EA041148B1 - COMPOSITIONS AND METHODS FOR THE TREATMENT OF PANCREATITIS AND PAIN USING DEATH RECEPTOR AGONISTS - Google Patents

Description

Cross-reference to related applications

This Application Claims the Benefits and Priority of a U.S. Provisional Application

62/319,454, filed April 7, 2016, which is hereby incorporated by reference in its entirety.

Information regarding research or development sponsored by the federal budget.

The present invention was made with government support under W81XWH-15-1-0301 and W81XWH-15-1-0302 provided by the US Department of Defense (DOD) and R21AA023855 provided by the US National Institutes of Health. The government has certain rights to the invention.

State of the art

The tumor necrosis factor receptor superfamily (TNFRSF) is a group of cytokine receptors that are distinguished by their ability to bind tumor necrosis factors (TNF) via an extracellular cysteine-rich domain. With the exception of nerve growth factor (NGF), all TNFs are homologous to the typical member of the family, TNF-alpha. In their active form, most TNF receptors form trimeric complexes in the plasma membrane. Accordingly, most TNF receptors contain transmembrane domains (TMDs), although some can be cleaved to form soluble forms (eg, TNFR1) and some have no TMD at all (eg, DcR3). In addition, most TNF receptors require a specific adapter protein such as TRADD, TRAF, RIP, and FADD for signaling. TNF receptors are primarily involved in apoptosis and inflammation, but they may also be involved in other signaling pathways such as proliferation, survival, and differentiation. TNF receptors are expressed in various mammalian tissues, especially in leukocytes.

The term death receptor refers to members of the TNF receptor superfamily that contain a death domain such as TNFR1, Fas receptor, DR4 and DR5. They were named for their role in apoptosis (programmed cell death), although they are now known to have other functions as well.

The term TNF receptor is often used to refer to typical members of the superfamily, namely TNFR1 and TNFR2, which recognize TNF-alpha. There are 27 family members including: tumor necrosis factor receptor 1, tumor necrosis factor receptor 2, lymphotoxin-beta receptor, OX40, CD40, Fas receptor, decoy receptor 3, CD27, CD30, 4-1BB, death receptor 4 (DR4) , death receptor 5 (DR5), decoy receptor 1, decoy receptor 2, RANK, osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, herpes virus entry mediator, nerve growth factor receptor, glucocorticoid-induced B-cell maturation antigen TNFR- related, TROY, death receptor 6, death receptor 3, and ectodysplasin-A2 receptor.

Pancreatitis, whether acute or chronic, is a significant contributor to the burden of gastrointestinal disease in the United States in several studies. Chronic pancreatitis (CP) is a severe consequence of alcohol abuse and is characterized by progressive and irreversible destruction of the structure and function of the pancreas. CP is accompanied by pancreatic fibrosis and persistent abdominal pain. Pain in CP is very difficult to treat. The lack of understanding of underlying biology has led to various empirical approaches that are often based solely on anatomical sites and are usually highly invasive.

Therefore, there is a significant unmet need for therapeutic strategies for the treatment of pancreatitis.

The object of the invention is to provide a composition for the treatment of pancreatitis, pancreatic fibrosis and pancreatic pain.

Another object of the invention is to provide methods for the treatment of pancreatitis, pancreatic fibrosis and pancreatic pain.

The essence of the invention

A method has been developed for the treatment of pancreatitis and related disorders such as pain using death receptor agonists, for example recombinant analogs of human TRAIL or death receptor 5 (DR5) agonist antibodies. Recombinant analogues of TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL) and anti-DR5 antibodies selectively target activated pancreatic stellate cells and reduce inflammation, fibrogenesis, and pain, and improve pancreatic function in acute and chronic pancreatitis in patients who need it.

Methods for treating pancreatitis or pancreatic pain and improving pancreatic function include administering to a subject suffering from or at risk of developing pancreatitis, pancreatic fibrosis, or a disorder such as pancreatic pain, a pharmaceutical composition containing an effective amount of a death receptor agonist. Suitable death receptor agonists include, but are not limited to, TRAIL-R2 (death receptor 5) agonists such as recombinant human (rh) TRAIL, rhTRAIL analogs, engineered TRAIL analogs, long acting TRAIL proteins modified, for example, with polymers such as polyethylene glycol , copolymers and branched analogues and biopolymers such as hyaluronic acid. TRAIL Long-acting Dosage Forms Including Fusion Proteins

TRAIL, anti-TRAIL-R2 agonist antibodies, and small molecule or peptide agonist molecules that bind TRAIL-R2. TRAIL-R2 (DR5), but not DR4, is the major receptor inducing selective apoptosis in activated pancreatic stellate cells, as demonstrated in Example 3.

In preferred embodiments, TRAIL is rhTRAIL (ie, recombinant human TRAIL) or a functional fragment or variant thereof, eg, the 281 amino acid fragment of human TRAIL. In preferred embodiments, the fragment has an amino acid sequence of 114 to 281 or 95 to 281 of the full length human form, 281 amino acids long. In preferred embodiments, the long-acting rhTRAIL is a PEGylated tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) protein, a PEGylated TRAIL derivative, or any combination thereof. In preferred embodiments, the anti-death receptor antibodies are anti-death receptor 5 agonist antibodies. In an exemplary aspect, the death receptor agonists comprise a PEGylated analog of TRAIL and anti-DR5 agonist antibodies.

Also provided herein are death receptor agonists and one or more polyethylene glycol (PEG) molecules or derivatives thereof. In some instances, the death receptor agonist includes a PEGylated analog of TRAIL or a derivative thereof.

In one aspect, the PEG molecule or derivative is selected from the group consisting of linear PEG, branched PEG, star PEG, comb PEG, dendrimeric PEG, succinimidyl propionate PEG, N-hydroxysuccinimide PEG, propionaldehyde PEG, maleimide PEG, linear methoxypolyethylene glycol (mPEG), branched mPEG , star mPEG, comb mPEG, dendrimer mPEG, mPEG succinimidyl propionate, mPEG N-hydroxysuccinimide, mPEG propionaldehyde and mPEG maleimide. In some instances, the branched PEG molecule or derivative includes monomeric, dimeric and/or trimeric PEG molecules or derivatives thereof. In some instances, the PEG molecule or derivative is a trimeric methoxy polyethylene glycol maleimide.

The PEG molecule has a molecular weight of at least 1000 daltons as measured by size exclusion chromatography or MALDI-VP mass spectrometry. In some cases, the PEG molecule includes a PEG molecule with an average molecular weight of about 1000-1000000 daltons, an average molecular weight of about 10000-500000 daltons, an average molecular weight of about 1000-100000 daltons, most preferably 5000-50000 daltons. In other instances, the PEG molecule includes a PEG molecule with an average molecular weight of about 200,00250,000 daltons, an average molecular weight of about 30,000-100,000 daltons, or a PEG molecule with an average molecular weight of about 40,000-80,000 daltons.

Also provided are compositions containing the anti-DR5 antibody conatumumab, tigatuzumab, lexatumumab, HGS-TR2J/KMTR-2, LBY135, drositumab, TAS266, DS-8273/DS-8273a, APG880, or RG7386.

Examples of diseases or disorders include acute or chronic pancreatitis, pancreatitis-associated pain and pancreatic fibrosis, and fibrosis-associated pain. In some cases, pancreatic fibrosis includes desmoplasia in the tumor microenvironment in the pancreas. In an exemplary embodiment, the fibrotic disorder is fibrotic-related pain. In some instances, the methods further comprise identifying a patient suffering from or at risk of fibrosis-related pain.

Suitable routes of administration include injection, including intravenous and subcutaneous, inhalation, pulmonary, nasal and possibly intraocular administration. The compositions may be administered at a dose of 0.001-100 mg/kg, e.g. , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg. Preferably the compositions are administered at a dose of 0.2-20 mg/kg or a dose of 0.001-20 mg/kg. Dosage forms are administered at a dose and for a period of time that provide protection of pancreatic tissue, reduction of fibrous tissue formation, regression of pancreatic fibrogenesis, reduction of pain, and preservation of healthy pancreatic tissue intact. In one aspect, treating a fibrotic disease or disorder includes reducing inflammation.

Brief description of the drawings

Fig. 1 is a diagram showing the role of death receptor (DR) agonists, such as TRAIL analogs or anti-DR agonist antibodies, in models of pancreatitis. Death receptor agonists selectively act on activated pancreatic stellate cells (PSCs) in the pancreas while leaving other tissues intact. Activated PCOs are one of the initiators of fibrosis and pain in the pancreas. By selectively blocking PKD activation and/or eliminating activated PZKs, death receptor agonists reduce fibrosis and pain, resulting in repair of pancreatic tissue.

Fig. 2 is a bar graph showing the percentage of viable cells (%) of primary human islet and rat acinar cells when cells were treated with 10, 100, 1000 ng/mL

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TRAIL-PEG. No toxicity to normal pancreatic cells, including primary human pancreatic islet and acinar cells, was found.

TRAIL-PEG (10, 100, 1000 ng/ml) was incubated with various cells for 24 h and cell necrosis was quantified using MTT assays.

Fig. 3A, 3B, 3C, 3D, 3E, and 3F are histograms showing the relative expression of these genes by culture-activated primary human PGCs on culture days 2, 4, and 7. In culture-activated primary human PGCs, Acta2 (αSMA, a marker for activated stellate cells), fibrogenic markers, and TRAIL receptors (DR5/DR4) are upregulated and become highly sensitive to TRAIL-induced apoptosis. qPCR analysis of resting (day 2) and activated PGCs (day 4 and day 7). *P<0.05, **P<0.01, ***P<0.001 compared to day 2.

Fig. 4 is a bar graph showing the percentage of dead cells (%) in culture-activated primary human PGCs on days 1, 2, 4 and 7 of culture. *P<0.05.

Fig. 5 is a bar graph showing the change (fold) in caspase 3/7 activity (apoptosis effector) in culture-activated PGCs that were treated with the indicated concentrations of mapatumumab (an anti-DR4 agonist antibody, 0-103 ng/mL) or conatumumab (an anti-DR4 agonist antibody against DR5, 0-103 ng/ml). Only conatumumab induces apoptosis in activated PGCs. This indicates that DR5, and not DR4, plays a critical role in TRAIL signaling in activated stellate cells. ***P<0.001 vs. untreated PZK.

Fig. 6A and 6B are graphs showing the cell membrane expression profiles of DR4 or DR5 of resting PGCs (day 2) and activated PGCs (day 7) measured by flow cytometry using PE-labeled death receptor antibodies. DR5 is predominantly expressed on the cell surface of activated PGCs compared to DR4.

Fig. 7A-7E are bar graphs showing that ethanol (EtOH) activated primary human PGCs are upregulated by Acta2 (α-SMA, an activated stellate cell marker), fibrogenic markers, and TRAIL (DR5/DR4) receptors. In FIG. 7A-7E show qPCR analysis of EtOH-activated PCOs (30 and 50 mM). *P<0.05, **P<0.01, ***P<0.001 compared to non-activated EtOH POCs.

Fig. 8A and 8B are bar graphs showing ratios of cell viability (%) as measured by MTT assay and caspase 3/7 (apoptosis effector) activity after treatment with TRAIL-PEG (1 μg/mL) activated EtOH (50 mM) PZK. Only alcohol-activated PGCs are sensitive to TRAIL-induced cell death. **P<0.01, ***P<0.001 compared to non-activated EtOH POCs.

Fig. 9 is a line graph quantifying caspase 3/7 (apoptosis effector) activity after treatment of activated EtOH (50 mM) PGCs with various concentrations of TRAILPEG, conatumumab (an anti-DR5 agonist antibody), and mapatumumab (an anti-DR4 agonist antibody). Only TRAIL-PEG and conatumumab induce apoptosis in EtOH-activated PGCs.

In FIG. 10A-10E show the effect of 48-hour incubation in conditioned medium (CM) culture derived from serum-free activated PZKs (PZK-KS) on the excitability of sensory neurons from dorsal root ganglia (DRGs) in vitro, as demonstrated by fixation. potential of the whole cell. Fig. 10A is a representative trace diagram showing increased spontaneous and induced action potentials in neurons cultured with PZK-KS. This is accompanied by a significant decrease in reobase (the amount of current required to excite an action potential) (see Fig. 10B). In FIG. 10C shows increased evoked action potentials. In FIG. 10D shows increased action potential amplitude. In FIG. 10E shows reduced Ia currents (Kv transients are important for maintaining excitability).

Fig. 11 is a bar graph showing the reduced number of MPO+ (a marker of neutrophils) cells in TRAILπ _EH -treated AP rats. Treatment with TRAILπ _EG significantly reduced inflammation and the number of infiltrating MPO+ cells in the pancreas. *P<0.05 vs. Cer-PBS (untreated OP rats).

Fig. 12 is a graph showing a timeline for a study design for testing TRAIL _PEG in a chronic pancreatitis (CP) rat model. An alcohol-induced CP model was developed in Sprague-Dawley (SD) rats fed an ethanol/liquid Liber-DeCarly diet for 43 days and five weekly injections of cerulein (20 μg/kg). Ethanol was added to the diet from 0 to 36% of total calories for one week and the final ethanol concentration was maintained from day 7 until the end of the study. Rats received cerulein (four hourly ip injections) on days 14, 21, 28, 35, and 41. TRAIL _PEG (4 mg/kg, iv) or PBS (control) were administered daily for 7 days starting on day 36.

Fig. 13A and 13B are histograms showing the positive area/field as quantitative analysis of digital images of Masson's trichrome stain (collagen stain) and α-SMA stain (activated PGC stain) stain.

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Fig. 14A-14I are bar graphs showing the expression of the indicated genes relative to GAPDH in cerulein-ethanol/Liber-DeCarly (EtOHCP) CP rats treated with PBS or TRAIL _PEG . The effect of TRAIL _PEG on various fibrogenic markers was shown. TRAIL _PEG downregulates various fibrosis-associated molecules at the mRNA (gene) level in rats with CP induced by cerulein and the ethanol/Liber-DeCarly diet (EtOH-CP). Fibrosis-associated marker gene expression levels, including: α-SMA, collagen-1, collagen-3, PDGFr, TIMP1, TIMP3, fibronectin, Pap, and TGFe, were reduced after treatment with TRAIL _PEG - Acta2 - the name of the alpha-SMA mRNA. TIMP is an MMP inhibitor (MMPs are responsible for the breakdown of collagen). Fibronectin is a marker of fibrosis. ##P<0.01, ###P<0.001 vs pair feeding (control), *P<0.05, **P<0.01, ***P<0.001 vs EtOH-CP /PBS.

Fig. 15 is a bar graph showing the concentration of hydroxyproline (µg/g) (a marker of collagen) in pancreas tissues of control and CP rats (EtOH-CP) treated with PBS or TRAILπe _G. TRAILπe _G significantly reduced pancreatic hydroxyproline levels in rats with CP induced by cerulein and the ethanol/Liber-DeCarly diet (EtOH-CP) compared to untreated EtOH-CP. #P<0.05 vs control group, ***P<0.001 vs EtOHCP/PBS.

Fig. 16 is a line graph showing the number of responses as a function of VFF strength in normal and CP (EtOH-CP) rats treated with PBS or TRAIL _PEG . TRAIL _PEG reduces pain associated with pancreatitis. The effect of TRAIL _PEG on nociception was evaluated in rat models of chronic pancreatitis (CP) by measuring the mechanical sensitivity of the abdomen by the VFF method (using von Frey threads). TRAIL _PEG shows strong antinociceptive efficacy in CP models.

Detailed description of the invention

I. Definitions.

By lessening the severity is meant the reduction, suppression, attenuation, reduction, cessation or stabilization of the development or progression of a disease.

By control or reference is meant a standard of comparison. As used herein, an altered control sample or subject should be understood as having a level that is statistically different from a normal, untreated or control sample. Control samples include, for example, cells in culture, one or more laboratory animals, or one or more patients. Methods for taking and analyzing control samples are known to those skilled in the art. The analyte can be a naturally occurring substance that is normally expressed or produced by a cell or organism (eg, an antibody, protein), or a substance produced by a reporter construct (eg, β-galactosidase or luciferase). Depending on the method used for detection, the amount and measurement of the change may vary. The definition of statistical significance is known to those skilled in the art, such as the value of the standard deviation from the means that constitute a positive result.

Detection refers to determining the presence, absence, or amount of an analyte to be detected.

As used herein, the term diagnosis refers to classifying a pathology or symptom, determining the severity of the pathology (eg, grade or stage), monitoring the progression of the pathology, predicting the outcome of the pathology, and/or determining the prospects for recovery.

By the terms "effective amount" and "therapeutically effective amount" of a dosage form or component of a dosage form is meant an amount of the dosage form or component, alone or in combination, sufficient to produce the desired effect. For example, by effective amount is meant the amount of a compound, alone or in combination, required to reduce the severity of one or more symptoms of a disease or disorder compared to an untreated patient. An effective amount for the therapeutic treatment of a disease varies depending on the route of administration, age, body weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and schedule for use.

Modulation refers to change (increase or decrease). Such changes are detected using standard known methods, such as those described in this application.

Fibrotic disease or disorder is a general term for the progressive formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. Fibrosis can appear in many body tissues, usually as a result of inflammation or injury. Examples of organs or tissues susceptible to fibrosis include, but are not limited to: lungs, pancreas, heart, liver, skin, fingers, joints, brain, bone marrow, penis, and intestines.

The term normal amount refers to the normal amount of the complex in a subject who has

- 4 041148 known to have not been diagnosed with a disease or disorder. The amount of the molecule can be measured in the test sample and compared to a normal reference level using methods such as reference limits, sensitivity limits, or risk thresholds to determine cut-off points and abnormal values (eg, in pancreatitis). A normal reference level means a level of one or more proteins (or nucleic acids) or total protein (or total nucleic acid) values, typically found in a subject who is not known to be suffering from prostate cancer. Such normal control levels and cut-off points may vary depending on whether the molecule is used alone or in a formula that combines other proteins into an index. In the alternative, the normal reference level may be a database of protein profiles of previously studied subjects who have not developed a disease or disorder for a clinically relevant period of time.

The level that is determined may be the same as the control level or threshold level or threshold, or may be raised or lowered relative to the control level or threshold level or threshold. In some aspects, a control subject is a matched control of one species, gender, ethnicity, age group, smoking status, body mass index (BMI), current status of a therapeutic regimen, medical history, or a combination thereof, but differs from the subject being diagnosed in that the control does not suffer from the disease in question or is not at risk of developing the disease in question.

The phrase pharmaceutically acceptable carrier is known in the art and includes a pharmaceutically acceptable material, composition, or diluent suitable for administering the compounds to patients in need thereof.

The term TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL) receptor agonist as used herein refers to those agents that bind to and activate death receptors (DR), TRAIL-R1 (DR4) and TRAIL-R2 (DR5). ). TRAIL or TRA receptor agonists include, but are not limited to, recombinant TRAIL, variants of recombinant TRAIL, derivatives of TRAIL, and anti-TRAIL receptor antibodies binding to TRAIL-R1 and/or TRAIL-R2, as well as small molecule or peptide agonist molecules that bind TRAIL -R1 and/or TRAIL-R2. In some embodiments, anti-TRAIL receptor antibodies include anti-TRAIL-R2 (DR5) antibodies.

In some embodiments, anti-TRAIL antibodies include, but are not limited to, anti-DR5 antibodies originally developed for cancer therapy, conatumumab, tigatuzumab, lexatumumab, HGS-TR2J/KMTR-2, LBY135, drozitumab, TAS266, DS-8273/DS-8273a , APG880, RG7386.

The term PEGylation refers to the process of covalently or non-covalently attaching or combining polyethylene glycol (PEG) polymer chains to molecules and macrostructures such as a drug, therapeutic protein, or vesicle.

The terms prevent, preventing, avoidance, and prophylactic treatment refer to the administration of an agent or composition to a clinically asymptomatic subject who is at risk of developing, susceptible to, or predisposed to a particular undesirable condition, disorder, or disease, and thus refers to preventing the onset of symptoms and/or their root causes.

Pharmaceutical compositions can be combined into kits or pharmaceutical systems for use in blocking the cell cycle in rapidly dividing cells, such as cancer cells. Kits or pharmaceutical systems may include a means of transport, such as a box, carton, or vial, in which one or more container means, such as vials, vials, ampoules, bottles, syringes, or pouches, are compactly placed. Kits or pharmaceutical systems of the present disclosure may also include accompanying instructions for use of the kit.

II. Compositions.

Useful compositions include death receptor (TRAIL receptor) agonists. Examples of death receptor agonists include purified TRAIL, isolated TRAIL, recombinant TRAIL, variants of recombinant TRAIL, derivatives of TRAIL, and antibodies against the TRAIL receptor that bind to TRAIL-R1 and/or TRAIL-R2, as well as agonistic small molecules or peptide molecules that bind TRAIL- R1 and/or TRAIL-R2. In some embodiments, anti-TRAIL receptor antibodies include anti-TRAIL-R1 (DR4) and TRAIL-R2 (DR5) antibodies.

A. Death receptor agonists.

The compositions are intended for use in methods of treating pancreatitis and fibrotic and pancreatic disorders using TRAIL-R1 (DR4) and TRAIL-R2 (DR5) death receptor agonists, such as a PEGylated analogue of TNF (tumor necrosis factor)-related ligand that induces apoptosis (TRAIL), and an anti-DR5 agonist antibody, to reduce inflammation, fibrogenesis, and pain, and improve pancreatic functions in the pancreas and

- 5 041148 for pancreatitis. Such a drug based on a PEGylated protein and an antibody against

DR5s have disease-modifying effects in pancreatitis and pancreatic fibrosis, as well as pain. They are safe, highly stable and active with an extended half-life.

1) TRAIL.

TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL) is a protein that functions as a ligand that induces a process of cell death called apoptosis. TRAIL is a cytokine that is synthesized and secreted by most normal tissue cells. It induces apoptosis primarily in tumor cells by binding to certain death receptors. TRAIL is also referred to as CD253 (cluster of differentiation 253) and TNFSF10 (tumor necrosis factor (ligand) superfamily, member 10).

In humans, the gene that codes for TRAIL is located on chromosome 3q26, away from other members of the TNF family. The genomic structure of the TRAIL gene spans approximately 20 kb and consists of five exon segments of 222, 138, 42, 106 and 1245 nucleotides and four introns of approximately 8.2, 3.2, 2.3 and 2.3 kb. The TRAIL gene lacks TATA and CAAT boxes, and the promoter region contains putative response elements for GATA, AP-1, C/EBP, SP-1, OCT-1, AP3, PEA3, CF-1, and ISRE.

TRAIL shows homology to other members of the tumor necrosis factor superfamily. It consists of 281 amino acids and has the properties of a type II transmembrane protein (i.e., it does not have a leader sequence and an internal transmembrane domain). The N-terminal endoplasmic domain is not conserved with family members, however the C-terminal extracellular domain is conserved and can be proteolytically removed from the cell surface. TRAIL forms a homotrimer that binds three receptor molecules.

TRAIL binds to the death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). The process of apoptosis is caspase-8 dependent. Caspase 8 activates a cascade of effector caspases, including pro-caspase-3, 6 and 7, which leads to the activation of specific kinases. TRAIL also binds DcR1 and DcR2 receptors that do not contain an endoplasmic domain (DcR1) or contain a truncated death domain (DcR2). DcR1 functions as a neutralizing TRAIL decoy receptor. The endoplasmic domain of DcR2 is functional and activates NFkappaB. In DcR2-expressing cells, TRAIL binding induces NFkappaB activation, leading to the transcription of genes known to antagonize the cell death signaling pathway and/or induce inflammation. TRAIL has been shown to interact with TNFRSF10B.

TRAIL can be obtained in native or genetically engineered (recombinant) form. TRAIL may include an amino acid zipper motif to promote trimer formation and/or an end group to facilitate its isolation and purification.

Suitable TRAIL proteins include TRAIL in human form, which has an amino acid sequence of 281 amino acids, SEQ ID NO: 1:

MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLK

EDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVA AHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQ TYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCFFWSKDAEYGLYSIYQGGIFELK ENDRIFVGS VTNEHLIDHEAS.

In preferred embodiments, TRAIL has the amino acid sequence arginine 114 (Arg, R) to glycine 281 (Gly, G) of the full length human form (1-281), and has the amino acid sequence of SEQ ID NO: 2:

RERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGE

LVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEY GLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEAS FFGAFLVG.

Typically, TRAIL is modified with ethylene glycol (EG) units, more preferably 2 or more EG (ie polyethylene glycol (PEG)) units at the N-terminal amino acid residue. The N-terminal amino acid residue includes, without limitation, lysine, cysteine, serine, tyrosine, histidine, phenylalanine, or arginine.

Generally, any TRAIL analogue may be suitable for PEGylation. Analogs include trimeric TRAIL in which at least one of the three monomers has the amino acid sequence of SEQ ID NO: 1 or 2, with one or more amino acid substitutions or deletions. TRAIL analogues can be prepared in vitro using standard molecular biology techniques.

TRAIL may be attached to a leucine or isoleucine zipper (ILZ) at its N-terminus. In preferred embodiments, the zipper motif is an isoleucine zipper (Kim et al., BBRC, 321:930-935 (2004)).

2) Agonistic antibodies to the death receptor.

In some embodiments, the death receptor agonist antibodies comprise, without

- 6 041148 limited to those listed, antibodies to DR5: conatumumab, tigatuzumab, lexatumumab, HGSTR2J/KMTR-2, LBY135, drozitumab, TAS266, DS-8273/DS-8273a, APG880, RG7386, or chimeric antibodies, with single, double or multiple antigenic or epitope specificity, as well as fragments such as F(ab')2 and the like, including hybrid fragments. Such antibodies and fragments can be prepared by methods known in the art and can be screened for specificity and activity according to general methods for producing antibodies and screening antibodies for specificity and activity (see, for example, Harlow and Lane. Antibodies, A Laboratory Manual). Cold Spring Harbor Publications, New York, (1988)).

Many non-human antibodies (eg, those obtained from mice, rats, or rabbits) are typically antigenic in humans and thus can lead to unwanted immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods described herein serves to reduce the likelihood that an antibody administered to a human will elicit an unwanted immune response. A humanized or chimeric death receptor agonist antibody may comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to CDR regions from a non-human immunoglobulin. (ie, a donor antibody) and all or substantially all of the framework regions are framework regions from a human immunoglobulin consensus sequence. Preferably, the death receptor agonist antibody also includes at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. The constant domains of death receptor agonist antibodies can be selected depending on the intended function of the antibody, in particular the effector function that may be desired. In some embodiments, the death receptor agonist antibody constant domains are (or include) human IgA, IgD, IgE, IgG, or IgM domains.

Antibody fragments include the binding and binding specificity of the antibody (and do not require a specific biological function of the antibody constant regions), such as a binding fragment specific for death receptors. Other regions of the antibody may be replaced with any heavy and light chains or portions thereof and/or modified based on any heavy and light chains or portions thereof, with the expectation that bispecific binding and binding specificity to the target death receptor will be retained. In the case of the antibody fragment and peptide forms, the death receptor specific binding moiety can be implemented in any of numerous forms of binding fragments and can be coupled by any suitable method, including any of the multivalent and multispecific methods used for antibody binding fragments. In the case of the disclosed antibodies, antibody fragments and polypeptides, such forms will be bispecific instead of (or in addition to) multivalent. Example forms of binding fragments include F(ab') ₂ , antigen binding fragment (Fab), semi-antibodies, single chain variable fragments (scFv), VhH domain, V-NAR domain, V _H domain, V _L domain, F(ab) ₃ bis -scFv, diabody, tribody, tetrabody, and minibody. Any of these forms can independently be used to implement a death receptor specific binding moiety and can then be combined or linked using any suitable linker or fusion. A death receptor specific binding moiety can also be used as part of the binding moiety of the multivalent and/or multispecific form of antibody fragments. Examples include F(ab')2, F(ab) ₃ bis-scFv, diabody, tribody, tetrabody, and minibody.

3) Analogues of death receptor agonists.

Death receptor agonists can be modified to include an additional molecule, thus producing analogues. The molecule can be a polymeric molecule, a polypeptide, a polysaccharide, a radioisotope label, and so on. The molecule may be non-covalently or covalently attached to death receptor agonists.

In some embodiments, the molecule is a polyalkylene oxide, such as polyethylene glycol (PEG). Polyethylene glycol (PEG) is a polyester compound used in many industries, from manufacturing to medicine. The PEG structure is as follows (note the repeating element in parentheses): H-(O-CH2-CH2)n-OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. PEG, PEO or POE refers to an ethylene oxide oligomer or polymer. The three names are chemically synonymous, but historically PEG is preferred in the biomedical field, while PEO is more common in the field of polymer chemistry. Because different applications require different polymer chain lengths, PEG is generally used below 20,000 g/mol molecular weight, PEO refers to polymers above 20,000 g/mol molecular weight, and POE refers to a polymer of any molecular weight. PEG and PEO are liquids or low melting solids, depending on their molecular weights. PEG is produced by the polymerization of ethylene oxide and is commercially available in a wide range of molecular weights, from 300 g/mol to 10,000,000 g/mol. Although PEG and PEO with different molecular weights have different applications and have different physical properties (e.g. viscosity) due to the effect of chain length, their chemical properties are almost

- 7 041148 identical. Various forms of PEG are also available, depending on the initiator used for the polymerization process, with the most common initiator being PEG monofunctional methyl ester or methoxypolyethylene glycol, mPEG for short. Lower molecular weight PEGs are also available as purer oligomers referred to as monodisperse, homogeneous, or discrete. Very high purity PEG has recently been shown to be crystalline, allowing the crystal structure to be determined by X-ray diffraction. Since purification and separation of pure oligomers is difficult, the cost at this quality is often 10-1000 times higher than the cost of polydisperse PEG.

PEGs are also available in various geometries. Branched PEGs have three to ten PEG chains extending from a central main group. Star-shaped PEGs have 10-100 PEG chains extending from a central core group. Comb PEGs have a plurality of PEG chains, usually grafted onto a polymer backbone. The numbers often included in the names of PEGs denote their average molecular weights (for example, a PEG with n=9 would have an average molecular weight of approximately 400 daltons and would be designated PEG400). Most PEGs include molecules with a molecular weight distribution (i.e., they are polydisperse). The size distribution can be characterized statistically by their weight average molecular weight (Mw) and their number average molecular weight (Mn), the ratio of which is called the polydispersity index (Mw/Mn). Mw and Mn can be measured using mass spectrometry.

PEGylation is the process of covalently attaching a PEG structure to another larger molecule, such as a therapeutic protein, which is then referred to as a PEGylated protein. PEGylated interferon alfa-2a or 2b is commonly used for the injectable treatment of hepatitis C. PEG is soluble in water, methanol, ethanol, acetonitrile, benzene and dichloromethane and insoluble in diethyl ether and hexane. It is crosslinked with hydrophobic molecules to form nonionic surfactants. PEGs contain potentially toxic impurities such as ethylene oxide and 1,4-dioxane. Ethylene glycol and its esters are nephrotoxic when applied to broken skin.

Polyethylene glycol is obtained by reacting ethylene oxide with water, ethylene glycol or ethylene glycol oligomers. The reaction is catalyzed by acidic or basic catalysts. Ethylene glycol and its oligomers are preferred as starting material over water because they allow the production of polymers with low polydispersity (narrow molecular weight distribution). The length of the polymer chain depends on the ratio of the reactants.

HOCH2CH2OH+P(CH2CH2O) —>HO(CH2CH2O)n+1H

Depending on the type of catalyst, the polymerization mechanism can be cationic or anionic. The polymerization of ethylene oxide is an exothermic process.

PEGylation (also often spelled pegylation) is the process of covalently and non-covalently joining or combining polyethylene glycol (PEG) polymer chains with molecules and macrostructures such as a drug, therapeutic protein, or vesicle, which are then referred to as PEGylated (pegylated). PEGylation is usually carried out by incubating the reactive PEG derivative with the target molecule. Covalent attachment of a PEG to a drug or therapeutic protein can mask the agent from the host immune system (reducing immunogenicity and antigenicity) and increase the hydrodynamic size (size in solution) of the agent, which increases its circulation time, decreasing renal clearance. PEGylation can also provide water solubility for hydrophobic drugs and proteins.

PEGylation is the process of attaching PEG polymer chains to molecules, typically peptides, proteins, and antibody fragments, which can improve the safety and efficacy of many therapeutic agents. This leads to a change in physico-chemical properties, including changes in structure, electrostatic bonding, hydrophobicity, etc. Such physical and chemical changes increase the systemic retention of the therapeutic agent. In addition, it may affect the binding affinity of the therapeutic molecule to cellular receptors and may alter absorption and distribution profiles.

PEG is the most attractive polymer for conjugation. The specific properties of PEG molecules suitable for pharmaceutical use are: water solubility, high mobility in solution, no toxicity and low immunogenicity, easy excretion from the body and altered distribution in the body.

The biological activity of the TRAIL derivative can be increased by selective PEGylation. The action of drugs can also be enhanced through the PEGylation process. The use of PEGylation increases molecular weight, protection of the metabolic cleavage site, and inhibition of the site of immunogenicity with increased half-life and in vivo stability and reduced immunogenicity. In addition, renal excretion of peptides and proteins associated with PEG is reduced due to the increase in molecular weights of peptides and proteins when PEG is used, so PEGylation has the benefit of increasing effects both pharmacokinetically and pharmacodynamically.

- 8 041148

Preferably, the polyethylene glycol or derivative thereof is linear or branched, or may be in the form of a dimer or trimer, with or without a linker to TRAIL and/or other PEG molecules. Representative polyethylene glycol derivatives include methoxy polyethylene glycol succinimidyl propionate, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide, or various branched types of these derivatives. Preferably, the polyethylene glycol derivative is a linear methoxy polyethylene glycol maleimide, a branched type methoxy polyethylene glycol maleimide or a trimeric methoxy polyethylene glycol maleimide, and more preferably a trimeric methoxy polyethylene glycol maleimide.

After PEGylating a TRAIL derivative with polyethylene glycol or derivatizing it, the molecular structure of the analog can be confirmed by mass spectrometry, liquid chromatography, X-ray diffraction analysis, polarimetry, and comparison of calculated values and measured values of representative elements in the PEGylated TRAIL formulation.

4) Auxiliary substances.

TRAIL, antibodies or derivatives can be included in the dosage form for administration. Typically, for injection, inhalation, pulmonary administration, or intraocular administration, a lyophilized form or spray-dried powder is used, which can be dissolved for administration in sterile water, a buffer, or other excipient, such as those listed in the Goodman and Gilman handbook. .

The PEGylated TRAIL derivative with polyethylene glycol or a derivative thereof can be prepared as a liquid or suspension, optionally including buffering agents, suspending agents, bacteriostatic agents, or viscosity modifiers, and then filled into an ampoule or vial in a single dose form. The composition is sterilized by filtration, irradiation or a gas such as ethylene oxide.

III. Methods for obtaining compositions.

A. Processes for making TRAIL and PEGylated TRAIL.

The first step of PEGylation is the functionalization of the PEG polymer at one or both ends. PEGs that are activated at each end with the same reactive group are known as homobifunctional, while if the functional groups present are different, then the PEG derivative is called 'Teterobifunctional'' or 'Teterofunctional''. Reactive or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.

The complete PEGylation processes for protein conjugation can be broadly classified into two types, namely solution-phase batch and column-fed batch. A simple and commonly used batch process involves mixing the reagents in a suitable buffer solution, preferably at a temperature of 4-6°C, followed by separation and purification of the desired product using a suitable technique based on their physico-chemical properties, including size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and membranes or aqueous two-phase systems.

The selection of an appropriate functional group for a PEG derivative is based on the type of reactive group available on the molecule to be coupled to the PEG. In the case of proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and the C-terminal carboxyl group can also be used as a site-specific site when conjugated to aldehyde-functional polymers. Techniques used to prepare first generation PEG derivatives typically involve reacting the PEG polymer with a group that is capable of reacting with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates and carbonates. Second generation PEGylation chemistry uses more efficient functional groups such as aldehyde, esters, amides, etc. for conjugation.

These heterobifunctional PEGs are very suitable for joining two molecules, requiring a hydrophilic, flexible and biocompatible spacer. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxyl groups and NHS esters. Third generation PEGylating agents are available where the polymer is branched, Y-shaped or comb-shaped, which have reduced viscosity and do not accumulate in organs. The unpredictability of the elimination period for PEGylated compounds can lead to the accumulation of high molecular weight compounds in the liver, leading to the formation of inclusion bodies with unknown toxicological consequences. In addition, changes in chain length can lead to unexpected in vivo clearance times.

B. Methods for the preparation of death receptor agonists.

Antibodies and antibody fragments can be obtained using any method known in the art for the production of polypeptides, eg, in vitro synthesis, recombinant DNA production, and the like. Antibodies can be produced using recombinant technology

- 9 041148

DNA. Agonistic antibodies against DR5 can be obtained using recombinant immunoglobulin expression technology. Recombinant production of immunoglobulin molecules, including humanized antibodies, is described in US Pat. No. 4,816,397 (Boss et al.), US Pat. UK GB 2,209,757. Methods for the recombinant expression of immunoglobulins, including humanized immunoglobulins, can also be found in Goeddel et al., Gene Expression Technology Methods in Enzymology Vol. 185 Academic Press (1991), and Borreback, Antibody Engineering, W. H. Freeman (1992). Additional information on the design, construction and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

Antibodies can also be obtained by immunizing animals with synthetic or purified monomeric, homomeric or heteromeric DR5. The immune serum is applied to a peptide affinity chromatography column to obtain a highly specific immunoreagent.

Human antibodies and humanized antibodies described in this application can be obtained by any known method. Examples of methods for producing human monoclonal antibodies include those described in Boerner et al., J. Immunol., 147(1), 86-95 (1991). Human antibodies described in this application (and fragments thereof) can also be obtained using phage display libraries (see, for example, Marks et al., J. Mol. Biol., 222, 581-597 (1991)). The human antibodies described in this application can also be obtained in transgenic animals. For example, transgenic mutant mice have been described that are capable of producing the full repertoire of human antibodies in response to immunization (see, for example, Jakobovits et al., PNAS, 90, 2551-255 (1993); and Jakobovits et al., Nature, 362, 255-258 (1993)).

Methods for humanizing non-human antibodies are known in the art. For example, humanized antibodies can be made by replacing rodent complementarity-determining regions (CDRs) or CDRs with corresponding human antibody sequences. Detailed procedures are disclosed in Jones et al., Nature, 321, 522-525 (1986); Riechmann et al., Nature, 332, 323-327 (1988); Verhoeyen et al., Science, 239, 1534-1536 (1988).

Methods that can be used to make humanized antibodies are also described in US Pat. No. 4,816,567; US Pat. No. 5,565,332; US Patent 5,721,367; US Patent 5,837,243; US Patent 5, 939,598; US Pat. No. 6,130,364; and US Patent 6,180,377.

IV. Application methods.

Death receptor agonists may be used to treat pancreatitis, pancreatic fibrosis, pancreatic pain, or any combination thereof. Death receptor agonists may be useful in reducing, arresting, or regressing pancreatic inflammation in the early stages of the inflammatory process, reducing, arresting, or regressing pancreatic fibrosis, and reducing or reversing pancreatic pain.

The human dose of a pharmaceutical composition containing a TRAIL derivative PEGylated with polyethylene glycol or a derivative thereof may vary depending on the age, body weight, sex, form of administration, health status and disease stage of the patients, and may be administered at the discretion of physicians or pharmacists, upon this preferably the dose is 0.01-200 mg/kg/day.

As described in the examples, the effect of TRAIL _PEG in primary human PGCs was examined. In particular, we investigated the antifibrotic efficacy and analgesic efficacy of intravenous TRAIL _PEG in various rat models of pancreatitis, including cerulein-induced acute pancreatitis (AP) and ethanol/cerulein/Libera DeCarly (LD)-induced chronic pancreatitis (CP). As described herein, TRAIL signaling plays an important role in pancreatic fibrogenesis as well as in the regulation of TGFe, which can directly sensitize nociceptors and cause pancreatic hyperalgesia. TRAILπ _E g reduced the progression of pancreatitis in the acute and chronic phases. Surprisingly, treatment with TRAILπ _Eg significantly reduced pain in rat models of CP.

As detailed below, TRAILπ _Eg selectively blocks PCO activation and eliminates activated PCO, CP effectors, leading to CP regression. In addition, by targeting PCOs that sensitize nociceptors as a result of TGFe upregulation, TRAILπ _Eg reduces HPAssociated severe pain without causing systemic toxicity.

The role of TRAIL signaling in pancreatic fibrogenesis and pain was investigated to determine whether a death receptor agonist (eg, TRAIL _PEG and anti-DR5 antibodies) could be used to treat pancreatitis in the outpatient setting. A new direction for the use of TRAIL signaling in the treatment of pancreatitis and new schemes based on TRAIL have been proposed.

A. Conditions to be treated.

Pancreatitis is an inflammation of the pancreas. The pancreas is a large organ behind the stomach that produces digestive enzymes. There are two main types - acute pancreatitis and chronic pancreatitis. Signs and symptoms of pancreatitis include

- 10 041148 pain in the upper abdomen, nausea and vomiting. The pain often radiates to the back and is usually sharp. Fever may occur during acute pancreatitis, and symptoms usually resolve within a few days. In chronic pancreatitis, weight loss, steatorrhea, and diarrhea may occur. Complications may include infection, bleeding, diabetes, or problems with other organs.

The most common causes of acute pancreatitis are gallstones and alcohol abuse. Other causes include direct trauma, certain medications, infections such as mumps, and tumors, among others. Chronic pancreatitis can develop as a result of acute pancreatitis. This usually occurs as a result of years of alcohol abuse. Other causes include high blood fat levels, elevated blood calcium, certain medications, and certain inherited conditions such as but not limited to cystic fibrosis. Smoking increases the risk of acute and chronic pancreatitis. The diagnosis of acute pancreatitis is based on a threefold increase in amylase or lipase in the blood. In chronic pancreatitis, these tests may be normal. Diagnostic imaging, such as ultrasound and CT scan, may also be helpful.

1) Acute pancreatitis.

Acute pancreatitis is usually treated with intravenous injections, pain therapy, and sometimes antibiotics. As a rule, food or drink is excluded, and a tube may be inserted into the stomach. A procedure known as endoscopic retrograde cholangiopancreatography (ERCP) may be performed to open the pancreatic duct if it is blocked. In individuals with gallstones, the gallbladder is often also removed. In chronic pancreatitis, in addition to the above, temporary feeding through a nasogastric tube may be used to ensure adequate nutrition. Long-term dietary changes and pancreatic enzyme replacement may be required, sometimes with partial pancreatic resection surgery.

Acute pancreatitis occurs in about 30 out of 100,000 people a year. New cases of chronic pancreatitis develop in about 8 out of 100,000 people a year and currently affect about 50 out of 100,000 people in the US. Globally, pancreatitis resulted in 123,000 deaths in 2013, up from 83,000 deaths in 1990. Pancreatitis is more common in men than in women. Often chronic pancreatitis begins at the age of 30-40 years, while it is rare in children. Acute pancreatitis was first described at autopsy in 1882, while chronic pancreatitis was first described in 1946.

The most common symptoms of pancreatitis are sharp, burning pain in the upper abdomen or left upper quadrant of the abdomen, radiating to the back, nausea, and vomiting that is aggravated by eating. In physical diagnosis, the results vary depending on the severity and presence of internal bleeding. Blood pressure may rise with pain or fall with dehydration or bleeding. The heart rate and respiration are often increased. The abdomen is usually soft, but to a lesser extent than when in pain. As is often seen in abdominal disease, bowel sounds may be muffled due to reflex paralysis of the bowel. Fever or jaundice may be present. Chronic pancreatitis can lead to diabetes or pancreatic cancer. Weight loss of undetermined etiology may occur as a result of pancreatic enzyme deficiency, which interferes with normal digestion.

Eighty percent of cases of pancreatitis are caused by alcohol or gallstones (stones). Gallstones are the single most common cause of acute pancreatitis. Alcohol is the single most common cause of chronic pancreatitis.

Some drugs are often associated with pancreatitis, usually corticosteroids such as prednisone, but also include HIV drugs didanosine and pentamidine, diuretics, the anticonvulsant valproic acid, the chemotherapy drugs L-asparaginase and azathioprine, estrogen by increasing triglycerides in the blood, as well as hypoglycemic agents such as metformin, vildagliptin and sitagliptin. Medicines used to treat conditions that are themselves associated with an increased incidence of pancreatitis may also be associated with pancreatitis. Examples include statins in the treatment of dyslipidaemia and gliptins in the treatment of diabetes. According to the US Food and Drug Administration's MedWatch monitoring system and published reports, atypical antipsychotics such as clozapine, risperidone, and olanzapine can also cause pancreatitis.

Other common causes include trauma, mumps, autoimmune disease, high blood calcium levels, hypothermia, and endoscopic retrograde cholangiopancreatography (ERCP). A divided pancreas is the most common congenital anomaly of the pancreas and may underlie some recurrent cases. Type 2 diabetes is associated with a 2.8-fold increased risk.

Less common causes include pancreatic cancer, pancreatic duct stones, vasculitis (inflammation of small blood vessels in the pancreas), Coxsackie virus infection, and porphyria, especially acute intermittent porphyria and erythropoietic protoporphyria.

There is a hereditary form that leads to the activation of trypsinogen in the pancreas, which leads to self-digestion. Genes involved may include Trypsin 1, which codes for trypsinogen, SPINK1, which codes for a trypsin inhibitor, or a transmembrane conductance regulator in cystic fibrosis.

Common causes of pancreatitis include alcohol/ethanol, gallstones, steroids, traumatic autoimmune pancreatitis, mumps, hyperlipidemia, hypothermia, hyperparathyroidism, scorpionism, endoscopic retrograde cholangiopancreatography, and drugs (usually azathioprine and valproic acid).

A number of infectious agents have been recognized as causes of pancreatitis, including: mumps, coxsackie virus, hepatitis B, cytomegalovirus, herpes simplex virus, and varicella zoster virus.

The differential diagnosis of pancreatitis includes, but is not limited to, cholecystitis, choledocholithiasis, perforated gastric ulcer, intestinal infarction, small bowel obstruction, hepatitis, and mesenteric ischemia. Diagnosis requires 2 of 3 of the following criteria: characteristic acute onset of epigastric pain or vague abdominal pain that may radiate to the back (see Signs and Symptoms above), serum amylase or lipase levels >3 times the upper limit of normal, and an imaging study with characteristic changes. CT, MRI, abdominal ultrasound, or endoscopic ultrasound may be used for diagnosis. Amylase and lipase are 2 enzymes produced by the pancreas. Elevated lipase is generally considered the best indicator of pancreatitis because it has greater specificity and a longer half-life. In the case of imaging, abdominal ultrasound is convenient, simple, non-invasive, and inexpensive. It provides higher sensitivity and specificity for gallstone pancreatitis than other imaging modalities. However, in 2535% of patients, observation of the pancreas is obstructed by intestinal gases, making evaluation difficult. A contrast-enhanced CT scan is usually performed more than 48 hours after the onset of pain to evaluate for pancreatic necrosis and excess pancreatic juice, and to predict disease severity. An earlier CT scan may be falsely reassuring. ERCP or endoscopic ultrasonography may also be used if a biliary etiology of pancreatitis is suspected.

Treatment for pancreatitis is supportive and depends on the severity. Morphine is usually suitable for pain control. There is a report that morphine can cause spasm of the sphincter of Oddi, but this is conflicting information. There are no clinical studies to suggest that morphine may aggravate or cause pancreatitis or cholecystitis. The treatment used for acute pancreatitis will depend on whether the diagnosis is a mild form of the condition, which does not cause complications, or a severe form, which can cause serious complications. Treatment of mild acute pancreatitis is successfully carried out upon admission to the general hospital department. Usually, people were not allowed to eat until inflammation resolved, however, more recent evidence suggests that eating early is safe and improves outcomes. Because pancreatitis can damage the lungs and affect normal lung function, oxygen is sometimes given through breathing tubes that are passed through the nose. The tubes can then be removed a few days after it is clear that the condition is improving. An episode of acute pancreatitis can cause dehydration, so fluids are given intravenously. The pain associated with even mild to moderate cases of acute pancreatitis can be acute, meaning that a narcotic analgesic may be needed.

Severe pancreatitis is associated with organ failure, necrosis, infectious necrosis, pseudocyst, and abscess. People who are diagnosed with severe acute pancreatitis should be admitted to a high-risk ward or intensive care unit. The body's fluid levels can drop significantly as it redirects fluids and nutrients in an attempt to repair the pancreas. Decreased fluid levels can lead to a decrease in blood volume in the body, which is known as hypovolemic shock. Hypovolemic shock can be life threatening because it can very quickly deprive the body of the oxygenated blood it needs to survive. To avoid the development of hypovolemic shock, fluids are administered intravenously. Oxygen is given through tubes through the nose, and mechanical ventilation equipment can be used to support breathing. Feeding tubes can be used to provide nutrition in combination with appropriate pain relief.

As with mild acute pancreatitis, it is necessary to treat the underlying causes - gallstones, stop taking medications, stop drinking alcohol, etc. If gallstones are the cause, an ERCP procedure or removal of the gallbladder may be recommended. The gallbladder should be removed at the first hospitalization or within two weeks of the onset of pancreatitis to limit the risk of recurrence of pancreatitis. If alcohol is the cause of pancreatitis, stopping alcohol use and treating alcohol dependence may cause the pancreatitis to regress. Even if the underlying cause is not related to alcohol use, doctors recommend avoiding it for at least six months, as it can cause further damage to the pancreas during the recovery process. Food intake, especially fat intake, is usually limited, but early enteral nutrition within 48 hours has been shown to improve clinical outcomes. Fluids and electrolytes are replenished intravenously. Clinical nutrition is initiated with tube feeding to bypass the part of the digestive tract most exposed to secreted pancreatic enzymes if there is no improvement in the first 72-96 hours of treatment.

Early complications include shock, infection, systemic inflammatory response syndrome, low blood calcium, high blood glucose, and dehydration. Blood loss, dehydration, and fluid leaking into the abdomen (ascites) can lead to kidney failure. Respiratory complications are often severe. An effusion is usually present in the pleural cavity. Shallow breathing caused by pain can lead to lung collapse. Pancreatic enzymes can attack the lungs, causing inflammation. Severe inflammation can lead to intra-abdominal hypertension and abdominal compression syndrome, further impairing kidney and airway function and potentially requiring open abdominal intervention to relieve pressure.

Late complications include recurrent pancreatitis and the development of pancreatic pseudocysts, cavities containing pancreatic secretions that are delimited by a wall of scar tissue. They can cause pain, become infected, rupture and bleed, block the bile duct and cause jaundice, or travel through the abdomen. Acute necrotizing pancreatitis can lead to pancreatic abscess, accumulation of pus caused by necrosis, tissue fusion, and infection. This occurs in about 3% of cases, or nearly 60% of cases with more than two pseudocysts and gas in the pancreas.

2) Chronic pancreatitis.

Chronic pancreatitis is a long-term inflammation of the pancreas that changes the normal structure and function of that organ. It may present with episodes of acute inflammation in a previously damaged pancreas or chronic damage with persistent pain or malabsorption. This is a pathological process characterized by irreversible damage to the pancreas, in contrast to reversible changes in acute pancreatitis. Annually, the number of newly registered cases of chronic pancreatitis is from 5 to 12 per 100,000 people, the total number of cases is 50 per 100,000 people. HP is more common in men than in women.

The symptoms of chronic pancreatitis are usually manifested by persistent abdominal pain or steatorrhea caused by impaired absorption of fats from food. Significant weight loss is often due to malabsorption and may remain a clinical problem as the disease progresses. The patient may also complain of pain when eating, especially in cases where the food contains a high percentage of fat and protein.

Causes of chronic pancreatitis include: alcohol, autoimmune disorders, intraductal obstruction, idiopathic pancreatitis, tumors, ischemia, and calcified stones. The relationship between etiological factors, genetic predisposition, and the rate of progression of the disease requires further clarification, although recent studies indicate that smoking may be a high risk factor for the development of chronic pancreatitis. In a study in a small group of patients, chronic pancreatitis was hereditary. Almost all patients with cystic fibrosis develop chronic pancreatitis, usually from birth. Gene mutations in cystic fibrosis have also been identified in patients with chronic pancreatitis who did not have other manifestations of cystic fibrosis. Obstruction of the pancreatic duct due to a benign or malignant process can lead to chronic pancreatitis.

The mechanism of chronic pancreatitis, considered from a genetic point of view, indicates an early onset of acute epigastric pain, starting in childhood. This autosomal dominant disease, chronic pancreatitis, was identified in the PRSS1 cationic trypsinogen gene with the R122H mutation. R122H is the most common mutation in hereditary chronic pancreatitis, with an arginine to histidine substitution at amino acid position 122 of the trypsinogen protein. Of course, there are other mechanisms - alcohol, malnutrition and smoking - each of which has its own effect on the pancreas.

The diagnosis of chronic pancreatitis is based on the study of the structure and function of the pancreas. Serum amylase and lipase may be moderately elevated in chronic pancreatitis, with amylase and lipase almost always elevated in the acute condition. The secretin stimulation test is considered the best test for diagnosing chronic pancreatitis. Other tests used to determine chronic pancreatitis are serum trypsinogen, computed tomography, ultrasound, and biopsy. In the case when chronic pancreatitis is caused by genetic factors, increases in ESR, IgG4, rheumatoid factor,

AHA and anti-smooth muscle antibodies.

The various treatments for chronic pancreatitis are medical measures, therapeutic endoscopy, and surgery. Treatment is directed, if possible, to the underlying cause, as well as to the elimination of pain and malabsorption. Insulin-dependent diabetes mellitus can occur and require long-term insulin therapy. Abdominal pain can be very severe and may require high doses of analgesics, sometimes opiates. Stopping alcohol consumption and dietary changes (low fat diet) are important to treat pain and slow down the calcification process. Antioxidants may help, but it is not clear if their effect is significant. Pacreatic enzyme replacement is often effective in the treatment of malabsorption and steatorrhea associated with chronic pancreatitis. Treatment for CP consists of administering a solution of pacreatic enzymes with meals. Some patients experience pain relief with enzyme replacement and, because they are relatively safe, enzyme replacement therapy for chronic pancreatitis is an acceptable treatment step for most patients. Treatment may be more successful in patients without major duct involvement and in patients with idiopathic pancreatitis. Surgery for the treatment of chronic pancreatitis is usually divided into two areas - resection and drainage procedures. Reasons for choosing surgery include the presence of a pseudocyst, fistula, ascites, or fixed obstruction.

3) Fibrosis of the pancreas.

Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. It can be a reactive, benign, or pathological condition. In response to injury, this is called scarring, and if the fibrosis originates from a single cell line, it is called a fibroma. Physiologically, fibrosis results in an overlay of connective tissue that can impair the structure and function of the underlying organ or tissue. Fibrosis can be used to describe the pathological condition of excessive fibrous tissue overlay as well as the process of connective tissue overlay during healing.

Fibrosis is similar to the scarring process in that in both cases there are stimulated cells overlaying connective tissue including collagen and glycosaminoglycans. Immune cells called macrophages, as well as any damaged tissue between surfaces called interstitium, secrete TGFe. There are many reasons for this, including inflammation of the surrounding tissue or a generalized inflammatory condition with elevated levels of circulating mediators. TGFe stimulates the proliferation and activation of fibroblasts that overlay connective tissue. Fibrosis can occur in many body tissues, usually as a result of inflammation or injury, and examples include: pancreas (chronic pancreatitis), liver (cirrhosis), lungs (pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis), heart (atrial fibrosis , endomyocardial fibrosis, previous myocardial infarction), brain (glial scar), arthrofibrosis (knee, shoulder, other joints), Crohn's disease (intestine), Dupuytren's contracture (hands, fingers), keloid (skin), mediastinal fibrosis (soft tissues mediastinum), myelofibrosis (bone marrow), Peyronie's disease (penis), nephrogenic systemic fibrosis (skin), progressive massive fibrosis (lungs); a complication of pneumoconiosis in coal workers, retroperitoneal fibrosis (retroperitoneal soft tissue), scleroderma/systemic sclerosis (skin, lungs), and some form of adhesive capsulitis (shoulder).

4) Methods for the treatment of pancreatitis or pancreatic pain syndrome.

a. Directed impact on the PZK.

On pathological examination, CP is recognized by significant fibrosis. Pacreatic fibrogenesis is mainly formed by pacreatic stellate cells (PSCs) (Erkan, M., et al., Gut, 2012. 61(2):172-178; Omary, M.B., et al., J Clin Invest, 2007. 117( 1):50-59; Pinzani, M., Gut, 2006. 55(1):12-14). When the pancreas is damaged or diseased, resting PGCs (pCCs) undergo activation and transform into proliferative, fibrogenic, and contractile myofibroblasts, which promote collagen deposition and result in fibrous tissue. By nature, activated PGCs (aPCDs) are the main target of antifibrotic therapies targeting the pancreas (Omary, MB, et al., J Clin Invest, 2007. 117(1):50-59, Apte, MV, et al., J Gastroenterol Hepatol, 2006. 21 Suppl 3:S97-S101). Thus, the elimination of aPCD is a logical strategy for preventing, stopping and/or regressing fibrogenesis and its complications, pain. The introduction of a molecularly targeted agent that can block the activation of pPPK in aPZK and cause selective induction of apoptosis of aPZK, but not pPZK, will provide a reliable anti-fibrotic effect in CP, since the effector of pancreatic fibrogenesis is removed. Regression of pancreatic fibrosis halts/reverses the progression of CP, which therefore causes a reduction in pain associated with CP and improves pancreatic function (FIG. 1).

b. Targeting the PZK-TGFe Axis With the use of death receptor agonists for the treatment of pain.

The transition from acute to chronic pain is an area of active research (Reichling, D.B. and Levine, J.D., Trends Neurosci, 2009. 32(12):611-618). Tissue inflammation initiates a cascade of events leading to peripheral sensitization, i.e. increased sensitivity of primary afferent neurons (nociceptors), whose bodies are located in the dorsal root ganglia (DNR), and the central synapse ends with second-order neurons in the spinal cord. However, little is known about the driving factors in the later stages of inflammation, when tissue fibrosis becomes noticeable. As described in this application, treatment of HDC neurons with TGFe caused changes in excitability and suppressed the specific voltage-dependent potassium current (IA), which is a characteristic feature of nociceptive excitability in chronic pancreatitis (Zhu, Y., et al., Mol Pain, 2012 8:65) . TGFe itself can sensitize nociceptors, induce pancreatic hyperalgesia, and contribute to the heightened behavioral response that accompanies CP. As described here, PGCs upregulate TGFe during the activation process and influence the excitability of HDC neurons. New facts about the role of TRAIL and TGFe in nociception are also relevant in other conditions characterized by inflammation and chronic pain Indeed, it has been pointed out that the transition from acute to chronic pain conditions may be the most important issue in research to improve the clinical management of debilitating pain (Reichling, DB and Levine, JD, Trends Neurosci, 2009 32(12):611-618).

C. Subjects to be treated.

Generally, subjects to be treated include subjects suffering from or at risk of developing pancreatitis, pancreatic fibrosis, and/or pancreatic pain.

In some embodiments, the subjects to be treated include subjects suffering from or at risk of developing pancreatitis, pancreatic fibrosis, and/or pancreatic pain, who also suffer from other chronic or acute conditions such as type 2 diabetes, liver fibrosis, cirrhosis. , liver cancer, pulmonary fibrosis, skin fibrosis, pancreatic cancer, metastatic cancer, autoimmune conditions including type 1 diabetes and rheumatoid arthritis.

In other embodiments, the subjects to be treated include subjects suffering from or at risk of developing pancreatitis, pancreatic fibrosis, and/or pancreatic pain, who do not suffer from additional chronic or acute conditions such as type 2 diabetes, liver fibrosis, cirrhosis. , liver cancer, pulmonary fibrosis, skin fibrosis, pancreatic cancer, metastatic cancer, autoimmune conditions including type 1 diabetes and rheumatoid arthritis.

D. Effective amounts of DR5 agonists.

The compositions are administered at a dose of 0.001 to 100 mg/kg, e.g. , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 10θ mg/kg. For example, the compositions are administered at a dose of 0.2 to 20 mg/kg, or at a dose of 0.001 to 20 mg/kg.

In some aspects, pancreatic tissues are protected, fibrous formation is reduced, pancreatic fibrogenesis is inverted, pain is reduced, and healthy pancreatic tissues are intact. In one aspect, treating a fibrotic disease or disorder includes reducing pain. The subject's pain is reduced by 1-100%, e.g. 99.5, 99.9 and 100%.

In one aspect, treating a fibrotic disease or disorder includes reducing pancreatic fibrosis. Fibrosis of the pancreas in the subject decreases by 1-100%, e.g. 99, 99.5, 99.9 and 100%.

In one aspect, treating a fibrotic disease or disorder includes reducing pancreatitis. Pancreatitis decreases by 1-100% e.g. , 99.9 and 100%.

1) Dose and regimens for combination therapy.

Methods of treatment generally include treating a disease or a symptom thereof, or a method of achieving a desired physiological change, comprising administering to a mammal, especially a human, an effective amount of a pro-apoptotic agent to treat pancreatitis or a symptom thereof, or producing a physiological change.

An effective amount of a death receptor agonist may be administered to a subject as a single dose, once or multiple times. Administration can be done once a day, twice a day, three times a day, once a week, twice a week, once every two weeks, or once a month.

An effective amount of the death receptor agonist may be administered as a unit dosage form (eg, as a dosage unit) or subtherapeutic doses administered over a finite time interval. Such unit doses may be administered daily for a finite period of time, for example up to 3, or up to 5, or up to 7, or up to 10, or up to 15, or up to 20,

- 15 041148 or up to 25 days.

In some embodiments, a unit dose is administered as the primary therapy. In other embodiments, the unit dose is administered with the second agent in combination therapy.

a. Combined therapy.

In some embodiments, the death receptor agonist is present in combination with an additional active ingredient. The death receptor agonists and the additional active agent may be administered together, for example, as part of the same composition, or administered separately and independently, at the same or different times (i.e. administration of the ligand or agonist and the second active agent separated by a finite period time apart). Therefore, the term combination or combined is used to refer to the joint, simultaneous or sequential administration of a ligand or agonist and a second active substance. Combinations may be administered together (eg, as a mixture), separately but simultaneously (eg, via separate intravenous catheters to the same subject; one agent is administered orally while the other agent is administered by infusion or injection, etc.), or sequentially (for example, first one agent is administered, then the second).

In some embodiments, administration of a death receptor agonist in combination with a second active agent provides a superior result than when the proapoptotic agent and the second active agent are administered separately or separately (i.e., the result obtained by administering the combination is greater than the sum of the results obtained by administering components separately). 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 separately. In some embodiments, the amount of one or both agents, when used in combination therapy, is sub-therapeutic when used alone.

A combination therapy regimen may include single or multiple administrations of a ligand or agonist. A combination therapy regimen may include one or more administrations of the second active ingredient.

In some embodiments, the proapoptotic agent is administered prior to the first administration of the second active ingredient. In other embodiments, the implementation of the ligand or agonist is administered after the first administration of the second active substance.

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 administration of the second active agent.

Dosing regimens or courses of agents may overlap completely or partially, or may be sequential. For example, in some embodiments, all such administration(s) of the pro-apoptotic agent occurs before or after administration of the second active agent. Alternatively, the administration of one or more doses of the pro-apoptotic agent may alternate in time with the administration of the second therapeutic agent to obtain a homogeneous or heterogeneous course of treatment, in which one or more doses of the pro-apoptotic agent are administered, then one or more doses of the second active substance, followed by the administration of one or over doses of death receptor agonists; or one or more doses of the second active ingredient are administered, followed by one or more doses of the proapoptotic agent, followed by one or more doses of the second active ingredient; and so on, all in accordance with whatever schedule is chosen or required by the researcher or healthcare provider conducting the therapy.

V. Kits.

Medical kits are also disclosed. Medical kits may include, for example, a dose reserve of a pro-apoptotic agent, preferably a TRAIL receptor agonist ligand or agonist, alone or in combination with a second therapeutic agent. When present in combination with a second therapeutic agent, the active ingredients may be supplied separately (eg in lyophilized form) or in a pharmaceutical composition (eg in a mixture). The active ingredients may be present in a unit dosage form or in a base form that needs to be diluted prior to administration. In some embodiments, the kit includes a supply of a pharmaceutically acceptable carrier. The kit may also include devices for administering active substances or compositions, such as syringes. The kits may include printed instructions for administering the compound for use as described above.

Hereinafter, the present invention will be described with reference to the following non-limiting examples. The contents of all references, patents and published patent applications cited throughout this application, as well as drawings, are incorporated herein by reference.

Examples

General methods.

To test the efficacy of TRAIL _P & _G in vivo, biological samples were tested as follows.

Biochemical analysis and liver lipid levels:

levels of serum amylase, lipidase, ALT, ACT, alkaline phosphatase, sodium, cholesterol, triglyceride,

- 16 041148 glucose, albumin, protein and urea nitrogen were determined using an IDEXX analyzer.

Histological examination and IHC:

fixed pancreas specimens were stained with H-E, Sirius red, α-SMA and counterstained with Mayer's hematoxylin. Immunofluorescence (IF) double staining was performed using the appropriate antibodies against α-SMA, active caspase-3, and the nuclei were stained with DAPI. TUNEL analysis with pancreatic tissues was also performed to determine DNA damage when exposed to apoptotic signaling cascades.

Real-time PCR:

gene expression levels were measured by PCR-PB (ABI7500) using suitable primers for DR5, α-SMA, Pdgf-r, Col1a1, Col1a2, Col1a3, Tgf-b1, Tgf-br2, Tgf-br3, Bmp7, Timp1/3 , Mmp2/3/7/9/13, as well as Bcl-2, Bcl-xl, Mcl-1, FLIP and cIAP.

Western blotting:

Western blot analyzes were performed on protein extracts from pancreatic homogenates for key markers including, but not limited to: DR5, α-SMA, collagen, Timp, TGFe, Mmps, caspase-8/-9/-3, cleaved PARP-1 , Bcl-2, Bcl-xl, Mcl-1, FLIP, FADD and t-Bid.

Volitional behavior when using the VFF test and electrical stimulation:

after treatment with TRAIL _PE g, 4 rats from each group of EtOH/cerulein/LD diet induced CP were used for VFF tests and electrical stimulation studies.

Data and statistical analysis:

six to ten animals in the anti-fibrosis group and four animals in the pain therapy group were required to observe the difference between the means of the treatment and control groups with a 5% significance level and 90% power. Tissue sections were examined by clinical laboratory assistants without prior acquaintance with the sample. The intensity and extent of staining was scored based on an accepted scoring system from 0 to 4. Immunohistochemical (IHC) images were analyzed using NIH Image J. Comparisons between controls and up/down regulated receptors, biomarkers, and between groups with and without TRAIL _PEG treatment, were performed. using two-tailed Fisher's exact test, one-way analysis of variance, or chi-square test, as appropriate. All data sets were analyzed by multiple comparisons in different control groups. All studies were carried out using Prism software (GraphPad). P-values less than 0.05 were considered statistically significant in all analyses.

Example 1. Effect of TRAILπ _EG on pancreatic cells.

PEGylation is the gold standard for increasing the half-life (ti/ ₂ ) of protein drugs and a highly effective commercial strategy (Harris et al., Nat Rev Drug Discov, 2(3):214-221 (2003)). More than ten PEGylated biologics have been approved by the FDA, with PEGylated proteins generally considered less immunogenic (Alconcel et al., Polymer Chemistry, 2(7):1442-1448 (2011)). Extremely short t _1/2 TRAIL (less than 5 minutes in rodents and 30 minutes in humans) (Kelley et al., J Pharmacol Exp Ther, 299(1):31-38 (2001); Ashkenazi et al., J Clin Oncol , 26(21):3621-3630 (2008)) makes it difficult to study the functions of TRAIL and test the effectiveness of drugs, especially in vivo. PEGylated TRAIL was prepared by stabilizing a trimeric TRAIL containing the isoleucine zipper (iLZ) amino acid motifs at the end of each monomer, which promotes the formation of a trimer at the N-terminus, with a 5 kDa PEG molecule (TRAILπ _EG ) (WO 2007/145457). TRAILπ _EG significantly increased stability and increased circulation half-lives in rats and monkeys compared to recombinant TRAIL such as dulanermin (Genentech) (Lemke, J., et al., Cell Death Differ, 2014. 21(9):1350- 1364), which has been clinically studied and has shown a good safety profile but poor efficacy.

Materials and methods.

To investigate potential pancreatic toxicity, TRAIL _PEG was tested in pancreatic acinar cells (AR42J, ATCC CRL-1492) (ATCC) and primary human islets (pancreas) (Celprogen, Torrance, CA). AR42J cells (ATCC) were maintained in RPMI 1640 medium (Corning cellgro, Manassas, VA, USA) supplemented with 20% fetal bovine serum (FBS; Sigma, St Louis, MO), 100 U/ml penicillin/streptomycin (Life Technology). Human pancreatic islets of Langerhans cells (#35002-04, Celprogen, Torrance, CA) were maintained in complete human extracellular matrix for primary cell culture of human islets of Langerhans (#E35002-04, Celprogen) and serum media (Celprogen #M35002-04). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Briefly, 2×10 ⁴ cells were cultured for 24 hours in a 96-well plate, then treated with TRAILπ _EG (0.101, 102, 103 ng/ml) for 3 hours and cell viability was examined in the MTT assay for apoptosis. After incubation, MTT solution was added to each well and incubated for 4 hours. Absorbance at 430 nm was determined using a microplate spectrophotometer (Bio-Tek Instruments Inc, Winooski, VT).

- 17 041148

Results.

As shown in FIG. 2, TIAGPeG showed no toxicity to acinar cells (AR42J) and primary human islets.

Example 2 Culture-activated pancreatic stellate cells become susceptible to TRAIL-induced apoptosis.

The results show that upon activation of primary human PGCs in culture, PGCs transformed into myofibroblast-like cells and upregulated DR4 and DR5 as well as other fibrogenic markers, and became very sensitive to TRAIL _PEG as a result of DR5/DR4 upregulation.

Activated primary human PZKs (aPZKs), non-resting PZKs (pPZKs) upregulate DR and become susceptible to TRAIL-mediated apoptosis.

Materials and methods.

TRAIL-induced apoptosis was investigated in primary human PGCs. Human PGCs (ScienceCell Research Lab) were grown in SteCM (ScienceCell) medium supplemented with 2% FBS, 1% stellate cell growth supplement, and 1% penicillin/streptomycin solution according to the manufacturer's instructions. 2x105 PGCs were cultured in 6-well poly-P-lysine coated plates and cultured for 2 (resting) days and 7 days (activated) and harvested for analysis. Unlike other cells, stellate cells, including hepatic stellate cells (HSCs) and pancreatic stellate cells (PSCs), can be activated and differentiated into activated stellate cells in successive cultures (eg, cultured for more than 5 days). The expression of DR5 and DR4, α-SMA, collagen and TGFe, as well as PDGFR and TGFe, was analyzed by Western blotting in resting and activated cells. Activation causes a simple cultivation for 7 days.

Results.

Activation caused morphological changes in PGC and significantly increased fibrogenic markers and, importantly, DR4 and DR5 (Fig. 3A-3F). When TIA1E cells were treated _{with PEG} (1 μg/mL) for 3 h in the resting or activated states, TRAIL-induced apoptosis was clearly observed in aPZK but not in pPZK, which was confirmed by the features of cell apoptosis and the quantification of cell apoptosis measured by the test MTT (Fig. 4).

Monitoring the regulation of TRAIL signaling molecules, fibrosis markers and apoptosis markers.

The regulation of TRAIL signaling molecules, fibrosis markers and apoptosis markers was monitored in primary human PGCs during activation. Safety was confirmed in primary human islets and pancreatic acinar cells.

Materials and methods.

Representative TRAIL-signaling and fibrosis-associated molecules were screened for protein and mRNA levels in primary human PGCs (ScienceCell). PGCs were activated in culture for 2, 4 and 7 days in SteCM medium and harvested. Expression of TRAIL-signaling molecules DR5, DR4, and FLIP and fibrosis markers α-SMA, Pdgf-r, type 1/2/3 collagen, Mmps, Timps, collagens, and TGFe, as well as apoptotic and anti-apoptotic markers, including caspase-8 , -9, -3 digested with PARP-1, BCL-2, BCL-XL, FLPI and cIAP were analyzed by qPCR and Western blotting. Changes in the morphology of the PGC were observed using microscopic examination. After confirming the expression profiles of TRAIL signaling molecules, the effect of TRAIL-induced apoptosis during PCO activation was examined. For molecular studies, resting and activated PGCs in culture (2 to 7 days) were treated with TRAIL _PEG at 1 µg/ml. Activated PGCs were treated with or without TRAIL _PEG (1 μg/ml) for 3 h and analyzed by qPCR.

Results.

Activated PGCs upregulated many anti-apoptotic proteins such as BCL-2, BCL-XL, X-IAP, however, cells remained susceptible to TRAIL-induced apoptosis, as evidenced by upregulation of cleaved (Cl)PARP-1, Cl Casp-8 and Cl Casp- 3. In the case of most primary cancer cells, such upregulated anti-apoptotic proteins potently inhibit TRAIL-induced apoptosis, causing resistance to TRAIL. Activated PGCs have also been shown to be difficult to kill when incubated with common toxic agents, including antitumor agents such as doxorubicin (DOX, 100 nM), cisplatin (CIS, 10 μM), or hydrogen peroxide (H ₂ O ₂ , 10 μM). When activated PGCs were incubated with TRAILπ _EG (1 µg/mL) for 3 h and toxic agents for 48 h, only TRAIL _PEG induced strong apoptosis, as TRAIL _PEG -treated cells upregulated Cl PARP-1, Cl Casp-8, Cl Casp-3 and Cl Casp-9.

Culture-activated primary human PGCs upregulate many anti-apoptotic proteins (BCL-XL, BCL-2, X-IAP) but remain susceptible to TRAIL-induced apoptosis as evidenced by upregulation of positively cleaved (Cl) PARP-1, Cl Casp-8 (caspase8) and Cl Casp-3 (caspase-3).

Example 3 An anti-DR5 antibody, but not an anti-DR4 antibody, induces selective apoptosis in activated pancreatic stellate cells.

- 18 041148

TRAIL induces apoptosis when bound to its cognate receptors, DR4 and DR5. To investigate whether both TRAIL receptors are required to induce apoptosis in activated PZKs, caspase 3/7 activity (apoptosis markers) was measured by treating activated PZKs with a human anti-DR4 agonist antibody (mapatumumab) or an anti-DR5 agonist antibody (conatumumab). For treatment with specific antibodies to DR4 or DR5, PGCs were activated in culture for 7 days in a 96-well plate (Corning), sequential concentrations (0.101, 102, 103 ng/mL) of mapatumumab (a human anti-DR4 antibody, Creative Biolabs) were added. ) or conatumumab (human anti-DR5 antibody, Creative Biolabs) with protein G (Thermo Fisher Scientific, Waltham, MA #21193) and incubated for 3 hours, after which the cells were tested for caspase activity. Caspase 3/7 activity was measured using the Caspase 3/7 Activity Kit (Promega) according to the manufacturer's protocol. The luminescence of each sample was measured on a plate spectrophotometer (Bio-Tek Instruments Inc) with a delay time of 1 min and a reading time of 0.5 s/well (n=4).

To investigate the expression profiles of DR4 and DR5 on the cell membrane of activated PGCs, human primary PGCs were activated in culture for 2 and 7 days, cells harvested, washed twice with cold PBS, and incubated for 30 min with PE anti-human CD261 (DR4) antibody. or anti-CD262 PV antibody (DR5) (eBioscience, San Diego, CA). A mouse FE-IgG1 K isotype control (eBioscience) was used as isotype control. Expression of TRAIL receptors on the cell surface was examined using flow cytometry (Accuri C6, BD Biosciences, San Jose, CA). Histographic results and mean fluorescence intensity (MFI) data were analyzed using the FlowJo software (FlowJo LLC, Ashland, OR).

Results.

As shown in FIG. 5, when activated PGCs were treated with conatumumab (antibody to DR5, 103 ng/ml), caspase-3/7 activity was increased by 10.79±1.42 times. In contrast, mapatumumab (103 ng/ml) did not increase caspase-3/7 activity (1.27±0.03-fold). Then, the expression of DR4 and DR5 on the PGC cell membrane was examined by flow cytometry analysis using DR4 or DRS-specific antibodies labeled with phycoerythrin (PE) in resting (Day 2) and activated PGC (Day 7). Surprisingly, a large change in mean fluorescence intensity (MFI) was observed only in activated PGCs treated with anti-DR5 antibody compared with anti-DR4 antibody (FIGS. 6A and 6B). This result indicated that DR5 was predominantly expressed on the cell surface of activated PGCs. In general, it has been shown that TRAIL-induced apoptosis in activated PCOs is predominantly mediated by DR5 rather than DR4.

Example 4 Alcohol-activated pancreatic stellate cells become susceptible to apoptosis induced by TRAIL and anti-DR5 antibody.

The main cause of chronic pancreatitis (in approximately 70% of all cases) is alcohol abuse. It has been shown that quiescent PGCs, when activated with alcohol, ethanol (EtOH), significantly increase TRAIL receptors DR4 and DR5 and become sensitive to TRAIL-induced apoptosis.

Materials and methods.

To investigate the effect of alcohol on PZK activation, 2x105 human primary PZKs were cultured in poly-L-lysine-coated 6-well plates for 24 h and treated with ethanol (EtOH) (0.30.50 mM) for 48 h. alcohol stimulation, cells were harvested for analysis by real-time qPCR and Western blotting. Investigated the expression of TRAIL signaling molecules, including DR4, DR5, and fibrogenic factors, including α-SMA (Acta2), collagens. To study TRAIL-induced apoptosis in alcohol-activated PGCs, 2x104 cells were cultured overnight in a 96-well plate and then activated with 50 mM EtOH for 48 h. ) TRAIL, TRAIL _n e _r , mapatumumab (human anti-DR4 antibody) or conatumumab (human anti-DR5 antibody) and incubated for 3 hours. Apoptosis was measured using MTT apoptosis assay and caspase 3/7 assay as described above.

Results.

As shown in FIG. 7A-7E, resting PGCs treated with EtOH (50 mM) for 48 h, sustainably upregulated α-SMA mRNA levels (Acta2, 3.7.5±0.29-fold), collagen, (Col1a2, 4.±0. 43 times), PDGF receptor (Pdgf-r, 1.9 ± 0.25 times), as well as TRAIL receptors (DR4; 2.1 ± 0.12, DR5 1.8 ± 0.09 times) compared with non-activated PZK. In addition, PGCs activated with EtOH (50 mM) demonstrated significantly increased apoptosis against TRAIL _n e _r (59.95+6.37% apoptosis) and caspase 3/7 activity (a marker of apoptosis, 10.45+1.60 times ) compared to non-activated PZK (FIGS. 8A and 8B).

When alcohol-activated PGCs were treated with various concentrations of TRAIL _n e _r /conatumumab (anti-DR5 agonist antibody) and mapatumumab (anti-DR4 agonist antibody), when alcohol was still present in culture, only TRAIL _ror and conatumumab induced strong dose-dependent apoptosis (Fig. 9) . As described in Example 3, mapatumumab, an anti-DR4 antibody, did not cause

- 19 041148 toxicity in alcohol-activated PZK. Studies demonstrate that only DR5 agonist antibodies induce apoptosis in activated pancreatic stellate cells. Human TRAIL and TRAIL _PEG bind to DR4 and DR5, but antibodies bind only to DR4 or only to DR5. These results confirm that human analogues of TRAIL and anti-DR5 agonist antibodies can be used to treat pancreatic fibrosis, pain and pancreatitis by targeting upregulated DR5 in the pancreas.

Example 5 Antinociceptive role of TRAIL signaling along the PZK-nociceptor-TGFe axis.

Activated PPCs were shown to upregulate TGFe (FIG. 3B). Therefore, activated PGCs may be the main cellular source of TGFe and may play a role in nociceptive sensitization in pain.

Materials and methods.

The effect of activated PZKs on the excitability of sensory neurons from HDKs (dorsal root ganglia) was studied in vitro by incubating isolated rat HDKs with conditioned medium obtained from PZKs activated in culture (7 days) in a serum-free conditioned medium (PZK-KS). Excitability was determined using whole cell electrophysiological potential fixation with an Axopatch 200B amplifier and digitized using a Digidata 1200.

Results.

As shown in FIG. 10A-10E, PZK-KS elicited a significant and comparable response from HDK, indicating that PZK activation plays a critical role in nociceptor sensitization. TRAIL _PEG treatment induced pain regression as demonstrated in models of chronic pancreatitis (FIG. 16, Example 6). Thus, activated PGCs sensitized nociceptors through the production of TGFe and stimulation of hyperalgesia, which was blocked by TRAILπ _EG .

Example 6. The use of TRAIL _PEG for antifibrotic and analgesic therapy in CP.

To confirm that death receptors are a significant target for antifibrotic and analgesic therapy and diagnostics, the efficacy of TRAIL _PEG as a potent antifibrotic, analgesic drug was investigated. WEAV _PEG showed strong antifibrotic efficacy in both acute (FIG. 11) and chronic pancreatitis (FIGS. 13A-15). Its analgesic efficacy was demonstrated in a rat model of CP (FIG. 16). The study demonstrates that, when administered systemically, TRAILπ _EG reduces the severity of both acute pancreatitis (AP) and chronic pancreatitis (CP) in animal models and causes a reduction in somatic referred hyperalgesia in CP models.

Materials and methods.

Acute pancreatitis (AP) was induced by hourly intraperitoneal injections of 20 μg/kg cerulein in rats (220-240 g) four times treated with PBS or TRAIL _PEG (iv, 4 mg/kg, single injection) 2 h after the last injection cerulein. Two control groups received PBS or TRAIL _PEG without cerulein. Rats with OP were sacrificed 24 hours after the administration of TRAIL _PEG .

An experimental alcohol-induced chronic pancreatitis (CP) model was developed in rats (Bertola, A., et al., Nat Protoc, 2013. 8(3):627-637; Deng, X., et al., Am J Pathol, 2005.166(1):93106). As shown in FIG. 12, four groups of rats (n=8, each group) received a Lieber-DeCarly (LD) liquid diet with a gradual increase in EtOH concentration from 0 to 36% over seven days, and then 36% EtOH for a maximum of six weeks. Rats were treated with cerulein (four hourly ip injections) on days 14, 21, 28, 35 and 41. TRAILπ _EG (4 mg/kg, iv) or PBS (control) were administered daily for 7 days starting on day 36. Two control groups without alcohol in the diet received intravenous PBS or TRAIL _PEG .

The von Frey filament (VFF) technique is an important tool for investigating the mechanisms of pain in rodents (Zhu et al., Mol Pain, 8:65(2012)). After treatment on day 43, the VFF test was used to study nociception. After the VFF test, the animals were sacrificed, pancreatic samples were taken and examined by IHC, qPCR and Western blotting. Levels of hydroxyproline (a marker of collagen) in pancreatic tissues were determined using a hydroxyproline assay kit (Sigma).

Results.

photographic images of pancreatic tissues stained with GE and immunostained for infiltrating neutrophils (MPO) demonstrated the anti-inflammatory efficacy of TRАIL _PEG (CER-TRAIL) when compared with OP groups treated with PBS (Cer-PBS). Intravenous TRAIL _PEG (CER-TRAIL) protects against acute pancreatitis (AP) in rats with cerulein-induced AP (Cer-PBS). The quantitative analysis of the results is shown in FIG. eleven.

In CP models, pancreatic fibrogenesis was demonstrated by high expression of collagen and α-SMA (a marker of activated PGCs) (FIGS. 13A and 13B), as well as various fibrogenic markers (FIGS. 14A-14I). TRAIL _PEG treatment significantly reduced collagen accumulation, downregulated α-SMA and PDGFRe, as well as other fibrogenic and pancreatitis markers, including

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Claims (10)

containing collagens (Col1a2, Col3a1), TIMPs (tissue inhibitor of metalloproteinases), fibronectin, Pap (pancreatitis associated protein) and TGFe (FIGS. 14A-14I). Hydroxyproline levels in CP models were greatly elevated. Treatment with TRAILπ _EG significantly reduced the content of hydroxyproline in the pancreas (Fig. 15). Cleaved caspase-8 was only significantly upregulated in CP-treated TRAILπ _EG , indicating that elimination of activated PGCs may be caused by TRAIL-mediated apoptosis. Importantly, as a result of double immunostaining of pancreatic tissues (aPZK - α-SMA staining, apoptosis - TUNEL staining and nucleus - DAPI staining), it could be confirmed that apoptosis (TUNEL staining) was observed specifically in αSMA+ aPZK. Systemic administration of TRAIL _PEG alone to normal rats did not cause significant toxicity. For example, TRAIL _PEG treatment significantly normalized ALT levels, a convenient marker of liver damage, compared to ALT levels in untreated CP rats. CP is accompanied by severe and constant abdominal pain. Surprisingly, TRAIL _PEG showed antinociceptive efficacy in CP models. TRAIL _PEG reduced somatic reflex hyperalgesia in rats with CP induced by ethanol/cerulein/LD diet. Treated with TRAILπ _EG (4 mg/kg, iv) animals (n=4) showed a significant reduction in somatic reflected hyperalgesia as measured in the VFF test (Fig. 16). It has been shown that treatment of HDC neurons with NGF (Nerve Growth Factor) or TGFe caused changes in their excitability and suppressed specific voltage-dependent potassium current (IA), which is a hallmark of nociceptive excitability in CP (Zhu et al., Mol Pain, 8:65 (2012)). TGFe itself can sensitize nociceptors, causing pancreatic hyperalgesia. Preliminary studies have confirmed that PGCs upregulate TGFe during activation, with conditioned media (CM) derived from aPGCs affecting the excitability of HDC neurons. Activated PZKs upregulate TGFe (Fig. 3B), and conditioned media (CM) derived from aPZKs affect the excitability of HDC neurons (Fig. 10). Thus, activated PZKs are the predominant cell type that should be responsible for nociceptive sensitization in the pancreas, with PZK activation playing an important role in nociceptor sensitization. Therefore, this technique represents a new way to reduce pain by selectively blocking the activation of PZK or depletion of activated PZK, one of the dominant cellular sources of TGFe and NGF (nerve growth factor), using death receptor agonists. CLAIM 1. A method for the treatment of acute pancreatitis or acute pancreatic pain syndrome, comprising administering to a subject suffering from acute pancreatitis or acute pancreatic pain syndrome, by intravenous or subcutaneous injection, intranasally, pulmonary or intraocularly, a composition comprising a death receptor agonist in an effective dose of 0.01-100 mg/kg, where the death receptor agonist is an agonist selected from the group consisting of the apoptosis-inducing ligand of the TNF family (TRAIL), death receptor 5 (DR5) agonists, PEGylated TRAIL, anti-death receptor (DR5) agonist antibodies, and combinations thereof. 2. The method of claim 1 wherein the death receptor agonist is a DR5 agonist selected from the group consisting of lexatumumab, tigatuzumab, conatumumab, drozitumab, HGSTR2J/KMTR-2 and LBY-135. 3. The method of claim 1 wherein the death receptor agonist is a multivalent DR agonist selected from the group consisting of TAS266 and scTRAIL-RBDs. 4. The method of claim 1, wherein the antibodies are full-length antibodies, functional fragments thereof that retain binding functionality, humanized antibodies, bifunctional or chimeric antibodies, or combinations thereof. 5. The method of claim 1 wherein the agonist is TRAIL or TRAIL modified with polyalkylene oxide. 6. The method of claim 5 wherein the polyalkylene oxide is a linear, branched, dimer or trimer of polyethylene glycol having a molecular weight of 5000-50000 daltons. 7. The method according to any one of claims 1 to 6, wherein acute pancreatitis or acute pancreatic pain syndrome is selected from the group consisting of acute pancreatitis and acute pain associated with pancreatic fibrosis. 8. The method of claim 7 wherein the acute pain associated with pancreatic fibrosis is pancreatitis associated acute pain. 9. The method of any one of claims 1 to 8, wherein the method further comprises identifying a patient suffering from or at risk of developing fibrosis-associated acute pain. 10. The method according to any one of claims 1 to 9, wherein the treatment of acute pancreatitis or acute pancreatic pain syndrome provides protection of pancreatic tissues, reduction of fibrosis, inversion of pancreatic fibrogenesis and/or reduction of pain, and preservation of healthy pancreatic tissues intact. -