Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
  • C07K14/8121—Serpins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• SERPINs Serine protease inhibitors
• LRP1 low-density lipoprotein receptor related protein
• SERPIN-derived peptides which were found to bind to LRP1 and exert healing and homeostatic function beyond its antiinflammatory function. See, e.g., US Patent Nos. 8,975,224; 9,951 ,104; 11 ,020,462; and US Patent Application Publication Nos. 2021/0188912 and 2021/0369822, the contents of which are incorporated herein by reference.
• the SERPIN-derived peptides such as SP16 and SP163M can be used to treat a number of conditions associated with LRP1 mediation.
• novel SERPIN peptide derivatives to further improve the stability, bioavailability, and/or efficacy of the existing SERPIN- derived peptides.
• a SERPIN peptide derivative comprising, consisting essentially of, or consisting of a pentapeptide having an amino acid sequence of FVFLM (SEQ ID NO: 1 ), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), or PFVFL[Nle] (SEQ ID NO: 9), and one or more of the following modifications: (i) a polar head added to the N-terminus of the pentapeptide, a polar tail added to the C- terminus of the pentapeptide, or both; (ii) one or more amino acid residues added to the N-terminus of the pentapeptide, C- terminus of the pentapeptide, or both such that the peptide derivative can be cyclized; (iii) one or more amino acid residues in the pentapeptide substituted by one or more amino acid residues having less hydrophobicity; (iv) one or more
• the SERPIN peptide derivative of the present technology is a linear peptide. In certain embodiments, the SERPIN peptide derivative of the present technology is a cyclized peptide. In certain embodiments, the SERPIN peptide derivative is cyclized by forming a disulfide bond between two Cys residues. In certain embodiments, the SERPIN peptide derivative is cyclized by a linker between two amino acid residues, for example, two amino acid residues outside the pentapeptide sequence. In certain embodiments, the polar head or the polar tail comprising two or more charged amino acids such as positively charged amino acids selected from the group consisting of Arg, Lys, and His.
• the SERPIN peptide derivative is fused to one or more other peptides include an epitope tag, a half-life extender, or both of an epitope tag and a half-life extender to form a fusion protein or fusion peptide.
• the SERPIN peptide derivative is conjugated to a permeability enhancer.
• compositions comprising the SERPIN peptide derivatives, fusions or conjugates of the present technology and one or more pharmaceutically acceptable carriers.
• the composition is formulated into a dosage form suitable for oral administration, transdermal administration, or parenteral administration.
• a method of treating various conditions or diseases associated with LRP1 binding such as respiratory viral or bacterial infections (e.g., COVID), and inflammatory diseases such as acute respiratory distress, asthma, atopic dermatitis, or eosinophilic esophagitis.
• Other conditions include those of the central and peripheral nervous system, such as peripheral nerve injury and neurodegenerative disease.
• the method entails administering an effective amount of one or more SERPIN peptide derivatives, fusions thereof, or a composition comprising one or more SERPIN peptide derivatives or fusions thereof of the present technology to a subject suffers from a condition associated with LRP1 binding.
• Figure 1 shows the crystal structure of cu-antitrypsin, including a pentapeptide sequence (SEQ ID NO: 8.).
• Figure 2 shows the distance between amino acids based on the crystal structure of ai -antitrypsin, including a pentapeptide sequence (SEQ ID NO: 8).
• Figure 3 illustrates ring closure strategy using Lys-pAla-Glu 13 C-C bonds as an example.
• Figures 4A-4B demonstrate the structure-activity relationship of SERPIN- derived peptide derivatives SA1-SA8.
• Figure 4A shows the activities in reducing NFKB activation for peptide derivatives SA1-SA8 at various concentrations, 0, 1 , 10, 50, and 100 pg/mL.
• Figure 4B compares the activities of SA3 and SA7 to those of SP163M and SP22. Compared to the SERPIN peptide SP163M previously disclosed, truncating the peptide while retaining the LRP1 binding site and adding arginine residues conferred improved activity in the NFKB reporter assay.
• Figures 5A-5B demonstrate the anti-inflammatory function of peptide derivatives A1-A15 in comparison to SP163M.
• the peptide derivatives (50 or 100 pg/ml) were tested in the NFKB reporter assay in response to LPS (5 ng/ml) and screened for TNFa secretion in IMG microglial cells in response to LPS (E.coli 01 11 : B4) stimulation (100 ng/ml, 24 hours).
• LPS LPS
• E.coli 01 11 : B4 LPS
• stimulation 100 ng/ml, 24 hours.
• SEAP NFKB inducible Secreted Embryonic Alkaline Phosphatase
• Figures 6A-6B demonstrate the anti-inflammatory activity of peptide derivatives A2-1 to A2-9 which include modifications to the ring structures and amino acid substitutions.
• Figure 6A shows reduction of TNFa in supernatant of IMG microglial cells after activation with LPS (100 ng/ml, 24 hours).
• Figure 6B shows the percentage of reduction in NFKB activation following LPS stimulation (5 ng/ml, 24 hours). SP163M, SA7, A5 and A15 peptides were included for comparison.
• Figures 7A-7B show dose response effects of the peptide derivatives on NFKB and TNFa activation.
• the peptide derivatives were tested at a concentration of 3.125, 6.25, 12.5, 25, and 50 pg/ml.
• SP163M, SP16 and SA7 were included at a concentration up to 100 pg/ml.
• the dose response concentrations are shown in log scale.
• Peptide derivatives A2-1 , A2-2, A2-3, A2-4, and A2-5 exhibited activities superior to SA7, SP16, and SP163M in a dose-dependent manner.
• Figure 8 shows the results of cytotoxicity assay on IMG cells (Microglial). IMG cells were treated with peptide derivatives at concentrations at or exceeding those tested for the activity assays ( Figures 5-7) for 24 hours. None of the peptides exhibited any cytotoxic effects.
• Figure 9 shows the effects of peptide derivatives A3-1 to A3-16, which include further optimization to the ring structure, on NFKB activation in response to LPS stimulation (5 ng/ml, 24 hours).
• the peptide derivatives were tested at concentrations between 1.56 pg/ml to 12.5 pg/ml.
• SP163M and A2-5 were included for comparison.
• SP163M was tested at concentrations between 12.5 pg/ml and 100 pg/ml. The dose response is shown in log scale.
• Figure 10 shows the activities of peptide derivatives A3-1 , A3-2, A3-3, A3- 5, A3-6, A3-7, A3-9, A3-14, A3-15, and A3-16 on reducing IL-6 secretion in IMG after LPS stimulation (100 ng/ml, 24 hours). IL-6 secretion was measured by ELISA. Peptide derivative A2-5 and SP163M were included for comparison. SP163M was tested at concentrations between 12.5 and 100 pg/ml. The peptide derivatives including A2-5 were tested at concentrations up to 12.5 pg/ml. The dose response is shown in log scale.
• Figures 11 A-11 B show the results of cytotoxicity assay on NFKB reporter cells (Figure 11A) and IMG microglial cells ( Figure 11 B).
• Peptide derivatives A3-1 , A3- 2, A3-5, A3-6, A3-7, A3-9, A3-14, A3-15, and A3-16 exhibited no cytotoxic effects when tested at concentrations that were effective in reducing both NFKB and cytokines IL-6 and TNFa (up to 12.5 pg/ml).
• SP163M was included for comparison and demonstrated no cytotoxic effects at concentrations up to 100 pg/ml.
• the cells were treated for 24 hours with SP163M or peptide derivatives A3-1 , A3-2, A3-5, A3-6, A3-7, A3-9, A3-14, A3-15, A3-16 or A2-5 at concentration indicated.
• Figure 12 shows that peptide derivatives A15 and A2-5 were tested for their ability to block capsaicin induced pain behaviors in mice.
• Mice were treated with the peptide derivatives by subcutaneous administration 1 hour before capsaicin injection (25 ng).
• SP163M was administered at a dose of 50 pg/mouse, while the peptide derivatives A15 and A2-5 were administered at a dose of 5 pg/mouse.
• n 4-9 mice/cohort.
• Figure 13 compares the cytokine profile tested in microglial cells treated with either SERPIN peptide derivative SA7 or SERPIN peptide SP163M.
• SA7 demonstrated significantly increased activity in reducing several cytokines including IL- 6 and IL-1 .
• Figures 14A-14B show that in an LPS induced neuroinflammatory model, the SERPIN peptide derivative SA7 reduced clinical scores (measured daily on a scoring system) ( Figure 14A), and reduced weight loss severity compared to both vehicle (LPS induced) and SP163M ( Figure 14B).
• SP163M and SA7 were administered by subcutaneous injection at a dose of 100 pg/mouse, 1 hour following LPS administration (1 mg/kg) for four consecutive days. Weights and clinical scores were taken daily, and the brain tissue was collected and processed at the end of the study for analysis.
• the score key is as follows: 0: bright, alert, responsive; 1-4: slightly scruffy/hunched, less active; 5-10: hunched, lethargic, weight loss; and >11 moribund.
• Figure 15 shows that in the neuroinflammatory model of Figure 14, animals treated with SA7 showed significant differences in the levels of many cytokines measured in the brain lysate, compared to both vehicle and SP163M treated animals, including IL-17A, IL-12, TNFa, and GM-CSF. IL-6 was the most substantially reduced with no detectable levels measured.
• Figures 16A-16B show that in brain lysate taken from the neuroinflammatory model previously disclosed, the western blot analysis for neurofilament light chain (NfL), a neuronal axon specific marker, and anti-glial fibrillary acidic protein (GFAP), a marker specific to astrocyte activation.
• the peptide derivative SA7 significantly increased expression of NfL ( Figure 16A), while GFAP expression was decreased significantly ( Figure 16B) compared to vehicle treated and SP163M animals.
• Figures 17A-17B show that SA7 regulated autophagy during inflammation similar to SERPIN peptide SP163M.
• Microglial cells were treated with SP163M or SA7 and then exposed to LPS.
• Western blot analysis of the LC3 II to LC3 1 ratio shows that SA7 increased autophagy ( Figure 17A) and LRP1 expression (Figure 17B) compared to LPS alone, comparable to SP163M.
• the LRP1 expression was also significantly higherwith SA7 without LPS treatment, compared to SP163M.
• Figure 18 shows the protease TMPRSS2 activity and IC50s of SP163M and peptide derivative SA7, with SA7 showing increased inhibition of TMPRSS2 activity in an overexpressing cell system compared to SP163M.
• Figures 19A-19B demonstrate that LRP1 was decreased in the esophagus of OVA induced mice and SP163M restored LRP1 protein expression. Further, SA7 inhibited the cytokine TSLP production in SPINK7 knockout cells more potently than SP163M and compared to control cells.
• Figures 20A-20C demonstrate that in a model of OVA induced allergic asthma, peptide derivative A2-5 reduces TSLP, total lung protein and TH-2 mediated cytokines IL-5, IL-4 and IL-13 in the lung homogenate to a greater extent than both OVA induced vehicle treated and SP163M mice.
• Figure 21 illustrates conjugates comprising a peptide derivative of the present technology (e.g., A2-5, SEQ ID NO: 42) and a permeability enhancer.
• a peptide derivative of the present technology e.g., A2-5, SEQ ID NO: 42
• a permeability enhancer e.g., A2-5, SEQ ID NO: 42
• the term “derivative” means a peptide shares amino acid sequence or structure similarity to the pentapeptide FVFLM (SEQ ID NO: 1 ), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), or PFVFL[Nle] (SEQ ID NO: 9), and contains one or more modifications including insertion, deletion or substitution to improve the stability, bioavailability, and/or biological activities or efficacy compared to the pentapeptide.
• the terms “derivative,” “variant,” and “analog” may be used interchangeably in this disclosure.
• SERPIN peptides were previously shown to (1 ) exert neurotrophic effects, (2) have regenerative and healing properties, (3) show analgesic effects, (4) have antiviral and anti-microbial properties, and/or (5) exert anti-allergic effects. This combination of activities provides a distinct mechanism in treating conditions associated with peripheral neuropathies such as diabetic peripheral neuropath, degenerative disorders, lung injury, allergic diseases and infectious disease.
• the SERPIN peptide analogs, and variants and derivatives thereof of the present technology have improved LRP1 binding activity, improved solubility, and/or improved pharmacokinetic properties an oral bioavailability.
• the SERPIN peptide derivatives of the present technology show improved anti-inflammatory effects and improved efficacy in a model of neuroinflammation.
• SERPIN peptide derivatives are SERPIN peptide derivatives, pharmaceutical compositions comprising the SERPIN peptide derivatives, and methods of using the same to treat a number of conditions where a dysregulated immune response or impaired endocytic function, or diseases in which LRP1 mediation contributes to pathology, such as in conditions associated with peripheral nerve injury and resulting pain, lung injury, infectious disease and allergic inflammation.
• the SERPIN peptide derivatives are synthetic peptides. In certain embodiments, the SERPIN peptide derivatives are cyclized. In certain embodiments, the SERPIN peptide derivatives comprise one or more hydrophilic amino acid substitutions. In certain embodiments, the SERPIN peptide derivatives comprise one or more hydrophobic amino acid substitutions. In certain embodiments, the SERPIN peptide derivatives comprise one or more positively charged amino acids at the N-terminus, at the C-terminus, or at both the N-terminus and the C- terminus.
• SERPIN peptide derivatives designed to target LRP1 with a higher affinity to exert more potent anti-inflammatory and cell regenerative effects. These peptide derivatives are modified from the original SERPIN- derived peptides based on structure to activity relationship studies and 3-D modeling of the peptide/LRP1 interaction. These derivatives overcome some of the challenges that are associated with peptide therapeutics such as solubility, plasma stability and oral bioavailability.
• the peptide derivatives of the present technology exhibit not only improved LRP1 activity but also improved solubility, pharmacokinetic properties, and bioavailability, in particular, oral and transdermal bioavailability.
• alpha-1 antitrypsin the prototypical SERPIN
• AAT alpha-1 anti-trypsin
• VKFNKPFVFLM SEQ ID NO: 6
• the derivatives do not contain the FNKP (SEQ ID NO: 7) motif that is highly conserved among SERPINS while retaining the LRP1 binding activity.
• the LRP1 binding motif is highly hydrophobic and unstable in solution, requiring modifications to the SERPIN peptide sequence.
• the “pentapeptide” refers to the FVFLM (SEQ ID NO: 1) sequence in SP16 or FVFL[Nle] (SEQ ID NO: 2) sequence in SPM163, where the Met residue is replaced with a Nle residue.
• the pentapeptide is responsible for most of the interaction with LRP1 .
• various modifications are made in the pentapeptide and/or the sequence surrounding the pentapeptide to obtain novel SERPIN peptide derivatives having improved properties.
• the sequence of the SP163M peptide is further modified by deletion, substitution, and/or cyclization to further improve anti-inflammatory activity, solubility, LRP1 binding activity, and/or oral bioavailability.
• the peptide derivatives of the present technology have a size of 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acid residues, preferably, 8, 9, or 10 amino acid residues.
• the peptide derivatives of the present technology have a size of 8 amino acid residues, 9 amino acid residues, or 10 amino acid residues.
• a polar head at the N-terminus comprising two or more amino acid residues having a charged side chain, a polar tail at the C-terminus comprising two or more amino acid residues having a charged side chain, or both of a polar head and a polar tail are added to the pentapeptide to improve solubility.
• the amino acid residues in the polar head or the polar tail are positively charged and include Arg, His, and Lys.
• a combination of the same charged amino acid residue or a combination of different amino acid residues can be used for the polar head or the polar tail.
• the polar head or the polar tail comprises an amino acid sequence of RR, RRR, KK, KKK, HH, HHH, KRR, KR, or RRK.
• one or more positively charged amino acid residues in the polar head or the polar tail has a reversed structure.
• reversed Lys means that the Lys residue is incorporated into the peptide backbone using the carboxylic acid group carried by the a-carbon and the £-amino group in the side chain rather than both of the amino groups and the carboxylic acid group carried by the a-carbon.
• Reversed Arg means that the Arg residue is incorporated into the peptide backbone using the guanidinium group carried by the a-carbon rather than the 5-carbon.
• two or three Arg residues are added to either or both termini of the pentapeptide.
• two or three Arg residues are added to the N-terminus of the pentapeptide.
• the peptide derivatives of the present technology are cyclized, for example, by forming a disulfide bond between two Cys residues or by a linker between two amino acid residues.
• two Cys residues can be added to both termini of the pentapeptide such that a cyclic peptide derivative can be obtained via a disulfide bond. It is within the purview of one of ordinary skill in the art to dispose the Cys residues at a selected location in the peptide derivative to achieve a desired cyclic structure with an optimized ring size.
• amino acid residues can be added to either or both termini of the pentapeptide such that a linker can be formed between these amino acid residues.
• the specific amino acid residues can be chosen and disposed at selected locations to achieve a desired cyclic structure with an optimized ring size.
• amino acid residue substitutions for cyclization can be chosen without significant loss of activity.
• amino acid residues having a carboxylic acid on its side chain or its C-terminal including but not limited to Asp, and Glu
• amino acid residues having an amino group on its side chain or its N-terminal including but not limited to Lys, Dab, and Dap
• Cys or any non-natural amino acid carrying a sulfhydryl group on its side chain can be used for -S- S- cyclization.
• the amino acids can be disposed at any desired locations of the peptide derivatives such that a ring of a desired size can be formed without substantially comprising the activity of the peptide derivative.
• a head-to-tail cyclization is formed.
• the linker comprises p-Ala. In some embodiments, the linker comprises 2-[(2-amino)-ethoxy]-ethoxy-acetic acid (AEEA). In some embodiments, the ring closing length between the amino acid residues is between 5 and 15 C-C bonds, for example, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, or about 15 C-C bonds.
• the peptide derivatives of the present technology comprise one or more substitutions in the sequence of the pentapeptide to enhance plasma stability and to achieve an increased binding affinity to the peptide’s cognate receptor.
• one or more amino acid residues in the pentapeptide having no interaction or minimal interaction with LRP1 can be replaced by one or more hydrophilic amino acid residues.
• one or more amino acid residues in the pentapeptide interacting with LRP1 can be replaced by one or more amino acid residues having similar but more pronounce physicochemical characteristics.
• the Phe residue has an aromatic ring on its side chain.
• the Phe residue can be substituted by Nal (Naphthylalanine) in peptide derivatives 1-5 and 1-6 or Trp which displays a naphthyl or indole ring instead of a phenyl ring. These substitutions greatly improve the aromatic character of the amino acid residue, allowing for more hydrophobic and more aromatic (pi stacking) interaction.
• one or more amino acid residues in the pentapeptide can be replaced by one or more natural or non-natural amino acid residues.
• one or more amino acid residues in the pentapeptide have a D-configu ration.
• the side chain of one or more amino acid residues in the pentapeptide is modified.
• peptide derivative A3-1 comprises a Vai to Thr substitution to retain some of the hydrophobicity while introducing some hydrogen bonding, and a Phe to Nal substitution to increase hydrophobic and aromatic interaction.
• peptide derivative A3-8 comprises a Nle to D-Ser substitution. Nle does not interact with LRP-1 based on studies of the crystal structure but rather being in the aqueous phase.
• the side chain of Nle is a hydrophobic linear hydrocarbon chain, which requires energy to solvate it. Replacing the Nle residue by a D-Ser having a hydrophilic side chain facilitates solvation by decreasing the enthalpic penalty which translates into stronger binding energy.
• the Nle residue of the pentapeptide is deleted.
• one or more of the hydrophobic residues in the pentapeptide are replaced by one or more less hydrophobic residues such as Ala, or by one or more neutral or hydrophilic residues such as Thr and Ser.
• one or more of the hydrophobic residues in the pentapeptide are replaced by one or more residues having more hydrophobicity such as Nal.
• the Nle residue of the pentapeptide is replaced by an amino acid residue having a D- configuration such as D-Dap, D-Lys, and D-Asp.
• the Met or Nle residue in the pentapeptide has a long linear hydrophobic side chain.
• a substitution of Met or Nle with a hydrophilic amino acid in a D- configuration greatly improves the binding activity of the peptide derivatives to LRP1.
• Substitutions with amino acid residues having a carboxylic acid in the side chain result in an improvement to some extent, while substitution with an amino acid having a short side chain presenting a hydroxy group (Ser) or an amino group (Dap) achieve the best result.
• the SERPIN peptide derivatives of the present technology can be further 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 polyethylene glycol (PEG).
• PEG polyethylene glycol
• a peptide derivative of the present technology comprises, consists essentially of, or consists of a pentapeptide having an amino acid sequence of FVFLM (SEQ ID NO: 1) or FVFL[Nle] (SEQ ID NO: 2), and a polar head added to the N-terminus of the pentapeptide, a polar tail added to the C- terminus of the pentapeptide, or both.
• the polar head or the polar tail comprises 2-9 charged amino acid residues such as Arg, Lys, and His.
• the polar head or the polar tail comprises 2 or 3 charged amino acid residues.
• peptide derivatives shown in Table 1 were used to investigate the role of positively charged amino acids in improving solubility and modulating NFKB activation, using SP163M and SP22 as positive controls.
• the peptide derivatives have various tripeptide sequences added to either or both termini of the LRP1 binding site.
• a peptide derivative comprises, consists essentially of, or consists of a peptide having an amino acid sequence of HHHPFVFLMHHH (SEQ ID NO: 10), HHHPFVFL[Nle]HHH (SEQ ID NO: 11 ), RRRPFVFL[Nle]RRR (SEQ ID NO: 12), KKKPFVFL[Nle]KKK (SEQ ID NO: 13), EEEVKFNKPFVFL[Nle]EEE (SEQ ID NO: 14), RRRCPFVFL[Nle]CRRR (SEQ ID NO: 15), RRRCPFVFL[Nle]C (SEQ ID NO: 16), CPFVFL[Nle]CRRR (SEQ ID NO: 17), RRRVKFNKPFVFLMRRR (SEQ ID NO: 18), or VKFNKPFVFL[Nle]IEQNTK (SEQ ID NO: 5).
• Example 1 when one or more positively charged amino acids such as arginine (R) residues are added to the LRP1 binding motif, the activity in NFKB reduction is increased. However, addition of one or more other charged amino acids such as positively charged amino acids, e.g., histidine (H) or lysine (K), or negatively charged amino acids, e.g., glutamic acid (E), flanking either side of the LRP1 binding site did not confer activity, indicating the importance and uniqueness of the arginine residues.
• positively charged amino acids e.g., histidine (H) or lysine (K)
• negatively charged amino acids e.g., glutamic acid (E)
• a peptide derivative of the present technology is a cyclic peptide and comprises, consists essentially of, or consists of a pentapeptide having an amino acid sequence of FVFLM (SEQ ID NO: 1 ) or FVFL[Nle] (SEQ ID NO: 2), and a polar head added to the N-terminus of the pentapeptide, a polar tail added to the C- terminus of the pentapeptide, or both.
• the cyclization can be between any two residues, for example, the cyclization can be a head-to-tail cyclization. Additional residues such as Cys can be inserted to facilitate the formation of S-S bond to connect two residues.
• one or more hydrophobic residues of F, M, or Nle in the pentapeptide are substituted with a neutral or hydrophilic residue. Accordingly, peptide derivatives having various combinations of positively charged amino acid residues addition and/or amino acid substitutions, some examples shown in Table 2, were designed to improve solubility, stability, and/or oral bioavailability without compromising their NFKB modulating activities.
• a peptide derivative of the present technology comprises a pentapeptide having an amino acid sequence of FVFLM (SEQ ID NO: 1) or FVFL[Nle] (SEQ ID NO: 2), and an additional proline (“P”) amino acid residue.
• the P amino acid residue is at the C-terminal end of the pentapeptide, such that the peptide derivative comprises an amino acid sequence of PFVFLM (SEQ ID NO: 8) or PFVFL[Nle] (SEQ ID NO: 9).
• the peptide derivative comprising an amino acid sequence of PFVFLM (SEQ ID NO: 8) or PFVFL[Nle] (SEQ ID NO: 9) may be a cyclized peptide.
• the peptide derivative comprising an amino acid sequence of PFVFLM (SEQ ID NO: 8) or PFVFL[Nle] (SEQ ID NO: 9) may by cyclized by further comprising Cys residues to facilitate the formation of S-S bonds, for example a peptide derivative having an amino acid sequence of CPFVFLMC (SEQ ID NO: 19) or CPFVFL[Nle]C (SEQ ID NO: 20).
• the peptide derivative having a sequence of CPFVFLMC (SEQ ID NO: 19) or CPFVFL[Nle]C (SEQ ID NO: 20) may further comprise a polar head, for example three R amino acid residues.
• the peptide derivative may comprise a sequence of RRRCPFVFLMC (SEQ ID NO: 21 ) or RRRCPFVFL[Nle]C (SEQ ID NO: 22).
• the peptide derivative having a sequence of RRRCPFVFLMC (SEQ ID NO: 21 ) or RRRCPFVFL[Nle]C (SEQ ID NO: 22) may be further acetylated.
• the peptide derivative may be peptide SA7.
• a peptide derivative of the present technology comprises, consists essentially of, or consists of an amino acid sequence of RCFVFL[Nle]C (SEQ ID NO: 23), RRCFVFL[Nle]C (SEQ ID NO: 24), RRCFVFL[Nle]C (SEQ ID NO: 25), RRRCFVFL[Nle]C (SEQ ID NO: 26), RRRCFVFT[Nle]C (SEQ ID NO: 27), RRRCFTFL[Nle]C (SEQ ID NO: 28), or RRRCTVFL[Nle]C (SEQ ID NO: 29).
• the underlined sequence denotes cyclization.
• a disulfide bond is formec between the C residues to cyclize the peptide.
• F, F, and M residues of the pentapeptide FVFLM point to one direction, while the V and L residues point to the opposite direction.
• the P residue at the N-terminus of the pentapeptide creates a turn in the structure but has no interaction with LRP1. It is unlikely that all five residues of the pentapeptide directly interact with LRP1.
• peptide derivatives A7-A9 are designed to replace one of the hydrophobic amino acid residues (F, V, and L) with a residue having a higher hydrophilicity such as T to investigate whether the solubility and/or activity can be improved.
• Table 3 shows some examples of the peptide derivatives having one of the F residues replaced by 1-naphylalanine, which is a non-natural amino acid with an additional aromatic ring.
• the naphylalanine (Nal) substitution increases hydrophobicity and may have stronger hydrophobic interaction with LRP1 .
• a peptide derivative of the present technology comprises, consists essentially of, or consists of an amino acid sequence of RRRCFV[Nal]L[Nle]C (SEQ ID NO: 30) or RRRC[Nal]VFL[Nle]C (SEQ ID NO: 31 ).
• Cys-Cys bridge can be replaced by a linker of a greater length to improve cyclization.
• the distance between the a-carbon of the amino acid on the N-terminus and the a-carbon of the amino acid on the C-terminus of the pentapeptide FVFLM was measured. Assuming 1 ,5Afor C-C bond’s length, the distance between the amino acids is converted to a number of C-C bonds which can be used to design a cyclization strategy.
• the distance between the a-carbons of K368 and I375 is 22.4A (approximately 15 C-C bonds) and between the a-carbons of P369 and I375 is 20.2A (approximately 13.5 C-C bonds).
• a linker of between 13 to 14 C-C bonds using a p-Ala can be used to connect the side chain of a Lys residue to the side chain of a Glu residue to cyclize the peptide derivative with a 13 C-C bond ring closing length. See Figure 3.
• a peptide derivative of the present technology comprises, consists essentially of, or consists of an amino acid sequence of RRRDFVFL[Nle][Dap] (SEQ ID NO: 32).
• RRRDFVFL[Nle][Dap] SEQ ID NO: 33.
• RRRDFVFL[Nle][Dap] SEQ ID NO: 34).
• RRREFVFL[Nle]K SEQ ID NO: 35).
• RRREFVFLfNIelK SEQ ID NO: 36
• RRRFVFL[Nle]D SEQ ID NO: 37).
• Table 4 illustrates some examples of peptide derivatives having various ring closure designs where a ring closing linker is used to replace the Cys-Cys bridge.
• Example 2 demonstrates that peptide derivatives A5, A8, A10, and A15 exhibited improved activities in reducing TNFa activation.
• Table 5 lists some examples of additional SERPIN peptide derivatives having similar modifications, as well as peptide derivatives having substitutions in the pentapeptide. For example, Nle in the pentapeptide is substituted with Ala or deleted to determine whether Nle is involved in the interaction with LRP1. Additionally, each residue of the pentapeptide is substituted with D-Ser (dS) to improve solubility and resistance to protease.
• dS D-Ser
• a peptide derivative of the present technology comprises, consists essentially of, or consists of an amino acid sequence of RRR[Dap]FT[Nal]L[Nle]D (SEQ ID NO: 38), RRRFT[Nal]L[Nle]D (SEQ ID NO: 39).
• RRRFVFLAD SEQ ID NO: 40
• RRRFVFLD SEQ ID NO: 41 ).
• RRRFVFL[dS]D (SEQ ID NO: 42), RRRFVF(dS1(Nle1D (SEQ ID NO: 43), RRRFV[dSlL[Nle1D (SEQ ID NO: 44), RRRF[dS]FL[Nle]D (SEQ ID NO: 45), or RRR[dS]VFL[Nle]D (SEQ ID NO: 46).
• the erlined sequence denotes cyclization
• a peptide derivative of the present technology comprises, consists essentially of, or consists of an amino acid sequence of RRRFTtNallLD (SEQ ID NO: 47), (rKIRRFTFNallLD (SEQ ID NO: 48), [rR]RRFT[Nal]LD (SEQ ID NO: 49), [rR][rR]FT[Nal]LD (SEQ ID NO: 50), RRR[Dap]FT[Nal]LD (SEQ ID NO: 51 ), RR[Dap]FT[Nal]LD (SEQ ID NO: 52), RRRKFT[Nal]LD (SEQ ID NO: 53), RR[Dap]FT[Nal]L[dS]D (SEQ ID NO: 54), RRRFVFL[dDap]D (SEQ ID NO: 56), RRRFVFL[dK]D (SEQ ID NO: 57), RRRFVFL[dD]D (SEQ ID NO: 58), RRRF
• Table 6 provides examples of SERPIN peptide derivatives that are further optimized by shortening the peptide length via deletion, optimizing the cyclization, and/or residue substitutions to further improve solubility, activities, and/or oral availability.
• one or more amino acid residues in the pentapeptide of the peptide derivative are deleted or substituted with one or more natural or non-natural amino acid residues.
• Nle residue can be substituted by a less hydrophobic amino acid such as Ala or Ser (D-Ser) to improve the agonist activity binding to LRP1.
• amino acids of D- configuration may be used to change the orientation of the amino acid in the 3-D structure of the peptide and/or to confer protease stability.
• two charged amino acid residues such as Arg and/or Lys are added to the N-terminus of the pentapeptide.
• three charged amino acid residues such as Arg and/or Lys are added to the N-terminus of the pentapeptide.
• the charged amino acid(s) can have a “reversed” chemical structure to optimize the ring size, as illustrated in the examples below:
• peptide derivatives of the present technology can be further modified in the amide bonds to improve protease stability and absorption, and these modifications include but are not limited to peptide bond isostere, N-methylation, and/or ⁇ -configuration amino acid substitution.
• a peptide derivative comprising, consisting essentially of, or consisting of the amino acid sequence X1 -X2-X3-X4-X5- X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein:
• XI is a hydrophilic amino acid residue or none
• X2 is a hydrophilic amino acid residue or none
• X3 is a hydrophilic amino acid residue or none
• X4 is a Cys amino acid residue or none
• X5 is a Pro amino acid residue or none
• X6 is a first hydrophobic amino acid residue
• X7 is a short-branched amino acid residue
• X8 is a second hydrophobic amino acid residue
• X9 is a saturated hydrophobic amino acid residue
• X10 is a hydrophilic amino acid of D configuration
• XI I is any amino acid residue which allows cyclization of the peptide derivative.
• SEQ ID NO: 3 further comprises one or more of the following: X4 is Cys, X5 is Pro, X7 is Thr, and X10 is a D-configured Lys residue.
• the first and second hydrophobic amino acid residues of X6 and X8 are aromatic amino acid residues.
• the short-branched amino acid residue of X7 is Vai or Thr.
• the saturated hydrophobic amino acid of X9 is Leu.
• the hydrophilic amino acid of D configuration of X10 is a D-configured Asp, Glu, Lys, Dap, or Cys residue.
• X6 is Phe or Nal.
• X8 is Phe or Nal.
• X6 is Ala, Phe, or Nal, and X4 is Nal.
• X7 is Asp, Glu, Lys, Dap, or Cys.
• the peptide derivative is linear or cyclized. In some embodiments, the peptide derivative has a size of less than 15 amino acid residues. In some embodiments, the peptide derivative has a size of 7, 8, 9, 10, 11 , or 12 amino acid residues.
• the peptide derivatives of the present technology acetylated at the N-terminus, amidated at the C-terminus, or both.
• the peptide derivative is cyclized with 10 C-C bonds or equivalent to close the ring between the a-carbon of X4 and X11 of SEQ ID NO: 3. Cyclization can also occur between X11 and an extra amino acid residue added between X4 and X5 of SEQ ID NO: 3. Cyclization can also occur between X1 1 and an extra amino acid residue added between X5 and X6 of SEQ ID NO: 3. In this situation the optimal ring is closed using 5 C-C bonds (or equivalent).
• the cyclization is a head-to-tail cyclization.
• the SERPIN peptide derivatives of the present technology has a size of between 5 and 30 amino acids, for example, 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, or 30 amino acids.
• the SERPIN peptide derivative of the present technology has a size of 20 amino acids or less. A longer peptide may have a decreased solubility, whereas a shorter peptide may have decreased stability.
• the peptide derivative has a size of 9 amino acid residues; however, a peptide derivative having a size of 8 amino acid residues in conjunction with an optimized cyclization strategy suffers only minimal activity loss.
• the SERPIN peptide derivatives of the present technology are fused to one or more other peptides or compounds to form a fusion peptide or fusion protein.
• one or more other peptides include an epitope tag such as ALFA-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag, NE-tag, a half-life extender such as polyethylene glycol) (PEG), Lipidation, FC fusion, or Albumin fusion, or both of an epitope tag and a half-life extender.
• an epitope tag such as ALFA-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag, NE-tag
• a half-life extender such as polyethylene glycol) (PEG), Lipidation, FC fusion, or Albumin fusion, or both of an epitope tag and a half-life extender.
• the peptide derivatives of the present technology can be conjugated to a permeability enhancer such as oleic acid, cholic acid, a cationic detergent, or lactam to further improve the transdermal, trans-epithelial, nasal, gastric or topical bioavailability, as illustrated in Figure 21 .
• a permeability enhancer such as oleic acid, cholic acid, a cationic detergent, or lactam to further improve the transdermal, trans-epithelial, nasal, gastric or topical bioavailability, as illustrated in Figure 21 .
• compositions comprising the SERPIN Peptide Derivatives
• this disclosure relates to a pharmaceutical composition
• a pharmaceutical composition comprising, consisting essentially of, or consisting of an effective amount of one or more SERPIN peptide derivatives or fusion peptides of the present technology.
• the pharmaceutical compositions of the present technology can be formulated into any suitable dosage form for transdermal, sublingual, nasal, oral, inhalation, rectal or ocular administration.
• the pharmaceutical composition further comprises one or more additional therapeutic agents, which are not the SERPIN peptide derivatives of the present technology.
• the pharmaceutical composition further comprises one or more permeability enhancers in an amount of 0.1 to 5% (w/w) to promote penetration of the peptide derivative or the fusion peptide into skin, mucosal membrane, nasal mucosa, or an epithelial layer.
• permeability enhancers include a fatty acid such as oleic acid, a bile acid such as cholic acid, a cationic detergent such as cetyltrimethylammonium, and a lactam such as laurocapram.
• the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof. Examples of acceptable carriers include physiologically acceptable solutions, such as sterile saline and sterile buffered saline.
• an effective amount refers to an amount of a composition that produces a desired effect.
• An effective amount of a composition may be used to produce a prophylactic or therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect.
• the effective amount of a composition is a “therapeutically effective amount,” “therapeutically effective concentration” or “therapeutically effective dose.”
• the precise effective amount or therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject or population of cells.
• composition including activity, pharmacokinetics, pharmacodynamics, and bioavailability
• physiological condition of the subject including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication
• an effective or therapeutically effective amount may vary depending on whether the composition is administered alone or in combination with another composition, drug, therapy or other therapeutic method or modality.
• the peptide derivatives or the pharmaceutical compositions of the present technology may be formulated for oral administration, parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration (bolus injection or through a device such as an infusion pump), intradermal administration, transdermal administration, topical administration, and intranasal administration.
• parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration (bolus injection or through a device such as an infusion pump), intradermal administration, transdermal administration, topical administration, and intranasal administration.
• a subcutaneous infusion pump can be used for delivery of the peptides or the pharmaceutical compositions of the present technology.
• the peptides or the pharmaceutical compositions may be administered more than once. More specifically, after the initial administration, one or more additional doses may be given as a booster.
• the SERPIN peptide derivatives, fusion peptides, or the pharmaceutical compositions of the present technology have various functions.
• of the present technology is a method of treating a subject in need thereof an effective amount of one or more SERPIN peptide derivatives, fusion peptides, or the pharmaceutical compositions disclose herein.
• the subject suffers from a disease or condition in which LRP1 mediation contributes to pathology, such as in conditions associated with peripheral nerve injury and resulting pain, lung injury, infectious disease and allergic inflammation such as eosinophilic esophagitis.
• the subject suffers from a disease associated with dysregulated immune response selected from the group consisting of peripheral neuropathies, neuropathic pain, COVID-19 infection, acute respiratory distress syndrome (ARDS), sepsis, SARS-CoV-2, Influenza, alphavirus infection, and cytokine storm.
• a disease associated with dysregulated immune response selected from the group consisting of peripheral neuropathies, neuropathic pain, COVID-19 infection, acute respiratory distress syndrome (ARDS), sepsis, SARS-CoV-2, Influenza, alphavirus infection, and cytokine storm.
• treating or “treatment” of a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. Treatment may also mean a prophylactic or preventative treatment of a condition.
• the term “subject” is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject has not received any prior treatment with serine protease inhibitors, such as alpha-1 -antitrypsin treatment before the treatment with the peptides of the present technology.
• serine protease inhibitors such as alpha-1 -antitrypsin treatment
• the peptides can be used to reduce the serum TNF- a levels in human individuals who have pathologically increased TNF-o levels.
• the peptide causes a 75% decrease in serum TNF-a levels when administered in an effective amount to a human subject.
• the peptide results in a 50% or 75% decrease in serum TNF-a levels when administered in an effective amount to a human subject compared to the levels before administration of the peptide.
• LRP-1 functions as an endocytic and cell signal transduction receptor and has several ligands that induce specific cell signaling cascades that can contribute to cell survival and anti-inflammatory mechanisms. 5 ' 18 ' 22 ' 25 LRP1 is ubiquitously expressed on many different organs, abundantly in brain, lung, heart and immune cells. Because of these unique capabilities and wide expression on both tissues and immune cells, it plays a critical role in regulating inflammation, cellular metabolism, and maintaining homeostasis.
• LRP1 regulates inflammatory signaling pathways such as NFKB and JNK pathways that induce the conversion of pro-inflammatory (M1) macrophages to the anti-inflammatory (M2) macrophage phenotype, regulates the cytokine output, and contributes to effective migration and phagocytosis. 22 ' 26 51 In neutrophils, LRP1 -dependent mechanisms lead to enhanced cell adhesion, chemotaxis, and antibacterial effects of these cells, thereby resisting immunosuppression 25 . During acute infection or injury, LRP1 also promotes inflammatory resolution through scavenging PAMPS and DAMPS from dying or injured tissue, to prevent the tissue injury cycle 25 .
• LRP1 was also shown to mediate autophagy during infection, an important metabolic process recently shown to play an important protective role in a variety of diseases 4 10 Therefore, because of its multifunctional ability to regulate inflammation, targeting LRP1 has substantial potential to mitigate several aspects of the immune response that contributes to the pathology of several diseases including neurological disorders, infectious diseases, and allergic inflammatory disease.
• LRP1 In terms of nerve injury and associated pain, injury to the peripheral nervous system induces an increase in the expression of LRP1 .
• LRP1 agonist are capable of promoting axonal growth in the CNS and are capable of inducing regeneration after spinal cord injury.
• 53 LRP1 is an endocytic receptor to a diverse number of ligands including tissue-type plasminogen activator (tPA), matrix metalloproteinase-9 (MMP-9), and activated a2-macroglobulin. 14 These ligands are capable of inducing anti-inflammatory activity, 39 activating the Schwann cell repair program 21 and transactivation of cell signaling pathways in neurons associated with axonal regeneration.
• LRP1 requires ligand-binding to activate cell-signaling, however, different ligands elicit distinct and sometimes opposing cell-signaling responses reflecting the ability of different ligands to assemble unique co-receptor complexes. Furthermore, many LRP1 ligands are multi-domain proteins with numerous effects on cell physiology that do not involve LRP1 -binding. For example, tissue-type plasminogen activator (tPA) binds to LRP1 to promotes Schwann Cell (SC) survival and migration. 23 Yet, by LRP1- independent activities, tPA elicits pain.
• tissue-type plasminogen activator binds to LRP1 to promotes Schwann Cell (SC) survival and migration. 23 Yet, by LRP1- independent activities, tPA elicits pain.
• EI-tPA promotes survival of human iPSC-derived neural progenitor cells (iNPCs) and transplanted EI-tPA activated iNPCs into rodents with severe spinal cord injury demonstrate improved motor functional recovery. 40 Imbalances in the microenvironment following nerve injury may have severe consequences, including the development of chronic neuropathic pain states. 12 In peripheral nervous system (PNS) injury, both inflammatory cytokines such as TNFa, IL-6 and IL-1 p and anti-inflammatory cytokines such as IL-10 have been shown to play a central role in axon regeneration and repair. 6
• PNS peripheral nervous system
• AD Alzheimer’s Disease
• LRPI neurofibrillary tangles
• LRP1 -mediated clearance of Ap across the blood brain barrier (BBB) is the key event in the regulation of Ap transcytosis from brain to periphery
• targeting LRP1 with one or more peptide derivatives of the present technology may serve as a novel treatment.
• tau protein aggregates forming NTFs plays a central role in the pathogenesis of Alzheimer’s disease.
• LRP1 functions to regulate tau protein endocytosis, accumulation and spread associated with worsened pathology.
• the SERPIN peptide derivatives of the present technology may serve as an intervention that remediates both plaque and tangle pathologies through mediation of LRP1 associated protein aggregation of tau and amyloid a p.
• the SERPIN peptide derivatives of the present technology can also act on neuroinflammation, which plays a critical role in neurodegenerative disease.
• LRP1 has been shown to be involved in the development of neurodegenerative diseases and in the regulation of the metabolism of amyloid-p peptides (Aps) in the brain and periphery.
• Neuroinflammation plays a critical role in neurodegenerative disease, such as Alzheimer’s Disease (AD).
• LRP1 is highly expressed in the cells of the CNS and shown to play a critical role for the survival of primary neurons under stressful conditions.
• Microglial cells act as the resident immune cell of the brain, serving to maintain homeostasis in the environment. Microglia are considered the prototypic tissue-resident macrophage-like innate immune cells of the CNS.
• Reactive glia cells (such as microglial cells) and associated neuroinflammation play a key role in both disease initiation and progression becoming activated through dysregulated clearance of beta amyloid and other damage associated molecular patterns (DAMPs).
• DAMPs beta amyloid and other damage associated molecular patterns 13 AB deposition and tau hyperphosphorylation contribute to microglial activation, NFKB inflammatory pathway activation and associated pro-inflammatory cytokines such as TNFa, IL-6 and IL-1 , which contribute to neuronal damage and loss.
• LRP1 has been shown to mediate healthy lysosomal processing associated with autophagy. Therefore, through LRP1 , the disclosed SERPIN peptide derivatives can mediate several aspects of AD including healthy cell metabolism to reduce the spread of protein aggregation, alleviate neuroinflammation and improve neuronal dysfunction leading to survival and possibly regeneration of these cells.
• Acute Lung Injury leading to Acute Respiratory Distress Syndrome (ARDS) can be initiated by a variety of inflammatory insults such as pneumonia, traumatic injury, and/or infection.
• a key initiation step in ALI is the dysregulated innate immune response to damage associated or pathogen associated molecular patterns (DAMPs or PAMPs, respectively).
• Alveolar macrophages are activated by the infectious microbes in Toll-like and Nod-like Receptor signaling pathways that lead to further macrophage and circulating neutrophil recruitment.
• Neutrophils accumulate in the lungs and release proinflammatory cytokines and other cytotoxic substances causing exacerbation of the injury.
• the lung epithelium is damaged by these cells and their secreted products cause pulmonary edema and potential respiratory distress (ARDS).
• ARDS pulmonary edema and potential respiratory distress
• An increase in proinflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IL-8, and IL-18 occurs resulting in a “cytokine storm” which is predictive of morbidity and mortality in sepsis.
• LRP1 was reported to be a critical player in sepsis and ARDS as it regulates lung inflammation and lung tissue repair. 49
• the SERPIN peptide derivatives of the present technology target LRP1 , leading to the precise coordination of the immune response in order to restore homeostasis.
• the disclosed peptide derivatives can have a significant impact to mitigate lung damage and improve survival through multiple mechanisms initiated by inducing specific LRP1-mediated signaling pathways. These mechanisms include rebalancing the cytokine output to promote inflammatory resolution, mediating autophagy to restore proper cell metabolic processes, restoring immune cell function and receptor scavenging to regulate the tissue injury cycle. These mechanisms help in infection clearance and allow the host to better fight infection. Based on these mechanisms, the disclosed SERPIN peptide derivatives can be used as immunomodulatory therapeutic agents to prevent or treat ARDS by mediating LRP1 signaling.
• ARDS Acute respiratory distress syndrome
• Clinical evaluation and retrospective studies out of Wuhan China show that all of the patients admitted to hospital presented with pneumonia, of those 29% developed ARDS and among critically ill patients who were non-survivors, a vast majority (81 %) had developed ARDS. 17 52 Therefore, patients who develop ARDS are at a substantially higher risk of death.
• Virally mediated activation of the innate immune response through the Toll-like Receptors (TLRs) initiates an inflammatory response that is necessary to eliminate the infection.
• TLRs Toll-like Receptors
• the initiating viral insults and triggers are highly amplified and uncontrolled, resulting in overstimulation of the immune cells and an aberrant cytokine release (known as a cytokine storm), resulting in tissue damage that can lead to organ failure and death.
• Cytokine regulation is a key factor in preventing the harmful effects of an overactive immune response and controlling the cytokine storm could have a significant impact on viral pneumonia progression to ARDS.
• SARS-CoV-2 emerging evidence suggests that for a subpopulation of patients with severe illness, the cytokine storm is a contributing factor to mortality.
• Kevzara an interleukin-6 inhibitor (IL-6)
• IL-6 is a biomarker associated with higher mortality rates in individuals with pneumonia. Those trials will provide critical insight into the effectiveness of single cytokine targeted therapeutics. However, historically, targeting a single cytokine or pathway in ARDS patients has not proved to be an effective approach.
• LRP1 signaling mediated by the SERPIN peptide derivatives of the present technology may have therapeutic potential as an immunomodulatory strategy to improve COVID-19 patient outcomes through regulating several mechanisms of lung inflammation including curbing the cytokine storm, improving cell survival, regulating autophagy and cell metabolism to clear infection and controlling tissue repair signaling pathways to promote proper healing and prevent fibrosis.
• LRP1 is widely known to regulate protease/antiprotease activity and mediate viral entry.
• the novel coronavirus utilizes proteases (TMPRSS2) to regulate cell entry and infection and treatment with its corresponding protease inhibitor such as SERPIN blocked lung cell infection.
• SARS-CoV-2 viral entry is dependent on a serine protease TMPRSS2 that primes the viral S protein involved in host cell entry, which is the first step in the viral replication cycle.
• Camostat mesylate is a natural SERPIN with protease inhibitor function of TMPRSS2 and was effective in reducing SARS-CoV-2 viral entry, which can limit both infection and spread of the disease.
• An alpha-1 antitrypsin derivative of the present technology such as SP163M is also capable of inhibiting TMPRSS2, potentially through LRP1 and may have effects in reducing SARS-CoV-2 viral replication.
• the disclosed SERPIN peptide derivatives may reduce SARS-CoV-2 replication via the mechanism of LRP1 control of the host protease/viral protein interaction to inhibit viral entry.
• VEEV Venezuelan
• EEEV eastern
• WEEV western equine encephalitis viruses
• CNS central nervous system
• VEEV infection is known to inhibit cellular transcription and translation in order to downregulate the innate immune response 45 48
• CNS central nervous system
• VEEV infection results in the upregulation of numerous genes in the inflammatory response and apoptotic pathway.
• pro-inflammatory cytokines including interleukin-1 p (IL-1 P), IL-6, IL-12, and tumor necrosis factor-a (TNF- a) play a role in VEEV pathogenesis.
• IL-1 P interleukin-1 p
• IL-6 IL-6
• IL-12 tumor necrosis factor-a
• TNF- a tumor necrosis factor-a
• ICAM-1 intracellular adhesion molecule-1
• the SERPIN peptide derivatives of the present technology can bind LRP1 and initiate the immune modulatory cascade. LRP1 expression increases during ischemia, tissue injury and viral infection. 2 ’ 22 ’ 25 43
• the binding of one or more disclosed peptide derivatives to LRP1 can inhibit the inflammatory response and induce prosurvival signaling through phosphorylation of protein kinase Akt. Therefore, targeting LRP1 has potential as a broad-spectrum therapeutic strategy for infectious disease.
• the SERPIN peptide derivatives can curb the harmful cytokine storm associated with severe COVID-19 disease, activate protective pathways to prevent lung damage, and/or clear infection faster and has direct impact on suppressing viral infection. Therefore, the dual antiinflammatory and antiviral mechanism of the SERPIN peptide derivatives can have effects in improving survival in patients with SARS-COV2 induced ARDS.
• Eosinophilic Esophagitis is a chronic, largely type-2 immune mediated allergic inflammatory response associated with esophageal dysfunction and disturbed epithelial barrier function.
• the esophageal inflammation results in trouble swallowing, persistent heartburn, chest and abdominal pain, weight loss and food impactions. It is characterized by a high number of eosinophils, proteases, cathelicidin, serine proteases including the kallikreins (KLK5), as well thymic stromal lymphopoietin (TSLP) - a cytokine and master regulator of allergic type-2 inflammatory responses in the local environment.
• KLK5 kallikreins
• TSLP thymic stromal lymphopoietin
• the SERPIN Alpha-1 antitrypsin is capable of inhibiting KLK5 activity in vitro and allergen-induced esophageal eosinophilia in vivo.
• the mechanism of the activity is not yet clear and may involve LRP1 , instead of or in addition to direct proteolytic inhibition.
• LRP1 deletion specifically of CD11 b and CD11 c dendritic cells in mice results in heightened allergic inflammatory response in an allergic airway disease model. 29 Mice with LRP1 deletion had increased antigen uptake and suffered increased eosinophilic inflammation, allergic sensitization, Th2 mediated cytokine production and a reduction in T-regulatory cells. 29 Therefore, LRP1 could aid in maintaining homeostasis of proteases/inhibitors in the esophageal environment, mediating the TH2 responses and inhibiting inflammatory signaling pathways (NFKB, JNK) resulting in repair of esophageal dysfunction.
• NFKB inflammatory signaling pathways
• the disclosed SERPIN peptide derivatives such as SA7, a potent LRP1 agonist with no protease inhibitor function, can reduce TMPRSS2 expression.
• TMPRSS2 expression was shown to be significantly increased in the nasal and airway epithelial cells in type 2 asthma and allergic rhinitis. Studies have also shown that TMPRSS2 expression is positively associated with TH2 mediated immune responses important in allergic responses.
• the disclosed SERPIN peptide derivatives may have significant implications in alleviating eosinophilic esophagitis by mediating control over LRP1 .
• a SERPIN peptide derivative comprising a core sequence of FVFLM (SEQ ID NO: 1 ), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), PFVFL[Nle] (SEQ ID NO: 9), and one or more of the following modifications:
• the SERPIN peptide derivative of any one of paragraphs 0096-0101 wherein the SERPIN peptide derivative has a size of less than 15 amino acid residues.
• a SERPIN peptide derivative comprising the amino acid sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein:
• XI is a hydrophilic amino acid residue or none
• X2 is a hydrophilic amino acid residue or none
• X3 is a hydrophilic amino acid residue or none
• X4 is a Cys amino acid residue or none
• X5 is a Pro amino acid residue or none
• X6 is a first hydrophobic amino acid residue
• X7 is a short-branched amino acid residue
• X8 is a second hydrophobic amino acid residue
• X9 is a saturated hydrophobic amino acid residue
• X10 is a hydrophilic amino acid of D configuration
• XI I is any amino acid residue which allows cyclization of the SERPIN peptide derivative.
• X2 is a basic residue displaying a positive charge or none
• X3 is a basic residue displaying a positive charge or none.
• X1 is a Arg, Lys or His residue or none
• X2 is a Arg, Lys or His residue or none
• X3 is a Arg, Lys or His residue or none.
• a SERPIN peptide derivative comprising a core sequence of FVFLM (SEQ ID NO: 1), FVFL[Nle] (SEQ ID NO: 2), PFVFLM (SEQ ID NO: 8), PFVFL[Nle] (SEQ ID NO: 9) and one or more of the following modifications:
• SERPIN peptide derivative of any one of paragraph 0119-0121 , wherein the SERPIN peptide derivative has a size of 7, 8, 9, 10, 11 , or 12 amino acid residues.
• a SERPIN peptide derivative comprising the amino acid sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 (SEQ ID NO: 3), wherein:
• X1 is a hydrophilic amino acid residue or none
• X2 is a hydrophilic amino acid residue or none
• X3 is a hydrophilic amino acid residue or none
• X4 is a Cys amino acid residue or none
• X5 is a Pro amino acid residue or none
• X6 is a first hydrophobic amino acid residue
• X7 is a short-branched amino acid residue
• X8 is a second hydrophobic amino acid residue
• X9 is a saturated hydrophobic amino acid residue
• X10 is a hydrophilic amino acid of D configuration; and X11 is any amino acid residue which allows cyclization of the SERPIN peptide derivative; wherein X4 is Cys, X5 is Pro, X7 is Thr, and/or X10 is a D-configured Lys residue.
• X1 is a basic residue displaying a positive charge or none
• X2 is a basic residue displaying a positive charge or none
• X3 is a basic residue displaying a positive charge or none.
• X1 is a Arg, Lys or His residue or none
• X2 is a Arg, Lys or His residue or none
• X3 is a Arg, Lys or His residue or none.
• a SERPIN peptide derivative comprising a sequence of SEQ ID NOs: IQ- 20, or 23-62, wherein the SERPIN peptide derivative comprises a polar head added to the N-terminus of the core sequence, a polar tail added to the C- terminus of the core sequence, or both.
• a SERPIN peptide derivative comprising a sequence of SEQ ID NOs: 15- 17, or 23-62 wherein one or more amino acid residues added to the N-terminus of the core sequence, C- terminus of the core sequence, or both such that the peptide derivative can be cyclized.
• a SERPIN peptide derivative comprising a sequence of SEQ ID NOs: 27- 29, 38, 39, or 42-62, wherein one or more amino acid residues in the core sequence substituted by one or more substitute amino acid residues having less hydrophobicity compared to the one or more amino acid residues in the core sequence that is substituted.
• a SERPIN peptide derivative comprising a sequence of SEQ ID NOs: 30 or 31 , wherein one or more amino acid residues in the core sequence substituted by one or more substitute amino acid residues having greater hydrophobicity compared to the one or more amino acid residues in the core sequence that is substituted.
• a SERPIN peptide derivative comprising a sequence of SEQ ID NOs: 41 or 47-53 wherein one or more amino acid residues in the core sequence are deleted.
• a SERPIN peptide derivative comprising a sequence of SEQ ID NOs: 10, 11 , 13, or 14, 35 or 36 wherein one or more Lys, Glu, or His residues added to the N- terminus of the core sequence, a polar tail added to the C- terminus of the core sequence, or both.
• a SERPIN peptide derivative comprising a sequence of SEQ ID NOs: 15- 17, or 23-31 wherein one or more amino acid residues added to the N-terminus of the core sequence, C- terminus of the core sequence, or both such that the SERPIN peptide derivative can be cyclized by forming a disulfide bond between two Cys residues.
• a SERPIN peptide derivative comprising a sequence of SEQ ID NOs: 27- 29, 38, 39, 47-55, or 59-62 wherein one or more amino acid residues in the core sequence substituted by one or more Thr amino acid residues.
• a fusion protein comprising the SERPIN peptide derivative of any one of paragraphs 0096-0144, and an epitope tag, a half-life extender, or both.
• a conjugate comprising the SERPIN peptide derivative of any one of paragraphs 0096-0144, and a permeability enhancer.
• a pharmaceutical composition comprising the SERPIN peptide derivative of any one of paragraphs 0096-0144, the fusion protein of paragraph 0145, or the conjugate of paragraph 0146.
• composition of paragraphs 0147 or 0148 further comprising a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof.
• a fusion protein comprising the SERPIN peptide derivative of any one of paragraphs 0119-0136, 0140, or 0142-0144, and an epitope tag, a half-life extender, or both.
• a conjugate comprising the SERPIN peptide derivative of any one of paragraphs 0119-0136, 0140, or 0142-0144, and a permeability enhancer.
• a pharmaceutical composition comprising the SERPIN peptide derivative of any one of 0119-0136, 0140, or 0142-0144, the fusion protein of paragraph 0152, or the conjugate of paragraph 0153.
• composition of paragraph 0154 or 0155 further comprising a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof.
• a method of treating a subject suffering from a disease or condition associated with LRP1 comprising administering to the subject an effective amount of the SERPIN peptide derivative of paragraphs 0096-0144, the fusion protein of paragraph 0145, the conjugate of paragraph 0146, or the pharmaceutical composition of any of paragraphs 0147-0151 , to treat the disease or condition associated with LRP1 .
• a method of treating a subject suffering from a disease or condition associated with LRP1 comprising administering to the subject an effective amount of the SERPIN peptide derivative of any one of paragraphs 0119-0136, 0140, or 0142- 0144, the fusion protein of paragraph 0152, the conjugate of paragraph 0153, or the pharmaceutical composition of any of paragraphs 0154-0158 to treat the disease or condition associated with LRP1.
• SERPIN peptide derivatives SA1-SA8 listed in Table 1 were tested fortheir anti-inflammatory effects using SP163M and SP22 as positive controls.
• the reporter cells THP1-XBIue-MD2-CD14 cells
• each peptide derivative SA1- SA8 as well as SP22 and SP163M (50 pg/ml) before being insulted with LPS (5 ng/ml) and incubated overnight.
• LPS 5 ng/ml
• SEAP NFKB inducible Secreted Embryonic Alkaline Phosphatase
• the SERPIN peptide derivatives that retain the shortest LRP1 binding sequence FVFL[Nle] and an RRR tripeptide as a flanker to improve solubility demonstrated improved activity in NFKB inhibition when compared to SP163M.
• the SERPIN peptide derivatives flanked with either HHH or KKK tripeptide or with negatively charged EEE tripeptide demonstrated minimal NFKB inhibition activity.
• NFKB reporter cells TNF1-XBIue-MD2-CD14 cells
• LPS NFKB inducible Secreted Embryonic Alkaline Phosphatase
• IMG Microglial cells were treated with each peptide derivative at various concentrations, up to 100 pg/ml before LPS (100 ng/ml) stimulation for 24 hours.
• An ELISA was used to measure TNFa in the supernatant (pg/ml).
• Figure 6 demonstrates that peptide derivatives A2-1 , A2-2, A2-3, A2-4, and A2-5 exhibited superior activities in reducing TNFa (Figure 6A) and NFKB (Figure 6B) activation.
• the activities of these peptide derivatives were dose-dependent and showed improved potency ( Figure 7).
• the peptide derivatives were tested at concentrations up to 50 pg/mL, while SP16 unmodified, SP163M and SA7 were tested at concentrations up to 100 pg/mL. None of the peptide derivatives show cytotoxicity (Figure 8).
• the cells were treated with either SP163M or peptide derivatives SA7, A5, A15, A2-1 , A2-2, A2-3, A2-4, A2-5, A2- 6, A2-7, A2-8, and A2-9 at concentrations up to 100 pg/mL for 24 hours and then cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay according to manufacturer’s instruction.
• Figure 9 demonstrates that peptide derivatives A3-10 and A3-14 exhibited superior activities in reducing NFKB activation
• Figure 10 demonstrates that peptide derivative A3-14 exhibited superior activities in reducing IL-6 secretion in IMG after LPS stimulation.
• Peptide derivative A3-10 was not included in the IL-6 assay due to issues related to synthesis. None of these peptide derivatives tested showed any significant cytotoxicity (Figure 11).
• SERPIN peptide derivatives A15 and A2-5 were tested for their ability to diminish pain related behaviors induced by capsaicin in mice in comparison to SP163M.
• SP163M 50 pg
• A15 5 pg
• A2.5 5 pg
• Capsaicin 25 ng
• both peptide derivatives at a low dose of 5 pg/mouse exhibited a level of pain blocking effect similar to that of SP163M at a much higher dose.
• the efficacy of the peptide derivative SA7 was explored in an in vivo model of neuroinflammation.
• the LPS model of neuroinflammation has been used in numerous studies to understand neurodegenerative disease. 60-63 These studies confirm that in C57BL/6 mice, LPS causes cognitive impairment. Further, acute systemic LPS causes activation of microglial cells in the brain, impaired amyloid beta clearance and an increase in blood and brain pro-inflammatory cytokines. Therefore, the LPS induced neuroinflammatory model was used to test the activity of the peptide derivative SA7.
• IL-6 For instance, moderate levels of IL-6 were measured in the brain homogenate of vehicle and SP163M, but no detectable levels of IL-6 were measured in any of the SA7 treated animals, while IL-17A, IL-12, TNFa, and GM-CSF, also showed significantly lower levels. This corresponds to the improvement in clinical signs that were seen with SA7 treatment.
• NfL neurofilament light chain
• GFAP glial fibrillary acidic protein
• Figure 16A shows that peptide derivative SA7 significantly increases NfL protein expression in the brain homogenate following LPS activated neuroinflammation, compared to both vehicle and SP163M, as measured by western blot. Abnormal expression of GFAP is indicative of reactive astrocytes and neuroinflammation.
• Figure 16B shows that peptide derivative SA7 significantly decreases GFAP expression in the brain lysate as measured by western blot analysis compared to both SP163M and vehicle treated animals.
• LRP1 has been shown to mediate healthy lysosomal processing associated with autophagy. Therefore, through LRP1 agonist such as derivatives derived from SERPIN peptides have the potential to mediate several aspects of AD including healthy cell metabolism to reduce the spread of protein aggregation, alleviate neuroinflammation and improve neuronal dysfunction leading to survival and possibly regeneration of these cells.
• LRP1 agonist such as derivatives derived from SERPIN peptides have the potential to mediate several aspects of AD including healthy cell metabolism to reduce the spread of protein aggregation, alleviate neuroinflammation and improve neuronal dysfunction leading to survival and possibly regeneration of these cells.
• SP163M and peptide derivative SA7 were tested in microglial cells.
• IMG microglial cells were treated with SP163M (100 pg/ml) or SA7 (100 pg/ml) before addition of LPS (100 ng/ml) for 24 hours. Lysates were collected and western blot analysis of both LRP1 and microtubule-associated protein light chain 3 (LC3 I and II) was performed. The LC3II/I ratio, a commonly used marker for autophagy indicating autophagic flux, was determined.
• Figure 17A shows that in LPS activated microglial cells, the autophagic flux was reduced, indicating impairment of normal lysosomal processing. Treatment with either SP163M or peptide derivative SA7 in the presence of LPS restored autophagic functioning to near baseline levels.
• LRP1 protein expression was measured and Figure 17B shows that LPS caused decreased levels (perhaps an indication of increased LRP1 shedding).
• SP163M and peptide derivative SA7 increased LRP1 protein expression in LPS activated microglial cells.
• treatment with SA7 significantly increased LRP1 expression compared to SP163M.
• SERPIN-derived peptide SP163M demonstrated anti-viral effects.
• SP163M was able to significantly reduce viral replication of neuroinflammatory alphaviruses such as Eastern Equine Encephalitis (EEEV).
• SP163M also suppressed viral replication of the novel coronavirus, SARS-CoV-2.
• SARS-CoV-2 As well as other viruses such as influenza use host cell proteases for viral entry.
• TMPRSS2 processes the S protein on the SARS-CoV2 envelope in a process called priming. Priming of the S protein is necessary for binding between the S protein and the host receptor ACE2.
• LRP1 is widely known to regulate protease/antiprotease activity.
• Protease inhibitors such as alpha-1 antitrypsin reduce proteolytic activity of TMPRSS2 preventing the priming of the S protein and therefore block virus entry.
• inhibition of TMPRSS2 prevents processing of ACE2, which decreases the infectivity of the coronavirus.
• Figure 18 shows the inhibitory effect of SP163M and peptide derivative SA7 against TMPRSS2 at various concentrations. The IC50 of SP163M was 1125 ng/mL, while the SA7 derivative was much more potent with an IC50 of 83 ng/mL.
• Eosinophilic Esophagitis A1AT has been shown to attenuate experimental EoE in a murine model and in vitro. It is unknown whether the A1AT effects are mediated by inhibition of proteolytic activity or through activating LRP1 signaling. Patients with eosinophilic asthma have lower LRP1 and in animal models, loss of LRP1 is associated with worsened allergic responses. SP163M does not contain any sequences for anti-protease activity and is likely working through mediation of LRP1 but not through proteolytic activity.
• TSLP thymic stromal lymphopoietin
• Th2 type 2 T cells
• SPINK7 knockout EPC2 cells Human esophageal epithelial cells
• SPINK7 KO cells and control cells were plated in high calcium and high density for 48 hours before being treated with either SP163M or derivative SA7 (200 pg/ml) and Poly l:C (5 pg/ml, or untreated) for 8 hours.
• TSLP production in the supernatant was measured by ELISA.
• mice were sensitized to ovalbumin by two intraperitoneal injections containing the adjuvant alum, a strong inducer of both innate and TH2 mediated immune responses and then challenged with ovalbumin given by intranasal instillation on four separate days, the mice treated with peptide derivative A2-5 showed a reduction in inflammatory mediators.
• Figure 20B shows that protein levels in the lung of mice treated with SP163M (SP163M/OVA), the steroid dexamethasone (Dex/OVA) and peptide derivative A2-5 significantly decreased compared to vehicle treated animals (p ⁇ 0.005). In A2-5 treated mice, this was associated with a reduction of TH-2 mediated cytokines IL-5, IL-4 and IL-13 in the lung tissue compared with vehicle treated mice ( Figure 20C).
• S. Pdhlmann (2020). “SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.” Cell. Hoffmann, M., H. Kleine-Weber, S. Schroeder, N. Kruger, T. Herder, S. Erichsen,
• the SEC receptor recognizes a pentapeptide neodomain of alpha 1 -antitrypsin- protease complexes. J Biol Chem 266(17): 11282-11288. Kawamura, A., D. Baitsch, R. Telgmann, R. Feuerborn, G. Weissen-Plenz, C. Hagedorn, K. Saku, S. M. Brand-Herrmann, A. von Eckardstein, G. Assmann and J. R. Nofer (2007). “Apolipoprotein E interrupts interleukin-1 beta signaling in vascular smooth muscle cells.” Arterioscler Thromb Vase Biol 27(7): 1610-1617. Kehn-Hall, K., A.
• Maheshwari (2008). “Venezuelan equine encephalitis virus infection causes modulation of inflammatory and immune response genes in mouse brain.”
• LRP1 A chameleon receptor of lung inflammation and repair.
• LRP1 modulates the microglial immune response via regulation of JNK and NF-KB signaling pathways.” Journal of Neuroinflammation 13(1 ): 304.
• Endotoxin induces a delayed loss of TH-IR neurons in substantia nigra and motor behavioral deficits.”
• Neurotoxicology 29(5): 864-870. Cazareth, J., A. Guyon, C. Heurteaux, J. Chabry and A. Petit-Paitel (2014). “Molecular and cellular neuroinflammatory status of mouse brain after systemic lipopolysaccharide challenge: importance of CCR2/CCL2 signaling.” Journal of Neuroinflammation 11 (1 ): 132. Meneses, G., G. Gevorkian, A. Florentino, M. A. Bautista, A. Espinosa, G. Acero, G. Diaz, A. Fleury, I. N.

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