TRAIL receptor agonists used to treat fibrosis

Abstract

This invention relates to the field of biomedicine, disclosing TRAIL receptor agonists for treating fibrotic diseases, as well as related compositions and methods. The compositions and methods can be used to selectively remove hepatic stellate cells, the origin of liver fibrosis and cirrhosis, and activated pancreatic stellate cells, the origin of pancreatic fibrosis and pancreatitis, and can effectively reduce or prevent further chronic fibrosis by simultaneously reducing multiple fibrosis-related molecules secreted or induced by these activated stellate cells.

Description

Cross-references

[0001] This application is a divisional application of Chinese patent application filed on April 17, 2015, with Chinese patent application number 201580021193.1 and the invention title "TRAIL receptor agonist for the treatment of fibrotic diseases", and claims priority to US61 / 982,207, US 61 / 990,530 and US 14 / 645,276.

[0002] The parent application claims priority and benefit to U.S. Provisional Patent Application No. 61 / 982,207, filed April 21, 2014, and U.S. Provisional Patent Application No. 61 / 990,530, filed May 8, 2014, and is a continuation-in-part of U.S. Utility Model Application No. 14 / 645,276, filed March 11, 2015, the disclosure of which is expressly incorporated herein by reference. Technical Field

[0003] This invention generally relates to compositions and methods for treating fibrotic diseases, and more specifically to TRAIL pro-apoptotic agents and methods for treating liver fibrosis and cirrhosis and their complications, as well as fibrosis in other organs such as the pancreas, for example by eliminating the cause of fibrosis, namely activated stellate cells, while simultaneously downregulating the activity of multiple fibrotic molecules. Background Technology

[0004] Fibrotic diseases, particularly those affecting the lungs, liver, pancreas, skin, and kidneys, account for up to 45% of deaths worldwide (Friedman, SL, et al, Science Translational Medicine, 5(167):167sr1 (2013)). For liver disease, there are no antifibrotic agents available for human liver fibrosis and cirrhosis. There is a clinical urgency for the treatment of liver fibrosis due to the increasing prevalence of viral, obesity-related, and alcohol-related fibrosis and cirrhosis, as well as the shortage of liver donors for transplantation.

[0005] In 1985, hepatic stellate cells (HSCs) were identified as a major culprit in the development of liver fibrosis through overexpression of extracellular matrix components (Friedman SL, et al., PNAS, 82(24):8681-5 (1985)). Pancreatic stellate cells (PSCs) are myofibroblast-like cells that play a crucial role in the development of pancreatic fibrosis, pancreatitis, and pancreatic cancer (Omary, MB, et al., J. Clin. Invest. 117(1):50-59 (2007)). In response to pancreatic injury or inflammation, quiescent PSCs are activated into myofibroblast-like cells and express smooth muscle actin, much like HSCs.

[0006] Existing treatments for liver fibrosis have several drawbacks. Some treatment regimens can affect HSCs. Many hepatoprotective drugs weaken or neutralize the upstream inflammatory response, thereby activating HSCs, as has been studied in vitro and in vivo. Vitamin E has been evaluated in clinical trials in non-alcoholic steatohepatitis (NASH) and has been shown to reduce histological liver damage, but without anti-fibrotic effects (Sanyal, AJ., et al., New Eng, J. Mede, 362(18):1675-85 (2010)).

[0007] Hepatocyte growth factor (HGF) has been reported to regulate hepatocyte proliferation, collagen synthesis, and TFG-β expression in hepatocytes (HSCs). Gene therapy or injection of recombinant proteins to deliver HGF can prevent the progression of experimental liver fibrosis. However, concerns remain regarding the use of HGF or HGF mimics, with their potential risks of stimulating hepatocyte growth and increasing tumorigenesis (Fallowfield, JA., American Journal of Physiology -Gastrointestinal and Liver Physiology , 300(5):G709-G15(2011)).

[0008] Agents that prevent HSC activation or proliferation have also been investigated. HSC activation is associated with low levels of PPAR-r expression. Upregulation of PPAR-r or the addition of PPAR-r ligands reverses HSC activation. Some PPAR-r ligands, thiazolidinones, have been tested in animal models, but they only slightly slow the progression of fibrosis in the early stages of the disease (Leclercq, IA, et al., Gut, 55(7):1020-9 (2006)).

[0009] It is also known that statin HMG-CoA reductase inhibitors can inhibit HSC proliferation in vitro and provide beneficial effects on portal hypertension and angiotensin II-induced inflammation in liver fibrosis models. For example, early atorvastatin treatment attenuates HSC activation and collagen deposition after bile duct ligation in rats; however, once fibrosis is established and treatment is initiated (Trebicka, J., et al., Journal of Hepatology, 53(4):702-12 (2010)), atorvastatin is ineffective, suggesting that it is beneficial only as a preventative agent rather than a therapeutic one.

[0010] The renin-angiotensin system plays a crucial role in liver fibrosis and portal hypertension. Studies have shown that angiotensin-converting enzyme inhibitors and AT1R antagonists (sartans) can reduce fibrosis (Yang, L., et al., Journal of Hepatology, 43(2):317-23 (2005)). Treatment of patients with chronic hepatitis C virus (HCV) with the AT1R antagonist losartan slowed the progression of fibrosis and pro-fibrotic genes (Colmenero, J., et al., American Journal of Physiology Gastrointestinal and Liver Physiology, 297(4):G726-34 (2009)).

[0011] TGF-β is a key effector in the pathogenesis of liver fibrosis. Reducing or inhibiting TGF-β synthesis and signal transduction is considered an important therapeutic target. Various strategies for inhibiting TGF-β activity include the use of TFG-β neutralizing antibodies, decoy receptors, siRNAs, and oligonucleotides. Some TGF-β-related molecules have shown anti-fibrotic effects in animal models; however, due to the widespread expression of the TGF-β receptor across all cell types, targeting HSCs can be challenging, and such inhibitors may trigger autoimmune diseases or cell dedifferentiation.

[0012] Chronic pancreatitis (CP) is a disease characterized by progressive and irreversible destruction of the structure and function of the pancreas (Braganza, JM, et al., Lancet, 377(9772):1184-97 (2011)). CP is accompanied by pancreatic fibrosis and persistent abdominal pain. The management of CP and CP-related pain is challenging because CP is currently an incurable condition. There are no drugs available for human use, resulting in a significant lack of medical resources for the CP patient population. CP is identified by significant fibrosis. Pancreatic fibrosis is primarily orchestrated by pancreatic fibrotic cells (PSCs). During pancreatic injury or pathogenesis, quiescent PSCs undergo activation and transform into proliferative, fibrotic, and contractile myofibroblasts, which promote collagen deposition and lead to fibrotic tissue. Therefore, activated PSCs are a major target for anti-fibrotic and analgesic therapies targeting the pancreas (Omary, MB, et al., J. Clin. Invest. 117(1):50-59 (2007)). However, like HSCs, the lack of methods to specifically target and influence activated PSCs in vivo hinders this strategy.

[0013] During fibrosis and after activation of the HSC or PSC, many fibrosis-related molecules are highly upregulated and contribute to the development of fibrosis and its complications. These molecules include, but are not limited to, PDGF, TGFβ, CTGF, MMP, TIMP, and collagen (Friedman, SL, Nat Rev Gastroenterol Hepatol. 7(8):425-36 (2010)). A common antifibrotic strategy is to inhibit the regulation of one of the many fibrosis-related molecules in the body. Inhibiting one fibrosis-related molecule will provide some antifibrotic efficacy; however, because fibrosis is a complex process involving multiple pathways involving many fibrotic molecules, this approach is not efficient in stopping or reversing fibrosis. Simultaneous inhibition or downregulation of multiple fibrosis-related molecules will exhibit strong antifibrotic efficacy; however, it is difficult to target multiple molecules simultaneously under physiological conditions, especially by utilizing a single drug molecule.

[0014] Therefore, the object of the present invention is to provide compositions and methods for treating fibrotic diseases, including liver fibrosis and pancreatic fibrosis.

[0015] Another object of the present invention is to provide compositions and methods for simultaneously inhibiting or downregulating multiple fibrosis-related molecules under physiological conditions.

[0016] Another object of the present invention is to provide compositions and methods for reducing, inhibiting or reversing liver fibrosis and other diseases such as cirrhosis and its complications.

[0017] Another object of the present invention is to provide methods and compositions for reducing or inhibiting liver inflammation.

[0018] Another object of the present invention is a method for treating fibrosis and related complications in other organs, such as pancreatic fibrosis, chronic pancreatitis and its complications such as pain. Summary of the Invention

[0019] Apoptotic agents, such as ligands and agonists of agonistic TRAIL receptors, have been found to induce or increase apoptosis in cells that contribute to fibrosis and underlying diseases, including liver diseases, pancreatic diseases, lung diseases, and skin diseases characterized by fibrosis, cirrhosis, or their complications. The compositions and methods disclosed herein can be used to selectively remove activated hepatic stellate cells (HSCs), the origin of liver fibrosis and cirrhosis, and activated pancreatic stellate cells (PSCs), the origin of pancreatic fibrosis and pancreatitis, and can simultaneously and effectively reduce the regulation of multiple fibrosis-related molecules induced by activated stellate cells. This will overall reduce or reverse fibrosis or prevent further fibrosis-related complications. The compositions typically and effectively target agonistic TRAIL receptors, such as TRAIL-R1 / DR4 and TRAIL-R2 / DR5, selectively expressed in activated HSCs and PSCs under physiological conditions. Ligands and agonists that can target agonistic TRAIL receptors include, but are not limited to, TRAIL-R1 and / or TRAIL-R2 agonists, such as recombinant human (rh)TRAIL, engineered TRAIL analogs, long-acting TRAIL proteins modified with polymers such as polyethylene glycol, copolymers, and branched analogs, and biopolymers such as hyaluronic acid. Long-acting TRAIL-based formulations include polymer systems; TRAIL fusion proteins; agonistic anti-TRAIL-R1 antibodies; agonistic anti-TRAIL-R2 antibodies; and agonistic small molecules or peptides that bind to TRAIL-R1 and / or TRAIL-R2. These agonists, alone or in combination with other therapeutic agents, can reduce or block the development of existing fibrosis in multiple organs or reverse existing fibrosis. Exemplary methods for treating fibrotic diseases include administering an effective amount of a pro-apoptotic agent to an individual in need to induce apoptosis in hepatic stellate cells, pancreatic stellate cells, fibromyoblast cells, fibromyeloblastic cells, activated endothelial cells, or activated epithelial cells, which produces or induces excessive extracellular matrix, leading to undesirable scarring of the liver, pancreas, or other organs. The pro-apoptotic agent may be a TNF-associated apoptosis-inducing ligand (TRAIL) agonist, such as PEGylated TRAIL or an agonist TRAIL antibody. Such antibodies have been clinically tested in cancer patients but have not shown great success. TRAIL may be a protein, for example, in a natural or genetically engineered (recombinant) form. In a preferred embodiment, TRAIL is human TRAIL or a functional fragment or variant thereof. For example, the functional fragment may be a fragment of 281 amino acids of human TRAIL. In a preferred embodiment, the fragment has an amino acid sequence of 114 to 281 or 95 to 281 in the full-length 281-amino acid human form (1-281).

[0020] In some embodiments, the polyethylene glycol-modified TRAIL comprises a trimer TRAIL including a zippered amino acid motif, more preferably an isoleucine zippered motif, which facilitates the formation of a trimer at its N-terminus, and PEG or a derivative thereof, wherein the PEG binds to the N-terminus of at least one monomer of the trimer TRAIL. The PEG or its derivative may be in linear, branched, or trimer form. Exemplary derivatives of PEG include methoxy polyethylene glycol succinimide propionate, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol aldehyde, methoxy polyethylene glycol maleimide, and multibranched polyethylene glycol. In some embodiments, the PEG or its derivative has a molecular weight of 1,000 to 100,000 Da, preferably 5,000 to 50,000 Da.

[0021] A key aspect of this technology is the simultaneous targeting of multiple fibrosis-related molecules by specifically eliminating the fibrosis-originating cells (such as activated hepatic and pancreatic stellate cells) under physiological conditions. This provides a means of treating pathological symptoms that induce excessive extracellular matrix leading to undesirable scarring. Representative conditions include liver fibrosis and cirrhosis, as well as chronic pancreatitis and fibrosis in other organs such as the lungs, skin, heart, and kidneys. It also reduces the amount of ascites, a major complication of cirrhosis, and pain, a major complication of chronic pancreatitis. Attached Figure Description

[0022] Figure 1 This is a bar graph showing apoptosis in the fibroblast-activated human primary hepatic stellate cells (HSCs) induced by TRAIL agonists, PEG-TRIAL, and agonistic TRAIL antibodies. Highly activated HSCs (on days 7 and 14) were more sensitive to TRAIL-induced apoptosis. Apoptosis is expressed as induced cell death (%), calculated as a percentage relative to untreated cells, and measured by a cell death assay. P<0.05 compared to day 1 P<0.01 relative to day 1.

[0023] Figure 2 This diagram illustrates the experimental design for injection regimens of CCl4, CCl4 and TRAIL, and CCl4 and PEG-TRAIL over several weeks of treatment. Regimen 1 was used to examine whether PEG-TRAIL prevented fibrosis, regimen 2 was used to examine whether PEG-TRAIL reversed liver fibrosis, and regimen 3 was used to investigate whether PEG-TRAIL improved cirrhosis.

[0024] Figure 3This is a bar chart showing the relative protein expression levels of α-SMA (α-SMA), a marker of stellate cell activation, in Western blots of isolated liver tissue treated with a medium, CCl4 alone, and CCl4 with PEG-TRAIL. When rats were treated with CCl4, α-SMA levels increased significantly; however, PEG-TRAIL decreased α-SMA expression. P < 0.001 relative to the medium, P<0.05 relative to CCl4.

[0025] Figure 4A and 4B Quantitative collagen deposition (Sirius red staining) in 20 regions of each liver sample. Figure 4A ) and α-SMA positive areas ( Figure 4B A dotted graph.

[0026] Figure 5 It comes from group 3 (treated with media, CCl4+ mediator and CCl4+ PEG-TRAIL). Figure 2 A dot plot of ascites volume (ml) in mice.

[0027] Figure 6 This shows the relative protein expression levels of α-SMA, markers of stellate cell activation, PDGFRβ, and fibrosis markers in Western blots of isolated pancreatic tissues from healthy rats (white bars), rats treated with an ethanol / frog dermalin / Lieber Decarli (LD) diet (black bars), and rats with alcohol-induced chronic pancreatitis (CP) treated with PEG-TRAIL (grey bars). Treatment of rats with ethanol / frog dermalin / LD significantly increased the levels of α-SMA and PDGFRβ; however, PEG-TRAIL reduced the expression of α-SMA and PDGFRβ. #P<0.05 relative to the vector. P < 0.001 relative to CP+ vector, P<0.05 relative to CP+ vector. Invention Details I. Definition As used herein, the term “treatment” includes the suppression, relief, prevention, or elimination of one or more symptoms or side effects associated with the disease, condition, or disorder being treated.

[0028] The terms “reduced,” “inhibited,” “mitigated,” or “decreased” are used relative to a control. Those skilled in the art will readily identify the appropriate control for each experiment. For example, a reduced response in individuals or cells treated with the compound is compared to a response in individuals or cells not treated with the compound.

[0029] As used herein, the term "effective dose" or "therapeutic effective dose" refers to a dose sufficient to treat, suppress, or alleviate one or more symptoms of the treated disease state or to otherwise provide the desired pharmacological and / or physiological effect. The precise dose will vary depending on a variety of factors, such as individual dependence variables (e.g., age, immune system health, etc.), the disease or disorder, and the treatment administered. The effect of an effective dose can be relative to a control. Such a control is known in the art and discussed herein, and can be, for example, the individual's condition before or without administration of the drug or combination of drugs, or, in the case of a combination of drugs, the effect of which can be compared to the effect of administration of only one drug.

[0030] As used herein, the term “combination therapy” refers to a method of treating a disease or its symptoms, or achieving a desired physiological change, including the administration of effective amounts of two or more chemical agents or components to treat the disease or its symptoms, or to produce a physiological change, wherein the chemical agents or components are administered together, for example, as part of the same composition, or individually and independently at the same time or at different times (i.e., the administration of each agent or component is separated from each other by a limited time interval).

[0031] As used herein, the term "dosing regimen" refers to the administration of a drug with regard to its formulation, route of administration, dosage, dosing interval, and duration of treatment.

[0032] As used herein, the term "peptide" includes proteins and fragments thereof. Peptides are disclosed herein as sequences of amino acid residues. Those sequences are written from left to right, from the amino group to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named with three-letter or single-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0033] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains its essential properties. A typical variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. Typically, the difference is limited, making the sequences of the reference polypeptide and the variant very similar overall and identical in many regions. Variants and reference polypeptides can differ in their amino acid sequences through one or more modifications (e.g., substitution, addition, and / or deletion). The substituted or inserted amino acid residues may or may not be residues encoded by the genetic code. Variants of a polypeptide can be naturally occurring, such as allelic variants, or they may be unknown naturally occurring variants.

[0034] The structure of the polypeptides disclosed herein can be modified and altered, and molecules with similar characteristics to the polypeptides (e.g., conserved amino acid substitutions) can still be obtained. For example, certain amino acids can replace other amino acids in the sequence without significant loss of activity. Because the biological functional activity of a polypeptide is defined by its interaction capabilities and properties, certain amino acid sequence substitutions can be made in the polypeptide sequence to obtain polypeptides with similar properties.

[0035] When making such changes, the hydropathic index of the amino acid can be considered. The importance of the amino acid hydropathic index in conferring biological function to peptide interactions is generally understood in the art. It is known that certain amino acids can substitute for other amino acids with similar hydropathic indices or fractions and still result in peptides with similar biological activities. Each amino acid has been assigned a hydropathic index based on its hydrophobic and charge properties. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0036] It is believed that the relative hydrophilicity of amino acids determines the secondary structure of the resulting polypeptide, which in turn limits the interaction of the polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, and antigens. It is known in the art that amino acids can be substituted with another amino acid having a similar hydrophilicity index, and functionally equivalent polypeptides are still obtained. In such modifications, substitution of amino acids with a hydrophilicity index within ±2 is preferred, substitution within ±1 is particularly preferred, and substitution within ±0.5 is even more particularly preferred.

[0037] Similar substitutions of amino acids can also be based on hydrophilicity. The following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine ​​(1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (2.3); phenylalanine (-2.5); tryptophan (-3.4). It should be understood that an amino acid can be substituted for another amino acid with a similar hydrophilicity value, and biological equivalents, particularly immunologically equivalent polypeptides, can still be obtained. In such modifications, the substitution of amino acids with a hydrophilicity value within ±2 is preferred, the substitution of amino acids with a hydrophilicity value within ±1 is particularly preferred, and the substitution of amino acids with a hydrophilicity value within ±0.5 is even more preferred.

[0038] As described above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering the various features described above are well known to those skilled in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). In particular, embodiments of the polypeptide may include variants having approximately 50%, 60%, 70%, 80%, 90%, and 95% sequence identity with the target polypeptide.

[0039] As is known in the art, "identity" refers to the relationship between two or more polypeptide sequences determined by comparing sequences. In the art, "identity" also refers to the degree of sequence correlation between polypeptides determined by matching such sequences between strings. "Identity" can also refer to the degree of sequence correlation between a polypeptide and the full length of a reference polypeptide. "Identity" and "similarity" can be readily calculated using known methods, including but not limited to those described in Computational Molecular Biology. Lesk, A. M ., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W ., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I Griffin, A.M., and Griffin, H.G. ., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology von Heinje, G ., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J ., Eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D ., SIAM J Applied Math., 48: 1073 (1988). The preferred method for determining identity is designed to give the maximum match between the tested sequences. The methods for determining identity and similarity are encoded in a publicly available computer program. The percentage of identity between two sequences can be determined by using... Needelman and Wunsch(J. Mol. Biol., 48: 443-453, 1970) Analysis software using algorithms (e.g., NBLAST and XBLAST) (i.e., the Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.). Default parameters are used to determine the identity of the peptides disclosed herein.

[0040] As an example, the polypeptide sequence may be identical to the reference sequence, i.e., 100% identical, or may include up to a specific integer number of amino acid changes compared to the reference sequence, such that the identity percentage is less than 100%. Such changes are selected from: at least one amino acid deletion, substitution (including conserved and non-conserved substitutions), or insertion, and said changes may occur at the amino or carboxyl terminus of the reference polypeptide sequence or at any of those terminus positions, either individually scattered among amino acids in the reference sequence or in one or more consecutive groups within the reference sequence.

[0041] The number of amino acid changes for a given percentage of identity is determined by multiplying the total number of amino acids in the reference polypeptide by the corresponding percentage of identity (divided by 100), and then subtracting the product from the total number of amino acids in the reference polypeptide.

[0042] As used herein, the term "operably linked" refers to a juxtaposition in which components are configured to perform their normal functions. For example, a control sequence or promoter operably linked to a coding sequence enables the expression of the coding sequence, and an organelle localization sequence operably linked to a protein helps the linked protein to be localized to a specific organelle.

[0043] As used herein, the term "cell type" is a way of grouping or classifying cells in the art. The term cell type refers to a group of cells whose biological characteristics are determined in part by the common biological functions, locations, morphology, structure, and expression of polypeptides, nucleotides, or metabolites.

[0044] As used herein, the term "cell state" refers to the condition of a cell type. Cells are dynamic throughout their lifespan and can achieve various states of differentiation, function, morphology, and structure. As used herein, cell state refers to a specific cell type within its lifespan.

[0045] As used herein, the term “cell surface marker” refers to any molecule present on or near the cell surface that is sufficient to uniquely identify a cell by type or state, such as fractions, peptides, proteins, carbohydrates, nucleic acids, antibodies, antigens, and / or metabolites.

[0046] II. Compositions for the treatment of liver and pancreatic diseases Ligands and agonists of agonistic TRAIL receptors It has been found that ligands and agonists of agonistic TRIAL receptors can be formulated to make the ligands effective in treating fibrosis and / or fibrosis-related complications.

[0047] In a preferred embodiment, the ligand or agonist does not require delivery of a carrier such as particles or matrix that will be effective. For example, although formulations comprising particles and other delivery carriers are provided, in some embodiments the ligand is stable in cycling and remains effective for at least one day, preferably at least two days, without relying on a time-release matrix, particles, or other time-released or degradable carrier.

[0048] The ligands and agonists are typically TRAIL conjugates, which include TRAIL peptides, analogs, or kinases linked to the conjugate molecule, preferably TRAIL or fragments, variants, or fusions thereof, which prolong the in vivo half-life of the TRAIL conjugate compared to TRAIL fragments, variants, or fusions in the absence of the conjugate molecule.

[0049] TRAIL-conjugated formulations and dosage regimens can target and eliminate the origin of fibrosis-inducing activated hepatic stellate cells (HSCs) and pancreatic stellate cells (PSCs) that contribute to fibrosis formation, rather than quiescent stellate cells. By eliminating these originating cells, multiple fibrosis-related molecules secreted or induced by stellate cell activation can be simultaneously inhibited or downregulated. For example, systemic administration of PEG-TRAIL removes activated HSCs or PSC populations and reduces and / or normalizes highly upregulated fibrogenic molecules at the protein and mRNA levels, including α-SMA, type I collagen, type III collagen 3, PDGFR, TGFβ, MMP-2, MMP-3, TIMP-1, TIMP-3, and BMP-7. As discussed in more detail below, the disclosed compositions are typically administered to individuals in need in amounts that effectively target and eliminate the originating cells of fibrosis while simultaneously reducing one or more fibrosis-related molecules.

[0050] A. TRAIL peptides and analogues TRAIL-conjugates include TRAIL domains attached to conjugate molecules. TRAIL domains are typically TRAIL peptides, analogs, or mimics, preferably TRAIL or fragments, variants, or fusions thereof.

[0051] TRAIL TRAIL / Apo2L ( TNFSF10 It was initially identified in a search of the EST database of genes homologous to known TNF superfamily ligands (Benedict et al., J. Exp. Med ., 209(11):1903-1906 (2012)). In humans, TRAIL binds to two pro-apoptotic death receptors (DRs), TRAIL-R1 and TRAIL-R2 (TNFRSF10A and 10B), as well as two other membrane receptors that do not induce death, and can act as a decoy for death signal transduction. The binding of TRAIL to its homologous DR induces the formation of a death-inducing signal transduction complex, ultimately leading to caspase activation and the initiation of apoptosis (Benedict et al., 209(11):1903-1906 (2012)). J. Exp. Med., 209(11):1903-1906 (2012)).

[0052] In some embodiments, the TRAIL conjugate includes a TRAIL peptide or an agonistic TRAIL receptor-binding fragment or a variant thereof.

[0053] The nucleic acid and amino acid sequences of human TRAIL are known in the art. For example, the amino acid sequence of human TRAIL is... MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG (SEQ ID NO:1, (UniProtKB database accession number P50591 (TNF10_HUMAN))). In some embodiments, the TRAIL conjugate comprises a TRAIL peptide containing or having the amino acid sequence of SEQ ID NO:1.

[0054] Preferably, the TRAIL is a soluble TRAIL. Endogenous full-length TRAILs include a cytoplasmic domain, a transmembrane domain, and an extracellular domain. Typically, a soluble TRAIL is a fragment of a full-length TRAIL lacking the cytoplasmic and transmembrane domains. Therefore, a soluble TRAIL can be an extracellular domain of a TRAIL (e.g., the extracellular domain of SEQ ID NO: 1) or a functional fragment thereof. The common extracellular domain of the TRAIL of SEQ ID NO: 1 is amino acids 39-281 of SEQ ID NO: 1. Therefore, in some embodiments, the TRAIL conjugate comprises a TRAIL peptide containing or having amino acids 39-281, 41-281, 91-281, 92-281, 95-281, and 114-281 of SEQ ID NO: 1, or a functional fragment or variant thereof.

[0055] In some embodiments, the TRAIL conjugate comprises a functional fragment or variant of SEQ ID NO: 1 that can be activated by TRAIL-R1 and / or TRAIL-R2. The fragment or variant of SEQ ID NO: 1 may have 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more than 99% sequence identity with SEQ ID NO: 1.

[0056] Preferably, the functional fragment or variant thereof comprises the extracellular domain of SEQ ID NO: 1 or a functional fragment thereof. It is believed that the C-terminal 150 amino acids of TRAIL comprise a receptor-binding domain. Therefore, in some embodiments, the functional fragment comprises amino acids 132-281 of SEQ ID NO: 1. In other specific embodiments, the fragment is amino acids 95-281 or 114-281 of SEQ ID NO: 1.

[0057] Variants may have one or more substitutions, deletions, or additions relative to SEQ ID NO: 1, or any combination thereof. In some embodiments, variants are naturally occurring alternative sequences, splice variants or substitution, addition, or deletion variants or their extracellular domains or functional fragments or alternative sequences, splice variants or substitution, addition, or deletion variants. Naturally occurring alternative sequences and variants are disclosed in UniProtKB database accession number P50591 (TNF10_HUMAN), version 140 (last modified January 22, 2014).

[0058] TRAIL analogues TRAIL can interact with its receptor as a trimer. Therefore, in some embodiments, the ligand or agonist used in the methods disclosed herein is a polymer or can form a polymer, preferably a trimer. The trimer can be a homotrimer or a heterotrimer.

[0059] All TRAIL proteins described herein can be prepared using standard techniques for isolating natural or recombinant proteins and chemically modified as described herein.

[0060] TRAIL conjugates may include TRAIL analogs, or agonistic TRAIL receptor-binding fragments or variants thereof. TRAIL analogs are known in the art. In a preferred embodiment, the analog has increased affinity or specificity for one or more agonistic TRAIL receptors (e.g., TRAIL-R1 (DR4) and / or TRAIL-R2 (DR5)) and decreased affinity or specificity for one or more antagonistic or decoy TRAIL receptors (e.g., receptors DcR1 and DcR2) or combinations thereof, compared to wild-type or endogenous TRAIL.

[0061] In some embodiments, the analogue is a DR4-selective mutant of wild-type TRAIL. DR-4-selective mutants are known in the art and disclosed, for example, in Tur, J. Biological Chemistry In , 283(29):20560-8(2008). In a particular embodiment, the analog is a variant of SEQ ID NO: 1 having a D218H or D218Y substitution, or a functional fragment thereof (e.g., an extracellular domain).

[0062] In some implementations, the analogue is a DR5-selective mutant of wild-type TRAIL. Specific DR-5-selective mutants include variants of SEQ ID NO: 1 having D269H, D269H / E195R, or D269H / T214R, along with functional fragments (e.g., extracellular domains). These variants are described in van der Sloot, Proc. Nat. Acad. Sci. U SA 103(23):8634-9 (2006).

[0063] TRAIL fusion protein The TRAIL conjugate can be a TRAIL fusion protein. The TRAIL fusion polypeptide has a first fusion partner comprising all or part of the TRAIL protein extracellular domain, which (i) fuses directly to a second polypeptide, or (ii) optionally fuses to a linker peptide sequence fused to the second polypeptide. The fusion protein optionally contains a domain for dimerizing or polymerizing two or more fusion proteins. The peptide / peptide linker domain can be a separate domain or may be contained within one of the other domains of the fusion protein (the TRAIL polypeptide or the second polypeptide). Similarly, the domain for dimerizing or polymerizing the fusion protein can be a separate domain or may be contained within one of the other domains of the fusion protein (the TRAIL polypeptide, the second polypeptide, or the peptide / peptide linker domain). In one embodiment, the dimerizing / polymerizing domain and the peptide / peptide linker domain are identical.

[0064] The fusion protein disclosed in this article can be of formula I: N-R1-R2-R3-C Where "N" represents the N-terminus of the fusion protein, "C" represents the C-terminus of the fusion protein, "R1" is the TRAIL peptide, "R2" is an optional peptide / peptide linker domain, and "R3" is the second peptide. Alternatively, R3 can be the TRAIL peptide, and R1 can be the second peptide.

[0065] Fusion proteins can be dimerized or polymerized. Dimerization or polymerization can occur between two or more fusion proteins through the addition or subtraction of dimerizing or multiplying domains. Alternatively, dimerization or polymerization of fusion proteins can occur through chemical cross-linking. The resulting dimers or polymers can be homodimers / homopolymers or heterodimers / heteropolymers.

[0066] The presence of a second polypeptide can alter the solubility, stability, affinity, and / or valence of the TRAIL fusion polypeptide. As used herein, “valence” refers to the number of available binding sites per molecule. In some embodiments, the second polypeptide contains one or more domains of the constant region of the immunoglobulin heavy chain, preferably having amino acid sequences corresponding to the hinge regions CH2 and CH3 of the human immunoglobulin Cγ1 chain or the hinge regions CH2 and CH3 of the mouse immunoglobulin Cγ2a chain. In certain dimer fusion proteins, the dimer is formed by the covalent bonding of Cys residues in the hinge regions of two Ig heavy chains having the same Cys residues linked by disulfide bonds in the dimerized normal Ig heavy chain.

[0067] In one specific implementation, the TRAIL fusion protein is a TRAIL-mimicking protein comprising three TRAIL-origin subsequences, called a single-chain TRAIL receptor-binding domain (scTRAIL-RBD), combined in a single polypeptide chain, such as Gieffers. Molecular Cancer Therapeutics As described in , 12(12):273547 (2013). Two so-called scTRAIL-RBDs (each with three receptor binding sites) can come into close proximity to produce a multimeric fusion protein with a hexavalent binding pattern. In some embodiments, multiplication is achieved by fusing the Fc portion of the C-terminus of a human immunoglobulin G1 (IgG1) mutant protein with a scTRAIL-RBD peptide, thereby generating six receptor binding sites per drug molecule.

[0068] Based on scFv linker modification of a targeted scTRAIL, which is primarily composed of a dimer (DbscTRAIL), forced dimerization of scFv-scTRAIL exceeds the activity of non-targeted scTRAILs in some target cell types by approximately 100-fold (Siegemund, ibid.). Increased DbscTRAIL activity was also demonstrated on target-negative cells, indicating that oligomerization, equivalent to at least the dimer assembly of the standard TRAIL itself, enhances apoptosis signaling in addition to targeting. Therefore, in a preferred embodiment, the TRAIL fusion protein has a multimerizing domain that can lead to, for example, dimer, trimer, or hexamer molecules, such as dimerizing or trimerizing domains or combinations thereof.

[0069] Another fusion protein that promotes trimer formation includes the receptor-binding fragment of TRAIL, whose amino terminus is fused to the trileucine or isoleucine zipper domain.

[0070] The TRAIL fusion protein and the results of using the fusion protein in functional assays are also described in Wahl. Hepatology ,57(2):625-36 (2013)

[0071] Methods for producing peptides The disclosed TRAIL peptides, their fragments, variants, and fusions can be prepared using conventional techniques known in the art. Isolated peptides can be obtained, for example, through chemical synthesis or by recombinant production in host cells. For recombinant production of peptides, nucleic acids containing nucleotide sequences encoding fusion proteins can be used for transformation, transduction, or transfection of bacterial or eukaryotic host cells (e.g., insect, yeast, or mammalian cells). Typically, nucleic acid constructs include regulatory sequences operatively linked to the nucleotide sequences encoding the peptide. These regulatory sequences (also referred to herein as expression control sequences) typically do not encode gene products but rather influence the expression of the nucleic acid sequences operatively linked to them.

[0072] Useful prokaryotic and eukaryotic systems for expressing and producing peptides are well known in the art, including, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

[0073] In eukaryotic host cells, many virus-based expression systems can be used to express peptides. Virus-based expression systems are well known in the art and include, but are not limited to, baculoviruses, SV40, retroviruses, or vaccinia-based viral vectors.

[0074] Mammalian cell lines that stably express peptides can be generated using expression vectors with suitable control elements and selectivity markers. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810 815) are suitable for expressing variant peptides in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Other suitable expression systems include the GS Gene Expression System™, available from Lonza Group Ltd.

[0075] After introducing the expression vector via electroporation, lipid transfection, co-precipitation with calcium phosphate or calcium chloride, DEAE dextran, or other suitable transfection methods, stable cell lines can be selected (e.g., through metabolic selection or antibiotic resistance to G418, kanamycin, or hygromycin). Transfected cells can be cultured to express the target peptide, and the peptide can be recovered from, for example, cell culture supernatant or from lysed cells. Alternatively, fusion proteins can be generated by (a) ligating the amplified sequence into a mammalian expression vector such as pcDNA3 (Invitrogen Life Technologies), and (b) in vitro transcription and translation using wheat germ extract or rabbit reticulocyte lysates.

[0076] Peptides can be separated using chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. In some embodiments, the peptide can be engineered to include additional domains containing an amino acid sequence that allows the peptide to be captured onto an affinity matrix. For example, Fc fusion peptides can be separated from cell culture supernatants or cytoplasmic extracts using a protein A column. Furthermore, tags such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid peptide purification. Such tags can be inserted anywhere within the peptide, including at the carboxyl or amino terminus. Other available fusions include enzymes that facilitate the detection of the peptide, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify peptides. Peptides can be further engineered to include a secretion signal (if no secretion signal is already present) that causes the peptide to be secreted by the cell from which it is produced. The secreted peptide can then be conveniently separated from the cell culture medium.

[0077] B. Antibody Composition and Preparation Method Purified TRAIL receptor peptides, fragments, fusions, or their antigens or epitopes can be used to prepare antibodies that specifically bind to the TRAIL receptor. Antibodies can be prepared using any suitable method known in the art. Subsequently, the functional activity (e.g., agonist or antagonist activity) of the antibodies can be screened using methods known in the art.

[0078] Antibodies can be produced in cell cultures, bacteriophages, or various animals. In one embodiment, the antibody is a mammalian antibody. Phage technology can be used to isolate an initial antibody or to generate variants with altered specificity or affinity characteristics. Such techniques are conventional and well known in the art. In one embodiment, the antibody is produced by recombinant methods known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector containing a DNA sequence encoding an antibody. One or more vectors can be used to transfect DNA sequences expressing at least one VL and one VH region in host cells. Exemplary descriptions of recombinant methods for antibody generation and production include Delves, Antibody Production: Essential Techniques (Antibody Production: Key Technologies) (Wiley, 1997); Shephard et al., Monoclonal Antibodies (monoclonal antibodies) (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles and Practice (Academic Press, 1993); Current Protocols in Immunology (Immunology Laboratory Guide) (John Wiley & Sons, latest version).

[0079] The disclosed antibodies can be modified using recombinant methods to increase their potency in mediating desired functions. Recombinant methods can be used to modify antibodies through substitution. Typically, the substitution will be a conserved substitution. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, for example, U.S. Patent Nos. 5,624,821, 6,194,551, WO 9958572; and Angal et al., Mol. Immunol. 30:105-08 (1993). Amino acid modifications include the deletion, addition, and substitution of amino acids. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Antibodies are typically labeled by covalently or non-covalently linking a substance that provides a detectable signal. Various labeling and conjugation techniques are known and widely reported in scientific and patent literature. These antibodies can be screened for binding to the TRAIL receptor. See, for example, Antibody Engineering: A Practical Approach Practice Methods (Oxford University Press, 1996).

[0080] Suitable antibodies with the desired biological activity can be identified by in vitro assays, including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo assays such as inhibition of tumor growth.

[0081] Antibodies that can be used in the disclosed compositions and methods include any class of intact immunoglobulins (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least an antigen-binding variable domain of the antibody. The variable domains differ in sequence between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not typically evenly distributed across the variable domains of the antibody. It is usually concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable domains. The more highly conserved portions of the variable domains are called frames (FRs). The variable domains of the native heavy and light chains each contain four FR regions, which are predominantly β-sheet conformations, linked by three CDRs to form loops, and in some cases form part of a β-sheet structure. The CDRs in each chain are closely clustered together by the FR regions and, together with CDRs from other chains, facilitate the formation of the antigen-binding site of the antibody.

[0082] Biologically active antibody fragments are also disclosed. These fragments, whether or not linked to other sequences, include insertions, deletions, substitutions, or other selective modifications of specific regions or amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to an unmodified antibody or antibody fragment.

[0083] The technology is also applicable to the generation of single-chain antibodies specific to the antigenic proteins of this disclosure. Methods for generating single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be generated by fusing variable domains of the heavy and light chains together using short peptide linkers, thereby reconstructing the antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs) have been developed in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via a peptide or linker of 15 to 25 amino acids without significantly disrupting antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together with their correct conformational orientation.

[0084] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by generating a single peptide chain with two VH and two VL regions, resulting in a tandem scFv. ScFvs can also be engineered with linker peptides that are too short for the two variable regions to fold together (approximately 5 amino acids), forcing the scFv to dimerize. This type is called a biantibody. Biantibodies have been shown to have dissociation constants up to 40 times lower than their corresponding scFvs, meaning they have much higher affinity for their targets. Shorter linkers (one or two amino acids) lead to the formation of trimers (tri-chain antibodies or tri-chains). Tetraantibodies have also been generated. They exhibit even higher affinity for their targets compared to biantibodies.

[0085] Monoclonal antibodies are derived from substantially homogeneous groups of antibodies, meaning that individual antibodies within the group are identical except for possible naturally occurring mutations that may exist in a small subset of antibody molecules. Monoclonal antibodies include “chimeric” antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired antagonistic activity.

[0086] Monoclonal antibodies can be prepared using any method for producing monoclonal antibodies. In the hybridoma approach, mice or other suitable host animals are typically immunized with an immunogen to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunogen. Alternatively, lymphocytes can be immunized in vitro.

[0087] Antibodies can also be prepared using recombinant DNA methods. The DNA encoding the disclosed antibody can be easily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies. Libraries of antibodies or active antibody fragments can also be generated and screened using phage display technology.

[0088] Human and humanized antibodies Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and can therefore elicit undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the described method is intended to reduce the chance that the antibody administered to humans will induce an undesirable immune response.

[0089] Transgenic animals (e.g., mice) capable of producing the complete components of human antibodies in the absence of endogenous immunoglobulin production after immunization can be used. For example, homozygous deletion of the antibody heavy chain linker region (J(H)) gene in chimeric and germline mutant mice has been described as resulting in complete suppression of endogenous antibody production. Transferring human germline immunoglobulin gene arrays into such germline mutant mice will result in the production of human antibodies after antigen challenge. Optionally, antibodies are produced in other species and “humanized” for human administration. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding sequence of antibodies) containing minimal sequences derived from non-human immunoglobulins. Humanized antibodies comprise human immunoglobulins (receptor antibodies) whose CDR residues from the recipient antibody are replaced by residues from the CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit, exhibiting the desired specificity, affinity, and capability. In some cases, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in the receptor antibody or in the imported CDR or framework sequence. Typically, humanized antibodies will substantially contain at least one, usually two, variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the FR regions are those of the human immunoglobulin common sequence. Most preferably, humanized antibodies will also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins.

[0090] Methods for humanizing nonhuman antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are often referred to as “input” residues and are usually derived from an “input” variable domain. Antibody humanization techniques generally involve using recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Thus, the humanized form of a nonhuman antibody (or fragment thereof) is a chimeric antibody or fragment in which a variable domain, substantially smaller than the whole human, has been replaced by a corresponding sequence from a nonhuman species. In practice, humanized antibodies are often human antibodies in which some CDR residues and possibly some FR residues are replaced with residues from similar sites in rodent antibodies.

[0091] To reduce antigenicity, the selection of human variable domains (light and heavy chains) used to prepare humanized antibodies is crucial. Following a "best-fit" approach, sequences of variable domains for rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent is then accepted as the human frame (FR) for the humanized antibody. Another approach uses a specific frame derived from a specific subgroup of the light or heavy chain, based on a common sequence of all human antibodies. The same frame can be used for several different humanized antibodies.

[0092] More importantly, the antibody is humanized, retaining its high affinity for the antigen and other favorable biological properties. To achieve this, humanized antibodies are preferably prepared by analyzing the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Available computer programs illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues affecting the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from common and input sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

[0093] Single-chain antibodies Methods for generating single-chain antibodies are well known to those skilled in the art. Single-chain antibodies are generated by fusing variable domains of the heavy and light chains together using short peptide linkers, thereby reconstructing antigen-binding sites on a single molecule. Single-chain antibody variable fragments (scFvs) have been developed in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via a peptide or linker of 15 to 25 amino acids without significantly disrupting antigen binding or binding specificity. Linkers are selected to allow the heavy and light chains to bind together with their correct conformational orientation. These Fvs lack the constant regions (Fc) present in the heavy and light chains of native antibodies.

[0094] Monovalent antibodies In vitro methods are also suitable for preparing monovalent antibodies. Antibody digestion can be performed using conventional techniques known in the art to produce its fragments, particularly Fab fragments. For example, papain can be used for digestion. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site and a residual Fc fragment. Pepsin treatment produces fragments called F(ab')2 fragments, which have two antigen-binding sites and are still capable of cross-linking antigens.

[0095] The Fab fragments produced during antibody digestion also contain a constant domain of the light chain and a first constant domain of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the heavy chain domain, including one or more cysteine ​​residues from the antibody hinge region. The F(ab')2 fragment is a divalent fragment containing two Fab' fragments linked by disulfide bonds through the hinge region. Fab'-SH is the name given to Fab' in this paper, where the cysteine ​​residues in the constant domain have free thiol groups. Antibody fragments are initially produced as Fab' fragment pairs with a hinge cysteine ​​residue between them. Other chemical couplings of antibody fragments are also known.

[0096] Hybrid antibodies Antibodies can be hybrid antibodies. In a hybrid antibody, one pair of heavy and light chains is homologous to a pair of heavy and light chains found in an antibody targeting one epitope, while another pair of heavy and light chains is homologous to a pair found in an antibody targeting another epitope. This results in a multifunctional valence, meaning that a bivalent antibody has the ability to bind to at least two different epitopes simultaneously. Such hybrids can be formed by the fusion of hybridomas that produce antibodies of the corresponding components or through recombinant techniques. Of course, such hybrids can also be formed using chimeric chains.

[0097] Methods for preparing antibodies using protein chemistry One method for generating proteins containing antibodies is to link two or more peptides or polypeptides together using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using Fmoc (9-fluorenylmethoxycarbonyl) or Boc (tert-butoxycarbonyl) chemistry (Applied Biosystems, Inc., Foster City, CA) using currently available laboratory equipment. Those skilled in the art will readily understand that, for example, peptides or polypeptides corresponding to antibodies can be synthesized via standard chemical reactions. For example, peptides or polypeptides can be synthesized and not cleaved from their synthetic resin, while other fragments of the antibody can be synthesized and subsequently cleaved from the resin, exposing functionally blocked terminal groups on the other fragments. These two fragments can be covalently linked at their carboxyl and amino ends, respectively, via peptide bonds to form an antibody or a fragment thereof. Alternatively, peptides or polypeptides can be synthesized independently in vivo as described above. Once separated, these independent peptides or polypeptides can be linked together via similar peptide condensation reactions to form an antibody or an antigen-binding fragment thereof.

[0098] For example, enzymatic ligation of cloned or synthetic peptide segments allows relatively short peptide fragments to be linked to produce larger peptide fragments, polypeptides, or complete protein domains. Alternatively, native chemical ligation of synthetic peptides can be used to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of two chemical steps. The first step is a chemoselective reaction of an unprotected synthetic peptide-α-thioester with another unprotected peptide segment containing an amino-terminal Cys residue, yielding a thioester-linked intermediate as the initial covalent product. Under unchanged reaction conditions, this intermediate undergoes a spontaneous, rapid intramolecular reaction to form a native peptide bond at the linker site.

[0099] C. Conjugates and Complexes The disclosed TRAIL conjugates also include a second conjugate molecule linked to the TRAIL domain or an antibody portion that does not bind to the TRAIL receptor.

[0100] Polyepoxides such as PEG Hydrophilic polymers such as polyepoxides or their copolymers, such as PLURONIC® sold by BASF, can be covalently bound to the molecule to improve the pharmacokinetic and pharmacodynamic properties of TRAIL (Kim, et al.). Bioconjugate Chem ., 22(8), pp 1631–1637 (2011)). Studies have shown that PEG-derived TRAIL analogs maintain anticancer activity while exhibiting higher metabolic stability in plasma, prolonged pharmacokinetic properties, and a longer circulating half-life (Chae, et al., 22(8), pp 1631–1637 (2011)). Molecular cancer therapeutics 9(6):1719-29 (2010); Kim, et al., Bioconjugate chemistry, 22(8):1631-7 (2011); Kim, et al., Journal of pharmaceutical sciences 100(2):482-91 (2011); Kim, et al., Journal of controlled release: official journal of the Controlled Release Society 150(1):639 (2011)).

[0101] Therefore, in some embodiments, the TRAIL domain is derivatized with one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)) or derivatives thereof. PEG derivatives include, but are not limited to, methoxy polyethylene glycol succinimide propionate, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol aldehyde, methoxy polyethylene glycol maleimide, and branched polyethylene glycol.

[0102] The precise number of EG or derivative units depends on the desired activity, plasma stability, and pharmacokinetic profile. For example, Kim et al. (ibid.) reported that 2, 5, 10, 20, and 30K-PEG-TRAILs resulted in longer circulating half-lives of 3.9, 5.3, 6.2, 12.3, and 17.7 hours, respectively, in mice, relative to a circulating half-life of 1.1 hours for TRAILs. In some embodiments, the molecular weight of PEG is between about 1 and 100 kDa, preferably between about 1 and 50 kDa. For example, PEG may have a molecular weight of “N” kDa, where N is any integer between 1 and 100. PEG may have a molecular weight of “N” Da, where N is any integer between 1,000 and 1,000,000. In a specific embodiment, the molecular weight of PEG is “N” Da, where “N” is between 1,000 and 50,000, or more preferably between 5,000 and 50,000.

[0103] Apoptosis-inducing agents can be conjugated with either straight-chain or branched-chain PEG. Some studies have shown that proteins derived from branched-chain PEG have a prolonged in vivo circulating half-life compared to straight-chain PEG-proteins, believed to be partly due to the larger hydrodynamic volume of branched-chain PEG-proteins. (Fee, et al.) Biotechnol Bioeng ., 98(4):725-3 (2007).

[0104] Peptide ligands can be derivatized at the C-terminus or preferably at the N-terminus using methods known in the art.

[0105] TRAIL-PEG conjugates can be represented by the following formula: XL-(PEG) n , in X represents the TRAIL protein. L indicates a connector. PEG stands for branched poly(ethylene glycol) chain, and n is an integer selected from 2, 3, 4, 5, 6, 7 or 8.

[0106] In some implementations, n is 2.

[0107] Polyepoxides are coupled to proteins via linkers. The linkers can be polyepoxides, and preferably two polyepoxide polymers are linked to the protein.

[0108] In one specific embodiment, the TRAIL-conjugate is a PEG-conjugate comprising a TRAIL domain and a PEG, wherein the TRAIL domain comprises a truncated form of human TRAIL, for example, the full-length form of human TRAIL (1-281) from arginine-114 to glycine-281, and the PEG has a molecular weight of 1,000 to 100,000 Daltons, preferably 5,000 to 50,000 Daltons.

[0109] N-terminal modified PEG-TRAIL conjugates can be obtained by reacting the N-terminal amine of the TRAIL domain with the aldehyde group of PEG in the presence of a reducing agent. PEG and TRAIL can be reacted in a molar ratio of 2 to 10, or preferably 5 to 7.5 (PEG / TRAIL).

[0110] In a preferred embodiment, the TRAIL conjugate includes a zippered amino acid motif, such as an isoleucine zippered motif, which allows the formation of a trimer among the three TRAIL conjugate monomers.

[0111] The PEG chains preferably, but not necessarily, have equal molecular weights. An exemplary molecular weight range for each PEG chain is about 10 kDa to 60 kDa, preferably about 20 kDa to 40 kDa. PEG40 is a synthetic branched PEG moiety having a molecular weight of 40 kDa: 20 + 20 kDa (per PEG chain).

[0112] The trimer PEG moiety can consist of branched PEG chains connected to the linker arms. A visual description of the trimer PEG moiety is provided below.

[0113] The following trimer PEGs were synthesized: YPEG42, YPEG43.5, YPEG45, YPEG50 and YPEG60.

[0114] YPEG42 is the trimer PEG moiety with a molecular weight of 42 kDa: (20 + 20 kDa) (branched PEG) + 2 kDa (linker arm).

[0115] YPEG43.5 is the trimer PEG moiety, which has a molecular weight of 43.5 kDa: (20 + 20 kDa) (branched PEG) + 3.5 kDa (linker arm).

[0116] YPEG45 is the trimer PEG moiety with a molecular weight of 45 kDa: (20 + 20 kDa) (branched PEG) + 5 kDa (linker arm).

[0117] YPEG50 is the trimer PEG moiety, which has a molecular weight of 50 kDa: (20 + 20 kDa) (branched PEG) + 10 kDa (linker arm).

[0118] YPEG60 is a trimer PEG moiety with a molecular weight of 60 kDa: (20 + 20 kDa) (branched PEG) + 20 kDa (linker arm).

[0119] Connector section The protein or peptide is covalently linked to the branched PEG moiety via a linker. The linker is a polymer and typically has an atomic length of at least 800 angstroms. Typically, the linker has atomic lengths of about 800 to about 2,000 angstroms, about 800 to about 1,500 angstroms, about 800 to about 1,000 angstroms, or about 900 to about 1,000 angstroms. It should be understood that the atomic distances listed above refer to the fully extended polymer, and when in a solid or solution state, the linker can fold or coil, such that the actual distance between the branched PEG and the protein or peptide is less than the atomic lengths listed above.

[0120] In some embodiments, the linker is a poly(ethylene glycol) derivative with a molecular weight of about 1 kDa to 30 kDa, preferably about 2 kDa to 20 kDa. The linker may also be a natural or non-natural amino acid with a length of at least 80 units.

[0121] PEG alternatives for connectors include synthetic or natural water-soluble biocompatible polymers such as polyethylene oxide, polyvinyl alcohol, and polyacrylamide; proteins such as hyaluronic acid and chondroitin sulfate; and celluloses such as hydroxymethyl cellulose, polyvinyl alcohol, and polyhydroxyalkyl (meth)acrylates.

[0122] Proteins and peptides can be covalently bound to linkers using conventional chemical methods. Primary amine groups, such as those found at the N-terminus or in lysine residues, will react with aldehydes and their equivalents under reducing conditions to yield amines. (Molineux, Current pharmaceutical design, 10(11): 1235-1244 (2004)). For example, the hydrogen sulfide (-SH) group found in cysteine ​​residues can undergo conjugated addition with various Michael acceptors, including acrylic acid and methacrylic acid derivatives and maleimides (Gong et al., British Journal of Pharmacology, 163(2):399-412(2011)). Other suitable nucleophiles found in peptides and proteins include disulfide bonds (Brocchini, et al., Nature protocols, 1:2241-2252 (2006)) and histidine residues (Cong, et al., Bioconjugate Chemistry 23(2):248-263 (2012)).

[0123] Linkers can be covalently attached to proteins or peptides using conventional chemical methods. For example, linker polymers can be derivatized at one end with an electrophilic group such as aldehydes, epoxides, halogens (chlorine, bromine, iodine), sulfonates (toluenesulfonates, methanesulfonates), Michael acceptors, or activated carboxylic esters, and then reacted with nucleophilic amine or thiol groups in the protein or peptide. Suitable Michael acceptors include acrylic acid and methacrylic acid derivatives, such as acrylamide, methacrylamide, acrylates, and methacrylates, as well as maleimides. Suitable activated carboxylic esters include nitrobenzene carbonate and NHS (N-hydroxysuccinate) esters. In other embodiments, peptides and proteins containing arginine residues can be covalently attached to linkers containing reactive 1,3-diketone functional groups.

[0124] Conjugates can be prepared by first linking the linker to a peptide or protein and then linking the linker to a branched poly(ethylene glycol), or by first linking the linker to a branched poly(ethylene glycol) and then linking the linker to a peptide or protein. The optimal order of bond formation is determined by the specific chemical transformations involved.

[0125] macromolecules In other embodiments, TRAIL can be derived into a long-acting TRAIL with an extended half-life using reported methods, employing biopolymers or peptides; for example, but not limited to, the use of chemically conjugated hyaluronic acid (Yang et al., Biomaterials 32(33);8722-8729 (2011), Stockpiled peptides (Amiram et al., Proc Natl Acad Sci USA, 110(8); 27922792 (2013), US Publication No. US 2013-0178416 A1) and TRAIL linked to an extended recombinant peptide (US Publication No. US 2010-0239554 A1).

[0126] complex The TRAIL domain can complex with a negatively charged portion. In some embodiments, the negatively charged portion can facilitate the loading of ligands or agonists into the nanoparticles for prolonged, sustained, or time-dependent release. In some embodiments, the negatively charged portion itself mediates the prolonged, sustained, or time-dependent release of the ligand or agonist. Preferably, the negatively charged portion substantially does not diminish the ability of the ligand or agonist to induce or enhance apoptosis in immune cells or synovial cells.

[0127] The formation of a complex between positively charged TRAIL and negatively charged chondroitin sulfate (CS) (CS / TRAIL) was developed and shown to promote the loading of TRAIL in poly(lactide-glycolic acid) copolymer (PLGA) microspheres (MS) without impairing the activity of TRAIL (Kim, et al., Journal of Pharmacy and Pharmacology , 65(1):11–21 (2013). A nanocomposite of approximately 200 nm was formed at pH 5.0 with a weight ratio of 2 TRAIL to CS(TC2), which exhibited a loading efficiency >95% higher than that of the natural TRAIL complex in PLGA MS prepared via a multi-emulsion method. Therefore, in some embodiments, ligands or agonists, particularly TRAIL peptides and variants, functional fragments and their fusion proteins or conjugates thereof such as PEG-conjugates, are complexed with chondroitin sulfate and optionally loaded into micron or nanoparticles, such as PLGA-based particles.

[0128] In other embodiments, ligands or agonists, particularly TRAIL peptides, their variants, functional fragments, and fusion proteins, or conjugates such as PEG-conjugates, are complexed with hyaluronic acid (HA). PEG-TRAIL and HA nanocomposites prepared by mixing positively charged PEG-TRAIL and negatively charged HA have shown sustained in vivo delivery with negligible loss of bioactivity compared to PEG-TRAIL (Kim, et al.). Biomaterials , 31(34):9057-64(2010)). Delivery was further enhanced by applying nanoparticles in a solution containing 1% HA.

[0129] D. Targeted Part TRAIL conjugates, compositions containing TRAIL conjugate reagents, and delivery carriers for TRAIL conjugate reagents may include a targeting portion. In some embodiments, the targeting portion increases the targeting or accumulation of the pro-apoptotic agent to a target organ or target cells.

[0130] In a preferred embodiment, the targeting portion increases the targeting or accumulation of the pro-apoptotic agent in the liver and pancreas, more preferably increasing hepatic stellate cells and pancreatic stellate cells. Compositions and methods for liver targeting are known in the art, see, for example, U.S. Publication No. 2013 / 0078216 and Poelstra, et al., J., which describe compositions and methods for targeting hepatocytes. Control Release , 161(2):188-97 (2012), which identifies target cells for each liver disease and examines strategies for drug delivery to these cells. The use of proteins, viruses, polymers, and liposomes can all be used to enhance targeting to the liver, or more preferably hepatic stellate cells. In some embodiments, the liver-targeting molecule is fused or conjugated to the pro-apoptotic agent itself, or conjugated to a composition containing the pro-apoptotic agent or a delivery carrier carrying the pro-apoptotic agent (e.g., a carrier such as microparticles or nanoparticles, liposomes, etc.).

[0131] The molecule can target proteins expressed in the liver or pancreas, or preferably proteins expressed on or in the microenvironment surrounding hepatic or pancreatic stellate cells. The target portion can target proteins used in the art to recognize hepatic or pancreatic stellate cells. Preferably, the pro-apoptotic agent, compositions thereof, and the medium for delivering them to treat liver diseases and liver fibrosis are (1) specifically targeted at activated HSCs and preferably do not bind to myofibroblasts present in other tissues or quiescent HSCs; (2) reachable to areas with active fibrosis formation; and (3) well tolerated by the immune system and not nonspecifically taken up by the reticuloendothelial system.

[0132] Exemplary targeting strategies include mannose-6-phosphate coupled to human serum albumin (M6P-HAS) that binds to the mannose-6-phosphate / insulin-like growth factor II receptor, and a cyclic peptide (pCVI-HAS) coupled to HAS that recognizes collagen type VI receptor (Beljaars). Hepatology , 29:1486-1493 (1999) and Beljaars, et al., J Biol Chem., 275:12743-12751 (2000)). The structure of these proteins allows for coupling with additional chemical entities, making selective delivery of antifibrotic agents to HSCs feasible. In another specific embodiment, the target is stromalin, a large secreted extracellular matrix glycoprotein expressed by hepatic stellate cells, and is histologically used to distinguish it from other myofibroblasts.

[0133] The targeting portion can be, for example, an antibody or antibody fragment that binds to an antigen expressed in the liver or, more preferably, on the surface of hepatocytes or in the microenvironment surrounding hepatic stellate cells, such as an immunoglobulin (antibody) single variable domain (dAb). The antibody or its antigen-binding fragment can be used to direct the conjugate to a cell type or cell state. In one embodiment, a ligand or agonist composition comprising a ligand or agonist or delivery carrier has an antibody-binding domain, such as from a protein known to bind antibodies, for example from Staphylococcus aureus (…). Staphylococcus aureus Proteins A and G of the target antibody are known to bind antibodies. Other antibody-binding domains are known in the art and can be substituted. The antibody-binding domain can facilitate the binding of the target antibody to a ligand or agonist, or a composition comprising a ligand or agonist or a delivery vector. In some embodiments, the antibody is polyclonal, monoclonal, linear, humanized, chimeric, or a fragment thereof. Representative antibody fragments are those that bind to the antibody-binding portion of a non-viral vector, including Fab, Fab', F(ab'), Fv biantibodies, linear antibodies, single-chain antibodies, and bispecific antibodies known in the art. In a preferred embodiment, the target antibody or a fragment thereof is specific to hepatic stellate cell surface markers and is generated to reduce potential immunogenicity to a human host, as known in the art. For example, transgenic mice containing the complete human immunoglobulin gene cluster can be used, which are capable of generating “human” antibodies. In one embodiment, a fragment of such a human antibody is used as a targeting signal. In a preferred embodiment, a single-chain antibody modeled on a human antibody is prepared in a prokaryotic cell culture.

[0134] E. Small molecules and peptides In some embodiments, the pro-apoptotic agent is a small molecule or peptide molecule that recognizes TRAIL-R1 and / or R2. Exemplary small molecules are known in the art and are described in Wang et al. Nature Chemical Biology This activity is discussed in , 9:84–89 (2013). It was initially discovered through high-throughput chemical screening of compounds intended to promote cell death in combination with small-molecule mimics of Smac (antagonists of apoptosis protein inhibitors). Structure-activity relationship studies yielded a more potent mimic known as bioymifi, which can act as a single agent to induce DR5 clustering and aggregation, leading to apoptosis.

[0135] Monovalent, divalent, and trivalent TRAIL-mimicking peptides are described in Pavet, et al. Cancer Research 70:1101-1110, (2010). Therefore, in some embodiments, the ligand or agonist of the agonistic TRAIL receptor is one or more mimics.

[0136] The dosage is expected to be in the same range as the aforementioned compounds.

[0137] F. Ligand conjugates In an alternative embodiment, the biopolymer or polysaccharide may be conjugated with a ligand or agonist. For example, as Yang et al., Biomaterials As described in , 32(33):8722-9 (2011), HA is conjugated with ligands or agonists. Yang described a coupling reaction between aldehyde-modified HA and the N-terminal group of IFNα, which can be used to conjugate HA with the pro-apoptotic agents disclosed herein. The IFNα content can be controlled in the range of 2-9 molecules per individual HA chain, with a bioconjugation efficiency greater than 95%. The conjugates exhibit improved activity and half-life in vivo and increase IFNα delivery to the liver by targeting the CD44, the HA receptor, which is overexpressed in the liver. HA can be used as a ligand for targeting liver disease and activated HSCs after conjugation to pro-apoptotic agents (Kim, et al., ACS Nano , 4(6):3005-14 (2010)).

[0138] In some implementations, the pro-apoptotic agent is modified to improve purification, tag removal, promote small molecule attachment, or a combination thereof. The tandem application of elastin-like peptides and SrtA (SrtA) transpeptidase provides a chromatographic-free purification method for recombinant proteins and, optionally, a method for site-specific conjugation of proteins to small molecules (Bellucci, et al.). Angewandte Chemie International Edition, 52(13):3703–3708(2013)). This system provides an efficient mechanism for producing bioactive proteins in high yield and purity.

[0139] Other tags and labels are known in the art and include, for example, SUMO tags, His tags, which typically comprise six or more usually consecutive histidine residues; FLAG tags, which typically comprise the sequence DYKDDDDK (SEQ ID NO: 2); hemagglutinin (HA), such as YPYDVP (SEQ ID NO: 3); and MYC tags such as ILKKATAYIL (SEQ ID NO: 4) or EQKLISEEDL (SEQ ID NO: 5). Methods for using purification tags to facilitate protein purification are known in the art and include, for example, chromatographic steps in which the tag reversibly binds to the chromatographic resin.

[0140] Purification tags can be located at the N-terminus or C-terminus of the fusion protein. The purification tag can be isolated from the target peptide in vivo (e.g., during expression) or ex vivo after protein isolation. Therefore, purification tags can also be used to remove the fusion protein from post-expression cell lysates.

[0141] Fusion proteins may also include amino acid sequences that are expressed or soluble. Exemplary amino acid sequences that are expressed or soluble include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUSA, ubiquitin (Ub), and small ubiquitin-associated modifier (SUMO).

[0142] In some embodiments, the fusion protein includes one or more linkers or spacers. In some embodiments, the linker or spacer is one or more polypeptides. In some embodiments, the linker includes a glycine-glutamic acid diamino acid sequence. The linker can be used to attach or connect two domains, regions, or sequences of the fusion protein.

[0143] G. Preparations In most cases, systemic delivery of TRAIL agonists is preferred, either by injection or by implantation, controlled-release matrix, or coating.

[0144] Pharmaceutical compositions containing an active agent, with or without a delivery carrier, are provided. The pharmaceutical compositions can be administered via parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous), enteric, or mucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerectible inserts, and can be formulated in dosage forms suitable for each route of administration.

[0145] In some embodiments, the composition is applied topically, for example by direct injection into the site of treatment (e.g., into the liver). In some embodiments, the composition is injected or otherwise applied directly to the vascular system in or near the intended site of treatment (e.g., adjacent to the liver). Typically, topical application results in an increased local concentration of the composition that is greater than that achievable through systemic administration.

[0146] Active agents and pharmaceutical compositions thereof can be administered via parenteral injection in aqueous solution. Formulations may also be in the form of suspensions or emulsions. Typically, a pharmaceutical composition comprising an effective amount of the active agent is provided, and the composition optionally includes pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions include dilute sterile water, multiple buffered contents (e.g., Tris-HCl, acetate, phosphate), buffered saline with appropriate pH and ionic strength; and optional additives such as detergents and solubilizers (e.g., TWEEN® 20, TWEEN® 80, also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimersol, benzyl alcohol) and fillers (e.g., lactose, mannitol). Examples of non-aqueous solvents or carriers are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters such as ethyl oleate. The formulation can be lyophilized and reconstituted / resuspended immediately before use. The formulation can be sterilized, for example, by filtering through a bacterial retention filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0147] Active agents can be formulated for pulmonary or mucosal administration, such as for treating pulmonary ischemia. In one embodiment, the compound is formulated for pulmonary delivery, such as intranasal administration or oral inhalation. The respiratory tract is the structure involved in gas exchange between the atmosphere and bloodstream. The lungs are branching structures that terminate in alveoli where gas exchange occurs. The alveoli have the largest surface area in the respiratory system and are the site of drug absorption. Alveoli are covered by a thin epithelium without cilia or a mucous blanket and secrete surfactant phospholipids. The respiratory tract includes the upper respiratory tract, which includes the oropharynx and larynx, followed by the lower respiratory tract, which includes the trachea, which then branches into bronchi and bronchioles. The upper and lower airways are called airway pathways. The terminal bronchioles then divide into respiratory bronchioles, which then lead to the final respiratory zone, alveoli, or deep lung. The deep lung or alveoli are the primary targets of inhaled therapeutic aerosols used for systemic drug delivery.

[0148] Pulmonary administration of therapeutic compositions containing low molecular weight drugs, such as β-androgen antagonists for the treatment of asthma, has been observed. Other therapeutic agents active in the lungs have been administered systemically and targeted via pulmonary absorption. Nasal delivery is considered a promising drug delivery technology for therapeutic agents for the following reasons: the nose has a large surface area available for drug absorption due to the numerous microvilli covering the epithelial surface; the subepithelial layer is highly vascularized; and venous blood from the nose enters the systemic circulation directly, thus avoiding drug loss due to first-pass metabolism in the liver. This provides lower doses, faster attainment of therapeutic blood levels, quicker pharmacological activity, fewer side effects, and lower dosage per cm³. 3 High total blood flow, porous endothelial basement membrane, and easy to obtain.

[0149] As used herein, the term aerosol refers to any formulation of fine mist particles, which may be a solution or suspension, whether or not it is produced using a propellant. Aerosols can be produced using standard techniques such as ultrasonic treatment or high-pressure treatment.

[0150] Carriers for pulmonary formulations can be categorized into carriers for dry powder formulations and carriers for solution administration. Aerosols for delivering therapeutic agents to the respiratory tract are known in the art. For administration via the upper respiratory tract, formulations can be formulated as solutions, such as water or buffered or unbuffered isotonic saline, or as suspensions, for intranasal administration as drops or sprays. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and have approximately the same pH, for example, from about pH 4.0 to about pH 7.4 or from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, by way of example only, phosphate buffers. For example, representative nasal decongestants are described as buffered to about pH 6.2. Those skilled in the art can readily determine the appropriate saline content and pH of a harmless aqueous solution for nasal and / or upper respiratory tract administration.

[0151] Preferably, the aqueous solution is water, a physiologically acceptable aqueous solution containing salts, and / or a buffer solution, such as phosphate-buffered saline (PBS), or any other acceptable aqueous solution for administration to animals or humans. Such solutions are well known to those skilled in the art and include, but are not limited to, distilled water, deionized water, pure or ultrapure water, saline, and phosphate-buffered saline (PBS). Other suitable aqueous carriers include, but are not limited to, Ringer's solution and isotonic sodium chloride. The aqueous suspension may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, and tragacanth gum, and wetting agents such as lecithin. Suitable preservatives for aqueous suspensions include ethylparaben and n-propylparaben.

[0152] In another embodiment, solvents with low toxicity organic (i.e., non-aqueous) Class III residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, diethyl ether, and propanol, can be used in the formulation. The solvent is selected based on its ability to readily atomize the formulation. The solvent should not react harmfully with the compound. A suitable solvent that dissolves the compound or forms a suspension of the compound should be used. The solvent should be sufficiently volatile to form an aerosol of the solution or suspension. Additional solvents or atomizing agents, such as Freon, may be added as needed to increase the volatility of the solution or suspension.

[0153] In one embodiment, the composition may contain a small amount of polymer, surfactant, or other excipients well known to those skilled in the art. In this context, "small amount" means the absence of excipients that could affect or mediate the uptake of the compound in the lungs, and the presence of excipients is in an amount that will not adversely affect the uptake of the compound in the lungs. Dry lipid powders can be directly dispersed in ethanol due to their hydrophobic properties. For lipids stored in organic solvents such as chloroform, the required amount of solution is placed in a vial, and the chloroform is evaporated under a nitrogen stream to form a dry film on the surface of the glass vial. The film readily expands when reconstituted with ethanol. The suspension is sonicated to completely disperse the lipid molecules in the organic solvent. A non-aqueous suspension of lipids can also be prepared in anhydrous ethanol using a reusable PARI LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, CA).

[0154] Dry powder formulations (“DPFs”) with large particle sizes offer improved flow characteristics, such as less aggregation, easier atomization, and potentially less phagocytosis. The average diameter of the dry powder aerosols typically produced for inhalation therapy is primarily in the range of less than 5 micrometers, although a preferred range is between 1 and 10 micrometers in aerodynamic diameter. Large “carrier” particles (without the drug) are co-delivered with the therapeutic aerosol to aid in effective atomization and other potential benefits.

[0155] III. Treatment Methods Compositions are typically administered by injection, but in some embodiments, they may be applied topically (e.g., during surgery) or applied to mucosal surfaces (rectum, vagina, oral, or lungs). These may be administered as solutions, implants, or gels, or as dry powders or reconstituted or resuspended.

[0156] The following examples illustrate that activated cells, such as hepatic and pancreatic stellate cells, can be specifically targeted and killed by TRAIL-R1 (DR4) and / or TRAIL-R2 (DR5) agonists that induce TRAIL-induced apoptosis. Importantly, by eliminating such activated stellate cells, highly upregulated fibrosis-related molecules were simultaneously downregulated in fibrosis in in vivo models. This suggests that the compounds may be used to treat pathological conditions in which activated fibroblasts, myofibroblasts, myelofibroblasts, and activated endothelial and epithelial cells produce or induce excessive extracellular matrix, leading to undesirable fibrosis or scarring. Scarring or fibrosis can occur in the liver, pancreas, lungs, heart, kidneys, intestines, skin, or arteries.

[0157] It also provides methods for specifically targeting and reducing, inhibiting, and / or removing activated fibroblasts, myofibroblasts, myelofibroblasts, and endothelial and epithelial cells that produce excessive extracellular matrix from organs.

[0158] As discussed in more detail below, the methods typically involve administering an effective amount of an apoptotic agent to an individual who has fibrosis or is likely to develop fibrosis to reduce fibrosis, usually by inducing or increasing apoptosis in cells that are likely to suffer from fibrosis, cirrhosis, or their complications such as ascites or pain. As used herein, “reduce” can mean a reduction in size, stiffness (as in scar tissue), or a combination of factors as understood by those skilled in the art.

[0159] A. Liver disease In a preferred embodiment, the composition and method are used to treat liver diseases. Liver fibrosis is characterized by excessive production of extracellular matrix, primarily type I collagen, as an inflammatory response to chronic liver injury. The main causes of liver fibrosis in industrialized countries include chronic hepatitis C virus (HCV) infection, alcohol abuse, and non-alcoholic steatohepatitis (NASH) (Bataller, et al., Clin. Inves., 115(2):209-18 (2005)). Progressive liver fibrosis ultimately leads to cirrhosis and vascular malformations, further resulting in liver failure, portal hypertension (PHT), hepatocellular carcinoma (HCC), and premature death. PHT can also trigger further complications such as gastrointestinal bleeding, ascites, encephalopathy, and decreased platelet levels or white blood cell counts. Treatment of liver fibrosis and cirrhosis can provide a higher standard of care and reduce complications directly associated with the fibrosis cascade. The progression of the fibrosis cascade can be slowed or even reversed after the removal of the damaging factors in the liver. Until 1985, hepatic stellate cells (HSCs) were identified as the primary cause of ECM overexpression in the liver, allowing for the investigation of its potential therapeutic applications.

[0160] During chronic liver injury or disease, quiescent HSCs undergo activation and transform from astrocytes rich in vitamin A into highly proliferating, myoblast-like vitamin A-deficient cells with fibrogenic properties (Bataller, et al.). Clin. Inves., 115(2):209-18 (2005); Friedman, et al., Proc. Nat. Acad. Sci. USA ; 82(24):8681-5 (1985); Senoo, Medical electron microscopy : official journal of the Clinical Electron Microscopy Society of Japan 37(1):3-15 (2004)). Activated HSCs express α-smooth muscle actin (α-SMA) and secrete type I collagen (Friedman, et al., Proceedings of the National Academy of Sciences of the United States of America ; 82(24):8681-5(1985); Rockey, et al., Journal of submicroscopic cytology and pathology , 24(2):193-203 (1992); Ramadori, et al., Virchows Archive B, Cell pathology including molecular pathology 59(6):349-57 (1990)). The identification of activated HSCs, previously known as adipocytes, Ito cells, or anterior sinusoidal cells as the main fibroblastic cell types in liver injury, and the recognition of key cytokines involved in this process, have provided numerous strategies for antifibrotic agents (Bataller, et al., Clin. Invest. 115(2):209-18 (2005)).

[0161] Several therapies have been tried to reduce the accumulation of activated HSCs to prevent excessive ECM, and these have been shown to be effective in experimental models (Wynn, et al.). Nature medicine, 18(7):1028-40 (2012); Cohen, et al., Ther. adv. gastroent. , 4(6):391-417 (2011); Kisseleva, et al., Best practice&research Clinical gastroenterology , 25(2):305-17 (2011)). For example, renin-angiotensin system blockers and antioxidants can reduce the accumulation of scar tissue, but their efficacy has only been shown in experimental models. There are currently many suggested strategies for treating liver fibrosis and cirrhosis (Table 1 from Friedman, In: Bruce A. Runyon ACT, ed. UpToDatecom, (2011)). Targeting activated HSCs or their activation, proliferation and function is an important anti-fibrotic strategy (Friedman, 25(2):305-17 (2011)). In: Bruce A Runyon ACT, ed. UpToDatecom , (2011);Breitkopf, et al., Clinical and Experimental Research , 29:121S-31S (2005); Kisseleva, et al., Journal of Gastroenterology and Hepatology HSC reversal has been shown to promote fibrosis regression in many animal models of hepatocellular injury (Kisseleva, et al., 21:S84-S87(2006)). Proc. Natl. Acad. Sci. USA , 109(24):9448-53 (2012);Troeger, et al., Gastroenterology 143(4):1073-83 (2012)). However, even with HSC activation and fibrosis termination, reversible HSCs showed higher responsiveness to recurrent fibrosis stimuli, indicating that these HSCs were not completely reversed to a quiescent state (Troeger, et al., Gastroenterology 143(4):1073-83 (2012)). Another proposed approach is to eliminate activated HSCs, which promote apoptosis rather than reverse it (Bataller, et al., Semin Liver Dis, 21(03):437-52 (2001); Friedman, Proc. Natl. Acad. Sci. USA, 109 (24):9230-1 (2012)).

[0162] This was further validated in a spontaneous recovery model of rat liver fibrosis, where apoptosis of activated HSCs was crucial for fibrosis regression (Iredale, et al.). J Clin Invest , 102(3):538-49 (1998)). Activated HSCs in this model are shown to be responsible for generating the fibrotic matrix and protecting it from degradation by producing tissue inhibitors of metalloproteinases (TIMPs). However, importantly, no therapeutic methods that promote specific apoptosis in hepatic stellate cells, such as those disclosed in this paper, have been reported in the literature.

[0163] B. Methods of treating liver disease Treatment methods typically involve administering an effective amount of a pro-apoptotic agent, such as one or more ligands or agonists of the TRAIL receptor, to the individual in need to induce or increase apoptosis in one or more target cell types, such as hepatic stellate cells, myofibroblasts, myelofibroblasts, activated endothelial cells, or activated epithelial cells, which produce or induce excessive extracellular matrix, leading to undesirable scarring of the liver in the individual. In a preferred embodiment, the target cells are hepatic stellate cells.

[0164] Typically, pro-apoptotic agents are administered to an individual in an effective amount to increase apoptosis in one or more target cell types. Preferably, the apoptosis level effectively reduces or inhibits the onset or progression of liver disease or one or more symptoms thereof. For example, in some embodiments, the pro-apoptotic agent is administered in an effective amount to reduce fibrosis or increase fibrosis regression, reduce the accumulation of scar tissue, reduce the progression of the fibrosis cascade, reduce the accumulation of extracellular matrix, reduce cirrhosis, or a combination thereof.

[0165] Various techniques can be used to assess the regression of hepatic stellate cell apoptosis and liver fibrosis in an individual. Overall improvement in an individual's liver disease can also be observed. An individual's condition and liver function can be assessed to monitor any reduction in the severity or complete disappearance of one or more symptoms associated with liver disease, particularly liver fibrosis. For example, changes in jaundice, fluid retention, susceptibility to bruising, frequency of nosebleeds, or the condition of the skin or nails can be assessed. An individual's general health can improve and can be assessed as an indicator of recovery. An individual may show an increase in appetite, a decrease in the incidence or severity of nausea, weight gain, and / or general feelings of strength and energy. An individual may also show a reduced incidence of hospitalization or the need for additional medical attention. Liver function can improve or increase. Liver function can be stable. This can be assessed in several ways. Liver biopsies or blood samples can be taken, and markers of liver function can be measured. The liver function markers that can be studied include hyaluronic acid, procollagen III N-peptide, procollagen IC-peptide, Undulin-collagen 16, 7S type IV collagen, MMP-2 and TIMP-1 levels.

[0166] An individual's liver may show reduced nodules, necrosis, inflammation, or a combination thereof. In particular, an individual's liver may show a reduction or stabilization in the amount of fibrosis within the liver. The presence of fibrotic material in the liver can be reduced, and this can be determined by staining sections from a liver biopsy using a staining agent such as Sirius red. The presence and amount of specific fibrotic extracellular matrix components, such as collagen, particularly collagen I and III, can be determined. Biochemical analyses can also be performed to determine the levels of TIMP and / or MMP and a reduction in TIMP expression in the individual.

[0167] Apoptosis of hepatic stellate cells in the liver can also be determined by liver biopsy. Any changes in the frequency of hepatic stellate cell apoptosis, especially any increase, can be measured. Apoptotic cells can be identified using a variety of well-known methods. Techniques such as TUNEL staining (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick labeling) can be used to identify apoptotic cells. TUNEL staining is particularly useful because it can be used for in situ identification of apoptotic cells. By co-staining, cells undergoing apoptosis can be examined to determine if they are hepatic stellate cells, for example, by staining cells expressing α-smooth muscle actin.

[0168] Other known techniques for identifying and / or quantifying apoptosis can be used, such as annexin V staining, antibodies against single-stranded DNA, caspase substrate assays, ligation-mediated PCR, and cell membrane permeability staining. DNA fragmentation can be analyzed by gel electrophoresis. Staining can also be used to determine morphological features associated with apoptosis, such as membrane bubbling and nuclear degradation. Acridine orange staining can be used to identify apoptotic cells. Propidium iodide can be used to stain cells to analyze DNA content. Tests such as trypan blue staining can be used to check if membrane cells are intact and if they are apoptotic rather than necrotic.

[0169] The effects of administration of the pro-apoptotic agent can be compared with those of a control. Suitable controls are known in the art, including, for example, matched untreated individuals or matched individuals who have received a therapeutic agent that does not induce or increase apoptosis of target cells.

[0170] The composition can be administered topically or systemically, as described above. In a specific embodiment, the composition is administered to an individual via percutaneous injection into the liver. The injection may be administered into and / or adjacent sites of fibrosis or scarring in the liver, sites of excessive extracellular matrix accumulation, sites of activated or proliferating HSCs, or sites of other biochemical, histological, or morphological markers of diseased liver. As discussed in more detail below, the composition can be administered alone or in combination with other active agents.

[0171] IV. Combination Therapy The one or more pro-apoptotic agents and combinations thereof disclosed herein can be administered alone or in combination with one or more additional active agents to individuals in need. In some embodiments, the second active agent is a pharmaceutical agent known in the art for treating fibrotic diseases, particularly liver fibrosis. In some embodiments, the second active agent is an agent that modulates hepatic stellate cells, such as reducing hepatic stellate cell proliferation, reducing hepatic stellate cell activation or activity, increasing stellate cell apoptosis, reducing deposition of extracellular matrix or its components, particularly collagen, increasing degradation of extracellular matrix or its components, particularly collagen, or any combination thereof. In some embodiments, the second active agent increases the efficacy of cells to ligands or agonists, enhances their effects, or otherwise improves the performance or sensitivity of cells to ligands or agonists.

[0172] In some embodiments, the second activator is independent of the regulation of hepatic stellate cells. For example, in some embodiments, the second agent reduces liver inflammation. In some embodiments, the second activator may be an agent that treats or reduces one or more symptoms of liver fibrosis without affecting the proliferation, activity, activation, or apoptosis of hepatic stellate cells.

[0173] Exemplary other therapeutic agents include, but are not limited to, glycyrrhizin, halofop-p-ethyl, hepatocyte growth factor (HGF), HOE 077, interferon-α, interferon-γ, interleukin-10, malotec, pentoxifylline, phosphatidylcholine, S-adenosyl-L-methionine (SAMe), saturated fatty acids, Sho-saiko-to, sylimarin, transforming growth factor β (TGF-β) inhibitors, TNP 470, tocopherol, trogopterin A, and urokinase-type plasminogen activator (uPA) (Bataller, et al., Semin Liver Dis. , 21(3) (2001).

[0174] A. Second activator 1. Antioxidants The second or subsequent activator can be an antioxidant. Exemplary antioxidants include, but are not limited to, vitamin E (α-tocopherol), silymarin (derived from silymarin), and other antioxidants. Silybum marianum It contains flavonoid antioxidants extracted from flavonoids, phosphatidylcholine (PPC), S-adenosyl-L-methionine (SAMe), retinoids (retinyl palmitate), and natural phenolic compounds (resveratrol and quercetin).

[0175] 2. Agents that inhibit HSC migration or interaction with the surrounding extracellular matrix. In some embodiments, the second or subsequent activator is an agent that reduces or inhibits the migration of hepatic stellate cells or the interaction between the cells and their microenvironment (e.g., the surrounding or underlying extracellular matrix). Stimulation of HSCs with platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-β1, and / or epidermal growth factor (EGF) increases migration capacity and upregulates matrix metalloproteinase (MMP)-2 activity (Yang, et al.). Gastroenterology , 124(1):147-59(2003)). PDGF-BB-induced migration is thought to be associated with increased proliferation, while TGF-β1 / EGF-induced migration appears to be proliferation-independent. Yang et al. (ibid.) also reported that COL-3 (an inhibitor of MMP-2 and MMP-9) inhibited HSC migration induced by direct activation of PDGF-BB or TGF-β1, but had no effect on migration induced by chemotactic stimuli without direct contact, showing two distinct MMP-dependent and MMP-independent mechanisms of PDGF-BB or TGF-β1-induced migration.

[0176] Therefore, in some embodiments, the second active agent is an agent that reduces or inhibits PDGF-BB-induced migration, TGF-β1-induced migration, or a combination thereof. An exemplary inhibitor is COL-3, which is the subject of clinical trials. Phase 1 trials included treatments starting at 36 mg / m². 2 Administering COL-3 to individuals at progressively increasing doses per day revealed a maximum tolerated dose of 98 mg / m². 2 / d, and at 70 mg / m 2 It is well tolerated at / d.

[0177] PDGF-BB, TGF-β1, and collagen I-induced migration can also be inhibited by α(1) and / or α(2)-integrin blocking antibodies and competitive RGD antagonists, and studies have shown that curcumin inhibits the migration and invasion of activated HSCs by reducing MMP-2 expression and activity (Huang, et al., Zhonghua Gan Zang Bing Za Zhi, 17(11):835-8(2009). Other evidence suggests that interferon α and interferon γ can also inhibit HSCs or increase their apoptosis (Weng, et al., J Hepatol. , 59(4):738-45 (2013) and (Glassner, et al., Lab Invest. , 92(7):967-77(2012)).

[0178] 3. Medications to suppress liver inflammation In some implementations, the second or subsequent active agent is a drug that reduces or inhibits liver inflammation. Exemplary anti-inflammatory agents include, but are not limited to, corticosteroids, colchicine, and malotec.

[0179] In some embodiments, the second or subsequent activator is an agent that reduces or inhibits the activity of pro-inflammatory factors or cytokines. For example, said agent may be an interleukin-1 receptor antagonist or a soluble tumor necrosis factor-α (TNF-α) receptor, which can reduce necrosis and inflammation in liver tissue. Additionally, IL-10 has been shown to downregulate pro-inflammatory Th1 responses. Patients with chronic HCV infection treated with recombinant interleukin-10 have shown not only improvement in liver inflammation but also regression of initial deposits of fibrous scarring (Louis, et al., Hepatology , 28:1607-1615 (1998)).

[0180] 4. Agents that inhibit TGF-β activity In some implementations, the second activator inhibits TGF-β activity. Methods for preventing TGF-β from binding to its receptor include the use of a dominant-negative type II TGF-β receptor, expression of the extracellular domain of a type II receptor fused to the Fc portion of human IgG, expression of a truncated type II receptor, and the construction of a soluble type II receptor. HGF, as a recombinant protein or through gene therapy, has also been effective in preventing the progression of liver fibrosis in various experimental models and can be used to regulate HSC proliferation, collagen formation, and TGF-β expression without the potential drawbacks and the risk of prolonged systemic or overall TGF-β inhibition.

[0181] In some implementations, the second activator is a microRNA or a mimic thereof. Members of the miR-17-92 cluster (19a, 19b, 92a) are significantly downregulated in activated HSCs (Lakner, et al.). Hepatology , 56(1):300-10(2012)). In particular, miR19b mimics the negative regulation of TGF-β signaling components, as demonstrated by reduced expression of TGF-β receptor II (TGF-βRII) and SMAD3, binding of miR19b to the 3' untranslated region of TGF-βRII, inhibition of TGF-β signaling, reduced expression of type I collagen, and blocking of TGF-β-induced expression of α1(I) and α2(I) procollagen mRNA. miR19b also inactivates the activated HSC phenotype and reduces smooth muscle α-actin expression by morphological assessment. Therefore, in a preferred embodiment, the microRNA is miR19b or a mimic thereof.

[0182] 5. Chemotherapy agents Ligands that stimulate the TRAIL receptor have been studied, both alone and in combination with conventional cancer therapies such as chemotherapy agents. Some reports indicate that chemotherapy drugs can sensitize cells to TRAIL-induced apoptosis, and some results suggest that the combination of two agents is more effective than the sum of their individual effects (Cuello, et al.). Gynecol Oncol ., 81(3):380-90 (2001) Wu, et al., Vitam Horm. , 67:365-83 (2004)). Therefore, in some embodiments, the individuals and diseases disclosed herein are treated with a combination of an agonist or ligand of the TRAIL receptor and a chemotherapeutic agent. In some embodiments, the individual does not have cancer.

[0183] Exemplary chemotherapy drugs include, but are not limited to, doxorubicin, 5-fluorouracil, cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, vincristine, vinorelbine, vindesin, paclitaxel and its derivatives, irinotecan, topotecan, acridine, etoposide, etoposide phosphate, teniposide, epipodophyllotoxin, trastuzumab (HERCEPTIN®), cetuximab and rituximab (RITUXAN® or MABTHERA®) or bevacizumab (AVASTIN®), and combinations thereof.

[0184] B. Dosage and treatment regimen for combination therapy The treatments disclosed herein generally include methods for treating diseases or their symptoms, or for achieving desired physiological changes, including administering an effective amount of apoptosis-promoting agent to animals, such as mammals, particularly humans, to treat liver diseases or their symptoms, or to produce physiological changes. In some embodiments, the apoptosis-promoting agent is combined with another active agent. The apoptosis-promoting agent and the other active agent may be administered together, for example as part of the same composition, or separately and independently at the same time or at different times (i.e., administration of the ligand or agonist and the second active agent is spaced finitely between each other). Therefore, the terms "combination" or "combined" are used to refer to the concurrent, simultaneous, or sequential administration of the ligand or agonist and the second active agent. The combination may be simultaneous (e.g., as a mixture), separately but simultaneously (e.g., administered to the same individual via separate intravenous lines; one agent is administered orally while the other is administered by infusion or injection, etc.), or sequentially (e.g., one agent is administered first, followed by the second agent).

[0185] In a preferred embodiment, administration of the pro-apoptotic agent in combination with the second activator achieves better results than when the pro-apoptotic agent and the second activator are administered alone or separately (i.e., the results achieved through combination are greater than the sum of the results obtained by the individual components alone). In some embodiments, the effective amount of one or both agents used in combination is lower than the effective amount of each agent when administered alone. In some embodiments, when used in combination therapy, the amount of one or both agents is subtherapeutic when used alone.

[0186] Combination therapy regimens may include single or multiple administrations of a ligand or agonist. Combination therapy regimens may also include single or multiple administrations of a second activator.

[0187] In some embodiments, the pro-apoptotic agent is administered before the first administration of the second activator. In other embodiments, the ligand or agonist is administered after the first administration of the second activator.

[0188] The ligand or agonist may be administered at least 1, 2, 3, 5, 10, 15, 20, 24 or 30 hours or days before or after the administration of the second activator.

[0189] The dosing regimens or cycles of the agents can be completely or partially overlapping, or they can be sequential. For example, in some embodiments, all such administrations of the pro-apoptotic agent occur before or after the administration of the second active agent. Alternatively, the administration of one or more doses of the pro-apoptotic agent can be staggered with the administration of the second therapeutic agent to form a homogeneous or non-homogeneous treatment process, thereby administering one or more doses of the pro-apoptotic agent, followed by one or more doses of the second active agent, and then one or more doses of the apoptotic agent; or one or more doses of the second active agent, followed by one or more doses of the pro-apoptotic agent, followed by one or more doses of the second active agent, etc., all according to any schedule chosen or desired by the investigator or clinician administering the treatment.

[0190] The effective amount of each reagent can be administered as a single unit dose (e.g., as a dose unit) or as a subtherapeutic dose administered over a limited time interval. Such a unit dose can be administered on a daily basis for a limited period of time, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days.

[0191] V. Reagent Kit Medical kits are also disclosed. These kits may include, for example, a dose supply of an apoptosis-promoting agent, preferably a ligand or agonist of an agonist of the TRAIL receptor, either alone or in combination with a second therapeutic agent. When combined with a second therapeutic agent, the active agent may be provided alone (e.g., lyophilized) or in a pharmaceutical composition (e.g., a mixture). The active agent may be a unit dose or in a stock solution to be diluted prior to administration. In some embodiments, the kit includes a supply of a pharmaceutically acceptable carrier. The kit may also include a device for administering the active agent or composition, such as a syringe. The kit may include printed instructions for administering the compound as described above.

[0192] The invention will be further understood by referring to the following non-limiting embodiments.

[0193] Example 1: Expression of TRAIL-R1 / DR4 and TRAIL-R2 / DR5 by activated human primary hepatic stellate cells (HSCs) and pancreatic stellate cells (PSCs) Materials and methods Human primary liver stellate cells Human primary HSCs, PSCs, and stellate cell culture medium (SteCM) were obtained from ScienCell Research Laboratories (Carlsbad, CA). Cells were cultured in SteCM medium supplemented with 2% FBS, 1% stellate cell growth supplement, and 1% penicillin / streptomycin solution in polylysine-coated plates. Cells were then cultured in 6-well plastic culture plates for 1, 4, 7, and 14 days to activate the primary stellate cells, and the cells were harvested. The expression of DR-4, DR-5, and α-SMA in the cultured stellate cells was determined by Western blotting and real-time PCR.

[0194] Comparative quantitative real-time RT-PCR Total RNA was extracted from cultured cells using TRIzol reagent (Life Technologies, Grand Island, NY) and reverse transcribed into cDNA using a Reverse Transcription System (Life Technologies, Grand Island, NY). Following the manufacturer's instructions, quantitative real-time PCR was performed in duplicate on each sample using SYBR Green Master Mix (Life Technologies, Grand Island, NY) and a StepOnePlus Real-Time PCR System (Life Technologies, Grand Island, NY). The expression levels of the target gene were normalized to GAPDH expression and compared based on the cycle threshold Ct method (2...). The expression levels of target genes were calculated. Primers for type 1 collagen and α-SMA, TGF-β, Timp-1, TRAILR1 / DR-4, and TRAILR2 / DR-5 were used for PCR.

[0195] Western blot analysis Cultured cells were washed three times with ice-cold PBS and collected in cold lysis buffer containing protease inhibitors (Santa Cruz Biotechnology, Dallas, TX). Cells were then sonicated and centrifuged, and the protein concentration in the supernatant was measured. α-SMA antibody (Sigma Aldrich, St. Louis, MO), TGF-β antibody (Cell signaling, Beverly, MA), DR-4 antibody (Santa Cruz Biotechnology, Dallas, TX), and DR-5 antibody (Abcam, Cambridge, MA) were used as markers of activated astrocytes and liver fibrosis. An antibody against cleaved PARP-1 (Cell signaling) was used as a marker of apoptosis. An antibody against β-actin (Sigma Aldrich, St. Louis, MO) was used as a protein loading control.

[0196] result Starting from plating, primary human HSCs and PSCs were gradually activated and progressively expressed DR4 and DR5. Real-time PCR was performed in cultured cells to confirm the gene expression of TRAIL-R1 / DR4 and TRAIL-R2 / DR5. As HSCs and PSCs were activated, the gene expression of DR4 and DR5 gradually increased. mRNA expression was assessed using real-time PCR. Based on real-time PCR analysis, α-SMA (a marker of astrocyte activation); type I collagen; TGF-β and MMP-2, MMP-9, MMP-13; and Timp-1 were upregulated in early-activated (day 4) and fully activated (days 7 and 14) astrocytes, but undetectable during the quiescence stage. Protein levels of TRAIL-R1 / DR4 and R2 / DR5, confirmed by Western blot analysis, were also induced in highly activated astrocytes, but not in quiescence cells. Similarly, αSMA (α-SMA) was undetectable in cultured cells on day 0, but significantly increased on days 4 and 7. β-actin was present to equal extent on days 0, 4, and 7. Expression of DR5 and α-SMA during HSC activation was confirmed (data not shown). This result suggests that HSCs and PSCs initiate expression of death receptors or overexpress existing death receptors during activation.

[0197] Example 2: Activated human primary HSCs and PSCs showed enhanced sensitivity to TRAIL agonist-induced apoptosis.

[0198] Materials and methods Immunofluorescence staining for apoptosis Cells were plated on glass coverslips in 35 mm culture dishes (MatTek, Ashland, MA) and grown for 1, 4, 7, and 14 days. On these days, cells were treated for 3 hours with or without a TRAIL agonist containing 1 μg / ml TRAIL, PEGTRAIL, or 50 ng / ml DR5 antibody (R&D systems, Minneapolis, MN). Cells were washed twice with cold PBS and fixed for 10 minutes in PBS containing 4% paraformaldehyde. For apoptosis detection, the TdT In Situ Apoptosis Detection Kit (TUNEL)-Fluorescein (R&D systems, Gaithersburg, MD) was used according to the manufacturer's instructions. In short, cells were incubated with proteinase K at room temperature for 15 minutes, washed twice with dry water (DW), immersed in TdT labeling buffer, and then incubated with the TdT labeling reaction mixture at 37°C for 1 hour. Next, cells were treated with TdT stop buffer to terminate the labeling reaction and washed twice with dry water (DW). Finally, Step-Fluor solution was added, and the cells were incubated at room temperature for 20 minutes, followed by washing twice with PBS. Fluorescence Mounting Medium (Vector Laboratories, Burlingame, CA) containing DAPI was used for the samples. Cell apoptosis was observed under a fluorescence microscope using a 495 nm filter, and DAPI levels were observed using a 358 nm filter.

[0199] Western blot analysis Cultured cells were washed three times with ice-cold PBS and collected in cold lysis buffer containing protease inhibitors (Santa Cruz Biotechnology, Dallas, TX). Cells were then sonicated and centrifuged, and the protein concentration in the supernatant was measured. An antibody against cleaved PARP-1 (cell signaling) was used as a marker of apoptosis. An antibody against β-actin (Sigma Aldrich, St. Louis, MO) was used as a protein loading control.

[0200] Cell viability (MTT assay) HSCs and PSCs were plated in 48-well plates and cultured for 1, 4, 7, and 14 days. On days 1, 4, 7, and 14, cells were incubated at 37°C for 3 hours with TRAIL, PEG-TRAIL, TRAIL agonist antibody, and anti-DR5 antibody. At the specified time points, MTT solution to a final concentration of 5 µg / ml was added to each well, and incubation was continued for 1 hour. After removing the culture medium, 200 ml of DMSO was added to each well to dissolve the formazan crystals. Absorbance at 590 nm was measured using a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). For each condition, three replicates were performed.

[0201] result Activated human primary HSCs and PSCs showed enhanced sensitivity to TRAIL-induced apoptosis. HSCs and PSCs were cultured in medium for 1, 4, 7, and 14 days, and then incubated for 3 hours with TRAIL, PEG-TRAIL, TRAIL agonist antibody, and anti-DR5 antibody. At days 7 and 14, TUNEL fluorescence observed by direct photography of cell culture plates using a bright-field microscope (Nikonmetrology, Brighton, MI) in TRAIL-treated cells was increased compared to control cells and apoptotic cells, indicating that highly activated HSCs and PSCs (on days 7 and 14) were more susceptible than less activated HSCs and PSCs (on days 1 and 4). Furthermore, Western blot analysis confirmed the protein level of cleaved PARP-1 (an apoptosis marker). The level of cleaved PARP-1 (an apoptosis marker) was observed as evidence of TRAIL-induced apoptosis in activated astrocytes when activated HSCs and PSCs were treated with TRAIL agonists on days 7 and 14.

[0202] To quantitatively analyze the TRAIL sensitivity of activated HSCs and PSCs to TRAIL agonists, TRAIL sensitivity was expressed as induced cell death (%), calculated as a percentage relative to untreated cells, and measured by an MTT assay after 3 hours of incubation. TRAIL, PEG-TRAIL, and TRAIL-agonist antibodies induced strong TRAIL-mediated apoptosis in activated HSCs and PSCs at days 7 and 14 (highly activated at these time points as shown in Example 1). In HSCs, when treated with TRAIL agonists on days 7 and 14 of activation, cell death induced by TRAIL, PEG-TRAIL, and anti-DR5 antibodies increased by 4.5-fold, 4.1-fold, and 4.4-fold, and 7.5-fold, 6.2-fold, and 5.2-fold, respectively, compared to cells treated on day 1. Figure 1Similarly, in PSCs, from day 1 to day 14, antibodies to TRAIL, PEG-TRAIL, and anti-DR5 induced 5.5-fold, 4.7-fold, and 5-fold increases in cell death, respectively. These results clearly demonstrate that activated HSCs and PSCs (the cause of fibrotic liver and pancreatic diseases) can be specifically targeted and removed by treating them with TRAIL agonists.

[0203] Example 3: TRAIL treatment for the prevention of liver fibrosis.

[0204] Materials and methods In rats via CC1 4 Induced liver fibrosis (Group 1) Six- to eight-week-old SD rats (Hanlim Experimental Animal Laboratory, Seoul, Korea) were divided into three groups (n=8-10 per group); i) carrier (olive oil), ii) 20% CCl4 in olive oil, and iii) CCl4 in olive oil, as well as TRAIL (group 1, ...). Figure 2 Rats were administered CCl4 (20% CCl4 in olive oil, 2 ml / kg) three times weekly via intraperitoneal injection, or olive oil as a control, for 4 weeks, while simultaneously treated with TRAIL administered intravenously at 4 mg / kg every 3 days, or, for the control group (group 1, ...). Figure 2 Treat with the same amount of salt water.

[0205] Histological and immunohistochemical analysis of liver fibrosis After treatment, the animals were euthanized, and the collected liver tissue was fixed in 10% buffered formaldehyde, embedded in paraffin, and cut into 4 µm thick sections. The sections were then stained with hematoxylin and eosin (H&E). To detect activated HSCs, immunohistochemistry was performed using α-SMA (DakoCytomation, Carpinteria, CA). All steps of the immunohistochemistry were performed using the Histostain-Plus Kit (Life Technology). Briefly, liver sections were dewaxed, hydrated, quenched in 3% hydrogen peroxide solution, and washed on slides. Blocking solution was applied to the slides, followed by the application of a first α-SMA antibody and a biotinylated second antibody, then an enzyme conjugation reagent. The liver sections were stained with 3,3'-diaminobenzidine (DAB) using a chromogen / substrate kit (Vector Laboratories, Burlingame, CA). To detect collagen deposition, liver sections were stained with Sirius red staining solution (Sigma, St. Louis, MO) and washed in 5% acetic acid water. The stained liver tissue was made visible under an optical microscope (Olympus America).

[0206] Immunofluorescence analysis of HSC cell apoptosis in fibrotic liver Liver sections were immunostained with a primary antibody (α-SMA antibody (Dakocytomation) and caspase-3 (Cell signaling)) and a secondary antibody (anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546), and counted using DAPI-containing fluorescent counting medium (Vector Laboratories, Burlingame, CA). The sections were observed under a fluorescence microscope and images were recorded.

[0207] result Immunohistochemical analysis of liver tissues from control, CCl4, CCl4-treated, and TRAIL-treated rats, including H&E, α-SMA immunohistochemical analysis, and Sirius red staining (a marker of collagen deposition), showed that TRAIL treatment significantly prevented and inhibited liver fibrosis. Liver tissues from rats treated with CCl4 in the absence of TRAIL showed strong α-SMA signaling and Sirius red staining, indicating fibrosis in the liver. In contrast, rats treated with both CCl4 and TRAIL simultaneously exhibited significantly reduced fibrosis, as evidenced by reduced α-SMA and collagen in the liver (data not shown). These results suggest that TRAIL agonists, such as TRAIL, effectively prevent the induction of liver fibrosis in vivo.

[0208] To confirm whether this preventative effect was induced by TRAIL-induced apoptosis in activated HSCs, immunofluorescence analysis was performed on α-SMA, caspase-3 (an apoptosis marker), and DAPI (nuclear marker) in liver tissues from rats treated with CCl4 and saline, CCl4 and TRAIL, and the control group. α-SMA (Alexa Fluor 488, green) was detected in both the CCl4 and saline and CCl4 and TRAIL groups, while caspase-3 (Alexa Fluor 546, red) was not detected in TRAIL-treated normal liver tissue or CCl4-treated fibrotic liver tissue. In contrast, a strong apoptotic signal from caspase-3 was observed in fibrotic liver tissue treated with TRAIL. Specifically, the expression of caspase-3, co-localized with α-SMA (an activated HSC marker), confirmed that TRAIL specifically induces apoptosis in activated HSCs.

[0209] Example 4: Treatment of liver fibrosis by PEGylated TRAIL Materials and methods CCI in rats 4 Induced liver fibrosis (Group 2) Six- to eight-week-old SD rats (Hanlim Laboratory Animals) were randomly divided into three groups (n=8-10 per group): i) a medium (olive oil), ii) olive oil with 20% CCl4, and iii) olive oil with CCl4 and PEG-TRAIL. Rats were administered CCl4 (2 ml / kg) or olive oil as a control group via intraperitoneal injection three times weekly for 4 weeks. After a total of 4 weeks of induction of liver fibrosis, rats were treated with PEG-TRAIL administered intravenously at 4 mg / kg every other day for 2 weeks, while continuing CCl4 or olive oil injections (group 2, ...). Figure 2 ).

[0210] Western blot analysis Rapidly frozen liver tissue was placed in a porcelain mortar and ground into a fine powder while still at liquid nitrogen temperature. The fine powder was then briefly dissolved by sonication in ice-cold PBS buffer (1 mM PMSF, with 1 μg / ml each of aprotinin, leuproprolactin peptide, and pepsin A). Cell lysates were clarified by centrifugation at 14,000 rpm at 4 °C. Protein concentration was measured using Bradford solution (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved by SDS-PAGE, and the proteins on the gel were transferred to nitrocellulose (Bio-Rad, Hercules, CA) using a semi-dry blot (Bio-Rad). Membranes were blocked with 3% BSA in TBST (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.5% Tween-20) and incubated overnight with primary antibody at 4 °C. Anti-DR4 (Abcam, Cambridge, MA), anti-DR5 (Abcam), anti-cysteine-8 (Cell Signaling Technology, Danvers, MA), anti-cleaved PARP-1 (Cell Signaling Technology), anti-cleaved cysteine-3 (Cell Signaling Technology), anti-cleaved cysteine-9 (Cell Signaling Technology), anti-αSMA (Sigma), anti-MMP-2 (Santa Cruz Biotechnology), anti-collagen 1 (Cell Signaling Technology), anti-TGF-β (Abcam), anti-TIMP-1 (Millipore, Billerica, MA), anti-PDGFR-β (Santa Cruz Biotechnology), anti-GAPDH (Santa Cruz Biotechnology), and anti-β-actin (Santa Cruz Biotechnology) were used for Western blot analysis. Immunoblots were visualized using enhanced chemiluminescence.

[0211] Real-time PCR quantification (qPCR) Total RNA from cultured cells and rat liver tissue was extracted using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. RNA concentration was measured spectrophotometrically using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA). 1–2 μg of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription System (Life Technologies). qPCR was performed in two or three replicates per sample using the Rapid SYBR Green Master Mix (Life Technologies) and StepOnePlus Real-Time PCR System (Life Technologies). Target gene expression levels were normalized to GAPDH expression and compared using the Ct method (2 t-tests). -ΔΔCt ) to calculate. qPCR was performed on rat liver samples using the RT2 qPCR primer set (Qiagen, Valencia, CA); Col1a2 (PPR56530A), Acta2 (PPR59337B), Mmp3 (PPR48487B), Col3a1 (PPR43017A), Mmp9 (PPR44728C), Mmp13 (PPR45162A), Timp1 (PPR48051C), Timp3 (PPR06533A), Gapdh (PPR06557B), Tgfb1 (PPR06430B), Tgfb3 (PPR06467C), Tgfbr2 (PPR06488E), and Bmp7 (PPR46571A) (RT2 qPCR Primer Assay, SABiosciences, Quiagen).

[0212] Other biomarker analysis Hydroxyproline, a producer of collagen deposits in the liver, was measured using a hydroxyproline assay kit (Sigma, MAK008-1KT) according to the manufacturer's instructions in liver tissue. Blood was collected from rats via cardiac puncture, incubated at room temperature for 2 hours, and centrifuged at 3000 rpm for 20 minutes. Routine liver function tests analyzed in serum included alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, alkaline phosphatase (ALP), total bilirubin, and direct bilirubin.

[0213] Immunohistochemistry of liver histology and liver fibrosis Liver tissue was fixed in 10% formalin buffer, embedded in paraffin, and cut into 4 μm thick sections. The sections were then stained with hematoxylin and eosin (H&E) and immunohistochemically. The immunohistochemical staining included α-SMA (DakoCytomation, Carpinteria, CA) for detecting HSC activation and Sirius red staining for detecting collagen deposition. The stained liver tissue was imaged under an optical microscope (Olympus America), and α-SMA or Sirius red positive areas were quantified in 20 regions of each sample using ImageJ software (NIH). For the detection of apoptosis, TUNEL-Fluorescein (R&D systems, Gaithersburg, MD) was used on the liver sections as described above according to the manufacturer's instructions.

[0214] result Western blot analysis showed that TRAIL-R (the TRAIL receptor in rats) was upregulated in both the CCl4-treated and CCl4-plus-PEG-TRAIL-treated groups. However, compared with the CCl4 and control groups, the expression of fibrosis markers, PAI-1, and α-SMA (α-SMA) was significantly reduced in the PEG-TRAIL group. Figure 3 Immunohistochemical analysis of α-SMA and Sirius red demonstrated that rats treated with PEG-TRAIL showed a significant reduction in fibrosis compared to rats treated with CCl4 without PEG-TRAIL (p<0.05). The analyzed images were quantified as the positive area (%) for each region. Figure 4A and 4B To verify whether the reduced α-SMA and collagen were attributable to TRAIL-induced apoptosis in activated HSCs, liver tissue was analyzed by TUNEL assay. TUNEL-positive cells were strongly detected only in the CCl4 and PEG-TRAIL combination group, but not in the other control, olive oil, saline, and CCl4 treatment groups. qPCR analysis of mRNAs obtained from PEG-TRAIL-treated liver tissue revealed a significant reduction in several highly upregulated fibrosis-related genes associated with activated HSCs, including TRAIL-R, α-SMA, collagen 1, collagen 3, TGF-β1, MMP-2, MMP-3, PDGFR, TIMP-1, TIMP-3, and BMP-7 (p < 0.05 compared to the non-PEG-TRAIL-treated CCl4 group). Western blot analysis confirmed decreased protein expression levels of these genes in the PEG-TRAIL-treated group.

[0215] Furthermore, hydroxyproline levels were lower in the PEG-TRAIL-treated group than in the untreated group, and these results were consistent with lower liver weight-to-body weight (LW / BW) ratios, alkaline phosphatase, and total bilirubin levels (p < 0.05 relative to the non-PEG-TRAIL-treated CCl4 group). Overall, these in vivo results clearly demonstrate that liver fibrosis can be reversed and / or inhibited by eliminating activated HSCs and simultaneously by treating fibrotic liver with TRAILs and their agonists to reduce multiple fibrosis-related molecules.

[0216] Example 5: Treatment with polyethylene glycol-modified TRAIL improved liver cirrhosis and reduced the incidence and volume of ascites.

[0217] Materials and methods CCl4 in rats 4 Induced cirrhosis (Group 3) Six- to eight-week-old SD rats (Hanlim Laboratory Animals) were randomly divided into three groups (n=8-10 per group): i) a medium (olive oil), ii) olive oil with 20% CCl4, and iii) olive oil with CCl4 and PEG-TRAIL. Initially, rats were administered CCl4 (2 ml / kg) or olive oil three times weekly via intraperitoneal injection as a control group for 8 weeks. At week 8, rats were treated with PEG-TRAIL intravenously at 4 mg / kg every other day for 2 weeks, or treated with the same amount of saline along with either CCl4 or olive oil as a control group (group 3). Figure 2 ).

[0218] Western blot analysis and qPCR As described above, the regulatory patterns of the above-listed TRAIL-R, α-SMA, and fibrosis-related molecules in liver tissue isolated at the protein and mRNA levels were analyzed by Western blotting and qPCR.

[0219] Immunohistochemistry of liver histology and liver fibrosis Liver tissue was fixed in 10% formalin buffer, embedded in paraffin, and cut into 4 μm thick sections. The sections were then stained with hematoxylin and eosin (H&E) and immunohistochemically. The immunohistochemical staining included α-SMA (DakoCytomation, Carpinteria, CA) for detecting HSC activation and Sirius red staining for detecting collagen deposition. The stained liver tissue was imaged under an optical microscope (Olympus America), and α-SMA or Sirius red positive areas were quantified in 20 regions of each sample using ImageJ software (NIH).

[0220] Collection and measurement of ascites fluid When ascites occurred during treatment, rats were euthanized and ascites was collected from the rat peritoneum using a sterile syringe. The volume, cell count, total protein, and albumin concentration of the ascites fluid were measured to determine the serum-ascites albumin gradient (SAAG), which has been proven in prospective studies to classify ascites.

[0221] result After 10 weeks of CCl4 treatment alone, all rats exhibited micronodular cirrhosis with discrete signs of inflammation. Ascites was found in 6 rats, ranging in volume from 4 to 65 ml. Dissected livers showed significant morphological damage. Strong signs of collagen deposition were detected in liver tissue by Sirus red staining compared to rats treated with CCl4 alone for 6 weeks. In contrast, rats treated with both CCl4 and PEG-TRAIL showed morphologically normal livers compared to rats treated with CCl4 alone. Furthermore, PEG-TRAIL-treated liver tissue clearly showed lower levels of fibrosis markers, namely α-SMA and collagen, as indicated by immunohistochemistry. Additionally, PEG-TRAIL treatment increased serum levels of total protein and albumin compared to rats treated with CCl4 alone, while significantly reducing bilirubin and hydroxyproline levels in liver tissue (p < 0.05 relative to the non-PEG-TRAIL-treated CCl4 group). As demonstrated in the liver fibrosis model, PEG-TRAIL treatment significantly downregulated molecules associated with fibrosis at both protein and mRNA levels. The relative fold changes in multiple expression levels of PEG-TRAIL in the multi-expressed CCl4 group, including TRAIL-R, α-SMA, collagen 1, collagen 3, TGF-β1, MMP-2, MMP-3, PDGFR, TIMP-1, and TIMP-3, were significantly lower than in the non-PEG-TRAIL-treated CCl4 group (p < 0.05). Ascites is one of the major complications of cirrhosis. 60% (6 / 10) of rats treated with CCl4 for 8–10 weeks developed ascites. In contrast, rats treated with both PEG-TRAIL and CCl4 showed only a 30% (3 / 10) reduction in the incidence of ascites. In particular, the volume of ascites fluid was significantly reduced in the PEG-TRAIL-treated group compared to the CCl4 group without PEG-TRAIL. Figure 5 In summary, treatment with PEG-TRAIL reversed and inhibited the progression of cirrhosis, while reducing the incidence of ascites in rats with cirrhotic liver disease.

[0222] Example 6: In an alcohol-induced rat model of chronic pancreatitis, treatment with PEGylated TRAIL reversed pancreatic fibrosis.

[0223] Materials and methods Chronic pancreatitis (CP) induced by ethanol / frog dermatin / LD liquid diet in rats Six- to eight-week-old SD rats (Hanlim Laboratory) were divided into three groups (n=8-10 per group): i) PBS, ii) CP rats treated with PBS, and iii) CP rats treated with TRAIL. An experimental alcohol-induced CP model was induced in rats as reported elsewhere (Deng, X., et at., Am. J. Pathol. 166(1):93-106 (2005)). Rats in all three groups were fed a liquid diet with gradually increasing ethanol concentrations from 0% to 36% for 7 days, followed by 36% ethanol for 3 weeks. Rats were injected intraperitoneally with 20 μg / kg (Σ) of scutellarin (Σ) once a week for 4 hours until day 28. From day 23 to day 28, rats were treated intravenously with PEG-TRAIL (4 mg / kg) or PBS daily for six days. The control group was treated with PBS. Pancreatic specimens were analyzed by immunohistochemistry and Western blotting after treatment. Pancreatic tissue was stained with H&E and Massons trichrome staining agents (collagen), and the regulation of biomarkers including α-SMA, PDGFRβ, cleaved caspase-8, and COX-2 was analyzed.

[0224] result In the CP model, pancreatic fibrosis was clearly observed by H&E staining and high collagen expression. Furthermore, α-SMA (an activated PSC marker) and fibrosis markers such as PDGFRβ were highly upregulated (6-fold and 4-fold, respectively, relative to the mediator, p < 0.05). Figure 6 PEG-TRAIL treatment significantly reduced collagen deposition, downregulated α-SMA and PDGFβ (1-fold and 2-fold, respectively, relative to the mediator), and other inflammatory markers, including COX-2, as demonstrated by Western blot analysis (p < 0.05, relative to the unPEG-TRAIL-treated CP group). Cleavage of caspase-8 was significantly upregulated only in PEG-TRAIL-treated CP (13-fold relative to the mediator, p < 0.05), indicating that eradication of activated PSCs is due to TRAIL-mediated apoptosis.

Claims

1. A method for treating an individual with fibrosis or liver disease, comprising administering to the individual an effective amount of an apoptotic agent to induce apoptosis in hepatic stellate cells, pancreatic stellate cells, myofibroblasts, myelofibroblasts, activated endothelial cells, or activated epithelial cells, thereby producing or inducing an excess of extracellular matrix that leads to undesirable scarring of organs or tissues, cirrhosis, or their consequences such as ascites or pain.

2. The method according to claim 1, wherein the undesirable scarring is liver fibrosis or cirrhosis.

3. The method according to claim 1, wherein the undesirable scarring is pancreatic fibrosis.

4. The method of claim 1, wherein the undesirable scarring is pulmonary fibrosis.

5. The method of claim 1, wherein the undesirable scarring is fibrosis of the skin.

6. The method according to any one of claims 1-6, wherein the pro-apoptotic agent comprises a TRAIL agonist incorporated or encapsulated in nanoparticles, microparticles, micelles, liposomes, synthetic lipoprotein particles, or carbon nanotubes, gels or suspensions, having a coating therewith or incorporating a TRAIL agonist, or a device, wherein the TRAIL agonist is covalently bound to or connected to the surface of the device, mixed or applied under a polymeric coating on the surface, or physically applied to the device at the time of implantation.

7. The method according to any one of claims 1-6, wherein the pro-apoptotic agent is formulated for delayed release, sustained release, or improved release.

8. The method according to any one of claims 1-6, wherein the pro-apoptotic agent comprises an agonistic TRAIL antibody, an analogue thereof, a derivative thereof, or a fragment thereof.

9. The method of claim 8, wherein the polyethylene glycol-modified TRAIL comprises a trimer TRAIL containing a zippered amino acid motif that facilitates the formation of a trimer at the N-terminus; and PEG or a derivative thereof, wherein the PEG is bound to the N-terminus of a monomer of the trimer TRAIL.

10. The method according to claim 8 or 9, wherein the PEG or its derivatives are in a linear or branched trimer form.

11. The method according to any one of claims 7-10, wherein the PEG or its derivatives are selected from the group consisting of: methoxy polyethylene glycol succinimide propionate, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol aldehyde, methoxy polyethylene glycol maleimide, and multi-branched polyethylene glycol.

12. The method according to claim 11, wherein the PEG or a derivative thereof is methoxy polyethylene glycol aldehyde.

13. The method according to any one of claims 8-12, wherein the PEG or its derivative has a molecular weight of 1,000 to 100,000 or a molecular weight of 5,000 to 50,000.

14. The method of claim 1, wherein the effective amount is applied before, during, or immediately after surgery.

15. The method of claim 1, wherein the effective amount is applied to a site of fibrosis or scarring in the liver and / or nearby, a site of excessive extracellular matrix accumulation, a site of activated or proliferating HSCs, or a site of biochemical, histological, or morphological markers in another diseased liver.

16. The method of claim 1, wherein the TRAIL agonist is applied topically.

17. The method of claim 14, wherein the TRAIL agonist is administered systemically.

18. The method according to any one of claims 1-17, wherein an effective amount of the TRAIL agonist is administered in one or more doses.

19. The method according to any one of claims 1-18, wherein an effective amount of the TRAIL agonist is present or released over a period of one or more days.

20. The method according to any one of claims 1-19, wherein the TRAIL agonist is administered in combination with an additional activator.

21. A dosage unit comprising the TRAIL agonist as described in any one of claims 1-20.

22. The dosage unit according to claim 21, which is formulated in an effective amount for treating liver or pancreatic diseases.

23. The dosage unit according to claim 21, which is formulated in an effective amount and formulation for treating pulmonary fibrosis.

24. The dosage unit according to claim 21, which is formulated in an effective amount and formulation for treating skin fibrosis.

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