Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/005—Enzyme inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders

Definitions

• Embodiments of the present invention relates generally to synthetic peptides and uses thereof for therapy and diagnostics, and more specifically to pharmaceutical formulations of the peptides including lipid-based formulations and formulations suitable for intravenous administration.
• the complement system an essential component of the innate immune system, plays a critical role as a defense mechanism against invading pathogens, primes adaptive immune responses, and helps remove immune complexes and apoptotic cells.
• Three different pathways comprise the complement system: the classical pathway, the lectin pathway and alternative pathway.
• Clq and mannose-binding lectin (MBL) are the structurally related recognition molecules of the classical and lectin pathways, respectively. Whereas IgM or clustered IgG serve as the principal ligands for Clq, MBL recognizes polysaccharides such as mannan. Ligand binding by Clq and MBL results in the sequential activation of C4 and C2 to form the classical and lectin pathway C3-convertase, respectively.
• alternative pathway activation does not require a recognition molecule, but can amplify C3 activation initiated by the classical or lectin pathways. Activation of any of these three pathways results in the formation of inflammatory mediators (C3a and C5a) and the membrane attack complex (MAC), which causes cellular lysis.
• C3a and C5a inflammatory mediators
• MAC membrane attack complex
• Naturally occurring peptides are essential signaling molecules that play critical physiological roles in human biology in the form of neurotransmitters, hormones, growth factors and anti-microbials [1], Given their intrinsic specificity and efficient properties, this class of molecules have received considerable attention as human therapeutics for a variety of disease indications, with over 60 approved for therapeutic use in the US, Europe and/or Japan and 155 currently in clinical development as of March, 2018 [2], The advantageous properties of peptides provides a significant advantage over small molecules ( ⁇ 500 Da) which often suffer from toxicity and off-target effects. Additionally, compared to large protein-based molecules such as humanized monoclonal antibodies, peptides typically enjoy low costs of manufacturing and in many cases can be synthesized chemically, thus avoiding costly and complex production and purification.
• complement regulators A need exists for complement regulators.
• the complement system is a vital host defense against pathogenic organisms.
• its unchecked activation can cause devastating host cell damage.
• autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, and multiple sclerosis
• only two anti-complement therapies have recently been approved for use in humans: 1) eculizumab (SolirisTM) and 2) ultomiris (RavulizumabTM) two humanized, long-acting monoclonal antibodies against C5 used in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS).
• PNH and aHUS are orphan diseases in which very few people are afflicted.
• no complement regulators are approved for the more common disease processes in which dysregulated complement activation plays a pivotal role.
• Dysregulated complement activation can play a role in both chronic disease indications and acute disease indications.
• Developing peptides to inhibit classical, lectin and alternative pathways of the complement system is needed, as each of these three pathways have been demonstrated to contribute to numerous autoimmune and inflammatory disease processes.
• Specific blockade of classical and lectin pathways is particularly needed, as both of these pathways have been implicated in ischemia reperfusion-induced injury among other diseases in many animal models.
• Humans with alternative pathway deficiencies suffer severe bacterial infections.
• a functional alternative pathway is essential for immune surveillance against invading pathogens.
• PIC1 also referred to as EPICC peptides
• the PIC1 peptides possess multiple anti-inflammatory properties including inhibition of the classical pathway of complement, myeloperoxidase (MPO) inhibition, neutrophil extracellular trap (NET) inhibition as well as intrinsic antioxidant and anti-microbial activity [3-8],
• MPO myeloperoxidase
• NET neutrophil extracellular trap
• the precursor to the PIC1 peptides were initially based upon the finding that the 787 amino acid capsid protein sequence of human astrovirus type 1, a non-enveloped icosahedral RNA virus that is an endemic pathogen causing gastroenteritis in human infants [9], could inhibit activation of the classical pathway of complement [10],
• the PIC1/EPICC family of molecules comprise a collection of rationally designed peptides with several anti-inflammatory functional properties including inhibition of the classical pathway of complement, myeloperoxidase inhibition, neutrophil extracellular trap inhibition and antioxidant activity.
• the original PIC1 peptide is a 15 amino acid peptide sequence, IALILEPICCQERAA (SEQ ID NO: 1), derived from a scrambled astroviral coat protein.
• the original PIC1 peptide has been modified with a C-terminal monodisperse 24-mer PEGylated moiety (IALILEPICCQERAA-dPEG24; PA-dPEG24; SEQ ID NO: 2), increasing its aqueous solubility.
• a sarcosine substitution scan of SEQ ID NO: 2 revealed that replacement of isoleucine at position 8 with sarcosine resulted in a peptide, IALILEP(Sar)CCQERAA (PA- I8Sar; SEQ ID NO: 3) that was water soluble without PEGylation (as described in U.S. Patent No. 10,005,818). Solubility studies showed that the PA-I8Sar peptide was amphipathic.
• Different formulations of the PA-18 Sar peptide were developed, including lipid-based formulations and formulations suitable for intravenous administration.
• Different formulations of PA-dPEG24 were also developed, including formulations suitable for intravenous administration.
• Embodiments of the present invention relate generally to synthetic peptides and more specifically to pharmaceutically useful formulations of the synthetic peptides, including for example and not limitation, lipid-based formulations of the synthetic peptides, particularly peptides that are present in or associated with lipid micelles, and formulations that are suitable for intravenous administration.
• the present invention provides a composition comprising a therapeutically effective amount of SEQ ID NO: 3 and a lipid-based carrier.
• the lipid- based carrier comprises lipid micelles.
• the lipid-based carrier comprises a lipid emulsion, e.g., Intralipid® carrier.
• the Intralipid® carrier is present in an amount of about 10% w/v to about 20% w/v.
• the present invention provides a composition comprising a therapeutically effective amount of SEQ ID NO: 2 or SEQ ID NO: 3 and at least one excipient.
• the at least one excipient is suitable for intravenous administration.
• the at least one excipient is selected from the group consisting of a citrate, an ascorbate, amino acids, and combinations thereof.
• the citrate comprises sodium citrate.
• the citrate is present in an amount of about 1% w/v to about 5% w/v.
• the ascorbate comprises sodium ascorbate.
• the ascorbate is present in an amount of about 1% w/v to about 5% w/v.
• the amino acids comprise L-methionine. In some embodiments, the amino acids are present in an amount of about 0.01% w/v to about 5% w/v.
• SEQ ID NO: 2 or SEQ ID NO: 3 is present in an amount of about 0.001 to about 200 milligrams per kilogram (mg/kg) of body weight. In some embodiments of any of the compositions described herein, SEQ ID NO: 2 and/or SEQ ID NO: 3 is present in an amount of about 5 to about 160 mg/kg. In some embodiments of any of the compositions described herein, SEQ ID NO: 2 or SEQ ID NO: 3 is present in an amount of about 1 mg/ml to about 100 mg/ml. In some embodiments of any of the compositions described herein, SEQ ID NO: 2 or SEQ ID NO: 3 is present in an amount of about 10 mg/ml to about 80 mg/ml.
• the present invention provides a method of altering cytokine expression comprising administering to a subject in need thereof any of the compositions described herein.
• the administration comprises parenteral administration.
• the administration comprises intravenous administration.
• the present invention provides a method of treating or preventing a disease or condition comprising administering to a subject in need thereof any of the compositions described herein.
• the administration comprises parenteral administration.
• the administration comprises intravenous administration.
• FIGS 2A-2B show that intravenous (IV) administration of lipo-RLS-0088 inhibited complement activation with a higher functionality over time relative to RLS-0088.
• Male Wistar rats with indwelling jugular catheters were administered 200 mg/kg RLS-0088 in Intralipid® or RLS-0088 in histidine buffer at 200 mg/kg of the compound as a single, bolus, IV infusion.
• an aliquot of blood was drawn and plasma isolated and frozen at -70C pending analysis. The Clq binding assay was then performed.
• Figure 2A shows the results of the Clq binding assay with a Y-axis maximum value of 6 mg/ml RLS-0088
• Figure 2B shows the same results with a Y-axis maximum value of 0.8 mg/ml RLS-0088.
• Figure 4 shows the appearance of different RLS-0088 formulations, where F denotes a freeze-thawed formulation and L denotes a lyophilized formulation.
• Figure 5 shows the appearance of different RLS-0071 formulations, where F denotes a freeze-thawed formulation and L denotes a lyophilized formulation.
• Embodiments of the present invention relate generally to synthetic peptides and more specifically to pharmaceutically useful formulations of the synthetic peptides, including for example and not limitation, lipid-based formulations of the synthetic peptides, particularly peptides that are present in lipid micelles, and formulations that are suitable for intravenous administration.
• reference to a component is intended also to include composition of a plurality of components.
• References to a composition containing “a” constituent is intended to include other constituents in addition to the one named.
• the terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denote the presence of “at least one” of the referenced item.
• the term “and/or” may mean “and,” it may mean “or,” it may mean “exclusive-or,” it may mean “one,” it may mean “some, but not all,” it may mean “neither,” and/or it may mean “both.”
• the term “or” is intended to mean an inclusive “or.”
• the term "about” should be construed to refer to both of the numbers specified as the endpoint (s) of any range. Any reference to a range should be considered as providing support for any subset within that range. Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and/or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and/or to the other particular value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
• “about” can mean within an acceptable standard deviation, per the practice in the art.
• “about” can mean a range of up to ⁇ 20%, preferably up to ⁇ 10%, more preferably up to ⁇ 5%, and more preferably still up to ⁇ 1% of a given value.
• the term can mean within an order of magnitude, preferably within 2-fold, of a value.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• substantially free of something can include both being “at least substantially free” of something, or “at least substantially pure”, and being “completely free” of something, or “completely pure”.
• the term “subject” or “patient” refers to mammals and includes, without limitation, human and veterinary animals. In a preferred embodiment, the subject is human.
• the term “combination” of a synthetic peptide according to the claimed invention and at least a second pharmaceutically active ingredient means at least two, but any desired combination of compounds can be delivered simultaneously or sequentially (e.g., within a 24 hour period). It is contemplated that when used to treat various diseases, the compositions and methods of the present invention can be utilized with other therapeutic methods/agents suitable for the same or similar diseases. Such other therapeutic methods/agents can be coadministered (simultaneously or sequentially) to generate additive or synergistic effects. Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.
• a “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.
• a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.
• the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
• terapéutica as used herein means a treatment and/or prophylaxis.
• a therapeutic effect is obtained by suppression, diminution, remission, or eradication of a disease state.
• the term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that when administered to a subject for treating (e.g., preventing or ameliorating) a state, disorder or condition, is sufficient to effect such treatment.
• the “therapeutically effective amount” will vary depending on the compound or bacteria or analogues administered as well as the disease and its severity and the age, weight, physical condition and responsiveness of the mammal to be treated.
• compositions of the invention refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human).
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
• pharmaceutical carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
• the pharmaceutical carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin.
• analog or “functional analog” refers to a related modified form of a polypeptide, wherein at least one amino acid substitution, deletion, or addition has been made such that said analog retains substantially the same biological activity as the unmodified form, in vivo and/or in vitro.
• sequence identity and “percent identity” are used interchangeably herein.
• sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence).
• the amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position.
• the two sequences are the same length.
• a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
• the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at www.accelrys.com/products/gc ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. These different parameters will yield slightly different results but the overall percentage identity of two sequences is not significantly altered when using different algorithms.
• a sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragments of the two sequences. Typically, the comparison will be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues
• Sequence identity refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical.
• Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G , Academic Press (1987); Sequence Analysis Primer, Gribskov, M.
• Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J.
• BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences.
• a polynucleotide having a nucleotide sequence having at least, for example, 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% “sequence identity” to a reference nucleotide sequence it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5, 4, 3, 2, 1, or 0 point mutations per each 100 nucleotides of the reference nucleotide sequence.
• a polynucleotide having a nucleotide sequence having at least 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity relative to the reference nucleotide sequence up to 5%, 4%, 3%, 2%, 1%, or 0% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5%, 4%, 3%, 2%, 1%, or 0% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
• a polypeptide having a given amino acid sequence having at least, for example, 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity to a reference amino acid sequence it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5, 4, 3, 2, 1, or 0 amino acid alterations per each 100 amino acids of the reference amino acid sequence.
• a given polypeptide sequence having at least 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity with a reference amino acid sequence up to 5%, 4%, 3%, 2%, 1%, or 0% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5%, 4%, 3%, 2%, 1%, or 0% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence.
• alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence.
• residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.
• immune response includes innate immune responses, T-cell mediated immune responses, and/or B-cell mediated immune responses.
• Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity, and B cell responses, e.g., antibody production.
• immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
• Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4+, CD8+, Thl and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes (e.g. neutrophils).
• “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intradermal (i d.) injection, or infusion techniques.
• prevention encompasses any activity which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease- related complications.
• a “variant” of a polypeptide according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and/or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag).
• the fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
• compositions according to the invention are used to refer to administration of a composition according to the invention and another therapeutic agent simultaneously in one composition, or simultaneously in different compositions, or sequentially (preferably, within a 24 hour period).
• a peptide with a C-terminal monodisperse 24-mer PEGylated moiety was found to be highly soluble and had strong inhibition of the complement system (IALILEPICCQERAA- dPEG24; SEQ ID NO: 2; PA-dPEG24).
• a sarcosine substitution of the non-PEGylated peptide was found to have similar solubility to the PEGylated peptide (lALILEP(Sar)CCQERAA; SEQ ID NO: 3; PA-I8Sar).
• PA-18 S Different formulations of PA-18 S ar were explored in order to develop formulations suitable for intravenous administration, including lipid-based formulations and formulations with excipients including citrate (e.g., trisodium citrate dihydrate), ascorbate (e g., sodium ascorbate), and/or amino acids (e.g., L-methionine).
• excipients including citrate (e.g., trisodium citrate dihydrate), ascorbate (e g., sodium ascorbate), and/or amino acids (e.g., L-methionine).
• Formulations of PA-dPEG24 with excipients including citrate (e.g., trisodium citrate dihydrate), ascorbate (e.g., sodium ascorbate), and/or amino acids (e.g., L-methionine) were also developed.
• peptide(s), refers to amino acid sequences, which may be naturally occurring, or peptide mimetics, peptide analogs and/or synthetic derivatives (including for example but not limitation PEGylated peptides) of about 15 amino acids based on SEQ ID NO: 2.
• the peptide may be less than about 15 amino acid residues, such as between about 10 and about 15 amino acid residues and such as peptides between about 5 to about 10 amino acid residues.
• Peptide residues of, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 amino acids are equally likely to be peptides within the context of the present invention.
• Peptides can also be more than 15 amino acids, such as, for example, 16, 17, 18, 19, and 20, or more amino acids.
• the disclosed peptides can be formulated in lipid-based carriers or with excipients suitable for intravenous administration.
• the lipid-based carrier can be Intralipid®.
• Intralipid® can be used for parenteral nutrition supplementation in humans to increase caloric intake intravenously and is composed of an emulsion of lipid micelles.
• the peptides can be associated with the lipid micelles, e.g., integrated into a layer of the lipid micelle.
• the excipients suitable for intravenous administration can comprise citrate (e.g., sodium citrate or sodium citrate dihydrate), ascorbate (e.g., sodium ascorbate), or amino acids (e.g., L-methionine).
• Substitutes for an amino acid within the peptide sequence may be selected from other members of the class to which the amino acid belongs.
• the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
• Amino acids containing aromatic ring structures include phenylalanine, tryptophan, and tyrosine.
• the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
• the positively charged (basic) amino acids include arginine and lysine.
• the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
• one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
• the peptide of the present disclosure comprises one or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue.
• conservative amino acid substitutions replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid
• Particularly preferred amino acid substitutions include: a) Ala for Glu or vice versa, such that a negative charge may be reduced; b) Lys for Arg or vice versa, such that a positive charge may be maintained; c) Ala for Arg or vice versa, such that a positive charge may be reduced; d) Glu for Asp or vice versa, such that a negative charge may be maintained; e) Ser for Thr or vice versa, such that a free — OH can be maintained; f) Gin for Asn or vice versa, such that a free NH2 can be maintained; g) He for Leu or for Vai or vice versa, as roughly equivalent hydrophobic amino acids; h) Phe for Tyr or vice versa, as roughly equivalent aromatic amino acids; and i) Ala for Cys or vice versa, such that disulfide bonding is affected.
• Substitutes for an amino acid within the peptide sequence may be selected from any amino acids, including, but not limited to alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine, N-formyl-L-methionine, sarcosine, or other N-methylated amino acids.
• sarcosine substitutes for an amino acid within the peptide sequence.
• the present invention provides lipid-based formulations of synthetic peptides that regulate the complement system and methods of using these peptides.
• the lipid-based formulations of the synthetic peptides can bind, regulate and inactivate Cl and MBL, and therefore can efficiently inhibit classical and lectin pathway activation at its earliest point while leaving the alternative pathway intact.
• These lipid-based formulations of peptides are of therapeutic value for selectively regulating and inhibiting Cl and MBL activation without affecting the alternative pathway and can be used for treating diseases mediated by dysregulated activation of the classical and lectin pathways.
• the lipid-based formulations of peptides regulate classical pathway activation but not lectin pathway activation.
• the lipid-based formulations of peptides are useful for various therapeutic indications, including for example and not limitation, diseases of organs with high lipid content such as the brain and pancreas (e.g., hypoxic ischemic encephalopathy (HIE), stroke, traumatic brain injury, pancreatitis).
• HIE hypoxic ischemic encephalopathy
• the inventive lipid-based formulations may also be used for subcutaneous depot dosing for chronic inflammatory conditions, such as for example and not limitation, lupus, antineutrophil cytoplasmic antibody-associated vasculitis (ANCA vasculitis), Behcet’s disease, autoimmune nephritis, and nephropathies.
• ANCA vasculitis antineutrophil cytoplasmic antibody-associated vasculitis
• Behcet’s disease autoimmune nephritis, and nephropathies.
• the present invention provides intravenous formulations of synthetic peptides that regulate the complement system and methods of using these peptides.
• the intravenous formulations of the synthetic peptides can bind, regulate and inactivate Cl and MBL, and therefore can efficiently inhibit classical and lectin pathway activation at its earliest point while leaving the alternative pathway intact.
• These intravenous formulations of peptides are of therapeutic value for selectively regulating and inhibiting Cl and MBL activation without affecting the alternative pathway and can be used for treating diseases mediated by dysregulated activation of the classical and lectin pathways.
• the intravenous formulations of peptides regulate classical pathway activation but not lectin pathway activation.
• the intravenous formulations of peptides are useful for various therapeutic indications.
• the invention provides synthetic peptides derived from human astrovirus coat protein, the peptides comprising the amino acid sequences and modifications of SEQ ID NO: 2, such as for example and not limitation, sarcosine substitutions of SEQ ID NO: 2.
• the invention provides lipid-based formulations of any of the peptides discussed herein, including for example but not limitation, SEQ ID NO: 3.
• the lipid-based formulation comprises lipid micelles.
• the lipid-based formulation comprises Intralipid®.
• the invention provides formulations of any of the peptides discussed herein, including for example but not limitation, SEQ ID NO: 3, that are suitable for intravenous administration.
• the invention provides intravenous pharmaceutical formulations of any of the peptides discussed herein, including for example but not limitation, SEQ IS NO: 2 and/or SEQ ID NO: 3, further comprising additional excipients suitable for intravenous administration, such as for example and not limitation, citrate (e.g., trisodium citrate dihydrate), ascorbate (e.g., sodium ascorbate), and/or amino acids (e g., L-methionine).
• citrate e.g., trisodium citrate dihydrate
• ascorbate e.g., sodium ascorbate
• amino acids e g., L-methionine
• the synthetic peptides are capable of altering cytokine expression.
• the invention provides a method of altering cytokine expression comprising administering to the subject in need thereof a composition comprising a therapeutically effective amount of a lipid-based formulation of a synthetic peptide comprising SEQ ID NO: 3.
• the lipid-based formulation comprises lipid micelles.
• SEQ ID NO: 3 is present within the lipid micelles.
• the lipid-based formulation comprises Intralipid® and SEQ ID NO: 3.
• the invention provides a method of altering cytokine expression comprising intravenously administering to the subject in need thereof a pharmaceutical formulation comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO: 2 or SEQ ID NO: 3.
• the pharmaceutical formulation can comprise excipients, carriers, and other ingredients that are suitable for intravenous administration.
• the pharmaceutical formulation comprises a therapeutically effective amount of SEQ ID NO: 2 or SEQ ID NO: 3 and a citrate (such as for example and not limitation, sodium citrate or sodium citrate dihydrate).
• the excipient is an antioxidant that is suitable for use in adult subjects (e.g., citrate, trisodium citrate, trisodium citrate trihydrate).
• the excipient is an antioxidant that is suitable for use in pediatric or infant subjects (e g , ascorbate such as sodium ascorbate, and/or amino acids such as L-methionine).
• the citrate can be present in an amount of about 1% w/v to about 5% w/v, including about 1% w/v to about 2.5% w/v.
• the pharmaceutical formulation comprises a therapeutically effective amount of SEQ ID NO: 2 or SEQ ID NO: 3 and an ascorbate (such as for example and not limitation, sodium ascorbate).
• the ascorbate can be present in an amount of about 1% w/v to about 5% w/v, including about 2.5% w/v to about 4.5% w/v.
• the pharmaceutical formulation comprises a therapeutically effective amount of SEQ ID NO: 2 or SEQ ID NO: 3 and an amino acid (such as for example and not limitation, L-methionine).
• the amino acid can be present in an amount of 0.01% w/v to about 5% w/v, including about 0.1% w/v to about 1% w/v.
• the disclosed peptides can selectively regulate Clq and MBL activation without affecting alternative pathway activity and are, thus, ideal for preventing and treating diseases mediated by the dysregulated activation of the classical and lectin pathways.
• Specific blockade of classical and lectin pathways are particularly needed, as both of these pathways have been implicated in ischemia-reperfusion induced injury in many animal models.
• the term “regulate,” as used herein, refers to i) controlling, reducing, inhibiting or regulating the biological function of an enzyme, protein, peptide, factor, byproduct, or derivative thereof, either individually or in complexes; ii) reducing the quantity of a biological protein, peptide, or derivative thereof, either in vivo or in vitro; or iii) interrupting a biological chain of events, cascade, or pathway known to comprise a related series of biological or chemical reactions.
• the term “regulate” may thus be used, for example, to describe reducing the quantity of a single component of the complement cascade compared to a control sample, reducing the rate or total amount of formation of a component or complex of components, or reducing the overall activity of a complex process or series of biological reactions, leading to such outcomes as cell lysis, formation of convertase enzymes, formation of complement-derived membrane attack complexes, inflammation, or inflammatory disease.
• the term “regulate” may refer to the measurable change or reduction of some biological or chemical event, but the person of ordinary skill in the art will appreciate that the measurable change or reduction need not be total to be “regulatory.”
• the present invention relates to therapeutically active peptides having the effects of regulating the complement system.
• compositions capable of regulating the complement system comprising at least one peptide, as discussed above, and at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient.
• Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. They can be solid, semi-solid, or liquid.
• the pharmaceutical compositions of the present invention can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, or syrups.
• compositions of the present invention are prepared by mixing the peptide having the appropriate degree of purity with pharmaceutically acceptable carriers, diluents, or excipients. Examples of formulations and methods for preparing such formulations are well known in the art.
• the pharmaceutical compositions of the present invention are useful as a prophylactic and therapeutic agent for various disorders and diseases, as set forth above.
• the composition comprises a therapeutically effective amount of the peptide.
• the composition comprises at least one other active ingredient effective in regulating the complement system.
• the composition comprises at least one other active ingredient effective in treating at least one disease associated with the complement system.
• composition comprises at least one other active ingredient effective in treating at least one disease that is not associated with the complement system.
• therapeutically effective amount refers to the total amount of each active component that is sufficient to show a benefit to the subject.
• the therapeutically effective amount of the peptide varies depending on several factors, such as the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the peptide employed, the duration of treatment, the co-therapy involved, and the age, gender, weight, and condition of the subject, etc.
• One of ordinary skill in the art can determine the therapeutically effective amount. Accordingly, one of ordinary skill in the art may need to titer the dosage and modify the route of administration to obtain the maximal therapeutic effect.
• the effective daily dose generally is within the range of from about 0.001 to about 200 milligrams per kilogram (mg/kg) of body weight, including about 5 to about 160 mg/kg, about 10 to about 160 mg/kg, about 40 mg/kg to about 160 mg/kg, and about 40 mg/kg to about 100 mg/kg.
• This dose can be achieved through a 1-6 time(s) daily dosing regimen.
• the effective dosage generally is within the range of about 1 mg/ml to about 100 mg/ml, including about 10 mg/ml to about 80 mg/ml.
• the effective dosage may depend on the additional ingredients included in the pharmaceutical composition. Alternatively, optimal treatment can be achieved through a sustained release formulation with a less frequent dosing regimen.
• the effective dosage may depend on the age of the subject. For example and not limitation, a pediatric or infant subject may require a lower dosage of the pharmaceutical composition than an adult subject. An effective dosage for a pediatric or infant subject may be about 10 mg/ml. An effective dosage for an adult subject may be about 80 mg/
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of a lipid-based formulation of SEQ ID NO: 3 and at least one pharmaceutically acceptable carrier, diluent, or excipient.
• the invention provides an intravenous pharmaceutical formulation comprising a therapeutically effective amount of SEQ ID NO: 2 or SEQ ID NO: 3 and at least one pharmaceutically acceptable carrier, diluent, or excipient that is suitable for intravenous administration.
• the excipient can comprise citrate (e.g., trisodium citrate dihydrate), ascorbate (e.g., sodium ascorbate), and/or amino acids (e.g., L-methionine).
• the excipient is an antioxidant that is suitable for use in adult subjects (e.g., citrate, trisodium citrate, trisodium citrate trihydrate).
• the excipient is an antioxidant that is suitable for use in pediatric or infant subjects (e.g., ascorbate such as sodium ascorbate, and/or amino acids such as L-methionine).
• compositions of the invention can comprise a carrier and/or excipient. While it is possible to use a compound of the present invention for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient and/or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• a suitable pharmaceutical excipient and/or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• the excipient and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Acceptable excipients and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A.R. Gennaro edit. 2005).
• Solid dosage forms for oral administration can also be used and can include, e.g., capsules, tablets, caplets, pills, troches, lozenges, powders, and granules.
• suitable excipients include, e.g., diluents, buffering agents (e.g., sodium bicarbonate), preservatives, stabilizers, binders, compaction agents, lubricants, dispersion enhancers, disintegration agents, antioxidants, flavoring agents, sweeteners, and coloring agents.
• the composition is formulated for delivery by a route such as, e.g., oral, topical, rectal, mucosal, sublingual, nasal, naso/oro-gastric gavage, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal, and intracheal administration.
• the composition is in a form of a liquid, foam, cream, spray, powder, or gel.
• the composition comprises a buffering agent (e g., sodium bicarbonate).
• Administration of the compounds and compositions in the methods of the invention can be accomplished by any method known in the art.
• useful routes of delivery include oral, rectal, fecal (by enema), and via naso/oro-gastric gavage, as well as parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal, and intracheal administration.
• the active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
• the useful dosages of the compounds and formulations of the invention can vary widely, depending upon the nature of the disease, the patient’s medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like.
• the initial dose may be larger, followed by smaller maintenance doses.
• the dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. It is contemplated that a variety of doses may be effective to achieve a therapeutic effect.
• a compound of the present invention for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e g., in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• the excipient, diluent and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A.R. Gennaro edit. 2005).
• compositions of the present invention can be selected with regard to the intended route of administration and standard pharmaceutical practice.
• oral delivery is preferred for delivery to the digestive tract because of its ease and convenience, and because oral formulations readily accommodate additional mixtures, such as milk, yogurt, and infant formula.
• Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• Solutions or suspensions can include any of the following components, in any combination: a sterile diluent, including by way of example without limitation, water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.
• a sterile diluent including by way of example without limitation, water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent
• antimicrobial agents such as benzyl alcohol and methyl parabens
• antioxidants such as ascorbic acid and sodium bisul
• solubilizing agents may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using co-solvents, such as, e.g., dimethylsulfoxide (DMSO), using surfactants, such as TWEEN®80, or dissolution in aqueous sodium bicarbonate.
• co-solvents such as, e.g., dimethylsulfoxide (DMSO)
• surfactants such as TWEEN®80
• dissolution in aqueous sodium bicarbonate such as sodium bicarbonate.
• Pharmaceutically acceptable derivatives of the agents may also be used in formulating effective pharmaceutical compositions.
• the composition can contain along with the active agent, for example and without limitation: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.
• a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose
• a lubricant such as magnesium stearate, calcium stearate and talc
• a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone
• Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active agent as defined above and optional pharmaceutical adjuvants in a carrier, such as, by way of example and without limitation, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
• a carrier such as, by way of example and without limitation, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
• the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
• auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
• auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextr
• composition or formulation to be administered will, in any event, contain a quantity of the active agent in an amount sufficient to alleviate the symptoms of the treated subject.
• the active agents or pharmaceutically acceptable derivatives may be prepared with carriers that protect the agent against rapid elimination from the body, such as time release formulations or coatings.
• the compositions may include other active agents to obtain desired combinations of properties.
• Parenteral administration generally characterized by injection, either subcutaneously, intramuscularly or intravenously, is also contemplated herein.
• Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
• Suitable excipients include, by way of example and without limitation, water, saline, dextrose, glycerol or ethanol.
• compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
• Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures or formulated as solids or gels.
• the sterile, lyophilized powder is prepared by dissolving an agent provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent.
• the solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder.
• Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
• the solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH.
• a buffer such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH.
• Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation.
• the resulting solution can be apportioned into vials for lyophilization.
• Each vial can contain, by way of example and without limitation, a single dosage (10-1000 mg, such as 100- 500 mg) or multiple dosages of the agent.
• the lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.
• a further embodiment of the invention provides a method of regulating the complement system, comprising administering to a subject a pharmaceutical formulation of the present invention.
• the pharmaceutical formulations of the present invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more therapeutic or prophylactic agent(s) that is(are) effective for regulating the complement system.
• the method of the present invention comprises administrating a pharmaceutical formulation of the present invention before, concurrently, and/or after one or more additional therapeutic or prophylactic agents effective in regulating the complement system.
• the pharmaceutical formulations of the present invention can be administered with additional agent(s) in combination therapy, either jointly or separately, or by combining the pharmaceutical formulations and the additional agent(s) into one composition.
• the dosage is administered and adjusted to achieve maximal regulation of the complement system.
• both the pharmaceutical formulations and the additional agent(s) are usually present at dosage levels of between about 10% and about 150%, more preferably, between about 10% and about 80%, of the dosage normally administered in a mono-therapy regimen.
• PA (IALILEPICCQERAA; SEQ ID NO: 1) was solubilized in 20% Intralipid®, which resulted in a homogeneous solution. Upon centrifugation, PA separated into lipid and aqueous layers. Interestingly, at a concentration of 2 mg/ml, 60% of PA was associated with the lipid layer, while only 40% of PA remained in the aqueous phase. If this mixture was allowed to sit at 4°C for 48 hours, the mixture became paste-like and it had enough viscosity that it could be applied topically.
• lipo-RLS-0088 the active pharmacologic agent is associated with the micelles, yet was able to interact with and inhibit activation of hydrophilic complement components in solution.
• the inhibiting effects of lipo-RLS-0088 was tested in vivo by IV injection into adult rats.
• the control was identical doses of RLS-0088 in solution injected into a different set of adult rats.
• the pharmacodynamics showed a 4-fold increase in functionality over time (measured by Clq binding assay) for the rats receiving IV lipo-RLS-0088 compared with those receiving the same dose of IV RLS-0088 ( Figures 2A-2B).
• human red blood cells (RBCs) from type AB donors were purified, washed, and standardized to 1.0 x 10 9 cells/ml.
• Human sera from type O donors at a 10% final concentration was combined with LOmM of the peptides and the volume was brought up to 0.250 ml with GVBS ++ and 5.0 x 10 7 RBCs.
• the samples were incubated for 1 hour at 37°C and then spun at 3,000 rpm for 5 minutes and the supernatant was collected and read at 412 nm.
• An Immunlon-2 HB ELISA plate was coated with 1 pg/ml Clq in bicarbonate buffer overnight at 4°C.
• the plates were washed with PBS-T (phosphate buffered saline + 0.1% Tween) and then blocked with 1% gelatin/PBS for 2 hours at room temperature. After washing, the plates were incubated with the plasma samples diluted in 1% gelatin/PBS for 1 hour at room temperature followed by washing.
• PBS-T phosphate buffered saline + 0.1% Tween
• PA-dPEG24 (RLS-0071) and PA-I8Sar (RLS-0088) have been successfully formulated in liquid containing histidine buffer. To develop formulations of these two molecules that can be utilized for intravenous administration to pediatric and adult patients, the following excipients were utilized to formulate PA-dPEG24 and PA-18 Sar:
• Citrate trisodium citrate dihydrate
• Ascorbate sodium ascorbate
• PA-dPEG24 and PA-I8Sar were formulated at a concentration of 80 mg/ml at pH 6.8.
• PA-dPEG24 it was noted that the preparation would start to gel if not kept on ice. This gelling behavior was not observed for PA-I8Sar.
• PA-dPEG24 and PA-I8Sar were formulated at a concentration of 10 mg/ml at pH 6.8.
• the formulated peptides were tested in a hemolytic assay to determine the ability of these molecules to inhibit complement activity.
• human red blood cells (RBCs) from type AB donors were purified, washed and standardized to I xlO 9 cells/ml, as previously described.
• PA-dPEG24 formulated in ascorbate, citrate or histidine had slightly increased complement inhibiting activity compared to peptide formulated in methionine (Figure 3 A).
• formulations comprising each of the four excipients demonstrated similar levels of activity ( Figure 3B).
• the F32V vehicles were prepared in 50 mL Falcon tubes according to Table 4 as follows. The tubes were vortexed to dissolve all solids.
• HPLC sample was prepared by transferring 100 mg of each sample into a 10 mL volumetric flask. QS to 5 mL by the diluent. Results.
• Figure 4 shows the vials of formulations containing RLS-0088 under the different storage conditions.
• Table 7 below shows the Accusizer results.
• F Freeze-thaw
• L lyophilize
• the F32 formulations were stable at 25°C for at least 24h and 2-8°C for at least 48h.
• the lyophilized-reconstituted and freeze-thaw Formulation F32 were all stable at 25°C for 24 h and 2-8°C for 48h.
• the sample was diluted 1: 1 v/v by DI water for measurement.
• the osmolality of F32 is 411 mOsm, which is acceptable for IV injection/infusion.
• the F32 formulation was stable at 25°C for at least 24h and 2-8 °C for at least 48h.
• the lyophilized-reconstituted and freeze-thaw Formulation F32 were all stable at 25°C for 24 h and 2-8°C for 48h. All F32 samples, regardless of whether they were freeze-thaw or lyophilized- reconstituted, met the particulate matter acceptance criteria in USP 26 ⁇ 788> after storing at 25°C for 24 and 2-8°C for 48h.
• the osmolality of F32 is 411 mOsm, which is acceptable for IV injection/infusion.
• the objective of this example was to prepare a stable liquid and lyophilized formulation of RLS-0071 for pediatric use by: (i) preparing F7, F8, F9, F10 formulations, (ii) lyophilizing the formulations in vials using an existing lyophilize cycle, and (iii) reconstituting the lyophile and thaw the frozen vials and test assay, impurity and particulate matter.
• Tables 11 and 12 show the composition of the different formulations.
• Formulations (F7 - F10) were prepared as follows. 400 mg of RLS-0071 was weighed out and transferred into falcon tubes for F7 - F10. Methionine, EDTA. Na2, Trehalose and sucrose were weighed out and transferred into corresponding falcon tubes. Formulations were brought to 40 g using WFI or Buffer. Each vial was vortexed to dissolve the solids. NaOH or HC1 solution was used to adjust each sample to the target pH values.
• Figure 5 shows the vials of formulations containing RLS-0071 under the different storage conditions.
• Tables 13 and 14 below shows the HPLC and particulate matter results.
• a pharmaceutical formulation comprising a therapeutically effective amount of a lipid- based formulation of SEQ ID NO: 3 is administered to a subject in need thereof to treat or prevent a disease or condition.
• the administration can be by any appropriate route (e g., injection, infusion, implantation).
• a pharmaceutical formulation comprising a therapeutically effective amount of a lipid- based formulation of SEQ ID NO: 3 is administered to a subject in need thereof to regulate the complement system in the subject.
• the administration can be by any appropriate route (e g., injection, infusion, implantation).
• a pharmaceutical formulation comprising a therapeutically effective amount of SEQ ID NO: 2 or SEQ ID NO: 3 in combination with an excipient that is suitable for intravenous administration is intravenously administered to a subject in need thereof to treat or prevent a disease or condition.
• the excipient may be selected based on the age of the subject.
• a pharmaceutical formulation comprising a therapeutically effective amount of SEQ ID NO: 2 or SEQ ID NO: 3 in combination with an excipient that is suitable for intravenous administration is intravenously administered to a subject in need thereof to regulate the complement system in the subject.
• the excipient may be selected based on the age of the subject.
• a composition comprising a therapeutically effective amount of SEQ ID NO: 3 and a lipid- based carrier.
• composition of embodiment 1, wherein the lipid-based carrier comprises lipid micelles.
• composition of embodiments 1 or 2 wherein the lipid-based carrier comprises a lipid emulsion. 4. The composition of embodiment 3, wherein the lipid-based carrier is present in an amount of about 10% w/v to about 20% w/v.
• composition comprising a therapeutically effective amount of SEQ ID NO: 2 or SEQ ID NO: 3 and at least one excipient.
• composition of embodiment 5, wherein the at least one excipient is suitable for intravenous administration.
• composition of embodiments 5 or 6, wherein the at least one excipient is selected from the group consisting of a citrate, an ascorbate, amino acids, and combinations thereof.
• composition of embodiment 7, wherein the citrate comprises sodium citrate.
• composition of embodiment 7, wherein the ascorbate comprises sodium ascorbate.
• composition of embodiment 7, wherein the amino acids comprise L-methionine.
• composition of embodiment 12, wherein the amino acids are present in an amount of about 0.01% w/v to about 5% w/v.
• a method of altering cytokine expression comprising administering to a subject in need thereof a composition according to any of embodiments 1-17.
• a method of treating or preventing a disease or condition comprising administering to a subject in need thereof a composition according to any of embodiments 1-17.
• Peptide Inhibitor of Complement Cl demonstrates antioxidant activity via single electron transport (SET) and hydrogen atom transfer (HAT).
• SET single electron transport
• HAT hydrogen atom transfer

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