PEPTIDE CONSTRUCTS COMBINED WITH STATIN DRUGS AND USE THEREOF

This application claims priority to U.S. Provisional Application No. 62/275,001, filed January 5, 2016, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to libraries of isolated aggregating peptides, peptide mimetics and repeat units that are formulated with inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase commonly known as statins to improve efficacy and or toxicity profile compared to naked drug.

BACKGROUND OF THE INVENTION

According to the CDC, there are over 1 million cases of sepsis— the host response to severe infection— each year, and it is the ninth-leading cause of disease-related deaths in the US. Sepsis generates direct medical costs exceeding $20 billion/yr (1). Novel therapies are sorely needed to complement advances in supportive care and antimicrobial therapy. Microcirculatory dysfunction may be a major determinant of death in sepsis (2, 3). Leaky microvessels promote end-organ dysfunction globally. In the lungs, this culminates in acute respiratory distress syndrome (ARDS). Methods to target this maladaptive host vascular response could have an outsized impact on sepsis, ARDS, and related disorders, a therapeutic space lacking effective drugs.

The Angiopoietin-Tie2 pathway has been identified as a molecular signaling axis that regulates vascular barrier function (4-11). Tie2 is an endothelial-enriched receptor tyrosine kinase whose activation by Angiopoietin-1 (Angpt-1) prevents vascular leakage against a broad spectrum of microbial and inflammatory mediators (12-14). Infections and systemic inflammation induce secretion and de novo synthesis in the vasculature of another Tie2 ligand called Angpt-2 that acts as a context-specific Tie2 antagonist (15-18). In human sepsis, falciparum malaria, polytrauma, and related critical illnesses, circulating Tie2 ligands tilt heavily toward Angpt-2— shown in >30 studies spanning >4000 subjects (12). Genetic deletion or targeted Angpt-2 inhibition confers protection against vascular leak, lung injury, and death in models of sepsis and ARDS (7, 19, 20). In applying an unbiased drug screen, statins have emerged as potent inhibitors of vascular Angpt-2 production (21). Given orally to treat hypercholesterolemia, statins undergo extensive first-pass hepatic metabolism, resulting in poor bioavailability with a peak plasma concentration in 1-2 hours post administration. Higher doses can harm liver, muscles, and kidneys, limiting their utility as clinical Angpt-2 suppressors. Statins applied to endothelial cells exert an on-target suppressive effect on Angpt-2 at nanomolar concentrations. Further, statins have been linked to FOXOl (also known as FKHR - forkhead in

rhabdomyosarcoma) belongs to the forkhead box O-class (FoxO) subfamily of the forkhead transcription factors. Mounting evidence suggests that the human FOXOl protein, a founding member of the FoxO family is likely involved in carcinogenesis, diabetes and other human diseases. (Huarui Lu and Haojie Huang; FOXOl: A potential target for human diseases, Curr Drug Targets. 2011 Aug; 12(9): 1235-1244.

Drug delivery strategies that alters the biodistribution of statins, could prove effective in targeting statin-responsive molecular pathways, enabling repurposing of statin drugs and novel application such as endothelium targeting of foxol in the vasculature (21). This application is directed to this goal and others.

SUMMARY OF THE INVENTION

A growing body of evidence supports the importance of the vasculature as an emerging therapeutic target in sepsis. The Angpt/Tie2 system consists of the endothelial-enriched receptor tyrosine kinase called Tie2 and its major circulating ligands, Angpt-1 and Angpt-2 (Fig 1) (24).

During health, Angpt-1 produced by peri-endothelial cells continually activates Tie2. During sepsis and other stressors, Angpt-2 is strikingly induced in the endothelium, displacing Angpt-1, and thus inhibiting otherwise tonic Tie2 signaling. Studies across thousands of humans show that Angpt-1/2 imbalance is a feature of diverse infectious and sterile etiologies of vascular leakage (summarized in (12)), suggesting that this pathway is a common effector of devastating clinical vascular

hyperpermeability. Moreover, numerous mammalian studies show that super-activation of Tie2 or inhibition of Angpt-2 rescues leakage while improving mortality in sepsis and related contexts (4, 7, 9, 11, 19-21, 25-28).

Peptide-based nanoparticles devices (PBND) are one interesting class of material that can be chemically synthesize in high yields and have been shown to form nanostructures, including nanoparticles capable of delivering small molecules, peptides, protein, and siRNA. These constructs can not only be used as passive drug carriers and excipients and in 2D and 3D cell culture systems, but also have potential to target and bind directly to cells as well as being incorporated into the extracellular matrix. S Zhang, X. Z., L Spirio. in Scaffolding in Tissue Engineering (ed Ma and Elisseeff) 217-238 (CRC Press, 2005); Branco, M. C, Sigano, D. M. & Schneider, J. P. Materials from peptide assembly: towards the treatment of cancer and transmittable disease. Current opinion in chemical biology 15, 427-434, doi: 10.1016/j.cbpa.2011.03.021 (2011). Compared to lipid and polymer constructs, peptide-based delivery systems are distinguished by unique efficiencies in accessing stable particles, drug loading, and targeting. In addition, human derived peptides that circulate in blood might not be immunogenic. Longer peptides increase immunogenicity, but also increase the chance for cross-reactivity. In contrast, short peptide sequences avoid complex tertiary structure that can increase immunogenicity short peptide-based drug carriers attractive. Utilizing peptide fragments that are inspired by tropoelastin fragments, we designed several formulations of peptide(s), peptide mimetics and repeat units, comprising various combinations of certain amino acids that can be combined with statins for administering to patients in need thereof.

A library of biocompatible and degradable self -assembling peptides were synthesized and screened for stable formulations of monodiperse nanoparticles. Selected peptides were found to entrap or encapsulate simvastatin and are active in human microvascular ECs treated cells. These same peptide constructs can be used to manipulate the pharmacological profile of simvastatin based on the ability to tune key features of peptide aggregates or nanostructures including size, surface charge, stability, drug loading and release kinetics. The peptides described herein can therefore be used as formulation vehicle for various hydrophobic and hydrophilic drugs including the simvastatin and rapamycin.

In one aspect, the instant application relates to a peptide-statin based formulations that demonstrate activity in mice subjected to either of two distinct models of sepsis. The peptide-statin construct comprises one or more peptide of the form VPGI or VPGY and a statin, or a

pharmaceutically acceptable salt thereof.

Accordingly, in some embodiments, the present application provides a composition comprising an aggregated peptide construct comprising a peptide having any ofhte amino acid sequences described in any of the embodiments supra and a statin drug, or a pharmaceutically acceptable salt thereof. In some embodiments, the aggregated peptide construct comprises one or more peptides of the form VPGI or VPGY and simvastatin, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present application provides a composition comprising an aggregated peptide construct comprising a peptide having the amino acid sequence VPGI or VPGY and a statin drug, or a pharmaceutically acceptable salt thereof. In some embodiments, the statin drug is selected from the group consisting of lovastatin, fluvastatin, lovastatin, pravastatin, simvastatin, rosuvastatin, atorvastatin, pitivastatin, cerivastatin, and fluvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the statin drug is lovastatin. In some embodiments, the statin drug is fluvastatin. In some embodiments, the statin drug is lovastatin. In some embodiments, the statin drug is pravastatin. In some embodiments, the statin drug is simvastatin. In some

embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the carrier is adapted for parenteral administration. In some embodiments, the aggregated protein construct is a mixture of a peptide having the amino acid sequence VPGY, a peptide having the amino acid sequence VPGI, and simvastatin.. In some embodiments, the aggregated protein construct is a 1 : 1 : 1 mixture of a peptide having the amino acid sequence VPGY, a peptide having the amino acid sequence VPGI, and simvastatin. In some embodiments, the present application provides a method of treating a mammal for sepsis comprising administrering to the mammal any of the compositions or aggregated peptide constructs described herein, which comprises a therapeutically effective amount of the statin drug, or a pharmaceutically acceptable salt thereof. In some embodiments, the present application provides any of the aggregated peptide constructs described herein, comprising a therapeutically effective amount of the statin drug for use in treating sepsis. In some embodiments, the present application provides use of any of the aggregated peptide constructs described herein, comprising a

therapeutically effective amount of the statin drug for use in preparation of a medicament for use in treatment of sepsis.

In some embodiments, the present application provides a method of treating a FOXOl related disease in a mammal in need thereof, comprising administering to the mammal any of the compositions or aggregated peptide constructs described herein, which comprises a therapeutically effective amount of the statin drug, or pharmaceutically acceptable salt thereof. In some

embodiments, the FOXOl related disease is selected from cancer, diabetes, obesity, muscle atrophy, insulin sensitivity, insulin resistance, impaired glucose tolerance, and an age-related disease. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is prostate cancer, renal cancer, hepatic cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, cancers of the head and neck, thyroid cancer, glioblastoma, Kaposi's sarcoma, Castleman's disease, melanoma, bone cancer (including osteosarcoma), prostate cancer, colorectal cancer, colon cancer, hepatocellular carcinoma, liver cancer, lung cancer (including non-small cell lung cancer), lymphoma or leukemia. In some embodiments, the diabetes is type 2 diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 show Angpt-2 induction inhibits Tie2 to potentiate vascular leakage.

Figure 2 shows Statins inhibit de novo Angpt-2 production, (clockwise from upper left), i.

Statins inhibit HMG-CoA reductase (heptagon), blocking translocation of Foxol (blue oval) into the nucleus, thereby inhibiting Angpt-2 transcription in endothelium, ii. Dose-response in ECs for simvastatin, iii. Parental simvastatin improves survival in CLP, but only when lung Angpt-2 expression is intact (siAngpt-2 reduces lung Angpt-2). iv. Parenteral simvastatin lowers lung Angpt-2 during LPS.

Figure 3 show Screening simvastatin-peptide formulations. Human microvascular ECs treated with vehicle (NT), lOmM or 20 mM naked simvastatin (10, 20), or a series of unique

VPGY:VPGI:simvastatin formulation (K series) loaded with 10 mM simvastatin.

Figure 4 shows schematic representation of in vivo evaluation of simvastatin nanoparticles in distinct models of sepsis.

Figure 5 shows VPGY:VPGI:simvastatin (Kl) formulation lowers 24 h Sepsis Score following LPS. Kl was compared to empty carrier (NT) and unencapsulated simvastatin injected at an equal dose of 400 mg/kg x 1.

Figure 6 shows Tie2 heterozygous mice suffer more leakage and death in sepsis. Tie2 heterozygotes (+/-) were compared to wildtype (+/+) littermate controls in the LPS and CLP model. (left to right) i. Lung stained with intravenously injected albumin-binding blue dye, indicative of extravasation from vascular leakage, ii. Survival following single LPS injection or (iii) CLP.

Figure 7 shows . Simvastatin reduces angiopoietin-2 (Angpt-2) transcription and binding of the transcription factor Foxol. A, Angpt-2 mRNA concentrations were measured via real-time polymerase chain reaction (RT-PCR) 24 hr after applying simvastatin (SIM) at indicated

concentrations to human umbilical vein endothelial cells (HUVECs). B, SIM (10 μΜ) was applied for 24 hr to HUVECs pretreated with control or Kriippel-like factor-2 (KLF2) siRNA and Angpt-2 mRNA was measured by RT-PCR. C, Putative Foxol binding sites were identified at -2,840 and - 1,660 in the three kilobases 5' to the translational start site of human ANGPT2 by TFsearch and crosschecked by aligning the consensus Foxol binding sequence (in bold). D, Nuclear extracts of HUVECs were incubated with biotin labeled DNA probes specific for the -2,840 and -1,660 sites of ANGPT2 in the presence (+) or absence (-) of six-fold excess unlabeled probes. Arrows indicate the specific band eliminated by competition. E, Nuclear extracts of HUVECs either treated with vehicle (-) or SIM (10 μΜ, +) for 24 hr were prepared and incubated with biotin-labeled DNA probes specific for the -2,840 and -1,660 sites of ANGPT2. Arrows indicate the band of interest and arrowhead indicates a non-specific band. F, HUVECs treated with SIM (10 μΜ) for 24 hr were gently lysed and chromatin immunoprecipitation was performed with anti-Foxol. Results of RT-PCR to quantify ANGPT2 promoter concentration are shown for the -2,840 and -1,660 sites. *p < 0.05, ***p < 0.001 (see Crit Care Med. 2015;43(7):e230-40).

Figure 8 shows Simvastatin prevents nuclear Foxol translocation by phosphorylation. A, Endothelial cells (ECs) treated with simvastatin (SIM, 10 μΜ) were stained for Foxol (red) and nuclei (4',6-diamidino-2-phenylindole, blue). White arrows indicate the edge of nuclear staining. B, Planimetric quantification of staining results by surveying 10 high-powered fields (A~40) per slide. *p < 0.05 and **p < 0.01. C, HUVEC lysates 24 hr after SIM treatment (10 μΜ) were immunoblotted with anti-pSer256 Foxol (pFoxo-1), anti-Foxol (tFoxo-1), and anti-GAPDH as a loading control. D, Densitometric quantification of the above results. E, Immunoblotting as described above for lysates of ECs infected with a virus encoding wild-type Foxol (AdFoxol) for 24 hr and treated with SIM (10 μΜ) for 24 hr or vehicle. F, Quantification of the above results. G, Immunoblotting as described above for lysates of ECs infected with a virus encoding triple-phosphorylation mutant Foxol (TM Foxol) before 24-hr treatment with SIM (10 μΜ) or vehicle. *p < 0.05 (see Crit Care Med.

2015;43(7):e230-40).

Figure 9 shows that Foxol phosphorylation is critical for the suppression of Angpt-2 by simvastatin. A, Real-time polymerase chain reaction for Angpt-2 24 hr after treating virally transduced human umbilical vein endothelial cells with simvastatin (SIM, 10 μΜ) or vehicle. " Gal" is a control virus expressing β-galactosidase; "AdFoxol" is a virus encoding wild-type Foxol; and "Ad-TM-Foxol" is a virus encoding the triple phosphorylation constitutively active mutant Foxol. B, ELISA for secreted Angpt-2 protein for the above conditions. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (see Crit Care Med. 2015;43(7):e230-40).

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, one aspect of the invention provides a statin and peptide(s) or statin and aggregated peptides construct comprised of chemically linked amino acid residues of the following sequence (X1-X2-X3-X4) wherein Xi is isoleucine (He) or a conservative substitution thereof; X2 is proline (Pro) or a conservative substitution thereof; X3 is glycine (Gly) or a conservative substitution thereof; X4 is tyrosine (Tyr) or a conservative substitution thereof that encapsulate or entrap a statin, such as simvastatin.

Accordingly, one embodiment of this invention includes a statin and peptide(s) or statin and aggregated peptides construct comprising chemically linked amino acid residues of the following sequence: Xi - X ₂ - X3 - X4; where Xi is an L- or D-amino acid; X2 is proline, or a conservative substitution thereof; X3 is selected from a group consisting of glycine or a conservative substitution thereof, a bond, and a non-coded, non-proteinogenic, or a non-standard amino acid linker; X ₄ is an L- or D-amino acid that encapsulate or entrap simvastatin.

Another further embodiment of this invention includes the statin and peptide(s) or construct, where said biological entity is selected from a group consisting of an amine, amide, imine, imide, azide, azo compound, carboxylic acid, carbonate, carboxylate, ester, alcohol, aldehyde, alkane, alkene, alkyne, halogens, ketone, acyl halide, boronic acid, boronic ester, borinic acid, borinic ester, hydroperoxide, peroxide, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, cyanates, nitrate, nitrile, nitrite, nitro compound, nitroso compound, pyridine, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, thial, phosphine, phosphonic acid, phosphate, phosphodiester, fatty acid, myristic acid, palmytolyl, Fluorenylmethyloxycarbonyl or FMOC carbamate, Z (CBZ), Boc, tert-Butyl, cell surface receptor ligand, an antibody, bispecific antibody, an antibody -like molecule, Fab, Fc, or a portion thereof, an aptamer, a cytokine, hormone, a lectin, a lipid, nucleic acid, a carbohydrate, enzyme, biotin, avidin, streptavidin, steroid, protein A, protein G, a plasma albumin, a ligand, a therapeutic agent, fluorescent molecule, a binding molecule, a biodegradable non-amino acid polymer and any combinations thereof.

In some embodiments, the following sequence (X1-X2-X3-X4) is repeated sequentially or randomly to form polypeptides of the following length: 11, 13, 17, 19, 22, 23, 26, 28, 29, 31, 33, 34, 36, 37, 38, 39, 41, 43, 44, 46, 47, 51, 52, 53, 57, 58, 59. In some embodiments, the statin and peptide(s) comprises a chemically linked amino acid residue having the sequence Xi - X ₂ - X3 - X4.

In some embodiments, the aggregated peptide construct comprises a chemically linked amino acid residue having the sequence Xi - X ₂ - X3 - X4.

In some embodiments, the aggregated peptide construct consists of a chemically linked amino acid residue has the sequence H- Xi - X ₂ - X3 - X4- OH. When a H is shown, it is part of the amino group of the amino acid residue, while OH is part of the carboxyl group of the amino acid residue.

In some embodiments, the aggregated peptide construct consists of a chemically linked amino acid residue has the sequence HO— Xi— X ₂ ^— X ₃ ^— X ₄ ^— H.

In some embodiments of the aggregated peptide construct comprises a chemically linked amino acid residue having the sequence Xi - X ₂ - X ₃ - X4; the aggregated peptide construct consisting of a chemically linked amino acid residue having the sequence Xi - X ₂ - X ₃ - X4; the aggregated peptide construct consisting of a chemically linked amino acid residue having the sequence H-Xi - X ₂ - X ₃ - X ₄ - OH; or the aggregated peptide construct consisting of a chemically linked amino acid residue having the sequence HO- Xi - X ₂ - X ₃ - X4-H: Xi is isoleucine (He), glutamic acid (Glu), tyrosine (Tyr), valine (Val), or lysine (Lys). In some embodiments, Xi is isoleucine (He). In some embodiments, Xi is glutamic acid (Glu). In some embodiments, Xi is tyrosine (Tyr). In some embodiments, Xi is valine (Val). In some embodiments, Xi is lysine (Lys). In some embodiments, Xi is isoleucine or a conservative substitution thereof. In some embodiments, X ₂ is proline. In some embodiments, X3 is glycine. In some embodiments, X4 is isoleucine (He), tyrosine (Tyr), histidine (His), or phenylalanine (Phe). In some embodiments, X4 is isoleucine (He). In some embodiments, X4 is tyrosine (Tyr). In some embodiments, X4 is histidine (His). In some embodiments, X4 is phenylalanine (Phe). In some embodiments, Xi is isoleucine (He), glutamic acid (Glu), tyrosine (Tyr), valine (Val), or lysine (Lys); X ₂ is proline; X3 is glycine; and X4 is isoleucine (He), tyrosine (Tyr), histidine (His), or phenylalanine (Phe). In some embodiments, Xi is isoleucine (He), X ₂ is proline, X3 is glycine, and X4 is isoleucine (He). In some embodiments, Xi is glutamic acid (Glu), X ₂ is proline, X3 is glycine, and X4 is isoleucine (He). In some embodiments, Xi is tyrosine (Tyr), X ₂ is proline, X3 is glycine, and X4 is isoleucine (He). In some embodiments, Xi is valine (Val), X ₂ is proline, X3 is glycine, and X4 is isoleucine (He). In some embodiments, Xi is lysine (Lys), X ₂ is proline, X3 is glycine, and X4 is isoleucine (He). In some embodiments, Xi is isoleucine (He), X ₂ is proline, X3 is glycine, and X4 is tyrosine (Tyr). In some embodiments, Xi is glutamic acid (Glu), X ₂ is proline, X3 is glycine, and X4 is tyrosine (Tyr). In some embodiments, Xi is tyrosine (Tyr), X ₂ is proline, X3 is glycine, and X4 is tyrosine (Tyr). In some embodiments, Xi is valine (Val), X ₂ is proline, X3 is glycine, and X4 is tyrosine (Tyr). In some embodiments, Xi is lysine (Lys), X ₂ is proline, X3 is glycine, and X4 is tyrosine (Tyr). In some embodiments, Xi is isoleucine

(He), X ₂ is proline, X3 is glycine, and X4 is histidine (His). In some embodiments, Xi is glutamic acid (Glu), X2 is proline, X3 is glycine, and X4 is histidine (His). In some embodiments, Xi is tyrosine (Tyr), X ₂ is proline, X3 is glycine, and X4 is histidine (His). In some embodiments, Xi is valine (Val), X2 is proline, X3 is glycine, and X4 is histidine (His). In some embodiments, Xi is lysine (Lys), X2 is proline, X3 is glycine, and X4 is histidine (His). In some embodiments, Xi is isoleucine (He), X2 is proline, X3 is glycine, and X4 is phenylalanine (Phe). In some embodiments, Xi is glutamic acid (Glu), X2 is proline, X3 is glycine, and X4 is phenylalanine (Phe). In some embodiments, Xi is tyrosine (Tyr), X2 is proline, X3 is glycine, and X4 is phenylalanine (Phe). In some embodiments, Xi is valine (Val), X2 is proline, X3 is glycine, and X4 is phenylalanine (Phe). In some embodiments, Xi is lysine (Lys), X2 is proline, X3 is glycine, and X4 is phenylalanine (Phe).

In some embodiments, the aggregated peptide construct is terminated with an 8-15 amino acid sequence, wherein the amino acids are selected from cysteine (C), glycine (Gly), histidine (His), arginine (Arg), serine (Ser), and phenylalanine (Phe). In some embodiments, the 8-15 amino acid sequence comprises a sequence -Cys-His-His-His-Arg-His-Ser-Phe.

In some embodiments, wherein the aggregated peptide construct is selected from:

H-Val-Pro-Gly-Tyr-OH;

H-Val-Pro-Gly-Phe-OH;

H-Val-Pro-Gly-Ile-OH;

H-Val-Pro-Gly-His-OH;

H-Val-Pro-Gly-Trp-OH;

H-Lys- Pro-Gly-Tyr-OH;

H-Glu Pro-Gly-Tyr-OH;

H-Lys-Pro-Gly-Phe-OH;

H-Glu-Pro-Gly-Phe-OH;

H-Ile-Pro-Gly-Tyr-OH;

H-Thr-Pro-Gly-Tyr-OH;

H-Ile-Pro-Gly-Phe-OH; and

H-Thr-Pro-Gly-Phe-OH

In some embodiments, the aggregated peptide construct is selected from:

VPGY-CHHHRHSF;

VPGY-G-CHHHRHSF;

VPGY-GS-CHHHRHSF; and

VPGY-GGGS-CHHHRHSF.

In some embodiments, the aggregated peptide construct is selected from:

H-Val-Pro-Gly-Tyr-Val-Pro-Gly-Tyr-Val-Pro-Gly-OH;

H-Ile-Pro-Gly-Tyr-Ile-Pro-Gly-Tyr-Ile-Pro-Gly-OH;

H-Val-Pro-Gly-Tyr-Val-Pro-Gly-Tyr-Val-Pro-Lys-OH; H-Ile-Pro-Gly-Tyr-Ile-Pro-Gly-Tyr-Ile-Pro-Lys-OH;

H-Val-Pro-Gly-Tyr-Val-Pro-Gly-Tyr-Val-Pro-His-OH;

H-Ile-Pro-Gly-Tyr-Ile-Pro-Gly-Tyr-Ile-Pro-His-OH;

H-Pro-Val-Gly-Tyr-Val-Pro-Gly-Phe-OH;

H-Val-Pro-Gly-Tyr-Pro-Val-Gly-Phe-OH;

H-Val-Phe-Pro-Gly-Tyr-Pro-Val-Gly-OH;

H-Gly-Pro-Val-Gly-Tyr-Val-Gly-Pro-Phe-Gly-OH;

H-Tyr-Gly-Val-Gly-Phe-Val-Gly-Pro-Gly-Pro-OH; and

H-Tyr-Gly-Pro-Val-Tyr-Gly-Pro-Val-OH.

In some embodiments, n is 2.

In some embodiments, the aggregated peptide construct is selected from:

H-Gly-Tyr-Gly-Pro-Val-OH; and

H-Gly-Phe-Gly-Pro-Val-Gly-Tyr-Gly-Pro-Val-OH.

Another embodiment of this invention includes the aggregated peptide construct, where the amino acid sequence is selected from the group consisting of:

IPGY;

VPGY;

LPGY;

IPGF;

VPGF;

LPGF;

VPGW;

IPGW;

LPGW;

IPGY-VPGY-VPG;

IPGY-IPGY-IPG;

VPGY-VPGY-VPK;

IPGY-IPGY-IPK;

VPGY-VPGY-VPH;

IPGY-IPGY-IPH;

VPGY-VPGF-VPGY-V;

VPGY-VPGY-VPGY-V;

VPGY-VPGY-VPGY-L;

VPGY-VPGY-VPGY-VPGY-V;

VPGY-VPGY-VPGY-VPGY-VPG;

VPGY-VPGY-VPGY-VPGY-VPGY-VP; VPGY-VPGY-VPGY-VPGY-VPGY-VPG;

VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VP;

VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VPGY;

VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-V;

VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VPG;

VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-V; and VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VPGY-VP.

In another embodiment, the invention provides an aggregated peptide construct, wherein the construct is a mixture of peptides, wherein the mixture is selected from:

VPGY:KPGY;

VPGY:EPGY;

VPGY:TPGY;

VPGY:KPGY:TPGY;

VPGY:VPGY-GGGS-CHHHRHSF;

KPGY:VPGY-GGGS-CHHHRHSF;

TPGY:VPGY-GGGS-CHHHRHSF;

VPGY:VPGY-CHHHRHSF;

VPGY:VPGY-G-CHHHRHSF;

VPGY: VPGY-GS-CHHHRHSF; and

VPGY:VPGY-GGGS-CHHHRHSF.

In some embodiments, the application provides any of the previous embodiments of aggregated peptide constructs and a statin. In some embodiments, the application provides any of the previous embodiments of aggregated peptide constructs and a statin selected from simvastatin, pravastatin, lovastatin, atorvastatin, fluvastatin, cerivastatin, rosuvastatin, pitavastatin, and mevastatin, or a pharmaceutically acceptable salt thereof.

In some embodiment of this invention includes the statin and peptide(s) formulations, wherein the amino acid sequence of the peptide is selected from the group consisting of:

IPGY: STATIN;

LPGY: STATIN;

VPGY: STATIN;

EPGY: STATIN;

KPGY: STATIN;

KPGI: STATIN;

TPGY: STATIN;

TPGI: STATIN;

VPGI: STATIN; VPGW: STATIN;

IPGW: STATIN;

LPGW: STATIN;

IPGY- VPGY- VPG: STATIN;

VPGI-VPGI- VPG: STATIN;

TPGY-TPGY-TPG: STATIN;

IPGY-IPGY-IPG: STATIN;

VPGY-VPGY-VPK: STATIN;

IPGY-IPGY-IPK: STATIN;

VPGY-VPGY-VPH: STATIN;

IPGY-IPGY-IPH: STATIN;

VPGY- VPGF-VPGY- V: STATIN;

VPGY-VPGY-VPGY- V: STATIN;

VPGY- VPGY-VPGY-L: STATIN;

VPGY-VPGY-VPGY- VPGY-V: STATIN;

VPGY-VPGY-VPGY- VPGY-VPG: STATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VP: STATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPG: STATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VP: STATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY- VPGY: STATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY-V: STATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY-VPG: STATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY- VPGY-V: STATIN; and

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY-VPGY- VP: STATIN;

and STATIN is a statin is selected from simvastatin, pravastatin, lovastatin, atorvastatin, fluvastatin, cerivastatin, rosuvastatin, pitavastatin, and mevastatin, or a pharmaceutically acceptable salt thereof.

In some embodiments, the STATIN in this embodiment is pravastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is lovastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is atorvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is fluvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the

STATIN in this embodiment is cerivastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is rosuvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is pitavastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is mevastatin, or a pharmaceutically acceptable salt thereof. In another embodiment, the invention provides an aggregated peptide construct, wherein the construct is a mixture of peptides and statin, wherein the mixture is selected from:

VPGY:VPGI:STATIN

KPGY:VPGI:STATIN

EPGY:VPGI: STATIN

TPGY:VPGI: STATIN

VPGY:EPGY: STATIN

VPGY:TPGY: STATIN

VPGY:VPGYG: STATIN

VPGY:VPGI:VPGYHHHRHSF:STATIN

VPGY:VPGYG:VPGIHHHRHSF: STATIN

VPGI:EPGI:VPGYHHHRHSF:STATIN

FSHRHHHVPGYG:VPGYG: STATIN

FSHRHHHVPGYG:VPGY: STATIN

FSHRHHHVPGYG:VPGI:VPGY: STATIN

FSHRHHHVPGY:VPGI:VPGY:STATIN;

and STATIN is a statin is selected from simvastatin, pravastatin, lovastatin, atorvastatin, fluvastatin, cerivastatin, rosuvastatin, pitavastatin, and mevastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is pravastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is lovastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is atorvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is fluvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is cerivastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is rosuvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is pitavastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the STATIN in this embodiment is mevastatin, or a pharmaceutically acceptable salt thereof.

In some embodiment of this invention includes the statin and peptide(s) formulations, where the amino acid sequence of the peptide is selected from the group consisting of:

IPGY: SIMVASTATIN;

LPGY: SIMVASTATIN;

VPGY: SIMVASTATIN;

EPGY: SIMVASTATIN;

KPGY: SIMVASTATIN;

KPGI: SIMVASTATIN; TPGY: SIMVASTATIN;

TPGI: SIMVASTATIN;

VPGI: SIMVASTATIN;

VPGW: SIMVASTATIN;

IPGW: SIMVASTATIN;

LPGW: SIMVASTATIN;

IPGY- VPGY- VPG: SIMVASTATIN;

VPGI-VPGI- VPG: SIMVASTATIN;

TPGY-TPGY-TPG: SIMVASTATIN;

IPGY-IPGY-IPG: SIMVASTATIN;

VPGY- VPGY-VPK: SIMVASTATIN;

IPGY-IPGY-IPK: SIMVASTATIN;

VPGY- VPGY-VPH: SIMVASTATIN;

IPGY-IPGY-IPH: SIMVASTATIN;

VPGY- VPGF-VPGY-V: SIMVASTATIN;

VPGY- VPGY-VPGY- V: SIMVASTATIN;

VPGY- VPGY-VPGY-L: SIMVASTATIN;

VPGY-VPGY-VPGY- VPGY-V: SIMVASTATIN;

VPGY- VPGY-VPGY- VPGY- VPG: SIMVASTATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VP: SIMVASTATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPG: SIMVASTATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VP: SIMVASTATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY:SIMVASTATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY-V:SIMVASTATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY- VPG: SIMVASTATIN;

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY- VPGY- V: SIMVASTATIN; and

VPGY-VPGY-VPGY- VPGY-VPGY-VPGY-VPGY- VPGY- VP: SIMVASTATIN.

In another embodiment, the invention provides an aggregated peptide construct, wherein the construct is a mixture of peptides and statin, wherein the mixture is selected from:

VPGY:VPGI:SIMVASTATIN

KPGY:VPGI:SIMVASTATIN

EPGY: VPGI: SIMVASTATIN

TPGY: VPGI: SIMVASTATIN

VPGY:EPGY: SIMVASTATIN

VPGY:TPGY: SIMVASTATIN

VPGY: VPGYG: SIMVASTATIN VPGY : VPGI : VPGYHHHRHSF : SIMVASTATIN

VPGY:VPGYG:VPGIHHHRHSF:SIMVASTATIN

VPGI:EPGI:VPGYHHHRHSF:SIMVASTATIN

FSHRHHHVPGYG:VPGYG: SIMVASTATIN

FSHRHHHVPGYG: VPGY: SIMVASTATIN

FSHRHHHVPGYG:VPGI:VPGY:SIMVASTATIN

FSHRHHH VPGY : VPGI : VPGY : SIMVASTATIN In some embodiments, the construct forms nanoparticles or microparticles. In some embodiments, the construct forms nanoparticles. In some embodiments, the construct forms microparticles.

In some embodiments, the construct allows for targeted delivery in said patient.

In some embodiments, the composition further comprises an additional therapeutic or targeting agent.

In some embodiments, the composition is suitable for intravenous or subcutaneous, administration to a patient.

Another embodiment includes a method of increasing the half -life of a drug during administration to a patient, comprising administering to said patient a composition as described herein.

Another embodiment includes a method of administering a drug to a patient, comprising administering to said patient a composition as described herein.

In some embodiments, the aggregated peptide construct is self -assembling.

In another aspect, the instant application provides a method of delivering a statin in a patient in need thereof, comprising administering to said patient any of the aggregated peptide constructs described herein, wherein said construct comprises one or more of any of the aggregated peptide constructs disclosed herein, and wherein said construct further comprises a statin drug, or a pharmaceutically acceptable salt thereof. In some embodiments, the construct forms nanoparticles or microparticles. In some embodiments, the construct allows for targeted delivery in said patient. In some embodiments, the statin drug, or a pharmaceutically acceptable salt thereof, is encapsulated by the construct. In some embodiments, the statin, or a pharmaceutically acceptable salt thereof, is encapsulated by the construct, and the statin is not covalently bound to the peptide. In some embodiments, the statin, or a pharmaceutically acceptable salt thereof, is encapsulated by the construct, and the statin is covalently bound to the peptide, optionally via a linker. In some embodiments, the statin, or a pharmaceutically acceptable salt thereof, is non-covalently or affinity bound to a receptor carrier. In some embodiments, the statin, or a pharmaceutically acceptable salt thereof, is modified by a targeting ligand. In some embodiments, the targeting ligand is selected from the group consisting of an antibody, a peptide, and a protein. In some embodiments, the statin, or a pharmaceutically acceptable salt thereof, is modified by a molecule to increase therapeutic window and blood circulation in said patient. In some embodiments, the statin, or a pharmaceutically acceptable salt thereof, is modified by a molecule selected from the group consisting of an antibody, PEG, and PLGA. In some embodiments, the statin is selected from the group consisting of simvastatin, pravastatin, lovastatin, atorvastatin, fluvastatin, cerivastatin, rosuvastatin, pitavastatin, and mevastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, the statin is simvastatin, or a pharmaceutically acceptable salt thereof.

In some embodiments, the patient suffers from a disease amenable to treatment by said statin, or a pharmaceutically acceptable salt thereof. In some embodiments, the disease is selected from the group consisting of atherosclerosis, cardiovascular disease, a lipid metabolism disorder,

hyperlipidemia, angina, heart attack, high cholesterol, peripheral neuropathy, stroke, fatty liver disease, peripheral vascular disease, claudication, sepsis, and acute respiratory distress syndrome (ARDS). In some embodiments, the disease is sepsis. In some embodiments, the disease is acute respiratory distress syndrome (ARDS).

As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed statin drugs, wherein the drug moiety is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of

Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

As used herein, the term "treating" or "treatment" refers to one or more of (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.

As used herein, the phrase "therapeutically effective amount" refers to the amount of active statin drug that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician. In some embodiments, the dosage of the compound, or a pharmaceutically acceptable salt thereof, administered to a patient or individual is about 1 mg to about 2 g, about 1 mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg to about 100 mg, about 1 mg to 50 mg, or about 50 mg to about 500 mg.

As used herein, the term "individual" or "patient," used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.

When employed as pharmaceuticals, the aggregated peptide constructs described herein can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.

Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump.

This invention also includes pharmaceutical compositions which contain, as the active ingredient, the compound of the invention or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers (excipients). In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container.

The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner.

EXAMPLES

Example 1: Design of exemplary aggregating peptide constructs comprising 4-11 amino acids

It was observed that tropoelastin monomer secreted by elastogenic cells such as smooth muscle cells, endothelial cells, and fibroblasts can aggregate into organized spheres that ultimately gets incorporated into growing elastin fibers. Toonkool P, Regan DG, Kuchel PW, Morris MB, Weiss AS. J Biol Chem. 2001;276:28042-28050 Wise SG, Mithieux SM, Raftery MJ, Weiss AS. J Struct Biol. 2005;149:273-281. A close analysis of the human tropoelastin sequence reveals a high abundance of the following amino acids: Glycine (Gly -28%), Alanine (Ala -21%), Valine (Val -14) Proline (Pro -12%) (see Table A below). Utilizing peptide fragments that are inspired by tropoelastin fragments, we designed several formulations of peptide(s), peptide mimetics and repeat units, comprising various combinations of certain amino acids that can be combined with statins for administering to patients in need thereof.

In particular, we discovered that certain key residues (e.g. Pro and Gly) that triggers clustering or controlled aggregation into well-defined nanostructures and microstructures for application delivery of statins and other drugs that promotes efficacy and reduce toxicity .

Table A

A library of highly scalable short linear peptides or their cyclic counterpart was designed that will aggregate in aqueous medium to form micro- and nano- aggregates (micro- and nano- particles) for use in drug delivery, tissue engineering and as formulation vehicle for various therapeutic agents that is both hydrophobic and hydrophilic drugs including the simvastatin and rapamycin. These peptides were shown to entrap simvastatin and can be formulated as homogeneous (one group of unique aggregating peptide sequence) or heterogeneous (two or more groups of unique aggregating peptide sequences) mixtures. Entrapment and is also possible with other statins, small molecule drugs, DNA, siRNA, mRNA, excipients, stabilizers, protein, drugs for reducing drug toxicity, improving pharmacokinetics (PK), enhancing drug efficacy, targeting agents selectively to disease sites, delivering drugs to intracellular targets, and any combinations thereof. In accordance with some embodiments of one aspect described herein, a diverse library of aggregating peptides and simvastatin, e.g., 4-11, amino acids total in length that can be combined with statins, Table 1-3, was designed. The aggregating peptides simvastatin construct can be formulated in aqueous media to generate a series of micro- and nano- structures (e.g., microparticles and nanoparticles) with a capability to control the size of the aggregates from nanometer to micrometer. These aggregating peptides can entrap statins, rapamycin and other therapeutic drugs for nanotherapeutics and nanomedicine, diagnostics, drug delivery, and tissue engineering applications.

For an experimental approach, a candidate peptide sequence such as VPGI, TPGY and so on can be synthesized as described herein, e.g., by solid-state peptide synthesis. The aggregating peptides sequences can also be produced as fusion peptides such as VPGICHHHRHSF,

VPGYCHHHRHSF or protein by recombinant gene expression in bacteria (e.g. Escherichia coli) or mammalian cell lines such as the Chinese hamster ovary (CHO), HEK and COS cell lines or any methods known in the art used to produce protein. The aggregating peptide or fusion peptide construct can then be combined with therapeutic drugs such as simvastatin (e.g. VPGY:simvastatin,

VPGY:VPGI:simvastatin) or rapamycin (e.g. VPGY: rapamycin, VPGY:VPGI: rapamycin) and other small molecules. Various aqueous solution including pure water or buffers are added to evaluate stability and drug entrapment. Characterization of any peptide aggregate, including peptide and statin nano or nanostructures formed, e.g., size, shape, stability can be performed using any methods known in the art or as described in the Examples below.

Exemplary aggregating peptide comprising linear sequences (4 -11 amino acid sequence) in three- and one- letter codes are shown in Tables 1-5. Each indicated amino acid sequence is designated with a number or letter to which is referred throughout the specification. The short aggregating peptide sequences having the general formula:

a) Xi - X ₂ - X3 - X4 (N-terminus to C-Terminus; N-C) or (C -terminus to N- Terminus; C-N)

b) X11 -X12 - X11 - X10 - X (the bond between Xn and X12 is formed from the alpha-carboxyl group of Xn and the alpha-amino group of X12) c) (Xs - X ₆ - X ₇ - Xs)„ (N-C and C-N)

d) random combinations of a, b,c ;

wherein Xi is an L- or D-amino acid; X2, Xe, and X10 is proline, or a conservative substitution thereof; X3, X7, and X11 is selected from a group consisting of glycine or a conservative substitution thereof, a bond, and a non-coded, non-proteinogenic, or a non-standard amino acid linker; X ₄ , Xs, and X12 is an L- or D-amino acid; and said peptide is terminated with chemical group, molecule, peptide blocking group, peptide, or biological entity. The 4 - 11 amino acids constructs reported here were designed to test the ability to control aggregation properties and stability (Tables 1-3). Each peptide in the Tables 1-3 was prepared, for example, by FMOC -based solid-phase peptide synthesis and all of the peptide sequences were verified for >70% purity before by HPLC. The ability of these short hydrophobic peptide sequences to self-organize in aqueous media was then evaluated. As described in detail in the following Examples, the short peptides (as shown in Tables 1-3) formed a particulate suspension spontaneously within seconds in aqueous media and size of aggregate measured by Dynamic light scattering (DLS). These aggregating peptides can be mixed in various combinations and ratios (Table 3-5) in the presence of statin or other therapeutic agent to form particulate suspension of well-defined sized as measured by DLS.

Each of the aggregating peptide in Tables 1-5 was prepared by FMOC solid-phase peptide synthesis and can be combined with simvastatin or rapamycin for delivery. For example, these peptides were synthesized on the acid sensitive Wang resin and cleaved from the resin with a solution mixture of trifluoroacetic acid/triisopropylsilane/water in a volume ratio of 9.5/2.5/2.5. Synthesized peptides were purified by reversed phase HPLC or flash column chromatography. The peptide sequences were purified by HPLC using a C18 5 μιη 120A 4.6 * 150mm column in 0.1% TFA/H20 (buffer A) and 0.09% TFA in 80% ACN/20 % H20 (buffer B).

In some embodiments, each of the peptides in Tables 1-5 are combined with a statin. In some embodiments, each of the peptides in Tables 1-5 are combined with a statin selected from lovastatin, fluvastatin, lovastatin, pravastatin, simvastatin, rosuvastatin, atorvastatin, pitivastatin, cerivastatin, and fluvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, each of the peptides in Tables 1-5 are combined with simvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments. In some embodiments, each of the peptides in Tables 1-5 are combined with pravastatin, or a pharmaceutically acceptable salt thereof. In some embodiments. In some embodiments, each of the peptides in Tables 1-5 are combined with lovastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, In some embodiments, each of the peptides in Tables 1-5 are combined with atorvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, In some embodiments, each of the peptides in Tables 1-5 are combined with fluvastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, each of the peptides in Tables 1-5 are combined with cerivastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, each of the peptides in Tables 1-5 are combined with rosuvastatin, or a

pharmaceutically acceptable salt thereof. In some embodiments, each of the peptides in Tables 1-5 are combined with pitavastatin, or a pharmaceutically acceptable salt thereof. In some embodiments, each of the peptides in Tables 1-5 are combined with mevastatin, or a pharmaceutically acceptable salt thereof.

Table 1. Design of aggregating peptide constructs comprising 4 amino acids

Table 2. Design of aggregating peptide constructs comprising 5-11 amino acids

Table 3. Design of aggregating peptide constructs comprising 8 -11 amino acids

Table 4. Aggregating peptide fusion sequence (i.e peptide covalently linked to therapeutic drug) with G GS and GGGS linker.

Table 5. Mixed aggregating peptide constructs mixed with peptide drug fusion in ratios limited to 1 : 1

Tables 6-8 shows aggregating amino acid sequences (4 -11 amino acid sequences) in three- and one-letter codes and simvastatin. Each indicated amino acid sequence is designated with an entry number or letter to which is referred throughout the specification. Table 9 shows one-letter code of aggregating fusion sequence (for example peptide carrier fused to peptide drug) with G, GS and GGS linker. Each indicated amino acid sequence is designated with an entry number or letter to which is referred throughout the specification. Table 10 shows one-letter code of combination of isolated aggregating peptide sequence combining different sequences. Each indicated amino acid sequence is designated with an entry number or letter to which is referred throughout the specification.

Table 6. Design of aggregating peptide simvastatin constructs comprising 4 amino acids

1-05 H-Val-Pro-Gly-His-OH:Simvastatin VPGH: Simvastatin

1-06 H-Val-Pro-Gly-Trp-OH: Simvastatin VPGW: Simvastatin

1-07 H-Ly s- Pro-Gly -Ty r-OH : Simvastatin KPGY: Simvastatin

1-08 H-Glu Pro-Gly-Tyr-OH: Simvastatin EPGY: Simvastatin

1-09 H-Ly s-Pro-Gly -Phe-OH : Simvastatin KPGF: Simvastatin

1-10 H-Glu-Pro-Gly-Phe-OH:Simvastatin EPGF: Simvastatin

1-11 H-Ile-Pro-Gly -Ty r-OH : Simvastatin IPGY : Simvastatin

1-12 H-Thr-Pro-Gly -Ty r-OH : Simvastatin TPGY: Simvastatin

1-13 H-Ile-Pro-Gly-Phe-OH:Simvastatin IPGF: Simvastatin

1-14 H-Thr-Pro-Gly-Phe-OH: Simvastatin TPGF: Simvastatin

Table 7. Design of aggregating peptide simvastatin constructs comprising 5-11 amino acids

Table 8. Design of aggregating peptide simvastatin constructs comprising 8 -11 amino acids

1-18 H-Ile-Pro-Gly-Tyr-Ile-Pro-Gly-Tyr-Ile-Pro-Lys- IPGYIPGYIPK:SI

OH: SIMVASTATIN MVASTATIN

1-19 H-Val-Pro-Gly-Tyr-Val-Pro-Gly-Tyr-Val-Pro-His- VPGYVPGYVPH

OH: SIMVASTATIN : SIMVASTATIN

1-20 H-Ile-Pro-Gly-Tyr-Ile-Pro-Gly-Tyr-Ile-Pro-His- IPGYIPGYIPH:SI

OH: SIMVASTATIN MVASTATIN

1-21 H-Pro-Val-Gly-Tyr-Val-Pro-Gly-Phe- PVGYVPGF:SIM

OH: SIMVASTATIN VASTATIN

1-22 H-Val-Pro-Gly-Tyr-Pro-Val-Gly-Phe- VPGYPVGF:SIM

OH: SIMVASTATIN VASTATIN

1-23 H-Pro-Val-Gly-Tyr-Pro-Val-Gly-Phe- PVGYPVGF:SIM

OH: SIMVASTATIN VASTATIN

1-24 H-Pro-Val-Gly-Tyr-Val-Pro-Phe-Gly- PVGYVPFG:SIM

OH: SIMVASTATIN VASTATIN

1-25 H-Val-Phe-Pro-Gly-Tyr-Pro-Val-Gly- VFPGYPVG:SIM

OH: SIMVASTATIN VASTATIN

1-26 H-Gly-Pro-Val-Gly-Tyr-Val-Gly-Pro-Phe-Gly- GPVGYVGPFG:S

OH: SIMVASTATIN IMVASTATIN

1-27 H-Tyr-Gly-Val-Gly-Phe-Val-Gly-Pro-Gly-Pro- YGVGFVGPGP:S

OH: SIMVASTATIN IMVASTATIN

1-30 H-Tyr-Gly-Pro-Val-Tyr-Gly-Pro-Val- YGPVYGPV:SIM

OH: SIMVASTATIN VASTATIN

Table 9. Aggregating peptide fusion sequence (i.e peptide covalently linked to therapeutic drug) with G, GS and GGGS linker and simvastatin.

Table 10. Mixed aggregating peptide simvastatin constructs mixed with peptide drug fusion in ratios but not limited to 1 : 1 Entry # Mixed sequences (1 : 1 or 1: 1 : 1)

K VPGY:VPGI:SIMVASTATIN

1-36 VPGY:EPGY: SIMVASTATIN

1-37 VPGY:TPGY: SIMVASTATIN

1-38 VPGY:VPGYG:VPGI: SIMVASTATIN

1-39 VPGY:VPGI:VPGYHHHRHSF:SIMVASTATIN

1-40 VPGY:VPGYG:VPGIHHHRHSF:SIMVASTATIN

1-41 VPGI:EPGI:VPGYHHHRHSF:SIMVASTATIN

1-42 FSHRHHHVPGYG:VPGYG: SIMVASTATIN

1-43 FSHRHHHVPGYG:VPGY: SIMVASTATIN

1-44 FSHRHHH VPGYG: VP GI : VPGY : SIMVASTATIN

1-45 FSHRHHH VPGY : VPGI : VP GY : SIMVASTATIN

Example 2. Angpt-2 induction inhibits Tie2 to potentiate vascular leakage.

The Angpt/Tie2 system consists of the endothelial-enriched receptor tyrosine kinase called Tie2 and its major circulating ligands, Angpt-1 and Angpt-2. During health, Angpt-1 is produced by peri- endothelial cells and platelets. Angpt-1 is highly matrix-bound and continually activates Tie2. During sepsis and other stressors such as inflammation, Angpt-2 induced in the endothelium through a least two mechanisms: (1) release of pre-formed protein stored in Weibel-Palade bodies and (2) de novo production via transcription of the ANGPT2 gene. Angpt-2 competes for Tie2 binding with Angpt-1, but antagonizes Tie2 in the endothelium. When Angpt-2 is induced, it therefore displaces Angpt-1 and inhibits otherwise tonic Tie2 signaling. Described below are formulations comprised of statin and peptide that demonstrates efficacy in sepsis models.

Example 3. Statins inhibit de novo Angpt-2 production.

Statins inhibit de novo Angpt-2 production, (clockwise from upper left), i. Statins inhibit HMG-CoA reductase (heptagon), blocking translocation of Foxol (blue oval) into the nucleus and binding of Foxol to the ANGPT2 promoter, thereby inhibiting its transcription in endothelium. Statins promote phosphorylation of Foxol at key serine and threonine residues (Thr-24, Ser-256, and Ser-319) in a manner dependent on PI3-kinase. ii. Dose-response in ECs for simvastatin. Endothelial cells in culture produce and secrete Angpt-2 protein tonically. Application of simvastatin at the indicated concentrations reduces Angpt-2 protein as detected in the conditioned media of cultured endothelial cells. The IC50 (fifty percent inhibitory concentration) of simvastatin is calculated from a classic "inhibitor" curve fit performed in GraphPad Prism with a strong correlation coefficient (r) as shown, iii. Parental simvastatin (200 mg/kg intraperitoneal doses injected -24 hrs, Ohr, and +12 hrs relative to induction of sepsis) improves survival in sepsis as induced by cecal ligation and perforation (CLP), but only when lung Angpt-2 expression is intact (siAngpt-2 reduces lung Angpt-2). iv.

Parenteral simvastatin (200 mg/kg intraperitoneal administered concurrently with LPS) lowers lung Angpt-2 during LPS endotoxemia (achieved by injecting 10 mg/kg 0111:B4 serotype Gram negative lipopoly saccharides intraperitoneal). Lungs were harvested for Angpt-2 mRNA measurement via semi-quantitative PCR 16 hrs after injection of simvastatin and LPS. Angpt-2 mRNA was measured on whole lung RNA isolated with TRIzol extraction followed by RNEasy cleanup.

Example 4. Screening simvastatin-peptide formulation. Human microvascular ECs treated with vehicle (NT), 10μΜ or 20 μΜ naked simvastatin (10, 20), or a series of unique

VPGY:VPGI:simvastatin formulation (K series) loaded with 10 μΜ simvastatin, (inset) Cellular uptake of dye-loaded VPGY nanoparticles. Peptides and simvastatin were formulated (1 : 1 or 1 : 1 : 1) for a final concentration of 5 mg/mL in 150mM NaCl. Each unique aggregating peptide constructs or simvastatin were dissolved in DMSO or ethanol (but not limited to these organic solvents- Isopropyl alcohol, ethanol, acetone, dioxane, acetonitrile, methanol, and THF) in separate vials at 50 - 800 mg/ml for peptides and 0.5 to 40 mg/mL for simvastatin. A fix volumes of each was combined then injected or mixed with an aqueous solution that can be water, acid, neutral or alkaline buffer solution (e.g., but not limited to, 150 mM NaCl, Sodium Citrate, Acetate, Phosphate) while stirring or mixing thus allowing for monodisperse particles of varying size and stability. The peptide concentration of the resulting mixture or suspensions are about 5 mg/mL to about 50 mg/mL (weight/volume, w/v). Nano and Micro aggregation can also be induced by adding a precipitation while stirring at about 100 rpm to about 500 rpm for about 1-20 mins. The stirring speed can be varied to control size homogeneity. Dynamic light scattering (DLS) analysis of these constructs confirmed nanostructures formation.

Example 6. VPGY:VPGI:simvastatin (Kl) formulation lowers 24 h Sepsis Score following LPS (Ol l l :B4 serotype injected intraperitoneally at 10 mg/kg). Kl was compared to empty carrier (NT) and unencapsulated simvastatin injected at an equal dose of 400 mg/kg intraperitoneal. The Sepsis Score is computed by a battery of physical observations (including core temperature, spontaneous physical activity, fur appearance, and ocular chemosis) conducted by an operator blinded to treatment group. Example 7. Tie2 heterozygous mice suffer more leakage and death in sepsis. Adult Tie2 heterozygous mice (+/-) were compared to wildtype (+/+) littermate controls for survival following two different models of sepsis: LPS endotoxemia (left) and cecal ligation perforation (right), i. To measure vascular leakage, anesthetized mice are injected intravenously with a blue dye that binds albumin, a large circulating protein that is normally excluded from appearing outside of blood vessels when abnormal leakage is absent. However, in the presence of elevated vascular leakage, the blue dye escapes the blood vessels to accumulate in tissue. Shown are example images of lungs harvested from septic mice after the dye injection procedure, indicative of elevated vascular leakage in Tie2 heterozygous mice. ii. Survival following two different models of sepsis: LPS endotoxemia (left) and cecal ligation perforation (right).

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The disclosure of International Patent Application Publication No. WO 2015/057820 and Crit Care Med. 2015;43(7):e230-40 is incorporated by reference herein in its entirety. All other patents or journal articles cited herein are also incorporated by reference herein in their entireties.