herein and by methods known in the art e.g., as described in Lyophilization: Introduction and Basic Principles, Thomas Jennings, published by Interpharm/CRC Press Ltd (1999, 2002).
  • the lyophilizate may optionally be milled ⁇ e.g., below 150 micron) or ground in a mortar.
  • the lyophilizate is, for example, milled before mixing of the hydrophilic fraction and the hydrophobic medium in order to produce batch-to-batch reproducibility.
  • Granulation may be carried out as described herein and by methods known in the art e.g., as described in Granulation, Salman et al , eds, Elsevier (2006) and in Handbook of Pharmaceutical Granulation Technology, 2nd edition, Dilip M. Parikh, ed., (2005).
  • binders may be used in the granulation process such as celluloses (including micro crystalline celluloses), lactoses (e.g., lactose monohydrate), dextroses, starch and mannitol and other binders as described in the previous two references.
  • celluloses including micro crystalline celluloses
  • lactoses e.g., lactose monohydrate
  • dextroses starch and mannitol
  • the aromatic-cationic peptide is formulated in an adsorbate before being contacted or associated with a substantially hydrophobic (lipophilic) medium.
  • An adsorbate includes the aromatic-cationic peptide and a substrate, and optionally an absorption enhancer.
  • the adsorbate is amorphous, meaning that the peptide is not crystalline as indicated by any conventional method, such as by powder X-ray diffraction (PXRD) analysis.
  • the peptide in the adsorbate is substantially amorphous, meaning that the amount of the peptide in amorphous form is about 70%-90%. In some embodiments, the peptide in the adsorbate is in a completely amorphous form.
  • the adsorbate also includes a high surface area substrate.
  • the substrate may be any material that is inert, meaning that the substrate does not adversely interact with the peptide to an unacceptably high degree and which is pharmaceutically acceptable.
  • the substrate also has a high surface area, meaning that the substrate has a
  • 2 2 2 2 2 2 2 2 surface area of about 20 m /g to 180 m /g, or about 40 m /g to 160 m /g, or about 60 m /g to
  • the surface area of the substrate may be measured using standard procedures.
  • the higher the surface area of the substrate the higher the peptide-to-substrate ratio that can be achieved and still maintain high concentration- enhancements and improved physical stability.
  • effective substrates can have surface areas of up to 200 m 2 /g, up to 400 m 2 /g and up to 600 m 2 /g or more.
  • the substrate may also be in the form of small particles ranging in size of from about 5 nm to about 1 ⁇ , preferably ranging in size from about 5 nm to about 100 nm. These particles may in turn form agglomerates ranging in size from 10 nm to 100 ⁇ .
  • the substrate is also insoluble in the process environment used to form the adsorbate. That is, where the adsorbate is formed by solvent processing, the substrate does not dissolve in the solvent. Where the adsorbate is formed by a melt or thermal process, the substrate has a sufficiently high melting point that it does not melt.
  • Exemplary materials which are suitable for the substrate include inorganic oxides, such as Si0 2 , Ti0 2 , Zn0 2 , ZnO, A1 2 0 3 , MgAl Silicate, Ca Silicate, A10H 2 , zeolites, and other inorganic molecular sieves; water insoluble polymers, such as cross-linked cellulose acetate phthalate, cross-linked hydroxypropyl methyl cellulose acetate succinate, cross-linked polyvinyl pyrrolidinone, (also known as cross povidone) microcrystalline cellulose, polyethylene/polyvinyl alcohol copolymer, polyethylene polyvinyl pyrrolidone copolymer, cross-linked carboxymethyl cellulose, sodium starch glycolate, cross-linked polystyrene divinyl benzene; and activated carbons, including those made by carbonization of polymers such as polyimides, polyacrylonitrile, phenolic resins, cellulose acetate, regenerated
  • the surface of the substrate may be modified with various substituents to achieve particular interactions of the peptide with the substrate.
  • the substrate may have a hydrophobic or hydrophilic surface.
  • the interaction between the peptide and substrate may be influenced. For example, where the peptide is hydrophobic, it may be desired to select a substrate having hydrophobic substituents to improve the binding of the peptide to the substrate.
  • the interaction of peptide with the substrate should be sufficiently high such that mobility of the aromatic-cationic peptide in the peptide/substrate adsorbate is sufficiently decreased such that the composition has improved stability.
  • the peptide/substrate interaction should be sufficiently low such that the peptide can readily desorb from the adsorbate when it is introduced to a use environment, resulting in a high concentration of peptide in solution.
  • the adsorbates are formed so as to form a thin layer of amorphous peptide on the surface of the substrate.
  • a "thin layer” is a layer that ranges in average thickness from less than one peptide molecule to as many as 10 molecules.
  • the peptide layer is generally termed a "monolayer.”
  • the composition of the present technology comprises a suspension which comprises an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of the aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH2, a phospholipid and a matrix forming polymer, and wherein the matrix forming polymer is present in the composition at an amount of about 3% or more by weight.
  • the aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH2
  • a phospholipid such as D-Arg-2'6'-Dmt-Lys-Phe-NH2
  • the matrix forming polymer is present in the composition at an amount of about 3% or more by weight.
  • the composition comprises a suspension which consists essentially of an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of an aromatic- cationic peptide, at least one salt of a phospholipid and a matrix forming polymer, and wherein the matrix forming polymer is present in the composition at an amount about 0.5% to about 10% by weight, or at an amount of about 1% to about 10% by weight, or at an amount of about 3% or more by weight.
  • the matrix forming polymer is present at an amount of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or about 20% or more, by weight.
  • the matrix forming polymer includes, but is not limited to, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), dextran, alginate salt, hyaluronate salt or polyacrylic acid salt or a combination thereof.
  • the matrix forming polymer is polyvinylpyrrolidone (PVP), Carbopol polymer or polyvinyl alcohol (PVA), ionic polysaccharides (for example alginic acid and alginates) or neutral
  • polysaccharides for example dextran and HPMC
  • polyacrylic acid and poly methacrylic acid derivatives for example polyvinyl alcohol
  • high molecular weight organic alcohols for example polyvinyl alcohol
  • the polyvinylpyrrolidone is present in the composition at an amount of about 2% to about 20% by weight, for example at an amount of about 3%) to about 18% by weight, or at an amount of about 5% to about 15% by weight, or at an amount of about 10% by weight.
  • the polyvinylpyrrolidone is PVP- 12 and/or has a molecular weight of about 3000.
  • the matrix forming polymer is polyvinylpyrrolidone (PVP), and the polyvinylpyrrolidone is present in the composition at an amount of about 0.5% to about 20% by weight or about 1% to about 18%, for example, at an amount of about 3% to about 18% by weight.
  • the matrix forming polymer is PVP at an amount of about 5% to about 1 % by weight, and in some embodiments, at an amount of about 10% by weight.
  • the polyvinylpyrrolidone is PVP- 12 and/or has a molecular weight of about 3000.
  • matrix forming polymers are believed to have a similar effects.
  • a range of matrix forming polymers can be substituted e.g., carbomers (Carbopol® polymers Lubrizol, 29400 Lakeland Boulevard, Wickliffe, OH.) or alginate or hyaluronate or polyacrylic acid sodium salt; glucosamine or glucose was also substituted.
  • the matrix forming polymers which produce a higher or similar bioavailability in the formulations of the present technology as PVP include but are not limited to Carbopol® polymer and PVA (polyvinyl alcohol); glucose may give results similar to PVP.
  • Carbopol® polymers are polymers of acrylic acid cross-linked e.g., with polyalkenyl ethers or divinyl glycol. In some embodiments, Carbopol® 934P may give higher bioavailability. Carbopol® 934P is a high molecular weight polymer of acrylic acid crosslinked with allyl ethers of sucrose. PVA is a water-soluble synthetic polymer of vinyl alcohol monomers.
  • replacing PVP-12 in the formulation by e.g., Carbopol® 934P or by PVA or by some of the other matrix forming polymers, may reduce the total amount of matrix forming polymer in the particle phase (i.e., the solid form) of the formulation (the hydrophilic fraction) and thus in some embodiments, may bestow the ability to load more API into the formulation, which may be desirable in order to achieve desired blood levels or reduce capsule size and number.
  • matrix forming polymers include, but are not limited to, cross-linked PVP (cross-povidones); linear polyacrylic acid polymers including polymethacrylic acid polymers; cross-linked polyacrylic acid polymers (carbomers); amino-polysaccharides (e.g., chitosans), S -containing polymers (thiomers) and combinations thereof.
  • cross-linked PVP cross-povidones
  • linear polyacrylic acid polymers including polymethacrylic acid polymers
  • cross-linked polyacrylic acid polymers carbomers
  • amino-polysaccharides e.g., chitosans
  • S -containing polymers thiomers
  • Carbomer is a generic name for cross-linked polymers of acrylic acid; carbomers may be homopolymers of acrylic acid, cross-linked with, for example, an allyl ether pentaerythritol, or allyl ether of sucrose or allyl ether of propylene or allyl sucrose or other sugars or allyl pentaerythritol or a polyalkenyl ether or divinyl glycol.
  • the matrix forming polymer is a cross-linked acrylic acid polymer (also termed carbomer).
  • Carbopol® polymers are examples of cross-linked polymers of acrylic acid.
  • the viscosity of the cross-linked acrylic acid polymer is about 2000-80000 cP, for example 4000-65000, most for example 25000- 45000 cP; the viscosity is measured in cP, 0.5% solution at pH 7.5.
  • the cross-linked acrylic acid polymer is an allyl sucrose-linked carbomer, of viscosity about 29000 to about 40000, e.g., Carbopol® 934P.
  • the cross-linked acrylic acid polymers are present in the composition at an amount between about 0.01% to about 0.1%, or between about 0.1% to about 1.0%, or between about 1% to about 5%, or between about 5% to about 10% by weight.
  • the matrix forming polymer is polyvinyl alcohol of molecular weight 10000-60000 Da, for example 20000-30000 Da.
  • the polyvinyl alcohol is polyvinyl alcohol of molecular weight of about 27000 Da, and may be present in the composition at an amount of about 0.1% to about 6% by weight, for example at an amount of about 0.5% to about 4% by weight, e.g., at an amount of about at an amount of about 1%, about 2%, or about 3% by weight.
  • Glucose and/or other sugars and/or mannitol may be substituted in certain embodiments instead of a matrix forming polymer.
  • the water soluble composition e.g., particle including at least one aromatic-cationic peptide, such as, e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH 2
  • the hydrophobic medium improves the selective translocation of the aromatic-cationic peptide across a biological barrier (e.g., a membrane) in the composition.
  • This capability can be assessed utilizing the "innocent bystander" assay, whereby an impermeable molecule is administered concomitantly to the composition by the same route of administration, and no translocation of the impermeable molecule can be detected.
  • Such an assay utilizing insulin as the impermeable molecule may be tested.
  • the aromatic- cationic peptides such as D-Arg-2'6'-Dmt-Lys-Phe-NH 2 and the phospholipid salt are in contact or association with a hydrophobic (lipophilic) medium.
  • a hydrophobic (lipophilic) medium For example, one or both may be coated, suspended, immersed or otherwise in association with a hydrophobic (lipophilic) medium.
  • Suitable hydrophobic mediums can contain, for example, aliphatic, cyclic or aromatic molecules.
  • Non-limiting examples of a suitable aliphatic hydrophobic medium include, but are not limited to, mineral oil (e.g., paraffin), fatty acid monoglycerides, diglycerides, triglycerides, ethers, esters, and combinations thereof.
  • a suitable fatty acid are octanoic acid, decanoic acid and dodecanoic acid, also C7 and C9 fatty acids and di-acidic acids such as sebacic acid and suberic acid, and derivatives thereof.
  • Non-limiting examples of triglycerides include, but are not limited to, long chain
  • triglycerides triglycerides, medium chain triglycerides, and short chain triglycerides.
  • the long chain triglyceride can be castor oil or coconut oil or olive oil
  • the short chain triglyceride can be glyceryl tributyrate and the medium chain triglyceride can be glyceryl tricaprylate.
  • Monoglycerides are considered to be surfactants and are described below.
  • Non- limiting exemplary esters include ethyl isovalerate and butyl acetate.
  • Non-limiting examples of a suitable cyclic hydrophobic medium include, but are not limited to, terpenoids, cholesterol, cholesterol derivatives (e.g., cholesterol sulfate), and cholesterol esters of fatty acids.
  • a non-limiting example of an aromatic hydrophobic medium includes benzyl benzoate.
  • the hydrophobic medium include a plurality of hydrophobic molecules.
  • the hydrophobic medium also includes one or more surfactants.
  • Exemplary surfactants include phospholipids such as lecithin or a block copolymer such as pluronic F-68
  • compositions including a surfactant in the hydrophobic medium comprises less than about 20% by weight of surfactant in the hydrophobic medium.
  • the hydrophobic medium generally comprises from about 30% to about 90%> by weight of the composition.
  • the hydrophobic medium comprises about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or higher by weight of the composition.
  • the hydrophobic medium also includes one or more adhesive polymers such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose (HPMC), or poly(acrylate) derivative CarbopolTM 934P (C934P).
  • adhesive polymers such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose (HPMC), or poly(acrylate) derivative CarbopolTM 934P (C934P).
  • HPMC hydroxypropylmethylcellulose
  • C934P poly(acrylate) derivative CarbopolTM 934P
  • the present technology provides compositions for penetration that specifically target various tissues, for example, those containing epithelial and endothelial cells, for the delivery of drugs and other the aromatic-cationic peptides across a biological barrier.
  • Existing transport systems known in the art are typically too limited to be of general application, and because they can be inefficient, they can alter the biological properties of the active substance, compromise the target cell, irreversibly destroy the biological barrier and/or pose too high of a risk to be used in human subjects.
  • Embodiments of the present technology include compositions containing an aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH 2 in a water soluble composition.
  • This complex can be optionally lyophilized and then immersed in a hydrophobic medium.
  • the immersion of the water soluble composition containing the aromatic-cationic peptide, or a lyophilizate thereof, in the hydrophobic medium results in an a unique association between the aromatic-cationic peptide and the penetration enhancing compounds, thereby enabling the aromatic-cationic peptide to efficiently translocate across a biological barrier.
  • the compositions of the present technology will be defined by their efficiency, and translocation of at least 5% (but for example 10%, 20%, 30%, 40%,50%, 60%,70%, 80% or more) of the aromatic-cationic peptide across an epithelial barrier is achieved.
  • compositions of the present technology selectively allow the translocation of an aromatic-cationic peptide across the biological barrier.
  • the hydrophobic medium serves as a shield, thereby preventing neighboring molecules, such as proteins, toxins, and other "bystander" molecules, from co-translocating through the biological barrier with the aromatic-cationic peptide.
  • microemulsions are thermodynamically stable dispersions of one liquid phase into another, that involve a combination of at least three components-oil, water, and a surfactant. Both water-in-oil (w/o) and oil-in- water (o/w) microemulsions have been proposed to enhance the oral bioavailability of drugs. They offer improved drug
  • the penetration compositions of the present technology contain aromatic-cationic peptides such as D-Arg-2'6'-Dmt-Lys-Phe-NH 2 in a water soluble composition immersed in a hydrophobic medium, which facilitates the effective translocation of the aromatic-cationic peptide across a biological barrier.
  • aromatic-cationic peptides such as D-Arg-2'6'-Dmt-Lys-Phe-NH 2
  • a water soluble composition immersed in a hydrophobic medium, which facilitates the effective translocation of the aromatic-cationic peptide across a biological barrier.
  • the water soluble composition can be dissolved either in water or in a non-aqueous medium such as, for example, mono-alcohols, di-alcohols, or tri-alcohols.
  • the water soluble composition according to the present technology can be totally evaporated, e.g., via lyophilization, prior to suspension in the hydrophobic medium.
  • the water soluble composition is totally evaporated, via lyophilization to provide a particle containing the aromatic-cationic peptide, which is, then suspended in the hydrophobic medium.
  • the compositions also include a membrane fluidizing agent. The membrane fluidizing agent is contained within the hydrophobic medium.
  • the penetration compositions of this present technology provide an oral delivery system whereby the addition of a surface active agent is optional.
  • the compositions contain less than about 1-30% by weight of a surface active agent (e.g., less than about 20% less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or is substantially free of surfactant).
  • the water soluble composition is suspended within a hydrophobic region, which contains a membrane fluidizing agent.
  • the water soluble composition is a particle (e.g., a lyophilized particle) comprising one or more aromatic-cationic peptide, such as, e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • the particles are from between about 10 nanometers and about 10 micrometers in diameter (e.g., from about 100 nanometers to about 1 micrometer in diameter).
  • the water soluble composition includes the aromatic-cationic peptide, and in some embodiments can include one or more additional agents, for example a stabilizer (e.g., a protein stabilizer), a surface active agent, one or more counter ions, a protective agent, or a viscosity adjusting agent.
  • the water soluble composition can include a stabilizer (e.g., a stabilizer of protein structure).
  • Stabilizers of protein structure are compounds that stabilize protein structure under aqueous or non-aqueous conditions and/or can reduce or prevent aggregation of the aromatic-cationic peptide.
  • an absorption enhancer can be added, for example during a drying process such as lyophilization or the processing step.
  • compositions of this present technology can deliver such aromatic-cationic peptides across biological barriers through non-invasive administration, including, for example oral, buccal, nasal, rectal, inhalation, insufflation, transdermal, or depository.
  • a further advantage of the compositions of the present technology is that they might be able to cross the blood-brain barrier, thereby delivering aromatic-cationic peptides to the central nervous system (CNS).
  • CNS central nervous system
  • compositions of this present technology facilitate the effective passage
  • Translocation may be detected and quantified by any method known to those skilled in the art, including using imaging compounds such as radioactive tagging and/or fluorescent probes or dyes incorporated into a hydrophobic composition in conjunction with a paracytosis assay as described in, for example, Scangegaarde, et al., Infect, and Immun., 68(8):4616-23 (2000).
  • imaging compounds such as radioactive tagging and/or fluorescent probes or dyes incorporated into a hydrophobic composition in conjunction with a paracytosis assay as described in, for example, Scangegaarde, et al., Infect, and Immun., 68(8):4616-23 (2000).
  • a paracytosis assay is performed by: a) incubating a cell layer with a composition described by this present technology; b) making cross sections of the cell layers; and c) detecting the presence of the aromatic-cationic peptides, or any other component of the compositions of this present technology.
  • the detection step may be carried out by incubating the fixed cell sections with labeled antibodies directed to a component of the compositions of this present technology, followed by detection of an immunological reaction between the component and the labeled antibody.
  • the peptide may be labeled using a radioactive label, or a fluorescent label, or a dye in order to directly detect the presence of the peptide.
  • a bioassay can be used to monitor the peptide translocation. For example, using a bioactive molecules such as erythropoietin, included in a penetration composition, the increase in hemoglobin or hematocrit can be measured. Similarly, by using a bioactive molecule such as insulin coupled with the aromatic-cationic peptide composition, the drop in blood glucose level can be measured
  • compositions of this present technology comprising aromatic-cationic peptides such as D-Arg-2'6'-Dmt-Lys-Phe-NH2, employ membrane fluidizing agents.
  • a membrane fluidizing agent may be a linear, branched, cyclical or aromatic alcohol.
  • suitable linear alcohols include, but are not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, and dodecanol.
  • Non- limiting examples of branched alcohols include geraniol and farnesol.
  • An example of a cyclical alcohol includes menthol.
  • suitable aromatic alcohols can include benzyl alcohol, 4-hydroxycinnamic acid, and phenolic compounds.
  • phenolic compounds can include phenol, m-cresol, and m-chlorocresol.
  • membrane fluidizing agents are medium chain alcohols which have a carbon chain length of from 4 to 15 carbon atoms (e.g., including 5 to 1 , 5 to 12, 6, 7, 8, 9, 10, or 1 1 carbon atoms).
  • a membrane fluidizing agent may be a linear (e.g., saturated or unsaturated), branched (e.g., saturated or unsaturated), cyclical (e.g., saturated or unsaturated), or aromatic alcohol.
  • linear alcohols examples include, but are not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, and pentadecanol.
  • butanol pentanol
  • hexanol hexanol
  • heptanol octanol
  • nonanol decanol
  • undecanol undecanol
  • dodecanol dodecanol
  • tridecanol tridecanol
  • tetradecanol examples include pentadecanol.
  • the membrane fluidizing agent includes 1-ocatanol
  • branched alcohols include geraniol, rhodinol, citronellol, and farnesol.
  • the membrane fluidizing agent includes geraniol.
  • Exemplary cyclical alcohol includes menthol, terineol, myrtenol, perilly alcohol.
  • suitable aromatic alcohols can include benzyl alcohol, 4-hydroxycinnamic acid, thymol, styrene glycol, and phenolic compounds.
  • phenolic compounds can include phenol, m-cresol, and m- chlorocresol.
  • the composition includes from about 1% to about 5% by weight of membrane fluidizing agent (e.g., from between about 5% to about 40% by weight of a membrane fluidizing agent or combinations thereof).
  • membrane fluidizing agents increase the fluidity and decrease the order of lipids in biological membranes. This alteration of membrane dynamics may be detected by the decrease in the steady state anisotropy of fluorescent membrane probes, such as l,6-diphenyl-l ,3,5-hexatriene.
  • fluorescent membrane probes such as l,6-diphenyl-l ,3,5-hexatriene.
  • Normal alcohols, or n-alkanols are known membrane fluidizing agents. Due to their amphipathic properties, they partition the membrane lipid bilayer with their hydroxyl moiety near the phospholipids polar headgroups, and their aliphatic chains intercalated among the fatty acyl chains of the phospholipids. Alkanols of increasing chain length penetrate the bilayer to increasing depths, and thus affect bilayer order and dynamics to a different extent. See Zavoico et ah , Biochim. Biophys Acta, 812:299-312 (1985).
  • one or more counter ions are combined with one or more aromatic-cationic peptide in the formulations of the present technology.
  • Counter ions according to this present technology include anionic or cationic amphipathic molecules, i. e. , those having both polar and nonpolar domains, or both hydrophilic and hydrophobic properties.
  • Anionic or cationic counter ions of this present technology are ions that are negatively (anionic) or positively (cationic) charged and can include a hydrophobic moiety. Under appropriate conditions, anionic or cationic counter ions can establish electrostatic interactions with cationic or anionic impermeable molecules, respectively. The formation of such a complex can cause charge neutralization, thereby creating a new uncharged entity, with further hydrophobic properties in case of an inherent hydrophobicity of the counter ion.
  • Suitable anionic counter ions are ions with negatively charged residues such as carboxylate, sulfonate or phosphonate anions, and can further contain a hydrophobic moiety.
  • anionic counter ions include sodium dodecyl sulphate, dioctyl
  • the anionic counter ion is sulfobutyl ether cyclodextrin, which is available as a sodium salt and can interact with the cationic peptide electrostatically and hydrophobically.
  • Anionic counter ions have the potential to protect the aromatic-cationic peptide from protease attack via charge neutralization and hydrophobic interaction (e.g., micellar solubilization or hydrophobic complex formation).
  • cyclodextrins can also be used, but these lack the anionic charge (e.g. , HPMCD, beta-DD, gamma-CD).
  • anionic counter ions include, but are not limited to, negatively charged, long chain hydrophobic or aromatic sulfate, phosphate, carbamate ester, and carbonyl acid.
  • the molar ratio of aromatic-cationic peptide to the anionic counter ion is about 1 : 10, about 1 :9, about 1 :8, about 1 :7, about 1 :6, about 1 :5, about 1 :4, about 1 :3, about 1 :2, or about 1 : 1.
  • the molar ratio of aromatic-cationic peptide to the anionic counter ion is about 1 :6.
  • Exemplary suitable cationic counter ions include quaternary amine derivatives, such as benzalkonium derivatives or other quaternary amines, which can be substituted by hydrophobic residues.
  • quaternary amines contemplated by the present technology have the structure: 1-R1-2-R2-3-R3-4-R4-N, wherein Rl, 2, 3, or 4 are alkyl or aryl derivatives.
  • quaternary amines can be ionic liquid forming cations, such as imidazolium derivatives, pyridinium derivatives, phosphonium compounds or
  • Ionic liquids are salts composed of cations such as imidazolium ions, pyridinium ions and anions, such as BF 4 and PFg, and are liquid at relatively low temperatures. Ionic liquids are characteristically in liquid state over extended temperature ranges, and have high ionic conductivity. Other favorable characteristic properties of the ionic liquids include non- flammability, high thermal stability, relatively low viscosity, and essentially no vapor pressure. When an ionic liquid is used as a reaction solvent, the solute is solvated by ions only, thus creating a totally different environment from that when water or ordinary organic solvents are used. This enables high selectivity, applications of which are steadily expanding.
  • Some examples are in the Friedel-Crafts reaction, Diels-Alder reaction, metal catalyzed asymmetric synthesis and others. Furthermore, some ionic liquids have low solubility in water and low polar organic solvents, enabling their recovery after reaction product is extracted with organic solvents. Ionic liquids are also used electrochemically, due to their high ion-conductivity, for example as electrolytes of rechargeable batteries.
  • imidazolium derivatives have the general structure of 1-R1-3-R2- imidazolium where Rl and R2 can be linear or branched alkyls with 1 to 12 carbons. Such imidazolium derivatives can be further substituted for example by halogens or an alkyl group.
  • imidazolium derivatives include, but are not limited to, l-ethyl-3- methylimidazolium, l-butyl-3-methylimidazolium, l-hexyl-3-methylimidazolium, 1-methyl- 3-octylimidazolium, l-methyl-3-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-imidazolium, 1,3-dimethylimidazolium, and l,2-dimethyl-3-propylimidazolium.
  • Pyridinium derivatives have the general structure of 1-R1-3-R2 -pyridinium where Rl is a linear or branched alkyl with 1 to 12 carbons, and R2 is H or a linear or branched alkyl with 1 to 12 carbons. Such pyridinium derivatives can be further substituted for example by halogens or an alkyl group. Pyridinium derivatives include, but are not limited to, 3 -methyl- 1 -propylpyridinium, l-butyl-3-methylpyridinium, and l-butyl-4- methylpyridinium.
  • enhancement of bioavailability may be achieved with one or more classes of enhancers selected from fatty acids, sugar esters of fatty acids, acyl carnitines and citrates. Some embodiments use combinations thereof, except that acyl carnitines and fatty acids are not used together because of undesirable interaction between them. Molecular structures regarding each class is discussed below.
  • fatty acids interact with peptides to desirably enhance their ability to penetrate cell membranes, thus enhancing transcellular transport.
  • the hydrophobic region of fatty acids is believed important to this function, and should desirably include as many consecutive carbon atoms as possible, consistent with water solubility, at least 8 consecutive carbon atoms, or 10-14 carbon atoms.
  • Illustrative fatty acids include but are not limited to lauric acid and oleic acid.
  • sugar esters of fatty acids may interact with cells in a manner that could alter their shape, increase pore size, and thereby desirably increase paracellular transport. They may also provide benefit in transcellular transport.
  • bioavailability may be especially enhanced by the combination of enhanced transcellular and enhanced paracellular transport.
  • the hydrophobic region may also include at least 8 consecutive carbon atoms, especially 10-14 carbon atoms.
  • the sugar moiety may aid water solubility.
  • Illustrative sugar esters of fatty acids include but are not limited to sucrose laurate, glucose laurate and fructose laurate. When used, concentration of sugar esters of fatty acids may be between 0.1 and 10.0 mg/ml, or between 0.5 and 5.0 mg/ml.
  • acyl carnitines are believed to enhance bioavailability, and in some embodiments are combined with a sugar ester of a fatty acid.
  • Illustrative acyl carnitines include but are not limited to lauroyl-1- carnitine and myristoyl carnitine. When used, concentration of acyl carnitine may be between 0.1 and 10.0 mg/ml, or between 0.5 and 5.0 mg/ml.
  • citrate -type bioavailability enhancing agents selected from the group consisting of citric acid, citric acid salt and mixtures thereof are used in
  • citrate-type enhancing agents may increase paracellular transport.
  • the concentration of all such citrate-type enhancing agents will be no lower than 5 mM and no higher than 50 mM, or in the range of 10-25 mM.
  • shelf stability may be undesirably reduced at higher citrate concentrations due to interaction of citrate with the active peptide at the amino terminus of the peptide, or at lysyl side chains.
  • the aromatic-cationic peptide formulations of the present technology are useful in treating any disease or condition that is associated with, for example, MPT.
  • diseases and conditions include, but are not limited to, ischemia and/or reperfusion of a tissue or organ, hypoxia, diseases and conditions of the eye, myocardial infarction and any of a number of neurodegenerative diseases.
  • Mammals in need of treatment or prevention of MPT are those mammals suffering from these diseases or conditions.
  • Ischemia in a tissue or organ of a mammal is a multifaceted pathological condition which is caused by oxygen deprivation (hypoxia) and/or glucose (e.g., substrate) deprivation.
  • Oxygen and/or glucose deprivation in cells of a tissue or organ leads to a reduction or total loss of energy generating capacity and consequent loss of function of active ion transport across the cell membranes.
  • Oxygen and/or glucose deprivation also leads to pathological changes in other cell membranes, including permeability transition in the mitochondrial membranes.
  • other molecules, such as apoptotic proteins normally
  • Ischemia or hypoxia in a particular tissue or organ may be caused by a loss or severe reduction in blood supply to the tissue or organ.
  • the loss or severe reduction in blood supply may, for example, be due to thromboembolic stroke, coronary atherosclerosis, or peripheral vascular disease.
  • the tissue affected by ischemia or hypoxia is typically muscle, such as cardiac, skeletal, or smooth muscle.
  • the organ affected by ischemia or hypoxia may be any organ that is subject to ischemia or hypoxia.
  • organs affected by ischemia or hypoxia include brain, heart, kidney, and prostate.
  • cardiac muscle ischemia or hypoxia is commonly caused by atherosclerotic or thrombotic blockages which lead to the reduction or loss of oxygen delivery to the cardiac tissues by the cardiac arterial and capillary blood supply.
  • Such cardiac ischemia or hypoxia may cause pain and necrosis of the affected cardiac muscle, and ultimately may lead to cardiac failure.
  • Ischemia or hypoxia in skeletal muscle or smooth muscle may arise from similar causes.
  • ischemia or hypoxia in intestinal smooth muscle or skeletal muscle of the limbs may also be caused by atherosclerotic or thrombotic blockages.
  • Reperfusion is the restoration of blood flow to any organ or tissue in which the flow of blood is decreased or blocked.
  • blood flow can be restored to any organ or tissue affected by ischemia or hypoxia.
  • the restoration of blood flow can occur by any method known to those in the art. For instance, reperfusion of ischemic cardiac tissues may arise from angioplasty, coronary artery bypass graft, or the use of thrombolytic drugs.
  • compositions e.g., formulations of the present technology comprising an aromatic-cationic peptide such as 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • neurodegenerative diseases associated with MPT include, for instance, Parkinson's disease, Alzheimer's disease, Huntington's disease and Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig's disease).
  • compositions of the present disclosure can be used to delay the onset or slow the progression of these and other neurodegenerative diseases associated with MPT.
  • the compositions of the present disclosure are useful in the treatment of humans suffering from the early stages of neurodegenerative diseases associated with MPT and in humans predisposed to these diseases.
  • the aromatic-cationic peptide formulations of the present disclosure may also be used in preserving an organ of a mammal prior to transplantation.
  • a removed organ can be susceptible to MPT due to lack of blood flow. Therefore, the oral formulation of the peptides can be administered to a subject prior to organ removal, for example, and used to prevent MPT in the removed organ.
  • the formulations of the present disclosure may also be administered to a mammal taking a different drug to treat a condition or disease.
  • a side effect of the drug includes MPT
  • mammals taking such drugs would greatly benefit from the oral formulations of aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Pfie-NH 2 ) of the present technology disclosed herein.
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) for the treatment or prevention of peripheral neuropathy or the symptoms of peripheral neuropathy.
  • the peripheral neuropathy is drug-induced peripheral neuropathy.
  • the peripheral neuropathy is induced by a chemotherapeutic agent.
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) for the treatment or prevention of peripheral neuropathy or the symptoms of peripheral neuropathy
  • chemotherapeutic agent is a vinca alkaloid.
  • the vinca alkaloid is vincristine.
  • the symptoms of peripheral neuropathy include hyperalgesia.
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) for the treatment or prevention of hyperalgesia.
  • the hyperalgesia is drug-induced.
  • the hyperalgesia is induced by a chemotherapeutic agent.
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) for the treatment or prevention of hyperalgesia.
  • the hyperalgesia is drug-induced
  • chemotherapeutic agent is a vinca alkaloid.
  • the vinca alkaloid is vincristine.
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to reduce CD36 expression in post-ischemic brain in a subject in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to reduce CD36 expression in renal tubular cells after unilateral ureteral obstruction (UUO) in a subject in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-N]3 ⁇ 4, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to reduce lipid peroxidation in a kidney after UUO.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-N]3 ⁇ 4, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2 ) to reduce tubular cell apoptosis in an obstructed kidney after UUO.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2 ) to reduce macrophage infiltration in an obstructed kidney induced by UUO.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2 ) to reduce interstitial fibrosis in an obstructed kidney after UUO.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2 ) to reduce infarct volume and hemispheric swelling in a subject suffering from acute cerebral ischemia.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2 ) to reduce the decrease in reduced glutathione (GSH) in post-ischemic brain in a subject in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2 ) to reduce up-regulation of CD36 expression in cold storage of isolated hearts.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2 ) to reduce lipid peroxidation in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged cold ischemia.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2 ) to abolish endothelial apoptosis in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged cold ischemia.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to preserve coronary flow in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged cold ischemia.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-N]3 ⁇ 4, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to prevent damage to renal proximal tubules in diabetic subjects.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-N]3 ⁇ 4, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to prevent renal tubular epithelial cell apoptosis in diabetic subjects.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to inhibit mitochondrial swelling and cytochrome c release.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to protect myocardial contractile force during ischemia-reperfusion in cardiac tissue.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2) that administered with a cardioplegic solution, which will enhance contractile function after prolonged ischemia in isolated perfused cardiac tissue (e.g., heart).
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to reduce spontaneous generation of hydrogen peroxide by mitochondria in certain stress or disease states.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to inhibit spontaneous production of hydrogen peroxide in mitochondria and hydrogen peroxide production stimulated by antimycin.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to decrease intracellular ROS (reactive oxygen species) and increase survival in cells of a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • ROS reactive oxygen species
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to prevent loss of cell viability in subjects suffering from a disease or condition characterized by mitochondrial dysfunction.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to decrease the percent of cells showing increased caspase activity in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to reduce the rate of ROS accumulation in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to inhibit lipid peroxidation in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to prevent mitochondrial depolarization and ROS accumulation in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to prevent apoptosis in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-N]3 ⁇ 4, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to significantly improve coronary flow in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged (e.g., 18 hours) cold ischemia.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-N]3 ⁇ 4, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2) to prevent apoptosis in endothelial cells and myocytes in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged (e.g., 18 hours) cold ischemia.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to improve survival of pancreatic cells in a subject in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to reduce apoptosis and increase viability in islet cells of pancreas in subjects in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2) to reduce oxidative damage in pancreatic islet cells in subjects in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-N]3 ⁇ 4, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to protect dopaminergic cells against MPP+ toxicity in subjects in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-N]3 ⁇ 4, or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to prevent loss of dopaminergic neurons in subject in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to increase striatal dopamine, DOPAC (3,4- dihydroxyphenylacetic acid) and HVA (homovanillic acid) levels in subjects in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • DOPAC 3,4- dihydroxyphenylacetic acid
  • HVA homovanillic acid
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to reduce oxidative damage in a mammal in need thereof.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • ROS and RNS examples include hydroxyl radical (HO ), superoxide anion radical (02 ' -), nitric oxide ( O ' ), hydrogen peroxide (H 2 0 2 ), hypochlorous acid (HOCI), and peroxynitrite anion (ONOO-).
  • a mammal in need thereof may be a mammal undergoing a treatment associated with oxidative damage.
  • the mammal may be undergoing reperfusion.
  • “Reperfusion” refers to the restoration of blood flow to any organ or tissue in which the flow of blood is decreased or blocked. The restoration of blood flow during reperfusion leads to respiratory burst and formation of free radicals.
  • a mammal in need thereof is a mammal suffering from a disease or condition associated with oxidative damage.
  • the oxidative damage can occur in any cell, tissue or organ of the mammal.
  • cells, tissues or organs affected by oxidative damage include, but are not limited to, endothelial cells, epithelial cells, nervous system cells, skin, heart, lung, kidney, eye and liver.
  • lipid peroxidation and an inflammatory process are associated with oxidative damage for a disease or condition.
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) for use in reducing oxidative damage associated with a neurodegenerative disease or condition.
  • the neurodegenerative disease can affect any cell, tissue or organ of the central and peripheral nervous system. Examples of such cells, tissues and organs include, the brain, spinal cord, neurons, ganglia, Schwann cells, astrocytes, oligodendrocytes and microglia.
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to effect oxidation state of muscle tissue.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to effect oxidation state of muscle tissue in lean and obese human subjects.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to effect insulin resistance in muscle tissue.
  • aromatic-cationic peptides e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH 2 , Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to treat, prevent or ameliorate a disease or condition comprising a neurological or neurodegenerative disease or condition, ischemia, reperfusion, hypoxia, atherosclerosis, ureteral obstruction, diabetes, complications of diabetes, arthritis, liver damage, insulin resistance, diabetic nephropathy, acute renal injury, chronic renal injury, acute or chronic renal injury due to exposure to nephrotoxic agents and/or radiocontrast dyes, hypertension, metabolic syndrome, an ophthalmic disease or condition such as dry eye, diabetic retinopathy, cataracts, retinitis pigmentosa, glaucoma, macular degeneration, choroidal
  • the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe- Lys-NH 2 , Phe-D-Arg-Phe-Lys-NH 2 , or D-Arg-2',6'-Dmt-Lys-Phe-NH 2 ) to treat, prevent or ameliorate macular degeneration, and/or the signs or symptoms of macular degeneration, wet or dry.
  • the macular degeneration is age related macular degeneration. Bioavailability and Delivery of Peptides
  • Aromatic-cationic peptides of the present technology may be chosen as an active ingredient for treatment of medical conditions and diseases as recited herein. Orally, nasally, inhaled, ophthalmic, topically, transdermally, intrathecally and parenterally (e.g.,
  • aromatic-cationic peptide will be effective against medical conditions or diseases such as those described herein.
  • delivery to the lungs via aerosolized e.g., lipid emulsion or micelle or suspension peptide formulations provides rapid absorption into the cardiovascular system and rapid uptake into the heart and lungs.
  • the same formulations e.g., a liquid preparation
  • inhalation e.g., aerosolized liquid preparation
  • Serum levels may be measured by HPLC or mass spectroscopy, according to methods known in the art.
  • the attending physician may monitor patient response, aromatic- cationic peptide blood levels, or surrogate markers of disease, especially during the initial phase of treatment. The physician may then alter the dosage somewhat to account for individual patient metabolism and response.
  • the bioavailability achievable in accordance with the present methods permits, for example, delivery of aromatic-cationic peptide into the blood while using between 10-200 micrograms, or between 200-500 micrograms, or between 500-1000 micrograms, or between 1000- 5000 micrograms, or between 5000- 10,000 micrograms, or between 10,000- 20,000 micrograms, or between 20,000- 30,000 micrograms, or between 30,000- 40,000 micrograms, or between 40,000- 50,000 micrograms of orally, ophthalmiclly, nasally, or inhaled administered aromatic-cationic peptides of the present technology.
  • the aromatic-peptide compositions of the present technology is give parenterally for example via intravenous (IV) infusion.
  • aromatic-peptide compositions of the present technology are delivered at between 10-200 micrograms, or between 200-500 micrograms, or between 500-1000 micrograms, or between 1000- 5000 micrograms, or between 5000-10,000 micrograms, or between 10,000- 15,000 micrograms.
  • a single dosage e.g., a capsule, tablet or liquid formulation for oral, inhalation, ocular, parenteral or nasal administration
  • a single dosage e.g., a capsule, tablet or liquid formulation for oral, inhalation, ocular, parenteral or nasal administration
  • an acid is able to reduce undesirable proteolytic attack on the peptide when the acid is released in close time proximity to release of the peptide.
  • Near simultaneous release is, in some embodiments, achieved by administering all components of the methods as a single pill or capsule.
  • the methods also include, for example, dividing the required amount of acid and enhancers, when used, among e.g., two or more capsules, tablets or doses (e.g., for nasal, inhalation or parenteral administration) which may be administered together such that they together provide the necessary amount of all ingredients.
  • Both man-made and natural peptides can be delivered orally, nasally, by inhalation, ophthalmic administration and/or parenterally, e.g., intravenously, in accordance with the methods and compositions disclosed herein.
  • the peptides for use in the methods may be in free form or in pharmaceutically acceptable salt or complex form, e.g. , in pharmaceutically acceptable acid addition salt form.
  • Such salts and complexes are known and tend to possess an equivalent degree of activity and tolerability to the free forms.
  • Suitable acid addition salt forms for use in accordance with the methods include for example the hydrochlorides and acetates.
  • a single tablet capsule or liquid oral dosage is used at each administration because a single dose of the product provides simultaneous release of the aromatic-cationic peptide of the present technology, pH-lowering agent and absorption enhancers.
  • the acid is best able to reduce undesirable proteolytic attack on the polypeptide when the acid is released in close time proximity to release of the polypeptide. Near simultaneous release, in some embodiments, can thus be achieved by administering all components of the present formulations as a single tablet or capsule or liquid oral dosage.
  • the present technology also includes, for example, dividing the required amount of acid and enhancers among two or more tablets or capsules or liquid dosages which may be administered together such that they together provide the necessary amount of all ingredients.
  • pharmaceutical composition includes a complete dosage appropriate to a particular administration to a human patient regardless of how it is subdivided so long as it is for substantially simultaneous administration.
  • a first oral pharmaceutical composition is administered in a capsule or tablet or liquid formulation which does not contain a protective acid stable vehicle, such that the components will be relatively rapidly released in the stomach and thus be available for immediate pain relief, e.g. , within about 10-20 minutes.
  • additional capsules or tablets formulated according to the methods with a protective vehicle may then be administered, resulting in bioavailability in the intestine of the active ingredient after the longer time interval that is required for gastric emptying, i. e. , typically around two hours.
  • a sufficient amount of the aromatic-cationic peptide is included in the oral formulation of the composition to achieve a serum level ( . e. , Cmax) of the aromatic-cationic peptide is from 200 ⁇ g ml to 20 ng/ml, or from 200 ⁇ g ml to 2 ng/ml.
  • Dosage levels of the aromatic-cationic peptide for achieving the above serum levels may range from 100 ⁇ g to 10 mg, or from 100 ⁇ g to 1 mg. With respect to the dosages recommended herein, however, the attending clinician should monitor the subject's response and adjust the dosage accordingly.
  • the dosage of the aromatic-cationic peptide of the present technology is identical for both therapeutic and prophylactic purposes. The dosage for each aromatic-cationic peptide discussed herein is the same, regardless of the disease being treated (or prevented).
  • dosages herein refer to weight of aromatic-cationic peptide unaffected by pharmaceutical excipients, diluents, carriers or other ingredients, although such additional ingredients are desirably included.
  • compositions of the present disclosure may be applied in accordance with the methods to the nasal mucosa, e.g., either in drop or in spray form.
  • compositions of the present disclosure may of course also include additional ingredients, in particular components belonging to the class of conventional pharmaceutically applicable surfactants.
  • the aromatic-cationic compositions of the present disclosure are formulated as liquid pharmaceutical compositions, and in some embodiments, contain a pharmaceutically acceptable diluent or carrier suitable for application to the nasal mucosa.
  • a pharmaceutically acceptable diluent or carrier suitable for application to the nasal mucosa.
  • Aqueous saline may be used for example.
  • the compositions of the present disclosure are formulated so as to permit administration via the nasal route.
  • they may also contain, e.g., minimum amounts of any additional ingredients or excipients desired, for example, additional preservatives or, e.g., ciliary stimulants such as caffeine.
  • a mildly acid pH will be used.
  • the compositions of the present disclosure have a pH of from about 3.0 to 6.5.
  • the compositions have a pH of from about 3 to 5, about 3.5 to about 3.9 or about 3.7.
  • adjustment of the pH is achieved by addition of an appropriate acid, such as hydrochloric acid.
  • the compositions of the present disclosure also possess an appropriate isotonicity and viscosity.
  • the compositions have an osmotic pressure of from about 260 to about 380 mOsm/liter.
  • the viscosity for the nasal spray is less than 0.98 cP.
  • the osmotic pressure is from 250 to 350 mOsm/liter.
  • compositions in accordance with the present disclosure may also comprise a conventional surfactant, such as a non-ionic surfactant.
  • a surfactant such as a non-ionic surfactant.
  • the amount present in the compositions will vary depending on the particular surfactant chosen, the particular mode of administration (e.g., drop or spray) and the effect desired. In general, however, the amount present will be of the order of from about 0.1 mg/ml to about 10 mg/ml, about 0.5 mg/ml to 5 mg/ml, or about 1 mg/ml.
  • the use of surface active agents generally in relation to the nasal application of aromatic-cationic peptides of the present technology may increase absorption via the nasal mucosa and hence improve obtained bioavailability rates.
  • a pharmaceutically acceptable preservative is included.
  • Many are known in the art, and have been used in the past in connection with aqueous nasal pharmaceuticals.
  • benzyl alcohol or phenylethyl alcohol or a mixture thereof may be employed.
  • 0.2% phenylethyl alcohol and 0.5% benzyl alcohol are used in combination.
  • the amount of peptide to be administered and hence the amount of active ingredient in the composition will, of course, depend on the particular peptide chosen, the condition to be treated, the desired frequency of administration and the effect desired.
  • the quantity of the total composition administered at each nasal application suitably comprises from about 0.05 to 0.15 ml, typically about 0.1 ml.
  • the compositions is provided in a container provided with means enabling application of the contained composition to the nasal mucosa, e.g., put up in a nasal applicator device.
  • Suitable applicators are known in the art and include those adapted for administration of liquid compositions to the nasal mucosa in drop or spray form. Because dosing should be as accurately controlled as possible, use of spray applicators for which the administered quantity is susceptible to precise regulation will generally be preferred.
  • Suitable administrators include, e.g., atomizing devices, pump-atomizers and aerosol dispensers. In the latter case, the applicator will contain a composition in accordance with the methods together with a propellant medium suitable for use in a nasal applicator.
  • the atomizing device will be provided with an appropriate spray adaptor allowing delivery of the contained composition to the nasal mucosa. Such devices are well known in the art.
  • the container e.g., nasal applicator
  • the container may contain sufficient composition for a single nasal dosing or for the supply of several sequential dosages, e.g., over a period of days or weeks. Quantities of individual dosages supplied may be as hereinbefore defined.
  • compositions comprising aromatic-cationic peptide as an active ingredient which meet the high standards of stability and bioavailability required for nasal application and which are, for example, eminently suitable for use in multiple dose nasal spray applicators, i.e., applicators capable of delivering a series of individual dosages over, e.g., period of several days or weeks, by the use of citric acid or a salt thereof in concentrations ranging from about 10 to about 50 mM as a buffering agent.
  • citric acid or a salt thereof at increasing concentrations confers, in some embodiments, beneficial advantages in relation to the nasal absorption characteristics of aromatic-cationic peptide containing compositions and hence enhance aromatic-cationic peptide bioavailability levels consequential to nasal application.
  • the use of citric acid or a salt thereof in concentrations ranging from about 10 to about 50 mM increase the stability of aromatic-cationic peptide compositions while at the same time higher concentrations of citric acid or salt thereof do not have the same stabilizing effect.
  • the aromatic-cationic peptide for use in the present methods may be in free form or in pharmaceutically acceptable salt or complex form, e.g. , in pharmaceutically acceptable acid addition salt form.
  • Such salts and complexes are known and possess an equivalent degree of activity and tolerability to the free forms.
  • Suitable acid addition salt forms for use in accordance with the methods include for example the hydrochlorides and acetates.
  • the aromatic-cationic peptide comprises D-Arg-2'6'-Dmt-Lys-Phe-NH2.
  • the peptide is provided as a salt, such as an acetate or trifluoroacetate salt.
  • compositions may be applied in accordance with the methods to the nasal mucosa, e.g., either in drop or in spray form. As hereinafter described however, they may be applied in spray form, i.e. , in the form of finely divided droplets.
  • the liquid pharmaceutical compositions of the present methods contain a pharmaceutically acceptable diluent or carrier suitable for application to the nasal mucosa, such as aqueous saline.
  • treatment may suitably comprise administration of dosages at a frequency of from about once daily to about three times daily. Dosages may be administered in a single application, i. e. , treatment will comprise administration of single nasal dosages of aromatic- cationic peptide of the present technology. Alternatively such dosages may be split over a series of 2 to 4 applications taken at intervals during the day. The total composition quantity administered at each nasal application will vary according to the condition being treated, the particular peptide being administered, and the characteristics of the subject.
  • the container e.g., nasal applicator, in some embodiments, may contain sufficient composition for a single nasal dosing or for the supply of several sequential dosages, e.g., over a period of days or weeks. Quantities of individual dosages supplied may be as hereinbefore defined.
  • the stability of the compositions may be determined in conventional manner.
  • the aromatic-cationic peptide content of the compositions will degrade less than 50 % in 15 days at 50°C as determined by standard analytical tests.
  • the aromatic-cationic peptides may be combined with one or more additional agents for the prevention or treatment of a disease or condition.
  • a synergistic therapeutic effect is produced.
  • a "synergistic therapeutic effect” refers to a greater-than-additive therapeutic effect which is produced by a combination of two therapeutic agents (e.g., an aromatic-cationic peptide and another agent), and which exceeds that which would otherwise result from individual administration of either therapeutic agent alone. Therefore, lower doses of one or both of the therapeutic agents may be used in treating or preventing a disease or condition, resulting in increased therapeutic efficacy and decreased side-effects.
  • the multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.
  • This example will demonstrate the effect of pH on the bioavailability of formulations comprising the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- NH 2 and D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • aromatic-cationic peptides described herein may be analyzed by a number of HPLC methods, including reverse phase HPLC, such as those described in Aguilar, HPLC of Peptides and Proteins: Methods and Protocols, Humana Press, New Jersey (2004).
  • reverse phase HPLC such as those described in Aguilar, HPLC of Peptides and Proteins: Methods and Protocols, Humana Press, New Jersey (2004).
  • MS methods such as those described in Sparkman, Mass Spectroscopy Desk Reference, Pittsburgh: Global View Pub (2000).
  • the absolute bioavailability or aromatic-cationic peptide (i.e., relative to an intravenous dose of aromatic-cationic peptide) will be calculated from the area under the curve obtained from plots of the plasma concentration of aromatic-cationic peptide as a function of time.
  • This example will demonstrate the effect of citric acid on the bioavailability of the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- N3 ⁇ 4 and D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • Formulations consisting of a fixed amount of taurodeoxycholic acid and two different amounts of citric acid will be prepared in a total volume of 0.5 ml. Mannitol will be included in the formulations as a marker to measure paracellular transport. The formulations will be administered to female Wistar rats as described in Example 1. Blood samples will be collected and bioavailability measured as described in Example 1.
  • This example will demonstrate the effect of absorption enhancers on the bioavailability of the aromatic-cationic peptides of the present technology, such as Phe-D- Arg-Phe-Lys- NH 2 and D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • Formulations consisting of citric acid, aromatic-cationic peptide, and various classes of enhancers will be prepared in a total volume of 0.5 ml. Mannitol will be included in formulation V as a marker to measure paracellular transport. The formulations will be administered to female Wistar rats as described in Example 1. Blood samples will be collected and bioavailability measured as described in Example 1. Anticipated trends in the effect of enhancers on the bioavailability of aromatic-cationic peptide are shown in Table 3. It is anticipated that formulations including an enhancer will result in increased
  • bioavailability of aromatic-cationic peptide relative to formulations lacking an enhancer.
  • the inclusion of a water soluble phospholipid is expected to increase the bioavailability of aromatic-cationic peptide by as much as four- fold.
  • the most effective enhancer is anticipated to be the sugar ester class (illustrative formulation V) in which the aromatic-cationic peptide bioavailability may be increased as much as eight- fold.
  • a mixture of bile acid and a cationic detergent (illustrative formulation III), a non-ionic detergent (illustrative formulation IV), or an acylcarnitine (illustrative formulation VI) are expected to increase the bioavailability of aromatic-cationic peptide as much as eight-fold compared to that achieved with illustrative formulation I.
  • Variations in the bioavailability of aromatic-cationic peptide administered with various classes of enhancers are expected to be minor compared to variations observed when the peptide is formulated with citric acid only and no enhancer.
  • Citric acid (77 mg) 8x 7x Cetylpyridinium chloride (5 mg)
  • Aromatic-cationic peptide (0.1 mg)
  • Citric acid 48 mg
  • 3x 5x Tween -20 (5 mg)
  • Citric acid 48 mg
  • 4x 4x Diheptanoylphosphatidylcholine 5 mg
  • Formulations consisting of lauroyl L-carnitine, aromatic-cationic peptide of the present technology, and various other compounds will be prepared in a total volume of 0.5 ml.
  • the formulations will be administered to female Wistar rats as described in Example 1. Blood samples will be collected and bioavailability measured as described in Example 1.
  • the ports Before and after the administration of aromatic-cationic peptide formulations into conscious dogs, the ports will be flushed with 2 ml of a mock formulation lacking aromatic-cationic peptide. Blood (2 ml) will be collected through angiocatheter tubes in the leg vein at 30, 15, and 0 minutes before administration of aromatic-cationic peptide, and at 5, 10, 20, 30, 40, 50, 60, and every 15 minutes thereafter for 2 hours after
  • bioavailability of aromatic-cationic peptide administered alone will be low compared to formulations that include taurodeoxycholic acid and/or citric acid. It is anticipated that including citric acid in the formulation (illustrative formulation II) will increase the bioavailability of the peptide by as much as 25-fold. It is anticipated that further including taurodeoxycholic acid in the formulation (illustrative formulation III) will increase the bioavailability of the peptide by as much as 50-fold.
  • This example will demonstrate the effect of citric acid lauroyl L-carnitine on the bioavailability of the aromatic-cationic peptides of the present technology, such as Phe-D- Arg-Phe-Lys- NH 2 and D-Arg-2'6'-Dmt-Lys-Phe-NH 2 , vasopressin, and insulin.
  • Formulations consisting of either [Arg 8 ]-vasopressin, aromatic-cationic peptide, or human insulin together with specified additives will be prepared in a total volume of 0.5 ml. The formulations will be administered to female Wistar rats as described in Example 1. Blood samples will be collected and bioavailability measured as described in Example 1.
  • Example 7 Effect of Enteric Coating on Absorption of formulations comprising Aromatic- Cationic Peptides of the Present Technology
  • This example will demonstrate the effect of enteric coating on absorption of illustrative formulations of the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- NH 2 and D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • Size 00 UPMC (hydroxypropylmethyl cellulose) capsules will each be filled with a powdered blend consisting of citric acid, lauroyl L-carnitine, and aromatic-cationic peptide.
  • Half the capsules will be coated with an enteric coating solution of EUDRAGIT L30D-55 (a methacrylic acid co-polymer with methacrylic acid methyl ester, ROUM Tech Inc., Maidan, Mass.), and the remaining capsules will not be coated.
  • EUDRAGIT L30D-55 a methacrylic acid co-polymer with methacrylic acid methyl ester, ROUM Tech Inc., Maidan, Mass.
  • the coating process will correspond to that taught in U.S. Pat. No. 6,086,918 at col. 11, line 50 to col. 12, line 11.
  • the average capsule content for the enteric coated and non-enteric coated capsules is shown in Table 7.
  • Example 8 Effects of Illustrative Formulations on Absorption of Aromatic-Cationic Peptide From Non-enteric Coated Capsules
  • Size 00 UPMC (hydroxypropylmethyl cellulose) capsules will each be filled with a powdered blend consisting of the indicated amount of citric acid, lauroyl L-carnitine, sucrose and aromatic-cationic peptide.
  • the average capsule content for the capsules is shown in Table 8.
  • Each week, eight fasted dogs will each be orally administered one uncoated capsule.
  • samples of blood will be taken at 15 minute intervals from an indwelling catheter for up to 4 hours.
  • the blood samples will be centrifuged and the resulting plasma supernatants will be stored frozen at -20° C.
  • the plasma samples will be subsequently analyzed for aromatic-cationic peptide by reverse phase HPLC chromatography and/or mass spectroscopy (MS).
  • Example 9 Effects of Illustrative Formulations Absorption of the Aromatic-Cationic Peptides of the Present Technology, such as Phe-D-Arg-Phe-Lys- NFL and D-Arg-2 f 6'-Dmt-Lys-Phe-
  • Size 00 UPMC capsules will each be filled with a powdered blend consisting of at least 500 mg citric acid, 50 mg lauroyl L-carnitine and 1.0 mg of the aromatic-cationic peptide Phe-D-Arg-Phe-Lys- NH2 or D-Arg-2'6'-Dmt-Lys-Phe-NFL; , or a pharmaceutically acceptable salt thereof, such as acetate or trifluoroacetate salt.
  • a pharmaceutically acceptable salt thereof such as acetate or trifluoroacetate salt.
  • Example 10 Effects of Enteric Coating on the Bioavailability of Aromatic-Cationic Peptides of the Present Technology.
  • aromatic-cationic peptide which include, in addition to the aromatic-cationic peptide, 0.5M citric acid and lauroyl L-carnitine (10 mg/ml). Samples of blood will be taken from the carotid artery through an indwelling catheter before and 5, 15, 30, 60 and 120 minutes after the administration of the respective formulations (i.e., formulated and unformulated).
  • the blood samples will be centrifuged and the resulting plasma supernatants will be stored frozen at -20° C.
  • the plasma samples will be subsequently analyzed for aromatic- cationic peptide by high-performance liquid chromatography (HPLC) through a 50 x 4.6 mm polysulfoethyl-aspartamid- e column with a mobile phase of 15.4 mM potassium phosphate (pH 3), 210 mM sodium chloride, and 25% acrylonitrile at a flow rate of 1.5 ml/min.
  • Peptide will be detected with an ultraviolet (UV) detector set at a wavelength of 210 nm.
  • a second series of tests will be carried out, as noted above, using beagle dogs.
  • the improved bioavailability of orally administered aromatic-cationic peptide will be demonstrated in this second series of tests by comparing the curves for (1) non-enteric coated salmon calcitonin (sCT) and (2) non-enteric coated aromatic-cationic peptide with the curves for (3) enteric coated sCT and (4) enteric coated aromatic-cationic peptide.
  • sCT non-enteric coated salmon calcitonin
  • size 00 HPLC capsules will be filled with 758 mg of a powdered blend consisting of citric acid (643 mg), lauroyl L-carnitine (66 mg), talc (33 mg), salmon calcitonin (sCT) (13 mg) and aromatic-cationic peptide (2.4 mg).
  • Half of the capsules will be coated with an enteric coating solution of L30D-55, while the remaining 50% of the capsules will not be coated.
  • Four fasted dogs will be each given 1 uncoated capsule, and 2 weeks later they will be each given an enteric coated capsule. After administration of each capsule, samples of blood will be taken at 15 minute intervals from an indwelling catheter for up to 4 hours.
  • the blood samples will be centrifuged and the resulting plasma supernatants will be stored frozen at -20°C.
  • the plasma samples will be subsequently analyzed for sCT by a direct ELISA, and for aromatic-cationic peptide by HPLC-mass spectrometry performed as set forth in Wan, H. and Desiderio, D., Quantitation of dmt-DALDA in ovine plasma by online liquid chromatography/quadrapole time-of-flight mass spectrometry, Rapid
  • results will be summarized as plasma peptide concentration normalized to a 1 mg dose as a function of time relative to the average Tmax, (i.e., the time at which the maximum amount of peptide is detected).
  • the results are expected to indicate that both peptides, i.e., sCT and aromatic-cationic peptide, are detected in dogs given uncoated or enteric coated capsules. It is expected that nearly three times as much aromatic-cationic peptide as sCT will be detected in dogs given uncoated capsules; whereas, nearly equal amounts of both peptides will be detected in dogs given enteric coated capsules.
  • the Cmax and AUC values for both sCT and aromatic-cationic peptide are expected to be significantly enhanced when the peptides are administered in enteric coated capsules versus in non enteric-coated capsules.
  • the Cmax of enteric coated aromatic-cationic peptide is expected to be 4-fold higher than that of non enteric coated aromatic-cationic peptide.
  • the bioavailability of both enteric coated and non-coated aromatic-cationic peptide is expected to be better than that of sCT. It would be expected that the bioavailability of a molecule such as aromatic-cationic peptide, which is positively charged and hydrophilic, would be extremely poor.
  • the data is expected to indicate that when the aromatic-cationic peptide is
  • the bioavailability is increased to the point where it is superior to that of sCT, a molecule that has previously been shown to be highly bioavailable when formulated according to the present methods.
  • Example 11 Effect of OmPA-MT3 on the Absorption of Aromatic-Cationic Peptides of the Present Technology from Rat Duodenum
  • Example 12 Effect of the HIV TAT Protein Transduction Domain as an MT on the
  • the first formulation (Fl) is prepared by blending 13g citric acid, 1.3g lauroyl L- carnitine, 0.65g talc and 0.03g aromatic-cationic peptide with a mortar and pestle.
  • the other formulation (F2) is prepared by blending the same mixture except that s aromatic-cationic peptide is replaced with an equivalent amount of MT3 -aromatic-cationic peptide. Both blends are used to fill size 00 gelatin capsules, and the capsules are coated with Eudragit L30D-55.
  • the resulting enteric-coated capsules contain approximately 1 to 2 mg of either aromatic- cationic peptide (Fl) or MT3-aromatic-cationic peptide (F2) per capsule.
  • the amount of aromatic-cationic peptide in plasma samples of dogs given either of the two formulations is HPLC using methods known in the art. Both formulations are expected to produce measurable amounts of aromatic-cationic peptide in the blood, the maximum concentration of aromatic-cationic peptide in the blood of dogs given Fl is expected to be in the range of 0.5 to 6.0 ng/ml, whereas the maximum concentration of aromatic-cationic peptide in dogs given F2 is expected to be at least 1 to 12 ng/ml.
  • the bioavailability of aromatic-cationic peptide in dogs given Fl is expected to be approximately 1%, whereas the bioavailability of aromatic-cationic peptide in dogs given F2 is expected to be at least 1.2%.
  • the in vivo cleavage of MT from aromatic-cationic peptide in dogs given F2 is proven by applying samples of plasma from dogs given Fl and F2 to an HPLC column and collecting the effluent in plastic tubes. The solvent in the tubes is removed under vacuum and analyzed for the presence of aromatic-cationic peptide by HPLC.
  • the in vivo cleavage of MT3-aromatic-cationic peptide is established by showing that the retention time of aromatic-cationic peptide in the plasma from dogs given F2 is the same as the retention time of aromatic-cationic peptide in the plasma of dogs given Fl .
  • Formulated aromatic-cationic peptide (5 ⁇ g per 25 ⁇ ) is administered intranasally through a micropipette tip inserted 8 mm into the rat's nostril. For single-dose studies, 5 ⁇ g of aromatic-cationic peptide is administered. In multiple dose studies, aromatic-cationic peptide is administered four times in a volume of 25 ⁇ each at 0, 30, 60 and 90 minutes for a total dose of 20 ⁇ g.
  • Each sample (0.5 ml) of blood is collected into a heparinized 1 ml syringes and then transferred to chilled 1.5 ml polypropylene tubes containing 10 ⁇ of heparin (500 U per ml).
  • the tubes are centrifuged at approximately 3000 rpm for 20 minutes at 2-8°C. and the plasma supernatant is transferred to microcentrifuge tubes that are stored at -20°C.
  • concentration of aromatic-cationic peptide in plasma is determined by HPLC using methods known in the art.
  • Cmax The values of Cmax are determined by inspection and the values for bioavailability (relative to an intravenous injection) are calculated from the areas under the curve that is obtained from plots of plasma aromatic-cationic peptide concentration as a function of time.
  • Example 14 Exemplary Formulation for Lipid Emulsion and Micelle Compositions
  • compositions and formulations provide formulations for lipid emulsion and micelle compositions of the present technology.
  • the tables are merely representative of formulations and should not be construed as limiting in any way.
  • the following compositions and formulations may also be used in any of the methods disclosed herein (e.g. , to treat a disease or condition, or be used in any of the disclosed test models).
  • Sodium caprate One or more of:
  • Adsorption enhancer for example lauroyl L- Mineral oil
  • Sodium caprate One or more of:
  • Adsorption enhancer for example lauroyl L- carnitine
  • Adsorption enhancer for example lauroyl L- Geranol
  • Adsorption enhancer for example lauroyl L- carnitine
  • Adsorption enhancer for example lauroyl L- Optionally, one or more of octanoic acid, carnitine) ricinoleic acid, ethyl octanoate.
  • Adsorption enhancer for example lauroyl L- Span-40
  • one or more of octanoic acid, ricinoleic acid, ethyl octanoate is optionally, one or more of octanoic acid, ricinoleic acid, ethyl octanoate.
  • Adsorption enhancer for example lauroyl L- carnitine
  • one or more of octanoic acid, ricinoleic acid , ethyl octanoate are optionally, one or more of octanoic acid, ricinoleic acid , ethyl octanoate.
  • Adsorption enhancer for example lauroyl L- Span-40
  • Adsorption enhancer for example lauroyl L- carnitine
  • Adsorption enhancer for example lauroyl L- Castor oil
  • Adsorption enhancer for example lauroyl L- carnitine
  • Table 25 Illustrative formulations
  • Adsorption enhancer for example lauroyl L- carnitine
  • Aromatic-Cationic peptide Glyceryl tributyrate Sodium octanoate Ethyl isovalerate PVP- 12/17/25 Glycerol monooleate Water Lecithin
  • Adsorption enhancer for example lauroyl L- Span-40
  • Aromatic-Cationic peptide Glyceryl tributyrate Sodium octanoate Ethyl isovalerate PVP- 12 Glycerol monooleate Water Lecithin
  • Adsorption enhancer for example lauroyl L- Span-40
  • Adsorption enhancer for example lauroyl L- carnitine
  • Adsorption enhancer for example lauroyl L- carnitine
  • Table 30 Illustrative formulations
  • Adsorption enhancer for example lauroyl L- Castor oil
  • Adsorption enhancer for example lauroyl L- Span-40
  • Adsorption enhancer for example lauroyl L- carnitine
  • Adsorption enhancer for example lauroyl L- Tween 80
  • Adsorption enhancer for example lauroyl L- carnitine
  • Adsorption enhancer for example lauroyl L- carnitine
  • Adsorption enhancer for example lauroyl L- carnitine
  • Table 38 Illustrative formulations
  • Adsorption enhancer for example lauroyl L- Glyceryl tricaprylate
  • Example 15 The use of Counter Anions in Compositions to Translocate Aromatic-cationic peptides Across an Epithelial Barrier
  • composition is prepared by the lyophilization of (1) an aromatic-cationic peptide, e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH2, (2) a counter anion, such as sodium dodecyl sulfate (SDS) or dioctyl sulfosuccinate (DSS),and (3) an absorption enhancer, for example lauroyl L-carnitine.
  • an aromatic-cationic peptide e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH2
  • a counter anion such as sodium dodecyl sulfate (SDS) or dioctyl sulfosuccinate (DSS)
  • an absorption enhancer for example lauroyl L-carnitine.
  • NMP N-methyl pirolidone
  • N-acetyl cysteine N-acetyl cysteine
  • NMP N-methyl pirolidone
  • N-acetyl cysteine N-acetyl cysteine
  • N-acetyl cysteine N-acetyl cysteine
  • N-acetyl cysteine N-acetyl cysteine
  • N-acetyl cysteine N-acetyl cysteine
  • N-acetyl cysteine N-acetyl cysteine
  • the composition is then administered to test animals, e.g. , mice, in two forms: rectally or by injection into an intestinal loop.
  • the experimental procedure involves male BALB/c mice, which are deprived of food, 18 hours prior to the experiment.
  • For intra- intestinal injection the mice are then anesthetized and a 2 cm long incision is made along the center of the abdomen, through the skin and abdominal wall. An intestine loop is gently pulled out through the incision and placed on wet gauze beside the animal. The loop remains intact through the entire procedure and is kept wet during the whole time.
  • the tested compound is injected into the loop, using a 26 G needle.
  • the mice are anesthetized and the penetration composition is then rectally administered to the mice, 100 ⁇ /mouse, using a plastic tip covered with a lubricant.
  • Penetration is assessed by direct measurement of aromatic-cationic peptide concentrations in the blood.
  • compositions disclosed herein are tested for their ability to cross an epithelial barrier.
  • a peptide formulation will be administered to rats and/or pigs im, rectally or nasally. Blood samples are taken at various times after administration, and peptide levels determined by methods known in the art.
  • Formulation 1 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 will be dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine, and phytic acid in double distilled water ("DDW") containing NaOH. The solution is then lyophilized and suspended with sodium dodecanoate (SD), octanol and geraniol in a mixture of mineral oil, medium chain triglyceride (MCT) oil and castor oil.
  • SD sodium dodecanoate
  • MCT medium chain triglyceride
  • Formulation 2 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 will be dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine, and phytic acid in DDW containing NaOH. The solution will be lyophilized and suspended with sodium dodecanoate (SD), octanol and geraniol in a mixture of mineral oil, medium chain triglyceride (MCT) oil and castor oil.
  • SD sodium dodecanoate
  • MCT medium chain triglyceride
  • Formulation 3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 is dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine, polyvinylpyrrolidone (PVP-40), sodium dodecanoate (SD) and methylcellulose (MC-400) in DDW containing NaOH. The solution is then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil, further containing sorbitan monopalmitate (Span- 40).
  • MCT medium chain triglyceride
  • Span- 40 sorbitan monopalmitate
  • Formulation 4 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 is combined with spermine, an absorption enhancer, for example lauroyl L-carnitine, polyvinylpyrrolidone (PVP-40), and sodium dodecanoate (SD) in DDW containing NaOH, octanol and geraniol.
  • the solution is then lyophilized and suspended with an additional amount of octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil further containing sorbitan monopalmitate (Span-40), methylcellulose (MC-400), and glyceryl monooleate (GMO).
  • MCT medium chain triglyceride
  • Span-40 sorbitan monopalmitate
  • MC-400 methylcellulose
  • GMO glyceryl monooleate
  • Formulation 5 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 is dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine, and sodium dodecanoate in DDW containing NaOH. The solution is then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil further containing sorbitan monopalmitate (Span-40), methylcellulose (MC-400), glyceryl monooleate, and pluronic (F-127).
  • MCT medium chain triglyceride
  • Span-40 sorbitan monopalmitate
  • MC-400 methylcellulose
  • glyceryl monooleate glyceryl monooleate
  • pluronic F-127
  • mice Five male CB6/F1 mice, 9-10 wks, are divided into 2 groups, and anesthetized by a solution of 85% ketamine, 15% xylazine, 0.01 ml/10 g of body weight. Each preparation is administered either i.p. (100 ul/mouse, containing 0.2 mg D-Arg-2'6'-Dmt-Lys-Phe-NH 2 ) or rectally (100 ul/mouse, containing 1 mg D-Arg-2'6'-Dmt-Lys-Phe-NH 2 ). Rectal administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 1 cm. Blood samples at various time intervals post administration are drawn from the tip of the tail into a glass capillary.
  • Formulation 6 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 is dissolved with spermine, polyvinylpyrrolidone (PVP-40, an absorption enhancer, for example lauroyl L-carnitine), and sodium dodecanoate (SD) in DDW containing NaOH. The solution is then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil further containing sorbitan monopalmitate (Span-40), methylcellulose (MC- 400), and glyceryl monooleate (GMO).
  • MCT medium chain triglyceride
  • Span-40 sorbitan monopalmitate
  • MC- 400 methylcellulose
  • GMO glyceryl monooleate
  • Rectal administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 2 cm. Blood samples are drawn from the jugular veins at various time intervals post administration. Serum was analyzed for detection of D-Arg-2'6'-Dmt-Lys-Phe-NH 2 .
  • Formulation 7 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 is dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine), polyvinylpyrrolidone (PVP-40), sodium dodecanoate, and methylcellulose (MC-400) in DDW containing NaOH. The solution is then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil further containing sorbitan monopalmitate (Span- 40). The control composition is prepared as described above, without the D-Arg-2'6'-Dmt- Lys-Phe-NH 2 .
  • an absorption enhancer for example lauroyl L-carnitine
  • PVP-40 polyvinylpyrrolidone
  • MC-400 methylcellulose
  • the control composition is prepared as described above, without the D-Arg-2'6'-Dmt- Lys-Phe
  • D-Arg-2'6'-Dmt-Lys-Phe-NH 2 a plastic canule protected by a soft coating
  • blood levels of the peptide are measured at various time intervals post administration, in blood samples drawn from the tip of the tail.
  • D-Arg-2'6'-Dmt-Lys-Phe-NH 2 will attenuate the rise in blood glucose and that the peptide will be detected in the blood stream.
  • parenterally administration of D-Arg-2'6'-Dmt-Lys-Phe-NH2 is anticipated to indicate absorption from the intestine into the blood stream.
  • Formulation 8 An exemplary composition used for mucosal delivery will contain a desired D-Arg-2'6'-Dmt-Lys-Phe-NH 2 , an absorption enhancer, for example lauroyl L- carnitine), and protein stabilizers, e.g. , spermine and phytic acid, which can be dissolved and then lyophilized together, along with additional components such as polyvinylpyrrolidone and a surface active agent, e.g.
  • composition can be administered nasally or orally to a subject in need of vaccination.
  • membrane fluidizing agents e.g., octanol and geraniol
  • a hydrophobic medium e.g., a mixture of MCT oil or glyceryl tributyrate and castor oil. Additional possible components of the composition have been described.
  • Such a composition can be administered nasally or orally to a subject in need of vaccination.
  • This method allows simple, efficient and rapid administration of a drug to a large populations in need thereof.
  • Formulation 9 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 and an absorption enhancer, for example lauroyl L-carnitine), are dissolved with CaCl 2 , polyvinylpyrrolidone (PVP-12), sodium dodecanoate (SD), sodium octanoate (SO) and silicon dioxide in DDW containing NaOH.
  • PVP-12 polyvinylpyrrolidone
  • SD sodium dodecanoate
  • SO sodium octanoate
  • silicon dioxide silicon dioxide
  • solution C phosphatidyl choline (PC), sorbitan monopalmitate (Span-40), octanol and geraniol, and ethyl isovalerate, glyceryl monooleate (GMO) in a mixture of glyceryl tributyrate and castor oil.
  • PC phosphatidyl choline
  • Span-40 sorbitan monopalmitate
  • octanol and geraniol octanol and geraniol
  • ethyl isovalerate glyceryl monooleate (GMO) in a mixture of glyceryl tributyrate and castor oil.
  • GMO glyceryl monooleate
  • Example 17 Composition for Mucosal Delivery Using a Counter Ion
  • a composition for oral delivery containing D-Arg-2'6'-Dmt-Lys-Phe-NH2 encapsulated with a counter ion, i.e., sodium dodecyl sulfate (SDS) or dioctyl sulfosuccinate (DSS), and a hydrophobic agent, i.e., tricaprin is formulated as follows.
  • the solid formulation can be further coated with protective polymers (e.g., HPMC) and subsequently overgranulated or coated with a trypsin inhibitor, e.g., citric acid (2 to 5 : 1 ratio with drug) or other required excipient, such as an absorption enhancer (e.g. , 3-0- Lauroyl-L-Carnitine) at a 1 : 1 ration with drug.
  • a trypsin inhibitor e.g., citric acid (2 to 5 : 1 ratio with drug
  • an absorption enhancer e.g. 3-0- Lauroyl-L-Carnitine
  • the particulates can be subsequently individually spray coated with an enteric coating polymer (e.g., Eudragit LlOO D-55) or can be co-compressed or encapsulated into tablets or capsules which can themselves be spray coated with the same enteric polymer.
  • compositions 41-46 Additional possible constituents of the pharmaceutical composition are exemplified in Tables 41-46. Such a composition can be administered to a subject in need thereof.
  • Phosphate buffered saline is comprised of 80 mM NaCl, 46.7 mM KH 2 P0 4 and 20 mM Na 2 HP0 4 adjusted to pH 6.5.
  • Fasted state simulated intestinal fluid is comprised of PBS with 0.5% w/v SIF powder (sodium taurocholate and phospholipid in a 4 to 1 molar ratio).
  • Aqueous solubility of lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH 2 was determined using the HPLC after stirring excess salt complex in water at room temperature overnight. Sample solutions were filtered through a 0.45 ⁇ nylon syringe filter.
  • Octanol solubility for lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH 2 complexes was determined using the undissolved material remaining in the flask from the aqueous solubility measurement, which was dried overnight. 1 -Octanol was added until the salt complex was visually dissolved. 50 of the octanol solution was diluted with 450 of methanol in an HPLC vial prior to analysis by HPLC.
  • Octanol/Water partitioning (LogP or Kow) was determined using the shake flask method with 5 mL each of 1 -octanol and water in a 20 mL scintillation vial shaken overnight, the layers allowed to settle/separate for at least 2 hours, and then each layer analyzed individually using the HPLC method. The octanol layer was diluted as described above. Partitioning was initially determined using the aqueous solubility solutions, which generally had low concentrations and did not partition in the octanol layer as expected, which was confirmed the octanol solubility results.
  • the caprate and laurate salts were retested by dissolving them in octanol and then adding water.
  • the subsequent Kow determinations were made using the octanol solubility samples diluted to 5 mL before adding the 5 mL of water.
  • Lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH 2 were tested for water solubility, octanol solubility, and octanol/water partitioning. These assays determined which counter ions resulted in complexes that were the most lipophilic, as indicated by low water solubility, high octanol solubility, and a high octanol/water partition coefficient. Lipophilic salt complexes will partition more preferentially into the oil phase of lipid formulations in the intestine, providing increased protection from trypsin digestion.
  • the octanol/water partition coefficient is defined as:
  • K oW [D-Arg-2'6'-Dmt-Lys-Phe-NH 2 Lipophilic Salt] oci , tota i
  • the total drug concentration in each phase is given by:
  • the effective logP represents some combination of the association constant between D-Arg-2'6'-Dmt-Lys-Phe-NH 2 and the counter ion and the logP of the resulting complexes at the concentration tested.
  • the acetate salt of D-Arg-2'6'-Dmt-Lys-Phe-NH 2 is highly water soluble (205 mg/mL) while the free base form of D-Arg-2'6'-Dmt-Lys-Phe-NH2 has a solubility of 0.13 mg/mL.
  • the free base form was prepared using a catch and release purification method with a polymer supported sulfonic acid ion exchange column. The resulting material was a clear oily liquid that was dissolved in water and measured by HPLC.
  • D-Arg-2'6'-Dmt-Lys-Phe-NH 2 acetate dissolved rapidly in FaSSIF, but was simultaneously degraded rapidly in the presence of trypsin.
  • FIG. 1 Greater than 90% of the D-Arg-2'6'-Dmt-Lys-Phe-NH 2 was dissolved at the first sampling time ( ⁇ 2 minutes) in the absence of trypsin (FIG. 1, triangles). However, in the presence of trypsin, no D-Arg-2'6'- Dmt-Lys-Phe-NH 2 is detected by the first sampling time (FIG. I , squares).
  • the trypsin digestion was significantly reduced for the 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complex relative to the D-Arg-2'6'-Dmt-Lys- Phe- H 2 acetate salt, while it is effectively eliminated in the case of the 1 :6 D-Arg-2'6'-Dmt- Lys-Phe-NH 2 docusate complex.
  • Other lipophilic counter ions that reduced trypsin digestion of D-Arg-2'6'-Dmt-Lys- Phe-NH 2 when compared to D-Arg-2'6'-Dmt-Lys-Phe-NH 2 acetate include, e.g. , napsylate (at 1 :3 and 1 :6), oleate (at 1 :3), and stearate (at 1 :3) (see FIGs. 3A, 3B, 4A, and 4B).
  • D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complexes were formulated into three different lipid particle formulations to assess the trypsin resistance of the lipid particles.
  • the lipid formulations were comprised of long chain triglycerides, monoglycerides, and a non- ionic surfactant (see Table 49).
  • the Type I lipid vehicle contains no surfactant and is not self-emulsifying.
  • the Type II and III lipid vehicles contain surfactant and are self- emulsifying drug delivery systems (SEDDS).
  • D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complexes were dissolved in Type I, II, or III lipid vehicles at 15 or 30 mgA (mg Active/mL)(FIG. 5A).
  • the saturated solubility of the 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complex in the Type III lipid vehicle was 30 mgA/mL.
  • the solubility of the 1 :3 and 1 :6 D-Arg-2'6'-Dmt-Lys- Phe-NH 2 docusate complexes is 29 and 19 mg/mL, respectively.
  • the solubility of the 1 :3 and 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 SLS complexes was 9 and 5 mg/mL, respectively and in the Type II lipid vehicle, the solubility of the D-Arg-2'6'-Dmt- Lys-Phe-NEL acetate salt is at least 15 mg/mL.
  • Lipid formulations were investigated for protection from trypsin degradation. This assay was performed similarly to the test described above for the D-Arg-2'6'-Dmt-Lys-Phe- NH 2 /lipophilic salt powders, but dosing was based on the lipid formulation. The test was either run by adding 10 mL FaSSIF (with or without trypsin) directly to the lipid formulation.
  • FaSSIF FaSSIF (with or without trypsin) would be added to 0.5 g of lipid formulation to achieve a final concentration of 0.71 mgA/mL in the test.
  • Type I and Type II lipid formulations of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH 2 docusate complex led to higher in vitro area under the curve (AUC) in the presence of trypsin as compared to non- lipid formulation 1 :6 D-Arg-2 ' 6 '-Dmt-Lys-Phe-NH 2 docusate complex (FIGs. 2B and 5A).
  • the total AUC in the presence of trypsin was higher in the case of the 1 :6 D-Arg-2 '6 '-Dmt-Lys- Phe-NH 2 docusate complex as compared to 1 :3 D-Arg-2 '6 '-Dmt-Lys-Phe-NH 2 docusate complex or for the D-Arg-2' 6' -Dmt-Lys-Phe-NH 2 triacetate salt complex (FIG. 5B).
  • each lipid formulation was subdivided into test vials such that there were three vials for each time point ( . e. , 3 test vials for day 5 and 3 test vials for day 11) for the potency assay.
  • the potency was determined in the absence of trypsin.

Abstract

La présente invention concerne des procédés et des compositions permettant d'augmenter la biodisponibilité et/ou la stabilité de peptides aromatiques-cationiques. Dans certains modes de réalisation, le peptide aromatique-cationique comprend D-Arg-2'6'-Dmt-Lys-Phe-NH2.
PCT/US2015/038598 2014-06-30 2015-06-30 Formulations de peptide aromatique-cationique, compositions et procédés d'utilisation WO2016004067A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3426675A4 (fr) * 2016-03-11 2019-10-16 Stealth BioTherapeutics Inc. Formes salines cristallines
US10676506B2 (en) 2018-01-26 2020-06-09 Stealth Biotherapeutics Corp. Crystalline bis- and tris-hydrochloride salt of elamipretide
US11034724B2 (en) 2017-04-05 2021-06-15 Stealth Biotherapeutics Corp. Crystalline salt forms of Boc-D-Arg-DMT-Lys-(Boc)-Phe-NH2
CN113795247A (zh) * 2018-12-14 2021-12-14 陈益祥 用于心脏手术的稳定心脏麻痹液
EP4166133A1 (fr) * 2021-10-13 2023-04-19 Chugai Seiyaku Kabushiki Kaisha Composition contenant un composé peptidique et un tensioactif
CN116059309A (zh) * 2023-04-06 2023-05-05 江西中医药大学 一种精油组合物及其制备方法

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US20070154559A1 (en) * 2003-12-24 2007-07-05 Chaul-Min Pai Nanoparticle compositions of water-soluble drugs for oral administration and preparation methods thereof
WO2013049697A1 (fr) * 2011-09-29 2013-04-04 Mayo Foundation For Medical Education And Research Peptides aromatiques cationiques et leurs procédés d'utilisation

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Publication number Priority date Publication date Assignee Title
US20070154559A1 (en) * 2003-12-24 2007-07-05 Chaul-Min Pai Nanoparticle compositions of water-soluble drugs for oral administration and preparation methods thereof
WO2013049697A1 (fr) * 2011-09-29 2013-04-04 Mayo Foundation For Medical Education And Research Peptides aromatiques cationiques et leurs procédés d'utilisation

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3426675A4 (fr) * 2016-03-11 2019-10-16 Stealth BioTherapeutics Inc. Formes salines cristallines
US11034724B2 (en) 2017-04-05 2021-06-15 Stealth Biotherapeutics Corp. Crystalline salt forms of Boc-D-Arg-DMT-Lys-(Boc)-Phe-NH2
US11773136B2 (en) 2017-04-05 2023-10-03 Stealth Biotherapeutics Inc. Crystalline salt forms of Boc-D-Arg-DMT-Lys-(Boc)-Phe-NH2
US10676506B2 (en) 2018-01-26 2020-06-09 Stealth Biotherapeutics Corp. Crystalline bis- and tris-hydrochloride salt of elamipretide
US11261213B2 (en) 2018-01-26 2022-03-01 Stealth Biotherapeutics Inc. Crystalline bis- and tris-hydrochloride salt of elamipretide
CN113795247A (zh) * 2018-12-14 2021-12-14 陈益祥 用于心脏手术的稳定心脏麻痹液
CN113795247B (zh) * 2018-12-14 2024-04-05 陈益祥 用于心脏手术的稳定心脏麻痹液
EP4166133A1 (fr) * 2021-10-13 2023-04-19 Chugai Seiyaku Kabushiki Kaisha Composition contenant un composé peptidique et un tensioactif
CN116059309A (zh) * 2023-04-06 2023-05-05 江西中医药大学 一种精油组合物及其制备方法
CN116059309B (zh) * 2023-04-06 2023-06-30 江西中医药大学 一种精油组合物及其制备方法

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