WO2014197524A2 - Peptido-thérapie pour traitement du déficit en alpha 1 antitrypsine et des pathologies associées - Google Patents

Peptido-thérapie pour traitement du déficit en alpha 1 antitrypsine et des pathologies associées Download PDF

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WO2014197524A2
WO2014197524A2 PCT/US2014/040776 US2014040776W WO2014197524A2 WO 2014197524 A2 WO2014197524 A2 WO 2014197524A2 US 2014040776 W US2014040776 W US 2014040776W WO 2014197524 A2 WO2014197524 A2 WO 2014197524A2
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peptide
inflammatory
alpha
amino acid
amino acids
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PCT/US2014/040776
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WO2014197524A3 (fr
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Soren Mogelsvang
Cohava Gelber
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Serpin Pharma, Llc
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    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans

Definitions

  • the present disclosure presents compositions for and methods of treatment of alpha- antitrypsin deficiency or dysfunction.
  • the present invention provides the use of a novel class of isolated and/or synthesized peptide fragments, and synthetic analogs based on the peptide fragments made based on the Serpin family of proteins for the treatment of alpha- antitrypsin deficiency.
  • the peptides can have preventative and therapeutic effects in human disease and/or associated pathologies and can be used in treatment of human disease.
  • Serine protease inhibitors represent a large (>1000) family of protease inhibitors, present in all branches of life and involved in a multitude of physiological processes. In mammals, such as humans, Serpins are important for homeostasis and although a certain level of promiscuity exists, each Serpin has a cognate serine protease(s). For example, alpha-1- antitrypsin (AAT) and alpha- 1 -antichymotrypsin (ACT) inhibit inflammatory proteases such as elastase, whereas antithrombin inhibits thrombin and plays a role in coagulation.
  • AAT alpha-1- antitrypsin
  • ACT alpha- 1 -antichymotrypsin
  • a number of specific AAT mutations are manifested in human disease, including COPD, thrombosis and Serpinopathies (cirrhosis and dementia).
  • COPD chronic obstructive pulmonary disease
  • thrombosis thrombosis and Serpinopathies
  • Serpinopathies cirrhosis and dementia.
  • AAT functions as a protease inhibitor similar to endogenous AAT, and is administered with the goal of restoring the balance between AAT and its cognate proteases.
  • AAT is the archetypical Serpin and shares tertiary structure with other Serpins.
  • Serpins have a ⁇ 20 amino acid (aa) exposed loop, called the reactive center loop (RCL), which serves as bait for the cognate proteases.
  • RCL reactive center loop
  • Alpha 1 -antitrypsin deficiency is a genetic disorder that causes defective production of alpha 1 -antitrypsin (A1AT), leading to decreased A1AT activity in the blood and lungs, and deposition of excessive abnormal A1AT protein in liver cells.
  • A1AT alpha 1 -antitrypsin
  • the invention is based, at least in part, on our finding that a short peptide, SP16 (SEQ ID NO: 1) derived from human alpha- 1 -antitrypsin shows anti-inflammatory and immune - modulatory properties similar to the much larger parent protein, alpha- 1 -antitrypsin, and can be used to treat the pathologies that arise from deficiency in the levels and/or activity of alpha- 1- antitrypsin.
  • a method of treating AAT deficiency and associated complications comprising administering to a human subject affected with AAT deficiency an anti- inflammatory peptide comprising, consisting essentially of, or consisting of the amino acid sequence Xl-Zl-F-N-K -P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 2), wherein
  • XI is V or L
  • X2 is V, L or M
  • X3 is M, I or V;
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids, and wherein the isolated anti-inflammatory peptide consists of 37 or fewer amino acids.
  • the anti-inflammatory peptide comprises, consists of, or consists essentially of the amino acid sequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein
  • XI is V or L
  • X2 is K or R
  • X3 is V, L or M
  • X4 is M, I or V
  • X5 is K or Q
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids.
  • described herein is a method of treating AAT deficiency and associated complications comprising administering to a human subject affected with AAT deficiency an antiinflammatory peptide comprising, consisting essentially of, or consisting of an amino acid sequence of any of SEQ ID NOs: 1-36.
  • the anti-inflammatory peptide comprises, consists of, or consists essentially of the amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1). In some embodiments, the anti-inflammatory peptide comprises, consists of, or consists essentially of the amino acid sequence RFNRPFLR (SEQ ID NO: 4). In some embodiments, the antiinflammatory peptide comprises, consists of, or consists essentially of the amino acid sequence KFNKPFLM (SEQ ID NO: 57). In some embodiments, the anti-inflammatory peptide comprises, consists of, or consists essentially of the amino acid sequence RRRFNRPFLRRR (SEQ ID NO: 8).
  • the anti-inflammatory peptide comprises, consists of, or consists essentially of the amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1). In some embodiments, the anti-inflammatory peptide comprises, consists of, or consists essentially of the amino acid sequence FNRPFL (SEQ ID NO: 10).
  • the isolated antiinflammatory peptide consists of, at most, 100, 35, 22, 21 or 9 additional amino acids. In some embodiments, the anti-inflammatory peptide consists of 35 amino acid residues or fewer. In some embodiments, the anti-inflammatory peptide consists of 22 amino acid residues or fewer. In some embodiments, the anti-inflammatory peptide consists of 21 amino acid residues or fewer.
  • the isolated anti-inflammatory peptide further comprises at least one other protein.
  • the combination of the at least two proteins can be referred to as a fusion protein.
  • the other protein can be selected from an epitope tag and a half-life extender.
  • the peptide can comprise both an epitope tag and a half-life extender.
  • the peptide can be modified to extend the shelf life and/or bioavailability using one or more non-natural peptide bonds or amino acids or by attaching to the peptide functional groups such as, e.g., polyethylene glycol (PEG).
  • the anti-inflammatory peptide comprises one or more D-amino acids.
  • the isolated anti-inflammatory peptide consists essentially of, or consists of the amino acid sequence of Z1-RFNRPFLR-Z2 (SEQ ID NO: 6) and Zl- RFNKPFLR-Z2 (SEQ ID NO: 7), wherein Zl and Z2 are independently 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10 or between 1 and 3, between 1 and 5, between 1 and 6, between 1 and 7, between 1 and 8, between 1 and 9, or between 1 and 10 basic amino acids.
  • the anti-inflammatory peptide comprises, consists of, or consists essentially of any one or a combination of the following peptides: SP40; SP43; SP46; and SP49 as set forth in Table B.
  • composition comprising the anti-inflammatory peptide may further comprise a carrier, such as a pharmaceutically acceptable carrier.
  • the subject is a mammal.
  • the mammal is a human.
  • the human has not been subjected to prior treatment with alpha- 1 antitrypsin, such as alpha- 1 -antitrypsin treatment before the treatment with the peptides of the invention.
  • the method further comprises administering to the subject alpha- 1 antitrypsin replacement therapy with alpha- 1 antitrypsin.
  • the antiinflammatory peptide is administered without alpha- 1 antitrypsin.
  • the anti-inflammatory peptide is administered intramuscularly, intravenously, subcutaneously or orally.
  • an anti-inflammatory peptide as described herein for the treatment of AAT deficiency and associated pathologies.
  • the use further comprises administering to the subject alpha- 1 antitrypsin replacement therapy with intravenous infusions of alpha- 1 antitrypsin.
  • the anti-inflammatory peptide is administered without alpha- 1 antitrypsin.
  • a method of screening for anti-inflammatory peptides comprising providing a candidate peptide comprising X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3- Q (SEQ ID NO: 2), wherein XI is V or L;
  • X2 is V, L or M
  • X3 is M, I or V
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids, and wherein the peptide comprises 37 or fewer amino acids; or
  • XI is V or L
  • X2 is K or R
  • X3 is V, L or M
  • X4 is M, I or V
  • X5 is K or Q
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids
  • the inflammatory marker or symptom is selected from the group consisting of the level of TNF-alpha; the level of C-reactive protein; the level of endotoxin; and the level of glycemic control.
  • the steps of comparing and identifying are performed by a non-human machine.
  • the candidate peptide further comprises at least one second peptide or protein.
  • the at least one other protein or peptide is attached to the peptide as a fusion peptide.
  • the at least one second peptide or protein is an epitope tag or a half-life extender or both.
  • the candidate peptide comprises one or more D-amino acids.
  • the candidate peptide consists of 35 amino acid residues or fewer.
  • the candidate peptide consists of 22 amino acid residues or fewer.
  • the candidate peptide consists of 21 amino acid residues or fewer.
  • Figure 1 is a table showing the sequences and homology of Serpin C-terminal peptides (SEQ ID NOs: 18-24, respectively, in order of appearance).
  • Figure 2 is a table showing peptides derived from truncations (SEQ ID NOs: 25-32, 1, 33-34, 1, 33, 38-51, 10, and 8, respectively, in order of appearance).
  • Figure 3 is a bar graph showing TNF-a levels in blood of mice injected with 0.5, 0.1 , 0.02 or 0.004 mg of various peptides (SP1-SP18 from left to right on the x-axis). Each peptide was administered in four different concentrations. The four bars for each of the peptide from left to right represent the concentrations from highest (0.5 mg) to the lowest (0.004 mg).
  • Figure 4 is a bar graph showing TNF-a levels in blood of mice injected with 0.004 mg of various peptides.
  • Figure 5 is a line graph showing cumulative paw scores for mock treated, dexamethasone treated and peptide (SEQ ID NO: 1, also referred to as SP16) in a collagen antibody induced arthritis (CAIA) rat model.
  • Figure 6 is a line graph showing cumulative paw scores for mock treated, dexamethasone treated and peptide (SEQ ID NO: 1, also referred to as SP16) in a collagen antibody induced arthritis (CAIA) rat model.
  • Figure 7 is a graph summarizing survival during a lethal endotoxemia study in mice.
  • Figures 8A-8F show graphs summarizing data from a study in the db/db T2DM model.
  • Five-week old db/db mice were assigned to groups of 10 animals, and IP injected with 0.6 mg/kg SP16 (biweekly), 15 mg/kg Rosiglitazone (biweekly), or vehicle control for 5 weeks.
  • HbAlc ( Figure 8A) and C-peptide (Figure 8B) levels were determined at the end of the study.
  • a glucose tolerance test was administered ( Figure 8C).
  • Non- fasted blood glucose was measured twice a week throughout the study and was significantly lowered in the SP16 (SEQ ID NO: 1) and Rosiglitazone groups.
  • Figure 8D summarizes the extent of islet hyperplasia in the db/db study as assessed by morphometry.
  • Figures 8E and 8F summarize serum CRP and TGF-beta levels in the db/db mouse model of type II diabetes. Decreased serum CRP levels are consistent with peptide treatment promoting an anti-inflammatory cytokine profile. Eight week old diabetic db/db mice were assigned to groups of 8. Group received Saline (Mock) or 0.6 mg/kg SP16 biweekly for 12 weeks.
  • FIG. 9 is a graph summarizing data from a study in the mouse CAIA Rheumatoid Arthritis model. The graph shows cumulative swelling scores for all paws at the peak of disease (Day 7) for groups of 5 animals. Balb/c mice were IV injected with a collagen antibody cocktail (MD Biosciences) on Day 0 and IP injected with LPS on Day 3. Normal Control Animals received no injections and served as disease-free baseline control. Daily SP16 injection provided protection equivalent to Dexamethasone.
  • FIG 10 shows TNF-alpha levels in LPS challenged mice.
  • the mice were treated with alanine scanned SP16 peptide.
  • Dexamethasone served as positive control of "effective treatment” and the peptides SP16, SP40, SP43, SP46 and SP49 all reduce TNF-alpha levels more than Dexamethasone.
  • the C- and N-terminal amino acids contribute to the anti-inflammatory effect of the SP16 peptide.
  • Figure 4 shows a graph summarizing serum TNF-alpha levels in a mouse inflammation model, LPS challenge, treated with different human Serpin derived peptides.
  • Serum TNF-alpha levels were determined by ELISA.
  • Several peptides provided the same level of protection as 1 mg/kg Dexamethasone.
  • FIG 11 shows that SP16 is a TLR2 agonist.
  • SP16 exhibited TLR-2 ligand properties, inducing TLR-2 signaling in a dose dependent manner.
  • the SP34 scrambled control peptide showed no TLR2 induction. *p ⁇ 0.05, compared to scrambled control (SP34).
  • Figure 12 shows structure activity relationship analysis for SP16.
  • TLR-2 indicator cell line HEK- BlueTM mTLR2, Invivogen
  • alanine scan alanine scan
  • Cells were incubated with 20 ⁇ g/ml of the indicated peptides for 24 hours.
  • TLR2 activation the cells secrete alkaline phosphatase which can be assayed.
  • the assay was done in triplicate and averages are plotted. Peptide sequences are shown in the following figure. *p ⁇ 0.05, compared with scrambled control (SP34).
  • Figure 13 shows Structure activity relationship analysis for SP16.
  • Table showing the amino acid sequences (SEQ ID NOS 1, 18, 12-17, 52-56, and 51, respectively, in order of appearance) of peptides that were tested using a TLR-2 indicator cell line (See data in previous figure).
  • the right side of the table summarizes the peptides' impact on TLR-2 signaling (* indicates low, ***** indicates high, N/A had no impact on signaling).
  • the data suggest the first three residues contribute to inducing TLR-2 signaling. If residues 1 -3 are substituted with alanines (SP37), the mutant peptide has no impact on TLR2. However, when substituted individually (SP52-SP54), the peptides retain the ability to stimulate TLR-2. Surprisingly, substitution of the phenyl alanine residue at position 3 with a smaller alanine residue enhances the ability to stimulate TLR-2 signaling compared to SP16.
  • Figure 14 shows a schematic of the different phases of Type II diabetes.
  • Figure 15 shows a graph summarizing data from an experiment with primary human synoviocytes.
  • Cells were incubated with 10 uM of the indicated peptides, as well as 0, 5, 10, or 30 ng/ml ILlb, for 48 hours.
  • the cells secrete the metaloprotease MMP1, which is involved in breaking down the cartilage in Arthritis.
  • the assay was done in triplicate and averages with standard deviations are plotted. SP16 lowered MMP1 secretion compared to the scrambled control peptides, at 20 ng/ml ILlb.
  • (*) indicates p ⁇ 0.05 compared to scrambled peptide control.
  • Figure 16 depicts a graph of the level of inflammation in the CAIA model following administration of SP16 orally or intraperitoneally.
  • the y-axis presents the clinical scores of inflammation.
  • Figure 17 depicts a graph of the level of IL6 secretion from RAW cells following addition of SP16.
  • Figure 18 depicts a graph of the percent of NOD mice which are diabetes free following administration of hAAT and SP16.
  • the present disclosure describes methods of treating AAT deficiency and associated pathologies by administering anti-inflammatory peptides described herein. As described herein, these peptides exhibit anti-inflammatory effects that can be advantageous in the treatment of AAT deficiency, alone or in combination with AAT replacement therapy. Specifically, the present invention provides isolated and/or synthesized peptides, and synthetic analogs based on these peptides, with preventative and therapeutic effects in human AAT deficiency.
  • the peptides described herein are specifically defined short isolated or synthesized C- terminal peptides based on Serpins and variants and derivatives thereof with surprisingly effective anti-inflammatory properties and with much more useful size for therapeutic applications compared to the native Serpin proteins.
  • the isolated anti-inflammatory peptides are shown in Figures 1-2.
  • Figure 1 shows the amino acid sequences of the C-terminal fragments of a variety of Serpins. Each peptide is marked with a SEQ ID NO: in column 2, immediately to the left of the peptide.
  • Figure 2 shows truncations of the C-terminal fragments shown in Figure 1 , as well as variants and derivatives thereof. Again, each peptide is marked with a SEQ ID NO: in column 2, immediately next to the peptide.
  • SP16 SEQ ID NO: 1 which is derived from human alpha- 1 antitrypsin exhibits anti-inflammatory and immune -modulatory properties similar to those of the parent protein, alpha- 1 -antitrypsin.
  • SP16 as described herein, is efficacious in the treatment of AAT deficiency and associated pathologies.
  • the peptides of the invention can provide a good safety profile, based on the good safety profile of the parent protein, alpha- 1 -antitrypsin.
  • the peptides of the invention are far easier and thus less expensive to produce as they are much smaller than the parent protein.
  • C-terminal peptides that result from a Serpin molecule's cleavage by one of its cognate serine proteases have intrinsic biologic function that is distinct from that of the protease inhibitor function of the parent, complete Serpin molecule.
  • the C-terminal peptides from AAT, antichymotrypsin and Kallistatin have varying degrees of anti-inflammatory effects.
  • these anti-inflammatory and/or immune modulating peptides that are a byproduct from the lifecycle of a Serpin molecule, represent a type of an immunological and inflammatory (homeostatic) "master switch.”
  • this anti-inflammatory activity and/or immune modulating activity is therapeutic in the context of AAT deficiency. That is, despite the lack of protease inhibitor function, the peptides described herein can, at least in part, replace a deficient or low level of the parent protein. This indicates a previously unappreciated aspect of the pathology of AAT deficiency.
  • Serpins are related to inhibiting the function of serine protease enzymes. A few Serpins inhibit other types of proteins, and several do not have an inhibitory function.
  • Serpins are a large family (>1000) of Serine Protease Inhibitors that are structurally similar but functionally diverse. They are involved in a multitude of physiological processes and are critical for homeostasis in mammals. Genetic mutations in individual Serpins are manifested in different human diseases, including COPD, thrombosis and emphysema.
  • Each serpin with an inhibitory role is responsible for blocking the activity of one or more proteins. Serpins bind to their target proteins to prevent them from completing any further reactions. Upon binding to a target, an irreversible change in the structure of a serpin protein occurs. Certain cells recognize when a Serpin is bound to its target and clear these attached proteins from the bloodstream.
  • Alpha- 1 -antitrypsin is the prototypical Serpin.
  • PROLASTIN® Telas
  • ZEMAIRA® Aventis Behring
  • ARALAST® Boxter
  • AAT is currently in clinical trials for treatment of new onset type I diabetes, graft vs. host disease and cystic fibrosis.
  • SERPiNAl jj serpin peptidase inhibitor clade A (alpha- 1 jj AAT, A1A, PI1, alpha- 1 -antitrypsin, jj jj antiproteinase, antitrypsin), member 1 ⁇ ⁇ 1 AT. alpha 1 AT.
  • SERPIN A4 serpin peptidase inhibitor, clade A alpha- 1 1 KST, KAL, KLST, kallistatin
  • SERPINAiO serpin peptidase inhibitor, clade A alpha- 1 jj PZI, ZPI
  • SERPINA13 jj serpin peptidase inhibitor, clade A alpha- 1 UNQ6121
  • SERP1NB6 jj scrpin peptidase inhibitor, clade B
  • SERP1NE2 jj serpin peptidase inhibitor clade E (nexin,
  • SERP1NF2 jj serpin peptidase inhibitor SERP1NF2 jj serpin peptidase inhibitor, clade F (alpha-2 ! j API, ALPHA-2 -PI, A2 AP, AAP iTable A
  • Table B shows peptides named SP16; SP40; SP43; SP46; and SP49 provided particularly good anti-inflammatory effect when administered to a mouse model of sepsis (See Figure 10).
  • any of isolated antiinflammatory peptides consisting of or consisting essentially of sequences set forth in SEQ ID NOs: 8, 10, 19-34, and 38-49 can be used to treat AAT deficiency and associated patholgies.
  • Table C presents additional exemplary peptides that were used to reduce TNF- alpha levels in mice subjected to an LPS challenge. See also Figure 10B. Also the peptides with capacity to reduce TNF-alpha levels as shown in Figure 10 are contemplated for the
  • compositions for use and treatment of AAT deficiency in the present invention.
  • SPl SEQ ID NO: 18
  • SP2 SEQ ID NO: 19
  • SP3 SEQ ID NO: 20
  • SP4 SEQ ID NO: 21
  • SP5 SEQ ID NO: 22
  • SP6 SEQ ID NO: 23
  • SP7 SEQ ID NO: 24
  • SP8 SEQ ID NO: 25
  • SP9 SEQ ID NO: 26
  • SP10 SEQ ID NO: 27
  • SPl 1 SEQ ID NO: 28
  • SP12 SEQ ID NO: 29
  • SP13 SEQ ID NO: 30
  • SP14 SEQ ID NO: 31
  • SP15 SEQ ID NO: 32
  • SP16 SEQ ID NO: 1
  • SP17 SEQ ID NO: 33
  • SP18 SEQ ID NO: 34
  • the phrase "consisting essentially of is herein meant to define the scope of the peptides to the specified material amino acids, and to only include additional amino acids or changes that do not materially affect the claimed invention's basic and novel characteristics, namely, the anti-inflammatory capacity of the short isolated or synthesized peptides.
  • the definition specifically excludes peptides that have a sequence of a complete Serpin protein, and the definition also specifically excludes peptide sequences that are equal to or longer than 37 amino acids of any naturally occurring Serpin protein.
  • Human AAT, antichymotrypsin, and kallistatin have been known to contain elements with anti-inflammatory properties. However, these elements have not been previously identified.
  • LPS induced endotoxemia mouse endotoxemia model
  • Formula I provides a composition comprising a peptide comprising, consisting essentially of or consisting of the amino acid sequence Xl-Zl-F-N-R -P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 1)
  • XI is V or L
  • X2 is V, L or M
  • X3 is M, I or V;
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids, wherein the peptide comprises 37 or fewer amino acids.
  • the isolated anti-inflammatory peptide causes a 50% or 75% decrease in serum TNF-a levels compared to the serum level as measured prior to administering the isolated anti-inflammatory peptide, when administered in an effective amount to a human subject.
  • the peptide further comprises a fusion protein.
  • the fusion protein can be selected from an epitope tag and a half-life extender or a combination thereof.
  • Formula II provides a composition comprising an isolated anti-inflammatory peptide comprising, consisting essentially of or consisting of the amino acid sequence X1-Z1-F-N-X2-P- F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein
  • XI is V or L
  • X2 is K or R
  • X3 is V, L or M
  • X4 is M, I or V;
  • X5 is K or Q
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids; and wherein the isolated anti-inflammatory peptide causes a 75% decrease in serum TNF-a levels compared to the serum levels prior to administering the isolated anti-inflammatory peptide when administered in an effective amount to a human subject.
  • the peptide comprising the amino acid sequence of Xl-Zl-F- N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3) as defined above, includes, at most, 35, 22 or 21 amino acid residues.
  • the peptide further comprises a fusion protein.
  • the fusion protein can be selected from an epitope tag and a half-life extender or a combination thereof.
  • the peptide is SP16.
  • the invention also provides an isolated anti-inflammatory peptide consisting of or consisting essentially of the amino acid sequence RFNRPFLR (SEQ ID NO: 4) and RFNKPFLR (SEQ ID NO: 5), which can also be used for the treatment of AAT deficiency and associated pathologies.
  • the peptide causes a 50% or 75% decrease in serum TNF-a levels compared to the serum TNF-a levels prior to administering the isolated antiinflammatory peptide when administered in an effective amount to a human subject.
  • the isolated anti-inflammatory peptide further comprises a fusion protein. Specifically the fusion protein can be selected from an epitope tag and a half-life extender.
  • the isolated anti-inflammatory peptide comprises, at most, 100, 35, 22, 21, 16 or 9 amino. In other embodiments, the isolated anti-inflammatory peptide comprises the amino acid sequence of Z1-RFNRPFLR-Z2 (SEQ ID NO: 6), and Zl- RFNKPFLR-Z2 (SEQ ID NO: 7) wherein Zl and Z2 are independently between 1, 2, 3, 4, 5, 6, 6,
  • the isolated antiinflammatory peptide consists essentially of or consists of the amino acid sequence of
  • composition comprising a peptide consisting essentially of or consisting of the amino acid sequence of FNRPFL (SEQ ID NO: 10) and FNKPFL (SEQ ID NO: 11).
  • the isolated anti-inflammatory peptide comprises 5 or more sequential amino acids from the amino acid sequence of FLMIEQNTK (SEQ ID NO: 36). These peptides can be used to treat or prevent AAT deficiency in a subject. In certain embodiments, the amount of TNF-a level in the serum is reduced compared to the amount of TNF-a in the serum prior to administering the isolated anti-inflammatory peptide.
  • the method of treatment of inflammation further comprises analysis of TNF-a serum levels prior to administering the isolated anti-inflammatory peptide and after administering the isolated anti-inflammatory peptide. If the TNF-a serum level is decreased less than 30%, the step of administering can be repeated with the same dose or with a larger dose of the peptide compared to the first dose.
  • Fragments of any of the peptides described above can vary in size.
  • these fragments can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37, amino acids in length.
  • the peptides described above are generally used to reduce inflammation.
  • the peptides exert anti-inflammatory and immune -modulating effects, and additionally, directly or indirectly stimulate beta cell regeneration.
  • these peptides reduce inflammation by reducing the activity or expression of TNF-a.
  • the activity of TNF-a can be reduced by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%.
  • the expression of TNF-a can be reduced 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%.
  • the peptide composition typically further comprises a pharmaceutically acceptable solution or carrier.
  • Serpinal (a-1 antitrypsin or AAT) is an inhibitor of neutrophil elastase produced by hepatocytes, mononuclear monocytes, alveolar macrophages, enterocytes, and myeloid cells. Individuals with mutations in one or both copies of the Serpinal gene can suffer from alpha- 1 anti-trypsin deficiency, which presents as a risk of developing pulmonary emphysema or chronic liver disease due to greater than normal elastase activity in the lungs and liver.
  • the deficiency in alpha- 1 antitrypsin is a deficiency of wildtype, functional alpha- 1 antitrypsin.
  • the individual is producing significant quantities of alpha- 1 antitrypsin, but a proportion of the alpha- 1 antitrypsin protein being produced is misfolded or contains mutations that compromise the functioning of the protein.
  • the individual is producing misfolded proteins which cannot be properly transported from the site of synthesis to the site of action within the body. Liver disease resulting from alpha- 1 antitrypsin deficiency can be caused by such misfolded proteins.
  • Mutant forms of alpha-1 antitypsin are produced in liver cells and in the misfolded configuration they are not readily transported out of the cells. This leads to a buildup of misfolded protein in the liver cells and can cause one or more diseases or disorders of the liver including, but not limited to, chronic liver disease, liver inflammation, cirrhosis, liver fibrosis, and/or hepatocellular carcinoma.
  • the lack of functional AAT in the lungs also leads to lung disease, e.g. emphysema.
  • Subjects with AAT deficiency may also develop necrotizing panniculitis, bronchiectasis, and/or prolonged neonatal jaundice.
  • Risk factors for developing AAT deficiency and/or lung or liver disease arising, at least in part, from AAT deficiency include a family history of such diseases, the presence of an inherited mutation in AAT, smoking, exposure to tobacco smoke, hepatitis infection, alcoholism, Some patients having or at risk of having a deficiency of alpha- 1 antitrypsin are identified by screening when they have family members affected by an alpha- 1 antitrypsin deficiency.
  • a patient treated in accordance with the methods described herein is first diagnosed has having AAT deficiency or an associated pathology arising from an AAT deficiency. That is, a subject has or is diagnosed as having a symptom or condition described herein, e.g. emphysema or liver fibrosis as well as a deficiency of AAT, e.g. a low level of AAT in the blood, an aberrant type of AAT in the blood, and/or the presence of a mutation that can cause AAT deficiency.
  • an AAT deficiency can be a level of AAT less than 100 mg/dL.
  • AAT present in serum can be phenotyped by isoelectric focusing, revealing whether the subject has the normal M type or the abnormal, disease-related S and Z types of AAT.
  • the Z type AAT comprises Glu342Lys (and E342K (c.l024G>A) relative to M type AAT.
  • the S type AAT comprises Glu264Val (c.791A>T), relative to M type AAT. Additional variations of the AAT polypeptide are known, but are present in the population at a low frequency.
  • AAT deficiency-related mutations and methods of detecting them are known in the art and described, e.g. in Brantly et al. Am J Med. 1988 84: 13-31 and Crystal et al. Chest 1989 95: 196-206; each of which is incorporated by reference herein in its entirety.
  • One of ordinary skill in the art can readily test for the presence or absence of such mutations and/or decreased levels of AAT.
  • tests for AAT deficiency are commercially available, e.g. the Serpinal Gene Sequence Test (Cat No. 1140; Ambry Genetics; Alisa Dahljo, CA), a series of tests from ARUP Laboratories (Cat Nos. 0051256, 0050001, and 0080500; Salt Lake City, UT) and the GenoTypeTM AAT test (Cat No. 251 ; Hain LifeScience; Nehren, Germany).
  • the disclosure further provides methods of treating inflammation comprising the step of administering any one of the peptides described herein or a combination thereof to a subject in need of treatment of AAT deficiency.
  • the subject has not been treated with alpha-antitrypsin prior to the treatment.
  • the method comprises a step of assaying whether the individual has increased serum TNF-a levels and if the subject has increased serum TNF-a levels then administering the peptide to the subject, and if not, then not administering the peptide to the subject.
  • the disclosure also enables methods of preventing development of symptoms of AAT deficiency comprising the step of administering any one of the peptides or a combination thereof to a subject in need of prevention of symptoms of AAT deficiency.
  • the subject has not been treated with alpha-antitrypsin prior to the treatment.
  • the method comprises a step of assaying whether the individual has increased serum TNF-a levels and if the subject has increased serum TNF-a levels then administering the peptide to the subject, and if not, then not administering the peptide to the subject.
  • the methods described herein can further comprise administering to the subject alpha- 1 antitrypsin replacement therapy with alpha- 1 antitrypsin.
  • alpha- 1 antitrypsin replacement therapy is know in the art and are described in, e.g. American Thoracic Society/European Respiratory Society Statement: Standards for the diagnosis and management of individuals with alpha- 1 antitrypsin deficiency, Am J Respir Crit Care Med 2003; 168: 818-900; Wewers MD, et al.New Eng J Med 1987; 316: 1055-1062; Seersholm N, et al, Eur Respir J 1997; 10: 2260-2263; and The Alpha-1 -Antitrypsin Deficiency Registry Study Group, Survival and FEV1 decline in individuals with severe deficiency of alpha-1 -antitrypsin, Am J Respir Crit Care Med 1998; 158: 49-59; each of which is incorporated by reference herein in its entirety.
  • AAT replacement therapy compositions are commercially available, e.g. GLASSIATM (Kamada Ltd), PROLASTINTM (Talecris); ARALAST NPTM (Baxter Healthcare); ZEMAIRATM (CSL Behring), and TRYPSONTM (Grifols).
  • the methods described herein relate to methods of treatment wherein the anti-inflammatory peptide is administered without alpha-1 antitrypsin (AAT), e.g. the subject has not previously been administered AAT, is not administered AAT during the period of time in which the subject is treated in accordance with the methods described herein, and/or the subject is not administered AAT after the treatment in accordance with the methods described herein is concluded.
  • AAT alpha-1 antitrypsin
  • wild type refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo.
  • mutant refers to any change in the genetic material of an organism, in particular a change (i.e., deletion, substitution, addition, or alteration) in a wild-type polynucleotide sequence or any change in a wild-type protein sequence.
  • a change in the genetic material results in a change of the function of the protein
  • the term - “mutant” refers to a change in the sequence of a wild-type protein regardless of whether that change alters the function of the protein (e.g., increases, decreases, imparts a new function), or whether that change has no effect on the function of the protein (e.g., the mutation or variation is silent).
  • mutation is used interchangeably herein with polymorphism in this application.
  • polypeptide and “protein” are used interchangeably to refer to an isolated polymer of amino acid residues, and are not limited to a minimum length unless otherwise defined.
  • Peptides, oligopeptides, dimers, multimers, and the like are also composed of linearly arranged amino acids linked by peptide bonds, and whether produced biologically and isolated from the natural environment, produced using recombinant technology, or produced synthetically typically using naturally occurring amino acids.
  • the polypeptide or protein is a "modified polypeptide" comprising non-naturally occurring amino acids.
  • the polypeptides comprise a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptides comprise only non-naturally occurring amino acids.
  • the peptides or modified peptides further comprise co-translational and post- translational (C-terminal peptide cleavage) modifications, such as, for example, disulfide -bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases), and the like to the extent that such modifications do not affect the anti-inflammatory properties of the isolated anti-inflammatory peptides or their capacity to improve glycemic control.
  • co-translational and post- translational (C-terminal peptide cleavage) modifications such as, for example, disulfide -bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases), and the like to the extent that such modifications do not affect the anti-inflammatory properties of the isolated anti-inflammatory peptides or their capacity to improve
  • the polypeptide is altered.
  • altered polypeptide refers to a peptide that includes alterations, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art, such as alanines), to the native sequence, as long as the protein maintains the desired activity, i.e., it anti- inflammaotry activity of capacity to improve glycemic control or reduce hyperglycemia.
  • Polypeptides or proteins are composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, have a well-defined conformation. Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids.
  • peptide typically refers to a sequence of amino acids made up of a single chain of amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length, unless otherwise desfined.
  • Modified peptide may include the incorporation of non-natural amino acids into the peptides of the invention, including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the peptides (or other components of the composition, with exception for protease recognition sequences) is desirable in certain situations.
  • D-amino acid- containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid- containing forms.
  • the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required.
  • D- peptides are resistant to endogenous peptidases and proteases, thereby providing better oral trans-epithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane -permanent complexes (see below for further discussion), and prolonged intravascular and interstitial lifetimes when such properties are desirable.
  • the use of D- isomer peptides can also enhance transdermal and oral trans-epithelial delivery of linked drugs and other cargo molecules.
  • D-peptides cannot be processed efficiently for major histocompatibility complex class II-restricted presentation to T helper cells, and are therefore less likely to induce humoral immune responses in the whole organism.
  • Peptide conjugates can therefore be constructed using, for example, D-isomer forms of cell penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides.
  • the peptides as disclosed comprise L and D amino acids, wherein no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 D-amino acids are included.
  • the peptides comprise more than 10 D-amino acids, and in certain aspects all the amino acids of the peptides are D-amino acids.
  • the peptides or fragments or derivatives thereof can be "retro- inverso peptides.”
  • a “retro-inverso peptide” refers to a peptide with a reversal of the direction of the peptide bond on at least one position, i.e., a reversal of the amino- and carboxy-termini with respect to the side chain of the amino acid.
  • a retro-inverso analogue has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence.
  • the retro-inverso peptide can contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, up to all of the amino acids being the D- isomer.
  • Partial retro-inverso peptide analogues are polypeptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Since the retro- inverted portion of such an analogue has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inverted portion are replaced by side -chain-analogous a-substituted geminal-diaminomethanes and malonates, respectively.
  • Retro-inverso forms of cell penetrating peptides have been found to work as efficiently in translocating across a membrane as the natural forms.
  • Synthesis of retro-inverso peptide analogues are described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, G. C, J. Chem. Soc. Perkin Trans. 1 :697-701 (1985); and U.S. Patent No. 6,261,569, which are incorporated herein in their entirety by reference.
  • Processes for the solid-phase synthesis of partial retro-inverso peptide analogues have been described (EP 97994-B) which is also incorporated herein in its entirety by reference.
  • Homology and identity can each be determined by comparing a position in each sequence which can be aligned for purposes of comparison. For example, it is based upon using a standard homology software in the default position, such as BLAST, version 2.2.14. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by similar amino acid residues (e.g., similar in steric and/or electronic nature such as, for example conservative amino acid substitutions), then the molecules can be referred to as homologous (similar) at that position.
  • Expression as a percentage of homology/similarity or identity refers to a function of the number of similar or identical amino acids at positions shared by the compared sequences, respectfully. A sequence which is "unrelated" or “non-homologous" shares less than 40% identity, though preferably less than 25% identity with the sequences as disclosed herein.
  • sequence identity means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide -by- nucleotide or residue -by- residue basis) over the comparison window.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T. C, G. U. or I) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85% sequence identity, preferably at least 90% to 95% sequence identity, more usually at least 99% sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which can include deletions or additions which total 20 percent or less of the reference sequence over the comparison window.
  • the reference sequence can be a subset of a larger sequence.
  • similarity when used to describe a polypeptide, is determined by comparing the amino acid sequence and the conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • the terms “homologous” or “homologues” are used interchangeably, and when used to describe a polynucleotide or polypeptide, indicates that two polynucleotides or polypeptides, or designated sequences thereof, when optimally aligned and compared, for example using BLAST, version 2.2.14 with default parameters for an alignment (see herein) are identical, with appropriate nucleotide insertions or deletions or amino-acid insertions or deletions, in at least 70% of the nucleotides, usually from about 75% to 99%, and more preferably at least about 98 to 99% of the nucleotides.
  • homolog or “homologous” as used herein also refers to homology with respect to structure and/or function. With respect to sequence homology, sequences are homologs if they are at least 50%, at least 60 at least 70%, at least 80%, at least 90%), at least 95% identical, at least 97% identical, or at least 99% identical. Determination of homologs of the genes or peptides of the present invention can be easily ascertained by the skilled artisan.
  • substantially homologous refers to sequences that are at least 90%, at least 95%) identical, at least 96%, identical at least 97% identical, at least 98% identical or at least 99% identical. Homologous sequences can be the same functional gene in different species.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482 (1981), which is incorporated by reference herein), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443-53 (1970), which is incorporated by reference herein), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show the percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J. Mol. Evol. 25:351-60 (1987), which is incorporated by reference herein). The method used is similar to the method described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53 (1989), which is incorporated by reference herein). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • BLAST algorithm Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described by Altschul et al. (J. Mol. Biol. 215:403-410 (1990), which is incorporated by reference herein). (See also Zhang et al., Nucleic Acid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res. 25:3389-402 (1997), which are incorporated by reference herein). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information internet web site.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990), supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • HSPs high scoring sequence pairs
  • Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (1993), which is incorporated by reference herein).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • an amino acid sequence is considered similar to a reference amino acid sequence if the smallest sum probability in a comparison of the test amino acid to the reference amino acid is less than about 0.1, more typically less than about 0.01, and most typically less than about 0.001.
  • variant refers to a peptide or nucleic acid that differs from the polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side -chain modifications, yet retains one or more specific functions or biological activities of the naturally occurring molecule.
  • Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art.
  • substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non- conventional amino acid.
  • amino acid substitutions are conservative.
  • polynucleotide or polypeptide refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild- type polynucleotide or polypeptide).
  • Variants can also be synthetic, recombinant, or chemically modified polynucleotides or polypeptides isolated or generated using methods well known in the art. Variants can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Variants can also include insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins.
  • conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties.
  • Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • a “conservative substitution” of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not reduce the activity of the peptide, (i.e. the ability of the peptide to penetrate the blood brain barrier (BBB)).
  • BBB blood brain barrier
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H.
  • substitutions suitable for amino acids on the exterior of a protein or peptide for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.
  • non-conservative amino acid substitutions are also encompassed within the term of variants.
  • derivative refers to peptides which have been chemically modified, for example but not limited to by techniques such as ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), lipidation, glycosylation, or addition of other molecules.
  • a molecule also a "derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half-life, etc.
  • the moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl., Easton, PA (1990), incorporated herein, by reference, in its entirety.
  • the peptides of the invention comprise peptide derivatives, such as pegylated peptides.
  • the term "functional” when used in conjunction with “derivative” or “variant” refers to a peptide of the invention which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the entity or molecule it is a functional derivative or functional variant thereof, i.e., anti-inflammatory activity in the context of the peptides described herein.
  • the term functional derivative is intended to include the fragments, analogues or chemical derivatives of a molecule.
  • insertions or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed can be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • substitution when referring to a peptide, refers to a change in an amino acid for a different entity, for example another amino acid or amino-acid moiety. Substitutions can be conservative or non-conservative substitutions.
  • an "analog" of a molecule such as a peptide refers to a molecule similar in function to either the entire molecule or to a fragment thereof.
  • the term “analog” is also intended to include allelic species and induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with natural peptides. Some analogs also include unnatural amino acids or modifications of N or C terminal amino acids.
  • unnatural amino acids are, for example but not limited to; disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ - trimethyllysine, ⁇ - ⁇ -acetyllysine, O- phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ - ⁇ - methylarginine. Fragments and analogs can be screened for prophylactic or therapeutic efficacy in transgenic animal models as described below.
  • covalently bonded is meant joined either directly or indirectly (e.g., through a linker) by a covalent chemical bond.
  • the fusion peptides are covalently bonded.
  • fusion protein refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by a nucleic acid sequence encoding one protein is joined to the nucleic acid encoding another protein such that they constitute a single open-reading frame that can be translated in the cells into a single polypeptide harboring all the intended proteins. The order of arrangement of the proteins can vary. Fusion proteins can include an epitope tag or a half-life extender. Epitope tags include biotin, FLAG tag, c-myc,
  • Half- life extenders include Fc domain and serum albumin.
  • subject and “individual” and “patient” are used interchangeably herein, and refer to an animal, for example a human or non-human animal (e.g., a mammal) , to whom treatment, including prophylactic treatment, with a pharmaceutical composition as disclosed herein, is provided.
  • subject refers to human and non-human animals.
  • non-human animals includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dogs, rodents (e.g.
  • Non-human mammals include mammals such as non-human primates, (particularly higher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits and cows.
  • the non-human animal is a companion animal such as a dog or a cat.
  • Treating" a disease or condition in a subject or “treating" a patient having a disease or condition refers to subjecting the individual to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease or condition is decreased or stabilized.
  • a pharmaceutical treatment e.g., the administration of a drug
  • treating AAT deficiency relates to decreasing and/or stabilizing a symptom of AAT deficiency, e.g. inflammation, emphysema, liver disease, cirrhosis, lung inflammation, or liver inflammation.
  • prevention is used in connection of prevention of symptoms or slowing down of symptom development from the time of asymptomatic state.
  • the peptide is administered preventively, it is administered to a subject who does not present imminent symptoms of AAT deficiency.
  • the subject is at risk of developing AAT deficiency due to the family history, laboratory results, genetic testing or life-style.
  • telomere binding a compound or antibody that recognizes and binds a desired polypeptide but that does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
  • Specific binding can be characterized by a dissociation constant of at least about 1x10-6 M or smaller. In other embodiments, the dissociation constant is at least about 1x10-7 M, 1x10-8 M , or 1x10-9 M.
  • Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
  • isolated it is meant that the polypeptide has been separated from any natural environment, such as a body fluid, e.g., blood, and separated from the components that naturally accompany the peptide.
  • substantially pure a polypeptide that has been separated and purified to at least some degree from the components that naturally accompany it.
  • a polypeptide is substantially pure when it is at least about 60%, or at least about 70%, at least about 80%), at least about 90%>, at least about 95%, or even at least about 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.
  • a decrease or “inhibition” used in the context of the level of, for example TNF- alpha levels refers to reduction of the amount of protein in the biological sample, such as blood or tissue sample, a cell, a cell extract, or a cell supernatant.
  • a decrease may be due to reduced RNA stability, transcription, or translation, increased protein degradation, or RNA interference.
  • this decrease is at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, or even at least about 90% compared to a reference value.
  • the term "reference value" in the context of the claims and the application refers typically to an abnormally high TNF-alpha level found in an individual affected with or suffering from inflammation.
  • the reference value is typically the amount of TNF-alpha in the individual prior to administering of the peptide of the invention.
  • the term “reference value” refers to the numeric values used in measuring glycemic control in a subject.
  • an “increase” in the expression or activity of a gene or protein is meant a positive change in protein or nucleic acid level or activity in a cell, a cell extract, or a cell supernatant.
  • such an increase may be due to increased RNA stability, transcription, or translation, or decreased protein degradation.
  • this increase is at least 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 100%), at least about 200%, or even about 500%> or more over the level of expression or activity under control conditions.
  • nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • recombinant as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.
  • Recombinant is also used herein to refer to, with reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) that the material has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).
  • material e.g., a cell, a nucleic acid, a protein, or a vector
  • a heterologous material e.g., a cell, a nucleic acid, a protein, or a vector
  • vectors refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked; a plasmid is a species of the genus encompassed by “vector”.
  • vector typically refers to a nucleic acid sequence containing an origin of replication and other entities necessary for replication and/or maintenance in a host cell.
  • vectors capable of directing the expression of genes and/or nucleic acid sequence to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility are often in the form of "plasmids" which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome, and typically comprise entities for stable or transient expression or the encoded DNA.
  • Other expression vectors can be used in the methods as disclosed herein for example, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages or viral vectors, and such vectors can integrate into the host's genome or replicate autonomously in the particular cell.
  • a vector can be a DNA or RNA vector.
  • expression vectors can also be used, for example self-replicating extrachromosomal vectors or vectors which integrates into a host genome.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • viral vectors refers to the use of viruses, or virus-associated vectors as carriers of a nucleic acid construct into a cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno- associated virus (AAV), or Herpes simplex virus (HSV) or others, including reteroviral and lentiviral vectors, for infection or transduction into cells.
  • AAV Adeno-associated virus
  • HSV Herpes simplex virus
  • the vector may or may not be incorporated into the cell's genome.
  • the constructs may include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors.
  • One aspect of the present invention relates to the use of peptides described herein and mutants, variants, analogs or derivatives thereof. Specifically, these methods relate to administering any one of the peptides as described herein or their pharmaceutically acceptable modifications in a pharmaceutically acceptable carrier to a subject, e.g., a mammal in need thereof, e.g., a human, i.e., a subject having AAT deficiency.
  • the human is first diagnosed as having one or more symptom of the disease before administering one or more of the peptides of the invention. In some embodiments, the human has not previously been administered AAT as a treatment for the symptoms.
  • the SP16 peptide can be safely administered using well-established preclinical safety studies. For example, we have also shown using FastPatch assay that, for example, the SP16 peptide does not impact hERG activity and we also were not able to identify any hits on human receptor panning study (GenSEP Explorer) for the SP 16 peptide.
  • the db/db mice carry a defective leptin receptor, which impairs their ability to regulate appetite and metabolism. The animals become obese at 3-4 weeks of age, and initially show insulin resistance, which is followed by hyperglycemia at about 4-8 weeks of age. Severe hyperglycemia is paralleled by depletion of the insulin-producing b-cells of the pancreatic islets and death by 10 months of age.
  • the db/db animals model the different phases of Type II Diabetes in humans ( Figure 14).
  • the peptides of the invention provide benefits at early and late stages of disease.
  • SP 16 improves glycemic control in the db/db model.
  • Figures 8B and 8D exemplify a graphs summarizing serum C-peptide levels (8B) and islet hyperplasia (8D) in the db/db mouse model of type II diabetes. Increased serum C-peptide levels are consistent with improved ⁇ -cell function in the treated groups.
  • Five-week old pre- diabetic db/db mice were assigned to groups of 10. Groups received Saline (Mock), 0.6 mg/kg SP16 or 25 mg/kg Rosiglitazone twice a week. Pooled serum C-peptide levels were determined for each group. . (*) indicates p ⁇ 0.05 and (**) p ⁇ 0.01 compared to the Vehicle control.
  • the preclinical CAIA model of Rheumatoid Arthritis is a short study where arthritis is induced in Balb/c mice. On Day 0, animals are intravenously injected with a collagen antibody cocktail (MD Biosciences) which initiates autoimmune destruction of the collagen in their joints. On Day 3, an intra-peritoneal injection of LPS is used to exacerbate the autoimmune reaction and inflammation. The readout in this model is paw swelling and histological assessment of joint erosion. Disease typically starts subsiding after 7-10 days. [00162] We demonstrated that SP16 shows efficacy in the preclinical CAIA mouse model. Specifically, we measured cumulative swelling scores for all paws at the peak of disease (Day 7) for groups of 5 animals.
  • mice were IV injected with a collagen antibody cocktail (MD Biosciences) on Day 0 and IP injected with LPS on Day 3. Normal Control Animals received no injections and served as disease-free baseline control. Daily SP16 injection of 12 ⁇ g/day provided protection equivalent to Dexamethasone and injections of 12 ⁇ g once every 3 days reduced the inflammation by almost 50% compared to the treatment with the vehicle control. See e.g, Figure 9.
  • LPS Lipopolysaccharide
  • gram negative bacteria leaking from the GI system contribute to a systemic inflammatory response syndrome and lethality.
  • SP16 increases survival in lethal endotoxemia thereby allowing us to extrapolate that the peptide would provide treatment in human burn victims or in human acute radiation syndrome.
  • Groups of 10 animals were injected as indicated, 2 hours before, at the time of, and/or 0.5 hours after induction of lethal endotoxemia. Endotoxemia was induced by injection of 15 mg/kg LPS.
  • the peptides of the invention are toll like receptor-2 agonists. Accordingly, without wishing to be bound by a theory, the peptides, such as SP16, act as an anti-inflammatory drug by promoting an anti-inflammatory cytokine profile. Also, without wishing to be bound by a theory, the peptides, such as SP16, also can act as an immune modulators by inducing expansion of tolerogenic and protective T-regulatory cells (T-regs).
  • T-regs T-regulatory cells
  • the peptides such as SP16, also can down- regulate autoimmune responses without inducing general immunological suppression thereby providing a superior treatment for autoimmune diseases compared to most of the currently available treatments which generally suppress the immune system exposing the treated individuals to a risk of infections while treated with the general immunosuppressants.
  • AAT has been shown to modulate T-cell proliferation and NF-kappa-B activation; impair NK target cell interaction; inhibit serine proteases activation of epithelial cell EGFR/TLR-4 signaling; be involved in TNF-alpha-induced gene expression and apoptosis or endothelial cells; prevent red blood cell haemolysis by E.
  • coli decrease circulating eosinophil cell count; inhibit neutrophil chemotaxis, NADHP oxidase and ANCA signaling; inhibit monocyte and macrophage cytokine release and regulation of CD 14 expression, and inhibit mast cell histamine release; and modulate B-cell proliferation and cytokine production.
  • the peptide is SP16, which may comprise one or more modifications typically performed to enhance peptide bioavailability and/or shelf life, such as pegylation and the like.
  • TLR- 2 assay We also performed a peptide optimization assay using an alanine scan with the TLR- 2 assay. Data was obtained using an experiment with an engineered TLR-2 indicator cell line (HEK-BLUETM mTLR2, Invivogen). Cells were incubated with the 20 ⁇ g/ml of the indicated peptides for 24 hours. Upon TLR2 activation, the cells secrete alkaline phosphatase which can be assayed. The assay was done in triplicate and averages are plotted. Peptide SP34 is a scrambled peptide control (Yellow), and PAM (Pam3CSK4; Red) is a positive control. See, e.g., Figure 12.
  • the SP16 safety profile also included hERG data.
  • the hERG FastPatch assay showed that SP16 does not inhibit hERG at up to 25 ⁇ doses. This data predicts that SP16 will not have cardiac safety issues in humans. The study was executed by Apredica, Boston, MA.
  • the GenSEP Explorer panel contains 111 in vitro assay targets carefully selected to assess drug/chemical biological activities. Assay categories include GPCRs, Voltage-Gated Ion Channels, Ligand- Gated Ion Channels, Neurotransmitter transporters, Nuclear Receptors and Steroids as well as a diverse set of biochemical targets including Phosphodiesterases, Kinases and other relevant enzymes. The study was executed by Caliper LifeSciences, and the results are summarized in the table below. It appears that SP16 has no effect on 111 human receptors, indicating that SP16 has an excellent human safety profile.
  • the methods of treatment described herein further comprise selection or diagnosis of a subject having AAT deficiency prior to administering a peptide as disclosed herein or a mutant, variant, analog or derivative thereof, to thereby treat the condition or dysfunction.
  • selection is performed by the skilled practitioner by a number of available methods, for instance, assessment of symptoms which are described herein. For example, one can assess the amount of TNF-alpha in the subject to determine the amount of inflammation present in the subject.
  • C-reactive protein is used to detect inflammation if there is a high suspicion of tissue injury or infection somewhere in the body.
  • CRP serves as a general marker for infection and inflammation and can be used to evaluate an individual for an acute or chronic inflammatory condition.
  • a high or increasing amount of CRP in the blood suggests the presence of inflammation.
  • a high CRP suggests the presence of one.
  • high levels of CRP suggest a flare-up or that treatment has not been effective.
  • Normal concentration in healthy human serum is usually lower than 10 mg/L, slightly increasing with aging.
  • the term "reference value" refers to the measurements of CRP when CRP is used as a diagnostic test for inflammation.
  • Successful or effective treatment is evidenced by amelioration of one or more symptoms of the condition or dysfunction as discussed herein.
  • Administering a peptide as disclosed herein or a mutant, variant, analog or derivative thereof in a subject in need thereof is expected to prevent or retard the development of the conditions and physical dysfunctions described herein (e.g., those arising from AAT deficiency).
  • the term "prevention” is used to refer to a situation wherein a subject does not yet have the specific condition being prevented, meaning that it has not manifested in any appreciable form. Prevention encompasses prevention or slowing of onset and/or severity of a symptom, (including where the subject already has one or more symptoms of another condition).
  • Prevention is performed generally in a subject who is at risk for development of a condition or physical dysfunction. Such subjects are said to be in need of prevention. For example, reduction in the TNF-alpha levels compared to the levels prior to administering the peptides of the invention, would be evidence of successful treatment.
  • the methods of prevention described herein further comprise selection of such a subject at risk for a condition, e.g., AAT deficiency, prior to administering a peptide or a mutant, variant, analog or derivative thereof, in the subject, to thereby prevent the condition or dysfunction.
  • a condition e.g., AAT deficiency
  • Such selection is performed by the skilled practitioner by a number of available methods. For instance, assessment of risk factors or diagnosis of a disease which is known to cause the condition or dysfunction, or treatment or therapy known to cause the condition or dysfunction. Subjects which have a disease or injury or a relevant family history which is known to contribute to the condition are generally considered to be at increased risk.
  • the terms “treat” or “treatment” or “treating” refers to therapeutic treatment measures, wherein the object is to prevent or slow the development of the disease, such as reducing at least one effect or symptom of a condition, disease or disorder associated with inflammation.
  • Treatment is generally “effective” if one or more symptoms are improved or clinical markers, such as TNF-alpha and/or CRP levels are within normal values or closer to the normal reference values than abnormal values reflecting inflammation, depending on the condition, as that term is defined herein.
  • treatment is “effective” if the progression of a disease is slowed down, exhibition of a symptom or a marker for a disease is reduced.
  • treatment includes the improvement of symptoms or markers, slowing of progress or slowing of worsening of at least one symptom that would be expected in absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include patients with one or more symptoms of AAT deficiency.
  • TNF-alpha levels can be assessed, for example, using any number of readily available commercial ELISA kits.
  • AIC test, FPG, and OGTT are commonly used to assess glycemic control in diagnosing and managing diabetes and pre-diabetes.
  • the invention relates to methods of preventing symptoms of AAT deficiency by administering the peptides as described to an individual not yet presenting symptoms of AAT deficiency.
  • the peptides can be administered to an individual at high risk of developing AAT deficiency, e.g. a subject having a mutation in AAT associated with AAT deficiency.
  • the term "effective amount” as used herein refers to the amount of a pharmaceutical composition comprising one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to decrease at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • the phrase "therapeutically effective amount” as used herein means a sufficient amount of the composition to treat a disorder, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the term “therapeutically effective amount” therefore refers to an amount of the composition as disclosed herein that is sufficient to effect a therapeutically or prophylatically reduction in a symptom or clinical marker associated with AAT deficiency.
  • a disease marker such as an inflammatory marker, e.g., TNF-alpha
  • a disease marker such as an inflammatory marker, e.g., TNF-alpha
  • reduction of more than 50% or more than 75% from the amount of TNF-alpha levels in the individual prior to administering the peptides of the invention is indicative of effective treatment.
  • a therapeutically or prophylactically significant reduction in a symptom is, e.g. at least about 10%>, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%), at least about 125%), at least about 150%) or more in a measured parameter as compared to a control or non-treated subject or the state of the subject prior to administering the peptide.
  • Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker, such as TNF-alpha, as well as parameters related to a clinically accepted scale of symptoms or markers for inflammation. It will be understood, however, that the total daily usage of the compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated, gender, age, and weight of the subject.
  • terapéuticaally effective amount refers to the amount that is safe and sufficient to delay the development of one or more symptoms and results in decrease in the amount of an inflammatory marker, e.g., TNF-a or CRP concentrations, compared to the amount of the inflammatory marker prior to administering the peptide.
  • the amount can thus improve or cause a decrease in at least one symptom of AAT deficiency or slow the course of disease progression, such as stabilizing the development of emphysema or cirrhosis.
  • the effective amount for the treatment of a disease depends on the type of disease, the species being treated, the age and general condition of the subject, the mode of administration and so forth.
  • an appropriate "effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • the efficacy of treatment can be judged by an ordinarily skilled practitioner, for example, efficacy can be assessed in known animal models of inflammation (e.g. LPS model) or auto-immune tissue destruction (e.g. CAIA model).
  • efficacy of treatment is evidenced when a reduction in a symptom of AAT deficiency is shown versus untreated animals.
  • the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof may be administered by any route known in the art or described herein, for example, oral, parenteral (e.g., intravenously or intramuscularly), intraperitoneal, rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or ocular.
  • the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof may be administered in any dose or dosing regimen.
  • the present invention contemplates all modes of administration, including intramuscular, intravenous, intraperitoneal, intravesicular, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose adequate to treat the AAT deficiency.
  • the therapeutic may be administered to the patient in a single dose or in multiple doses.
  • the doses may be separated from one another by, for example, one hour, three hours, six hours, eight hours, one day, two days, one week, two weeks, or one month.
  • the therapeutic may be administered for, e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. For example, the dosage of the therapeutic can be increased if the lower dose does not provide sufficient therapeutic activity.
  • therapeutically effective amounts of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof may be provided at a dose of 0.0001, 0.01, 0.01 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg/kg or ⁇ g/kg. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test bioassays or systems.
  • Dosages for a particular patient or subject can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol).
  • a physician may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the dose administered to a patient is sufficient to effect a beneficial therapeutic response in the patient over time, or, e.g., to reduce symptoms, or other appropriate activity, depending on the application.
  • the dose is determined by the efficacy of the particular formulation, and the activity, stability or serum half- life of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose is also determined by the existence, nature, and extent of any adverse side- effects that accompany the administration of a particular vector, formulation, or the like in a particular subject.
  • Therapeutic compositions comprising one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, such as models of AAT deficiency to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art.
  • dosages can be initially determined by activity, stability or other suitable measures of treatment vs. non-treatment (e.g., comparison of treated vs. untreated cells or animal models), in a relevant assay.
  • Formulations are administered at a rate determined by the LD50 of the relevant formulation, and/or observation of any side-effects of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof. Administration can be accomplished via single or divided doses.
  • the efficacy and toxicity of the compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions, which exhibit large therapeutic indices are preferred.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration that works for small peptides, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • compositions - "pharmaceutically acceptable carriers"
  • administration of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof may be by any suitable means that results in a concentration of the protein that treats the disorder.
  • the compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1 -95% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for the oral, parenteral (e.g., intravenously or intramuscularly), intra-peritoneal, rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or ocular administration route.
  • the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols.
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A.R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, incorporated, herein, by reference in its entirety).
  • compositions according to the invention may be formulated to release the active compound immediately upon administration or at any predetermined time or time period after administration.
  • the latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create substantially constant concentrations of the agent(s) of the invention within the body over an extended period of time; (ii) formulations that after a predetermined lag time create substantially constant concentrations of the agent(s) of the invention within the body over an extended period of time; (iii) formulations that sustain the agent(s) action during a predetermined time period by maintaining a relatively constant, effective level of the agent(s) in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the agent(s) (sawtooth kinetic pattern); (iv) formulations that localize action of agent(s), e.g., spatial placement of a controlled release composition adjacent to or in the diseased tissue or organ; (v) formulations that achieve convenience of dosing, e.g., administering the
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings.
  • the protein is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the protein in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.
  • parenteral administration and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.
  • systemic administration "administered systemically", “peripheral administration” and
  • administered peripherally as used herein mean the administration therapeutic compositions other than directly into a tumor such that it enters the animal's system and, thus, is subject to metabolism and other like processes.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in maintaining the activity of or carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • the pharmaceutical formulation comprising the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof in combination with one or more pharmaceutically acceptable ingredients.
  • the carrier can be in the form of a solid, semi-solid or liquid diluent, cream or a capsule.
  • These pharmaceutical preparations are a further object of the invention.
  • the amount of active compounds is between 0.1-95% by weight of the preparation, preferably between 0.2-20%> by weight in preparations for parenteral use and preferably between 1 and 50%> by weight in preparations for oral administration.
  • targeted delivery composition of the invention is formulated into
  • compositions or pharmaceutical formulations for parenteral administration e.g., intravenous; mucosal, e.g., intranasal; enteral, e.g., oral; topical, e.g., transdermal; ocular, e.g., via corneal scarification or other mode of administration.
  • the pharmaceutical composition contains a compound of the invention in combination with one or more pharmaceutically acceptable ingredients.
  • the carrier can be in the form of a solid, semi-solid or liquid diluent, cream or a capsule.
  • pharmaceutically acceptable carriers is intended to include all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its functional derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc;
  • excipients such as cocoa butter and suppository waxes
  • oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil
  • glycols such as propylene glycol
  • polyols such as glycerin, sorbitol, mannitol and polyethylene glycol
  • esters such as ethyl oleate and ethyl laurate
  • agar buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
  • compositions or formulations that usually comprise an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to mammals, and preferably humans or human cells.
  • excipient such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to mammals, and preferably humans or human cells.
  • Such compositions can be specifically formulated for administration via one or more of a number of routes, including but not limited to, oral, ocular parenteral, intravenous, intraarterial, subcutaneous, intranasal, sublingual, intraspinal, intracerebroventricular, and the like.
  • compositions for topical e.g., oral mucosa, respiratory mucosa
  • oral administration can form solutions, suspensions, tablets, pills, capsules, sustained-release formulations, oral rinses, or powders, as known in the art are described herein.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and preservatives For examples of carriers, stabilizers and adjuvants, University of the Sciences in Philadelphia (2005) Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st Ed.
  • the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
  • pharmaceutically acceptable salts, esters, amides, and prodrugs refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use of the compounds of the invention.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like
  • nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylanunonium, tetraethyl ammonium, methyl amine, dimethyl amine, trimethylamine, triethylamine, ethylamine, and the like (see, e.g., Berge S. M., et al. (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).
  • esters refers to the relatively nontoxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. The term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters.
  • salts or prodrugs are salts or prodrugs that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.
  • prodrug refers to compounds that are rapidly transformed in vivo to yield the functionally active one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof.
  • a thorough discussion is provided in T. Higachi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A. C. S. Symposium Series, and in Bioreversible Carriers in: Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference.
  • a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • a prodrug of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be designed to alter the metabolic stability or the transport characteristics of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to mask side effects or toxicity, to improve the flavor of a compound or to alter other characteristics or properties of a compound.
  • prodrugs of the compound see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, N. Y., pages 388-392). Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of
  • prodrugs ed. H. Bundgaard, Elsevier, 1985. Suitable examples of prodrugs include methyl, ethyl and glycerol esters of the corresponding acid.
  • the pharmaceutical composition may be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • injection, infusion, or implantation subcutaneous, intravenous, intramuscular, intraperitoneal, or the like
  • suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • the formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation.
  • compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below).
  • the composition may be in form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the composition may include suitable parenterally acceptable carriers and/or excipients.
  • the active agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release.
  • the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.
  • the pharmaceutical compositions according to the invention may be in a form suitable for sterile injection.
  • the suitable active agent(s) are dissolved or suspended in a parenterally acceptable liquid vehicle.
  • acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3- butanediol, Ringer's solution, dextrose solution, and isotonic sodium chloride solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).
  • a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10- 60% w/w of propylene glycol or the like.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium
  • antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like
  • metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • Formulations of the present invention include those suitable for intravenous, oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil- in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • compositions of this invention suitable for parenteral administration comprise one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions comprising one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide- polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly( anhydrides). Depot injectable formulations are also prepared by entrapping the drug, such as one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof in liposomes or microemulsions which are compatible with body tissue.
  • biodegradable polymers such as polylactide- polyglycolide.
  • Depot injectable formulations are also prepared by entrapping the drug, such as one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof in liposomes or microemulsions which are compatible with body tissue.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical
  • compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of ordinary skill in the art.
  • Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions.
  • the composition may also be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.
  • Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2- hydroxyethyl-L-glutamnine), poly(lactic acid), polyglycolic acid, and mixtures thereof.
  • biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2- hydroxyethyl-L-glutamnine), poly(lactic acid), polyglycolic acid, and mixtures thereof.
  • Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies.
  • Materials for use in implants can be nonbiodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters)) or combinations thereof.
  • Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients, and such formulations are known to the skilled artisan (e.g., U.S. Patent Nos.: 5,817,307; 5,824,300; 5,830,456; 5,846,526;
  • excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, eth
  • the tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period.
  • the coating may be adapted to release the protein in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the agent(s) until after passage of the stomach (enteric coating).
  • the coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose).
  • a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate, may be employed.
  • the solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active substances).
  • the coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical Technology, supra.
  • compositions of the invention may be mixed together in the tablet, or may be partitioned.
  • a first agent is contained on the inside of the tablet, and a second agent is on the outside, such that a substantial portion of the second agent is released prior to the release of the first agent.
  • Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin
  • water or an oil medium for example, peanut oil, liquid paraffin, or olive oil.
  • Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus, or spray drying equipment.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and g
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • a solution of resolvin and/or protectin or precursor or analog thereof can be administered as eye drops for ocular neovascularization or ear drops to treat otitis.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • Dosage forms for the topical or transdermal administration of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Transdermal patches have the added advantage of providing controlled delivery of the compounds (resolvins and/or protectins and/or precursors or analogues thereof) of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • biodegradable or absorbable polymers can provide extended, often localized, release of polypeptide agents.
  • the potential benefits of an increased half-life or extended release for a therapeutic agent are clear.
  • a potential benefit of localized release is the ability to achieve much higher localized dosages or concentrations, for greater lengths of time, relative to broader systemic administration, with the potential to also avoid possible undesirable side effects that may occur with systemic administration.
  • Bioabsorbable polymeric matrix suitable for delivery of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be selected from a variety of synthetic bioabsorbable polymers, which are described extensively in the literature.
  • Such synthetic bioabsorbable, biocompatible polymers which may release proteins over several weeks or months can include, for example, poly-a-hydroxy acids (e.g. polylactides, polyglycolides and their copolymers), polyanhydrides, polyorthoesters, segmented block copolymers of polyethylene glycol and polybutylene terephtalate (PolyactiveTM), tyrosine derivative polymers or polyester- amides).
  • bioabsorbable polymers to be used in manufacturing of drug delivery materials and implants are discussed e.g. in U.S. Pat. Nos. 4,968,317, 5,618,563, among others, and in "Biomedical Polymers” edited by S. W. Shalaby, Carl Hanser Verlag, Kunststoff, Vienna, New York, 1994 and in many references cited in the above publications.
  • the particular bioabsorbable polymer that should be selected will depend upon the particular patient that is being treated.
  • One or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be effectively used in treatment by gene therapy. See, generally, for example, U.S. Pat. No. 5,399,346, which is incorporated herein by reference.
  • the general principle is to introduce the polynucleotide into a target cell in a patient.
  • Entry into the cell is facilitated by suitable techniques known in the art such as providing the polynucleotide in the form of a suitable vector, or encapsulation of the
  • polynucleotide in a liposome in a liposome.
  • a desired mode of gene therapy is to provide the polynucleotide in such a way that it will replicate inside the cell, enhancing and prolonging the desired effect.
  • polynucleotide is operably linked to a suitable promoter, such as the natural promoter of the corresponding gene, a heterologous promoter that is intrinsically active in liver, neuronal, bone, muscle, skin, joint, or cartilage cells, or a heterologous promoter that can be induced by a suitable agent.
  • a suitable promoter such as the natural promoter of the corresponding gene, a heterologous promoter that is intrinsically active in liver, neuronal, bone, muscle, skin, joint, or cartilage cells, or a heterologous promoter that can be induced by a suitable agent.
  • Expression vectors compatible with eukaryotic cells can be used to produce recombinant constructs for the expression of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, including fusion proteins with one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment.
  • vectors can be viral vectors such as adenovirus, adeno-associated virus, pox virus such as an orthopox (vaccinia and attenuated vaccinia), avipox, lentivirus, murine moloney leukemia virus, etc.
  • viral vectors such as adenovirus, adeno-associated virus, pox virus such as an orthopox (vaccinia and attenuated vaccinia), avipox, lentivirus, murine moloney leukemia virus, etc.
  • pox virus such as an orthopox (vaccinia and attenuated vaccinia), avipox, lentivirus, murine moloney leukemia virus, etc.
  • plasmid expression vectors can be used.
  • Viral vector systems which can be utilized in the present invention include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus.
  • the vector is an adenovirus. Replication-defective viruses can also be advantageous.
  • the vector may or may not be incorporated into the cells genome.
  • the constructs may include viral sequences for transfection, if desired.
  • the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors.
  • operably linked is meant that a nucleic acid molecule and one or more regulatory sequences (e.g., a promoter) are connected in such a way as to permit expression and/or secretion of the product (e.g., a protein) of the nucleic acid molecule when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
  • regulatory sequences e.g., a promoter
  • operatively linked refers to the functional relationship of the nucleic acid sequences with regulatory sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
  • operative linkage of nucleic acid sequences, typically DNA, to a regulatory sequence or promoter region refers to the physical and functional relationship between the DNA and the regulatory sequence or promoter such that the transcription of such DNA is initiated from the regulatory sequence or promoter, by an RNA polymerase that specifically recognizes, binds and transcribes the DNA.
  • RNA polymerase that specifically recognizes, binds and transcribes the DNA.
  • An operatively linked polynucleotide which is to be expressed typically includes an appropriate start signal (e.g., ATG) and maintains the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence.
  • an appropriate start signal e.g., ATG
  • promoter refers to a segment of a nucleic acid sequence, typically but not limited to DNA or RNA or analogues thereof, that controls the transcription of the nucleic acid sequence to which it is operatively linked.
  • the promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter.
  • the promoter region includes sequences which modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be ds-acting or may be responsive to trans-acting factors. Promoters, depending upon the nature of the regulation may be constitutive or regulated.
  • regulatory sequences is used interchangeably with “regulatory elements” herein refers element to a segment of nucleic acid, typically but not limited to DNA or RNA or analogues thereof, that modulates the transcription of the nucleic acid sequence to which it is operatively linked, and thus act as transcriptional modulators. Regulatory sequences modulate the expression of gene and/or nucleic acid sequence to which they are operatively linked. Regulatory sequence often comprise “regulatory elements” which are nucleic acid sequences that are transcription binding domains and are recognized by the nucleic acid-binding domains of transcriptional proteins and/or transcription factors, repressors or enhancers etc.
  • Typical regulatory sequences include, but are not limited to, transcriptional promoters, inducible promoters and transcriptional elements, an optional operate sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences to control the termination of transcription and/or translation. Included in the term “regulatory elements" are nucleic acid sequences such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operatively linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended.
  • the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of a protein.
  • the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required for initiating transcription of a specific gene.
  • Regulatory sequences can be a single regulatory sequence or multiple regulatory sequences, or modified regulatory sequences or fragments thereof.
  • Modified regulatory sequences are regulatory sequences where the nucleic acid sequence has been changed or modified by some means, for example, but not limited to, mutation, methylation etc.
  • Regulatory sequences useful in the methods as disclosed herein are promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type- specific, tissue-specific or inducible by external signals or agents (e.g. enhancers or repressors); such elements may be located in the 5' or 3' regions of the native gene, or within an intron.
  • tissue-specific promoter means a nucleic acid sequence that serves as a promoter, i.e., regulates expression of a selected nucleic acid sequence operably linked to the promoter, and which selectively affects expression of the selected nucleic acid sequence in specific cells of a tissue.
  • Muscle-specific expression can be achieved, for example, using the skeletal muscle MKC promoter (as disclosed in U.S.
  • Patent Application WO2007/100722 which is incorporated herein by reference
  • muscle-specific promoters such as a-myosin heavy chain, myosin light chain-2 (which is specific for skeletal muscle (Shani et al., Nature, 314;283- 86, 1985), gonadotrophs releasing hormone gene control region which is active in the hypothalamus (Mason et al, Science, 234; 1372-78, 1986), and smooth muscle promoter SM22a, which are all commonly known in the art.
  • the term "constitutively active promoter” refers to a promoter of a gene which is expressed at all times within a given cell.
  • Exemplary promoters for use in mammalian cells include cytomegalovirus (CMV), and for use in prokaryotic cells include the bacteriophage T7 and T3 promoters, and the like.
  • CMV cytomegalovirus
  • inducible promoter refers to a promoter of a gene which can be expressed in response to a given signal, for example addition or reduction of an agent.
  • Non- limiting examples of an inducible promoter are "tet-on” and “tet-off ' promoters, or promoters that are regulated in a specific tissue type.
  • viral vectors that contain nucleic acid sequences encoding the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof are used.
  • a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599
  • retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473
  • sequence encoding one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be inserted into a retroviral vector which contains the sequences necessary for the efficient expression of the metabolic regulators (including promoter and/or enhancer elements which can be provided by the viral long terminal repeats (LTRs) or by an internal promoter/enhancer and relevant splicing signals), sequences required for the efficient packaging of the viral RNA into infectious virions (e.g., a packaging signal (Psi), a tRNA primer binding site (-PBS), a 3' regulatory sequence required for reverse transcription (+PBS)), and a viral LTRs).
  • the LTRs contain sequences required for the association of viral genomic RNA, reverse transcriptase and integrase functions, and sequences involved in directing the expression of the genomic RNA to be packaged in viral particles.
  • the vector DNA is introduced into a packaging cell line.
  • Packaging cell lines provide viral proteins required in trans for the packaging of viral genomic RNA into viral particles having the desired host range (e.g., the viral-encoded core (gag), polymerase (pol) and envelope (env) proteins).
  • the host range is controlled, in part, by the type of envelope gene product expressed on the surface of the viral particle.
  • Packaging cell lines can express ecotrophic, amphotropic or xenotropic envelope gene products.
  • the packaging cell line can lack sequences encoding a viral envelope (env) protein.
  • the packaging cell line can package the viral genome into particles which lack a membrane- associated protein (e.g., an env protein).
  • a membrane-associated protein e.g., an env protein
  • the packaging cell line containing the retroviral sequences can be transfected with sequences encoding a membrane- associated protein (e.g., the G protein of vesicular stomatitis virus (VSV)).
  • VSV vesicular stomatitis virus
  • the transfected packaging cell can then produce viral particles which contain the membrane- associated protein expressed by the transfected packaging cell line; these viral particles which contain viral genomic RNA derived from one virus encapsidated by the envelope proteins of another virus are said to be pseudotyped virus particles.
  • Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells.
  • adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys.
  • Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
  • MVA Modified Virus Ankara
  • avipox such as fowl pox or canary pox.
  • lentiviral vectors are used, such as the HIV based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
  • Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); and U.S. Pat. No. 5,436,146, which are incorporated herein by reference).
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
  • U.S. Patent No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposome carriers, into mice.
  • U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals.
  • a gene or nucleic acid sequence can be introduced into a target cell by any suitable method.
  • one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof constructs can be introduced into a cell by transfection (e.g., calcium phosphate or DEAE-dextran mediated transfection), lipofection, electroporation, microinjection (e.g., by direct injection of naked DNA), biolistics, infection with a viral vector containing a muscle related transgene, cell fusion, chromosome -mediated gene transfer, microcell-mediated gene transfer, nuclear transfer, and the like.
  • a nucleic acid encoding one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be introduced into cells by electroporation (see, e.g., Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-87 (1982)) and biolistics (e.g., a gene gun; Johnston and Tang, Methods Cell Biol. 43 Pt A:353-65 (1994); Fynan et al., Proc. Natl. Acad. Sci. USA 90: 11478-82 (1993)).
  • electroporation see, e.g., Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-87 (1982)
  • biolistics e.g., a gene gun; Johnston and Tang, Methods Cell Biol. 43 Pt A:353-65 (1994); Fynan et al., Proc. Natl. Acad. Sci
  • a gene or nucleic acid sequence encoding one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be introduced into target cells by transfection or lipofection.
  • Suitable agents for transfection or lipofection include, for example, calcium phosphate, DEAE dextran, lipofectin, lipfectamine, DIMRIE C, Superfect, and Effectin (Qiagen), unifectin, maxifectin, DOTMA, DOGS (Transfectam;
  • dioctadecylamidoglycylspermine DOPE (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP (l,2-dioleoyl-3- trimethylammonium propane), DDAB (dimethyl dioctadecylammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB (N- n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene, poly(ethylenimine) (PEI), and the like.
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOTAP l,2-dioleoyl-3- trimethylammonium propane
  • DDAB dimethyl dioctadecyl
  • Methods known in the art for the therapeutic delivery of agents such as proteins and/or nucleic acids can be used for the delivery of a polypeptide or nucleic acid encoding one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, e.g., cellular transfection, gene therapy, direct administration with a delivery vehicle or pharmaceutically acceptable carrier, indirect delivery by providing recombinant cells comprising a nucleic acid encoding a targeting fusion polypeptide of the invention.
  • Various delivery systems are known and can be used to directly administer therapeutic polypeptides such as the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof and/or a nucleic acid encoding one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, and receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction can be enteral or parenteral and include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes.
  • the agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • compositions of the invention may be desirable to administer locally to the area in need of treatment; this may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., by injection, by means of a catheter, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, fibers, or commercial skin substitutes.
  • the active agent can be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249: 1527-1533).
  • the active agent can be delivered in a controlled release system.
  • a pump may be used (see Langer (1990) supra).
  • polymeric materials can be used (see Howard et al. (1989) J. Neurosurg. 71 : 105).
  • the disclosure also contemplates an article of manufacture which is a labeled container for providing the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof.
  • An article of manufacture comprises packaging material and a
  • the pharmaceutical agent in an article of manufacture is any of the compositions of the present invention suitable for providing the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof and formulated into a pharmaceutically acceptable form as described herein according to the disclosed indications.
  • the composition can comprise the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof or a DNA molecule, which is capable of expressing such a peptide.
  • the article of manufacture contains an amount of pharmaceutical agent sufficient for use in treating a condition indicated herein, either in unit or multiple dosages.
  • the packaging material comprises a label which indicates the use of the pharmaceutical agent contained therein.
  • the label can further include instructions for use and related information as may be required for marketing.
  • the packaging material can include container(s) for storage of the pharmaceutical agent.
  • packaging material refers to a material such as glass, plastic, paper, foil, and the like capable of holding within fixed means a pharmaceutical agent.
  • the packaging material can be plastic or glass vials, laminated envelopes and the like containers used to contain a pharmaceutical composition including the pharmaceutical agent.
  • the packaging material includes a label that is a tangible expression describing the contents of the article of manufacture and the use of the pharmaceutical agent contained therein.
  • a method of screening for anti-inflammatory peptides comprising providing a candidate peptide comprising X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3- Q (SEQ ID NO: 2), wherein
  • XI is V or L
  • X2 is V, L or M
  • X3 is M, I or V
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids, and wherein the peptide comprises 37 or fewer amino acids; or
  • XI is V or L
  • X2 is K or R
  • X3 is V, L or M
  • X4 is M, I or V
  • X5 is K or Q
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids
  • contacting a cell or subject with the peptide detecting the level of an inflammatory marker or symptom; comparing the level of the inflammatory marker or symptom to the level in the absence of the peptide; and identifying a peptide as an anti-inflammatory peptide when the level of the inflammatory marker or symptom is decreased compared to the level in the absence of the peptide.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one cell.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, delivery to an in vitro scaffold in which cells are seeded, e.g., via perfusion or injection, or other delivery method well known to one skilled in the art.
  • the candidate peptide can be administered to a subject, e.g. an animal model, e.g. a mouse, or any of the mouse models of diseases described herein.
  • the inflammatory marker or symptom can be any marker or symptom of
  • inflammatory markers or symptoms can include the level of TNF-alpha; the level of CRP; the level of endotoxin; and the level of glycemic control. Methods of measuring inflammatory symptoms and markers are described elsewhere herein.
  • the level of glycemic control can be measured using, e.g., the AlC test, fasting plasma glucose test (FPG), and the oral glucose tolerance test (OGTT).
  • the candidate peptide can be according to any of the embodiments of the aspects of anti-inflammatory peptides described herein.
  • the candidate peptide further comprises at least one second peptide or protein.
  • the at least one second protein or peptide is attached to the peptide as a fusion peptide.
  • the at least one second peptide or protein is an epitope tag or a half-life extender or both.
  • the candidate peptide comprises one or more D-amino acids.
  • the candidate peptide consists of 35 amino acid residues or fewer. In some embodiments, the candidate peptide consists of 22 amino acid residues or fewer. In some embodiments, the candidate peptide consists of 21 amino acid residues or fewer.
  • the steps of comparing and identifying are performed by a non-human machine.
  • the level of the marker or symptom in the absence of the candidate peptide can be, e.g. the level in a parallel, untreated cell or subject, the level in the cell or subject prior to contact with the candidate peptide, and/or a level in a population of cells or subjects not contacted with a peptide, e.g. a pre-determined level.
  • a level of a marker of symptom which is higher or lower than a reference level can be a level which is statistically significantly different than the reference level.
  • a level that is lower than a reference level can be 90% or less of the reference level, e.g. 90%> or less, 80% or less, 70% or less, 60%> or less, 50%) or less, 25% or less, or 10%> or less of the reference level.
  • a level that is higher than a reference level can be 1.5x or more of the reference level, e.g. 1.5x or more, 2x or more, 3x or more, 5x or more, or lOx or more of the reference level.
  • a method of treating alpha 1 -antitrypsin deficiency and associated pathologies comprising administering to a human subject affected with alpha 1 -antitrypsin deficiency an antiinflammatory peptide comprising the amino acid sequence set forth in formula:
  • XI is V or L
  • X2 is V, L or M
  • X3 is M, I or V
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids, and wherein the peptide comprises 37 or fewer amino acids.
  • the anti-inflammatory peptide further comprises at least one second peptide or protein.
  • the at least one second peptide or protein is an epitope tag or a half-life extender or both.
  • anti-inflammatory peptide comprises one or more D-amino acids.
  • anti-inflammatory peptide comprises an amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1).
  • a method for treating alpha- 1 antitrypsin deficiency comprising administering to a human subject diagnosed with alpha- 1 antitrypsin deficiency therapy comprising an antiinflammatory peptide, wherein the anti-inflammatory peptide consists essentially of the amino acid sequence
  • XI is V or L
  • X2 is K or R
  • X3 is V, L or M
  • X4 is M, I or V
  • X5 is K or Q
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids.
  • the at least second peptide or protein comprises an epitope tag or a half-life extender or both.
  • the anti-inflammatory peptide comprises one or more D-amino acids.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • a method for treating alpha-1 antitrypsin deficiency comprising administering to a human diagnosed with alpha-1 antitrypsin deficiency a treatment comprising an anti-inflammatory peptide consisting essentially of the amino acid sequence RFNRPFLR (SEQ ID NO: 4).
  • a method for treating alpha-1 antitrypsin deficiency comprising administering to a human diagnosed with alpha-1 antitrypsin deficiency a treatment comprising an anti-inflammatory peptide consisting essentially of the amino acid sequence of RRRFNRPFLRRR (SEQ ID NO: 8).
  • a method for treating alpha-1 antitrypsin deficiency comprising administering to a human diagnosed with alpha-1 antitrypsin deficiency a treatment comprising an anti-inflammatory peptide consisting essentially of the amino acid sequence of VKFNKPFVFLMIEQNTK (SEQ ID NO: 1).
  • a method for treating alpha-1 antitrypsin deficiency comprising administering to a human diagnosed with alpha-1 antitrypsin deficiency a treatment comprising an anti-inflammatory peptide consisting essentially of the amino acid sequence of FNRPFL (SEQ ID NO: 10).
  • a treatment comprising an anti-inflammatory peptide consisting essentially of the amino acid sequence of FNRPFL (SEQ ID NO: 10).
  • the anti-inflammatory peptide further comprises at least one second peptide or protein.
  • the at least one second peptide or protein is an epitope tag or a half-life extender or both.
  • the anti-inflammatory peptide is administered intramuscularly, intravenously, subcutaneously or orally.
  • composition comprising an anti-inflammatory peptide for the treatment of alpha-1 antitrypsin deficiency, wherein the anti-inflammatory peptide comprises the amino acid sequence set forth in formula:
  • XI is V or L
  • X2 is V, L or M
  • X3 is M, I or V
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids, and wherein the peptide comprises 37 or fewer amino acids.
  • anti-inflammatory peptide further comprises at least one second peptide or protein.
  • anti-inflammatory peptide comprises one or more D-amino acids.
  • anti-inflammatory peptide comprises an amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1).
  • anti-inflammatory peptide for the treatment of alpha-1 antitrypsin deficiency, wherein anti-inflammatory peptide consists essentially of the amino acid sequence
  • XI is V or L
  • X2 is K or R
  • X3 is V, L or M
  • X4 is M, I or V
  • X5 is K or Q
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids.
  • paragraph 34-37 further comprising at least one second protein or peptide.
  • paragraph 38 wherein the at least second peptide or protein comprises an epitope tag or a half-life extender or both.
  • paragraph 34-40 wherein the anti-inflammatory peptide comprises one or more D- amino acids.
  • composition further comprises a pharmaceutically acceptable carrier.
  • an anti-inflammatory peptide for treating alpha-1 antitrypsin deficiency wherein the anti-inflammatory peptide consists essentially of the amino acid sequence of
  • an anti-inflammatory peptide for treating alpha-1 antitrypsin deficiency wherein the anti-inflammatory peptide consists essentially of the amino acid sequence of
  • VKFNKPFVFLMIEQNTK SEQ ID NO: 1.
  • a method of screening for anti-inflammatory peptides comprising:
  • XI is V or L
  • X2 is V, L or M
  • X3 is M, I or V
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids, and wherein the peptide comprises 37 or fewer amino acids; or
  • XI is V or L
  • X2 is K or R
  • X3 is V, L or M
  • X4 is M, I or V
  • X5 is K or Q
  • Zl is any amino acid
  • Z2 is a sequence of any two amino acids
  • Z3 is a sequence any five amino acids
  • detecting the level of an inflammatory marker or symptom comparing the level of the inflammatory marker or symptom to the level in the absence of the peptide;
  • TNF-alpha The level of TNF-alpha; the level of C-reactive protein; the level of endotoxin; and the level of glycemic control.
  • the candidate peptide further comprises at least one second peptide or protein.
  • a method of treating alpha 1 -antitrypsin deficiency and associated pathologies comprising administering to a human subject affected with alpha 1 -antitrypsin deficiency an antiinflammatory peptide comprising an amino acid sequence selected from any of SEQ ID NOs: 1-36.
  • anti-inflammatory peptide further comprises at least one second peptide or protein.
  • the anti-inflammatory peptide comprises one or more D-amino acids.
  • an anti-inflammatory peptide comprising an amino acid sequence selected from any of SEQ ID NOs: 1-36, comprising administering the peptide to a human subject affected with alpha 1 -antitrypsin deficiency or related pathology.
  • anti-inflammatory peptide further comprises at least one second peptide or protein.
  • Example 1 Testing of anti-inflammatory potential in a mouse sepsis model.
  • SP16 was tested in a lethal endotoxemia model, which models endotoxemia following acute radiation exposure, or during severe infection, where Gram negative bacteria leaking from the gastro- intestinal system can cause lethal endotoxemia.
  • peptide treatment improved survival (Figure 7).
  • SP16 lowers LPS-induced NFKB activation in THP1 cells, consistent with the in vivo anti-inflammatory effect.
  • mice were injected with 0.5, 0.1, 0.02 and 0.004 mg of each peptide. Mice were also treated with dexamethasone as a positive control and vehicle as a negative control. Two hours later each mouse was injected with LPS. Samples were taken 90 minutes after LPS injection.
  • Example 2 Testing of alanine substituted peptides in a mouse sepsis model
  • the mouse LPS challenge model is an established model for inflammation and thus we conclude that peptides wherein the amino acids 1-3 and 15-17 are present, can be effectively used in humans to treat or prevent inflammation and/or sepsis in conditions, such as burns or acute radiation, which expose humans to a high risk of developing endotoxemia.
  • Example 3 Testing of anti-inflammatory potential in a collagen antibody induced arthritis (CAIA) model of rheumatoid arthritis.
  • CAIA collagen antibody induced arthritis
  • mice were injected with a collagen antibody cocktail on Day 0 and received an LPS boost on Day 3. Following the LPS boost, the paw swelling was scored for each paw. For the results shown in Figure 5, animals were dosed with 0.2mg/kg of the peptide set forth in SEQ ID NO: l (also referred to as SP16), daily. In Figure 6, Balb/c mice were intravenously injected with a collagen antibody cocktail (MD Biosciences) on Day 0 and intraperitoneally injected with LPS on Day 3.
  • a collagen antibody cocktail MD Biosciences
  • Paw swelling was determined on Days 0, 3, 4, 5, 6 and 7, and the graph shows cumulative scores for all paws of each experimental group of 5 animals. Untreated control did not receive the CAIA cocktail or LPS. The mock group received both the antibody cocktail and LPS boost. Dexamethasone was administered daily at 1 mg/kg. The peptide set forth in SEQ ID NO: l was dissolved in water and administered intraperitoneally, at 0.6 mg/kg daily, or a one-time dose of 0.6 mg/kg on day 3.
  • Figure 9 shows efficacy of SP16 in the preclinical CAIA mouse model of RA.
  • Graph summarizes data from a study in the mouse CAIA RA model. The graph shows cumulative swelling scores for all paws at the peak of disease (Day 7) for groups of 5 animals.
  • Balb/c mice were injected intravenously with a collagen antibody cocktail (MD Biosciences) on Day 0 and injected intraperitoneally with LPS on Day 3. Normal control animals received no injections and served as disease-free baseline control.
  • Normal control animals received no injections and served as disease-free baseline control.
  • VKFNKPFVFLMIEQNTK (SEQ ID NO: l) is an effective anti-inflammatory and/or immune-modulating agent in mouse models of inflammation, severe sepsis and rheumatoid arthritis.
  • Example 4 SP16 improves glycemic control in the db/db model type II diabetes
  • TLR-2 Toll-like receptor 2
  • TLR-2 plays a role in the immune system and it is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity.
  • TLR-2 gene is expressed most abundantly in peripheral blood leukocytes, and has been shown to mediate host response to Gram-positive bacteria and yeast via stimulation of NF-kappaB.
  • SP16 is a TLR2 agonist, as demonstrated in an experiment with an engineered TLR-2 indicator cell line (HEK-BLUETM mTLR2, Invivogen) (Figure 11). Cells were incubated with the indicated concentrations of peptide for 24 hours. Upon TLR2 activation, the cells secrete alkaline phosphatase which can be assayed. The assay was done in triplicate and averages with standard deviations are plotted. SP16 exhibited TLR-2 ligand properties, inducing TLR-2 signaling in a dose dependent manner. The a scrambled control peptide (SP34) showed no TLR2 induction. *p ⁇ 0.05, compared with scrambled control (SP34).
  • SP34 scrambled control
  • Figure 12 shows additional structure activity relationship analysis for SP16.
  • alanine scan alanine scan
  • Cells were incubated with 20 ⁇ g/ml of the indicated peptides for 24 hours.
  • TLR2 activation the cells secrete alkaline phosphatase which can be assayed.
  • the assay was done in triplicate and averages are plotted.
  • Peptide sequences are shown in the following figure. *p ⁇ 0.05, compared with scrambled control (SP34).
  • Figure 13 shows the peptide sequences used for studying the structure activity relationship analysis for SP16. Table showing the amino acid sequences of peptides that were tested using a TLR-2 indicator cell line (See data in Figure 12). The right side of the table summarizes the peptides' impact on TLR-2 signaling (* indicates low, ***** indicates high, N/A had no impact on signaling).
  • Figure 16 depicts the level of inflammation (as measured by clinical scores) in the CAIA model of rheumatoid arthritis following administration of the SP16 peptide. Oral administration was shown to be efficacious in reducing inflammation, as compared to intraperitoneal administration.
  • RAW cells macrophage model
  • IL6 secretion after being contacted with SP16 consistent with the in vivo data described herein, as demonstrated in Figure 17.

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Abstract

L'invention concerne des peptides et leurs utilisations pour le traitement d'un dysfonctionnement ou d'un déficit en alpha 1 antitrypsine.
PCT/US2014/040776 2013-06-06 2014-06-04 Peptido-thérapie pour traitement du déficit en alpha 1 antitrypsine et des pathologies associées WO2014197524A2 (fr)

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WO2017040287A1 (fr) 2015-08-28 2017-03-09 Serpin Pharma, Llc Méthodes de traitement de maladies
US10006025B2 (en) 2014-06-17 2018-06-26 Arrowhead Pharmaceuticals, Inc. Compositions and methods for inhibiting gene expression of alpha-1 AntiTrypsin
US10450565B2 (en) 2017-01-10 2019-10-22 Arrowhead Pharmaceuticals, Inc. Alpha-1 antitrypsin (AAT) RNAi agents, compositions including AAT RNAi agents, and methods of use
EP4003391A4 (fr) * 2020-07-06 2023-02-22 Serpin Pharma, LLC Peptides et leurs méthodes d'utilisation
US11752203B2 (en) 2013-07-08 2023-09-12 University Of Utah Research Foundation Methods for treatment of and prophylaxis against inflammatory disorders

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US7850970B2 (en) * 2003-08-26 2010-12-14 The Regents Of The University Of Colorado Inhibitors of serine protease activity and their use in methods and compositions for treatment of bacterial infections
WO2011126882A2 (fr) * 2010-03-30 2011-10-13 American Type Culture Collection Peptides thérapeutiques et leurs dérivés et utilisations thérapeutiques de ceux-ci
EP2802338B1 (fr) * 2012-01-09 2018-03-07 Serpin Pharma, LLC Peptides et leurs méthodes d'utilisation

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US11752203B2 (en) 2013-07-08 2023-09-12 University Of Utah Research Foundation Methods for treatment of and prophylaxis against inflammatory disorders
US11384355B2 (en) 2014-06-17 2022-07-12 Arrowhead Pharmaceuticals, Inc. Compositions and methods for inhibiting gene expression of alpha-1 AntiTrypsin
US10006025B2 (en) 2014-06-17 2018-06-26 Arrowhead Pharmaceuticals, Inc. Compositions and methods for inhibiting gene expression of alpha-1 AntiTrypsin
IL257764A (en) * 2015-08-28 2018-04-30 Serpin Pharma Llc Methods of treating diseases
WO2017040287A1 (fr) 2015-08-28 2017-03-09 Serpin Pharma, Llc Méthodes de traitement de maladies
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CN111909258B (zh) * 2015-08-28 2021-09-17 赛品制药有限责任公司 用于疾病治疗的方法
CN113429455A (zh) * 2015-08-28 2021-09-24 赛品制药有限责任公司 用于疾病治疗的方法
US10450565B2 (en) 2017-01-10 2019-10-22 Arrowhead Pharmaceuticals, Inc. Alpha-1 antitrypsin (AAT) RNAi agents, compositions including AAT RNAi agents, and methods of use
US11203756B2 (en) 2017-01-10 2021-12-21 Arrowhead Pharmaceuticals, Inc. Alpha-1 antitrypsin (AAT) RNAi agents, compositions including AAT RNAi agents, and methods of use
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