Peptides and Methods of Use

Abstract

The present invention provides peptides that are synthetic modifications of Polar Assortant (PA) peptides that include C-terminal PEGylation. The present invention further provides methods of using at least one synthetic peptide to modulate the complement system and interact with neutrophils to alter their binding and activity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 279,423, filed November 15, 2021, and U.S. Provisional Application No. 63 / 195,401, filed June 1, 2021, the disclosures of which are incorporated by reference in their entireties herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on May 25, 2022, is named 251110_000164_SL.txt, and is 1,196 bytes in size. [Background technology]

[0003] 2. Background of the Invention Aspects of the present invention relate generally to synthetic peptides and their uses for therapy, and more specifically to synthetic peptide modifications that can prevent, treat, and / or ameliorate toxicity, particularly intestinal necrosis or injury, caused by checkpoint inhibitors.

[0004] complement system The complement system, an essential component of the innate immune system, plays a key role as a defense mechanism against invading pathogens, stimulating the adaptive immune response and helping to remove immune complexes and apoptotic cells. Three distinct pathways comprise the complement system: the classical pathway, the lectin pathway, and the alternative pathway. C1q and mannose-binding lectin (MBL) are structurally related recognition molecules of the classical and lectin pathways, respectively. IgM or clustered IgG serve as the main ligands for C1q, whereas MBL recognizes polysaccharides such as mannan. Ligand binding by C1q and MBL leads to sequential activation of C4 and C2 to form the C3 convertase of the classical and lectin pathways, respectively. In contrast, activation of the alternative pathway does not require a recognition molecule but can amplify C3 activation elicited by the classical or lectin pathways. Activation of any of these three pathways leads to the formation of inflammatory mediators (C3a and C5a) and the membrane attack complex (MAC), which triggers cell lysis.

[0005] While the complement system plays a key role in many protective immune functions, complement activation is a critical mediator of tissue damage in a wide range of autoimmune and inflammatory disease processes (Ricklin and Lambris, "Complement-targeted therapeutics." Nat Biotechnol 2007; 25(11):1265-75).

[0006] There is a need for complement regulators. On the one hand, the complement system is a vital host defense against pathogenic organisms. On the other hand, its unchecked activation can cause devastating host cell damage. Currently, despite the known morbidity and mortality associated with complement dysregulation in many disease processes, including autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, and multiple sclerosis, only two anti-complement therapies have been recently approved for use in humans: (1) eculizumab (Soliris™) and (2) ultomiris (Ravulizumab™), two humanized long-acting monoclonal antibodies against C5 used in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). PNH and aHUS are rare diseases that only a very small number of people suffer from. Currently, no complement regulators are approved for the more common disease processes in which dysregulated complement activation plays a central role. Dysregulated complement activation may play a role in both chronic and acute disease manifestations.

[0007] There is a need to develop peptides to inhibit the classical, lectin and alternative pathways of the complement system, because each of these three pathways has been shown to contribute to a number of autoimmune and inflammatory disease processes.Specific blockade of the classical and lectin pathways is particularly needed, because both of these pathways are involved in ischemia-reperfusion-induced injury and other diseases in many animal models.Humans with alternative pathway deficiency suffer from severe bacterial infections.Therefore, a functional alternative pathway is essential for immune surveillance against invading pathogens.

[0008] The PIC1 molecule family (also referred to herein as the EPICC family or EPICC peptides) comprises a collection of rationally designed peptides based on a scrambled astrovirus coat protein, which have several anti-inflammatory functional properties, including inhibition of the classical pathway of complement, myeloperoxidase inhibition, neutrophil extracellular trap (NET) inhibition, and antioxidant activity. The original compound has a 15 amino acid peptide sequence: TIFF2024522988000001.tif4128, and has a monodisperse 24mer PEGylated moiety at the C-terminus that enhances its aqueous solubility. TIFF2024522988000002.tif4128. PA-dPEG24 is a peptide inhibitor of the classical and lectin pathways, as well as myeloperoxidase activity and NETosis, the major effectors of neutrophils [6-8]. A sarcosine substitution scan of SEQ ID NO:2 revealed a peptide that is water soluble without PEGylation by replacing the isoleucine at position 8 with sarcosine: It has been found that this results in TIFF2024522988000003.tif4128 (as described in U.S. Patent No. 10,005,818).

[0009] Further features of the PA-dPEG24 and PA-I8Sar molecules are described herein.

[0010] Checkpoint Therapeutics and Associated Toxicity Intestinal necrosis is a potentially life-threatening medical condition that can result from a variety of causes, including bacterial sepsis and systemic inflammatory response syndrome (SIRS). The etiology of intestinal necrosis includes vascular injury, e.g., venous thrombosis, chronic ischemia, mechanical obstruction, and non-occlusive mesenteric ischemia [1], as well as a variety of disease processes that can result in toxic megacolon due to autoimmune inflammatory bowel disease, including Crohn's disease and ulcerative colitis, or C. difficile colitis. In addition, intestinal necrosis due to severe inflammatory responses is a severe, treatment-limiting adverse event associated with cancer checkpoint inhibitor drugs [2].

[0011] Intestinal necrosis generally compromises the intestinal lumen, leading to extravasation of enteric bacteria, and ultimately to massive leakage of intestinal contents, including bacteria, toxins, and other microbial products, into the peritoneum and systemic circulation. This process can subsequently lead to bacterial sepsis, hypotension, disseminated intravascular coagulation, multiple organ failure, and often death. Primary therapeutic interventions for the treatment of intestinal necrosis include antibiotics to kill bacterial pathogens in the peritoneum and bloodstream. The use of anticomplement therapy, such as eculizumab, can render subjects immunocompromised, increasing the risk of invasive N. meningitidis infection. Furthermore, persistent neutropenia is associated with a high risk of life-threatening bacterial sepsis. Thus, there is concern that modulation of the complement system and neutrophil effector function could potentially exacerbate the risk of uncontrollable bacterial infection due to intestinal necrosis.

[0012] There is a need in the art for peptide-based inhibitors of different pathways of the complement system. There is also a need in the art for therapeutic peptides to prevent, treat, and / or mitigate the toxic side effects associated with checkpoint inhibitors, particularly intestinal necrosis or damage. Summary of the Invention

[0013] BRIEF SUMMARY OF THE INVETION As stated in the background section, there is a great need in the art to identify techniques for peptide-based inhibitors of different pathways of the complement system and to use this knowledge to develop novel therapeutic peptides. The present invention meets this and other needs. Aspects of the present invention generally relate to synthetic peptides, more specifically synthetic peptides that are pegylated or contain sarcosine substitutions, and their use in methods of modulating the complement system and in methods of preventing, treating, and / or mitigating toxic side effects associated with checkpoint therapeutics, particularly intestinal necrosis or damage.

[0014] In one aspect, the present invention provides synthetic peptides that regulate the complement system and methods of using these peptides. In particular, in some embodiments, the synthetic peptides can bind to C1 and MBL, regulate C1 and MBL, and inactivate C1 and MBL, thus effectively inhibiting the activation of the classical and lectin pathways at their earliest points in the complement cascade, while leaving the alternative pathway intact. These peptides are of therapeutic value because they selectively regulate and inhibit the activation of C1 and MBL without affecting the alternative pathway, and can be used to treat diseases mediated by dysregulated activation of the classical and lectin pathways. In other embodiments, the peptides regulate the activation of the classical pathway, but not the activation of the lectin pathway. The peptides are useful for various therapeutic indications.

[0015] In any embodiment, the synthetic peptides can prevent, treat, and / or mitigate the toxic side effects of checkpoint inhibitors.

[0016] In any embodiment, the synthetic peptides can prevent, treat, and / or alleviate intestinal necrosis and / or damage, such as necrosis or damage caused by severe inflammatory responses, intestinal infarction (i.e., ischemia-reperfusion injury of intestinal tissue), autoimmune inflammatory bowel disease (IBD) and related therapies, and chemotherapy-induced or toxin-induced intestinal necrosis.

[0017] In one embodiment, the present invention is based on the identification and modification of a 15 amino acid peptide from Polar Assortant (PA) peptide (SEQ ID NO:1), derivatives of this peptide, and methods of their use. The PA peptide is a scrambled peptide derived from a human astrovirus protein called CP1. The PA peptide is also known as PIC1 (peptide inhibitor of complement C1), AstroFend, AF, or SEQ ID NO:1. The PIC1 peptide was originally named as such because it was found to be associated with diseases mediated by the complement system. A pegylated form of the PIC1 peptide, called PA-dPEG24 or RLS-0071 (SEQ ID NO:2), has 24 PEG units at the C-terminus of the peptide and has been shown to have improved solubility in aqueous solutions. A sarcosine substitution scan of SEQ ID NO:2 revealed the following peptide, which is water soluble without pegylation, by replacing the isoleucine at position 8 with sarcosine: It has been found that this results in TIFF2024522988000004.tif4128 (as described in U.S. Patent No. 10,005,818).

[0018] In one aspect, the present invention provides a method for preventing, treating, and / or ameliorating toxic side effects of checkpoint inhibitors comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0019] In one aspect, the present invention provides a method of preventing, treating, and / or alleviating intestinal necrosis and / or damage, such as necrosis or damage caused by severe inflammatory responses, intestinal infarction (i.e., ischemia-reperfusion injury of intestinal tissue), autoimmune inflammatory bowel disease (IBD), and related therapies, and chemotherapy-induced or toxin-induced intestinal necrosis, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0020] In one aspect, the present invention provides a method of preventing, treating, and / or alleviating intestinal necrosis and / or damage in a subject undergoing, having been treated, and / or to be treated with at least one checkpoint inhibitor comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0021] In one embodiment of any of the foregoing methods, the composition further comprises at least one pharma- ceutically acceptable carrier, diluent, stabilizer, or excipient. In one embodiment of any of the foregoing methods, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 10 mg / kg to about 160 mg / kg. In one embodiment of any of the foregoing methods, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 20 mg / kg to about 160 mg / kg. In one embodiment of any of the foregoing methods, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 40 mg / kg to about 160 mg / kg. In any embodiment, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered in at least one dose, the first dose comprising about 1 mg / kg to about 160 mg / kg of SEQ ID NO:2 and / or 3. In any embodiment, a second dose comprising a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered, the second dose comprising from about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, e.g., from about 10 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3. In any embodiment, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered in two doses, the first dose comprising from about 1 mg / kg to about 160 mg / kg of SEQ ID NO:2, e.g., from about 10 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, and the second dose comprising from about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, e.g., from about 1 mg / kg to about 40 mg / kg of SEQ ID NO:2 and / or 3. In any embodiment, the second dose is administered 30 seconds to 10 hours after the first dose is administered, e.g., about 8 hours after the first dose is administered. In any embodiment, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered in multiple doses over a period of about 1 week to about 2 weeks, each dose containing about 1 mg / kg to about 160 mg / kg of SEQ ID NO:2 and / or 3, e.g., about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, administered every 4 to 10 hours, e.g., about every 8 hours.

[0022] In any embodiment, at least one loading dose of about 10 mg / kg to about 160 mg / kg of SEQ ID NO:2 and / or 3, e.g., about 10 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, is administered, followed by at least one maintenance dose of about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, e.g., about 1 mg / kg to about 40 mg / kg of SEQ ID NO:2 and / or 3. In any embodiment, the first maintenance dose is administered 4-10 hours after the last loading dose. In any embodiment, the maintenance doses are administered every 4-10 hours for a period of about 1 week to about 2 weeks. In any embodiment, the first maintenance dose is administered 8 hours after the last loading dose, and the maintenance doses are administered every 8 hours for a period of about 1 week to about 2 weeks.

[0023] In any embodiment of any of the foregoing methods, the composition is formulated for subcutaneous, intravenous, intraperitoneal, or intramuscular administration. In one embodiment, the composition further comprises a pharma- ceutically acceptable carrier and / or excipient.

[0024] These and other objects, features, and advantages of the present invention will become more apparent from a reading of the following specification in conjunction with the accompanying description, claims, and drawings. [Brief description of the drawings]

[0025] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1]FIG. 1 shows that PA-dPEG24 (also referred to herein as RLS-0071) increases survival in rats after cecal ligation. Kaplan-Meier survival curve assessment. The red line shows the outcome after 75% CLP in untreated animals (n=6), while the red curve represents the outcome after animals received a single dose of 40 mg / kg RLS-0071 (n=9). [Diagram 2] Figure 2 shows that RLS-0071 reduces free DNA levels in blood. Plasma was isolated from animals subjected to CLP with (n=4) and without (n=3) a single dose of RLS-0071 before surgery (prebleed) (n=7) and 24 hours after surgery. Plasma samples were incubated with PicoGreen. Fluorescence was read in a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Data are mean and standard error of the mean. [Diagram 3] Figure 3 shows that RLS-0071 reduces IL-6 levels in blood. Plasma was isolated from animals subjected to CLP with (n=3) and without (n=5) a single dose of RLS-0071 before surgery (prebleed) (n=8) and 24 hours after surgery. Plasma samples were analyzed by IL-6 ELISA according to the manufacturer's instructions. Data are mean and standard error of the mean. [Figure 4] Figure 4 shows that multiple doses of RLS-0071 increase survival in rats after cecal ligation by Kaplan-Meier survival curve assessment. The black line shows the outcome after 75% CLP in untreated animals (n=12), while the green line represents the outcome after animals received a dose of 40 mg / kg RLS-0071 at 0.5, 24, 28, and 72 hours after surgery (n=12). [Diagram 5]Figure 5 shows that multiple doses of RLS-0088 increase survival in rats after cecal ligation by Kaplan-Meier survival curve assessment. The red line shows the outcome after 75% CLP in animals receiving saline only (n=3), while the green line represents the outcome after animals received a 40 mg / kg dose of RLS-0071 at 0.5, 24, 28, and 72 hours after surgery (n=5). SID=once daily dosing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Invention As stated in the background section, there is a great need in the art to identify techniques for peptide-based inhibitors of different pathways of the complement system and to use this knowledge to develop novel therapeutic peptides. The present invention meets this and other needs. Aspects of the present invention generally relate to synthetic peptides, more specifically synthetic peptides that are pegylated or contain sarcosine substitutions, and their use in methods of regulating the complement system and mitigating toxic side effects associated with checkpoint therapeutics, particularly intestinal necrosis or damage.

[0027] In order to facilitate understanding of the principles and features of various embodiments of the present invention, various exemplary embodiments are described below. Although exemplary embodiments of the present invention are described in detail, it should be understood that other embodiments are contemplated. Thus, it is not intended that the present invention be limited in scope to the details of the configuration and arrangement of components described in the following description or examples. The present invention is capable of other embodiments and can be practiced or carried out in various ways. Moreover, in describing the exemplary embodiments, specific terminology is used for the sake of clarity.

[0028] It should also be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, a reference to a component is also intended to include a composition of multiple components. A reference to a composition including "a" component is intended to include the other components in addition to the one specified. In other words, the terms "a," "an," and "the" do not denote a limitation of quantity, but rather denote the presence of "at least one" of the referenced item.

[0029] As used herein, the term "and / or" can mean "and", can mean "or", can mean "exclusive or", can mean "one", can mean "some but not all", can mean "neither", and / or it can mean "both". The term "or" is intended to mean an inclusive "or".

[0030] Furthermore, in describing the exemplary embodiments, terminology is used for clarity. Each term is intended to contemplate its broadest meaning as understood by a person skilled in the art and to include all technical equivalents that operate in a similar manner to achieve a similar purpose. It should be understood that the disclosed technology embodiments can be practiced without these specific details. In other cases, well-known methods, structures, and techniques have not been shown in detail so as not to obscure the understanding of this description. References to "one embodiment," "an embodiment," "exemplary embodiment," "several embodiments," "certain embodiments," "various embodiments," and the like indicate that the disclosed technology embodiments so described may include a particular feature, structure, or characteristic, but not all embodiments necessarily include the particular feature, structure, or characteristic. Furthermore, repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may.

[0031] As used herein, the term "about" should be construed to refer to both the numbers specified as the endpoints of any range. Any reference to a range should be considered as supporting any subset within the range. Ranges can be expressed herein as "about" or "approximately" or "substantially" from one particular value and / or "about" or "approximately" or "substantially" to another particular value. When such a range is expressed, other exemplary embodiments include from one particular value and / or to the other particular value. Furthermore, the term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation according to convention in the art. Alternatively, "about" can mean within a range of ±20%, preferably ±10%, more preferably ±5%, and even more preferably ±1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, preferably within two-fold, of a value. Where a specific value is described in this application and claims, unless otherwise stated, the term "about" is implicit and means within an acceptable error range for the particular value in this context.

[0032] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within the range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within the range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0033] Similarly, as used herein, a characterization such as "substantially free" or "substantially pure" of something can include both "at least substantially free" or "at least substantially pure" of something and "completely free" or "completely pure" of something.

[0034] "Comprising" or "containing" or "including" means that at least the specified compounds, elements, particles, or method steps are present in a composition or article or method, and does not exclude the presence of other compounds, elements, particles, method steps, even if other such compounds, elements, particles, method steps have the same function as the one specified.

[0035] Throughout this description, various components may be specified with specific values ​​or parameters, but these items are provided as exemplary embodiments. Indeed, the exemplary embodiments do not limit the various aspects and concepts of the present invention, since many comparable parameters, sizes, ranges, and / or values ​​may be implemented. The terms "first", "second", "primary", "secondary", etc. do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0036] It should be noted that terms such as "particularly," "preferably," "typically," "generally," and "often" are not utilized herein to limit the scope of the claimed invention or to imply that a particular feature is critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are intended merely to highlight alternative or additional features that may or may not be utilized in certain embodiments of the invention. It should also be noted that terms such as "substantially" and "about" are utilized herein to indicate the inherent degree of uncertainty that may result from any quantitative comparison, value, measurement, or other representation.

[0037] Dimensions and values ​​disclosed herein should not be understood to be strictly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "50 mm" is intended to mean "about 50 mm."

[0038] It should also be understood that the recitation of one or more method steps does not preclude the presence of additional or intervening method steps between those steps expressly identified.Similarly, it should also be understood that the recitation of one or more components in a composition does not preclude the presence of additional components other than those expressly identified.

[0039] The materials described below as constituting various elements of the present invention are intended to be exemplary and not limiting. Many suitable materials that would perform the same or similar functions as the materials described herein are intended to be encompassed within the scope of the present invention. Such other materials not described herein may include, but are not limited to, materials developed after the development of the present invention. Any dimensions listed in the various figures are for illustrative purposes only and are not intended to be limiting. Other dimensions and proportions are contemplated and are intended to be within the scope of the present invention.

[0040] As used herein, the term "subject" or "patient" refers to a mammal, including, but not limited to, humans and veterinary animals. In a preferred embodiment, the subject is a human.

[0041] As used herein, the term "combination" of the synthetic peptide according to the claimed invention and at least one second pharmacoactive ingredient means that at least two, but any desired combination of compounds are delivered simultaneously or sequentially (e.g., within 24 hours). When used to treat various diseases, it is contemplated that the compositions and methods of the present invention can be utilized with other therapeutic methods / agents suitable for the same or similar diseases. Such other therapeutic methods / agents can be co-administered (simultaneously or sequentially) to cause additive or synergistic effects. For reasons of additive or synergistic effects, the appropriate therapeutically effective dosage for each agent can be lowered.

[0042] A "disease" is a state of a subject's health in which the subject is unable to maintain homeostasis and in which the subject's health continues to deteriorate if the disease is not improved. In contrast, a "disorder" in a subject is a state of a subject's health in which the subject is able to maintain homeostasis, but in which the subject's health is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further decline in the subject's health.

[0043] The term "treating" a situation, disorder, or condition or "treatment" of a situation, disorder, or condition includes: (1) preventing or delaying the appearance of at least one clinical or subclinical symptom of the situation, disorder, or condition occurring in a subject who may be afflicted with or susceptible to the situation, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the situation, disorder, or condition; or (2) inhibiting the situation, disorder, or condition, i.e., arresting, reducing, or delaying the occurrence of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the situation, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to the subject being treated is either statistically significant or at least perceptible by the patient or physician.

[0044] As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. The therapeutic effect is achieved by suppressing, reducing, ameliorating, or eradicating the disease state.

[0045] As used herein, the term "therapeutically effective" as applied to a dose or amount refers to the quantity of a compound or pharmaceutical composition that, when administered to a subject to treat (e.g., prevent or ameliorate) a condition, disorder, or state, is sufficient to effect such treatment. A "therapeutically effective amount" will vary depending on the compound or bacterium or analog administered, as well as the disease and its severity, and the age, weight, physical condition, and responsiveness of the mammal being treated.

[0046] When used in connection with the compositions of the present invention, the phrase "pharmaceutical acceptable" refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a mammal (e.g., a human).Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency for use in mammals, more particularly humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0047] The term "pharmaceutical carrier" or "pharmaceutical acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Alternatively, a pharmaceutical carrier may be a solid dosage form carrier, including, but not limited to, one or more of binders (for compressed pills), glidants, encapsulating agents, flavoring agents, and coloring agents. "Suitable Pharmaceutical Carriers" is described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0048] The term "analog" or "functional analog" refers to a related modified form of a polypeptide in which at least one amino acid substitution, deletion, or addition has been made such that the analog retains substantially the same biological activity in vivo and / or in vitro as the unmodified form.

[0049] The terms "sequence identity" and "percent identity" are used interchangeably herein. In the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, it is defined herein that the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid or nucleotide residues at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions common to these sequences (i.e., identity %=number of identical positions / total number of positions (i.e., overlapping positions)×100). Preferably, the two sequences are the same length.

[0050] There are several different computer programs available for determining the degree of identity between two sequences. For example, the comparison of sequences between two sequences and the determination of percent identity can be achieved using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) incorporated in the GAP program of the Accelrys GCG software package (available at www.accelrys.com / products / gcg), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. These different parameters will likely produce slightly different results, but the overall percent identity of two sequences will not change significantly when using different algorithms.

[0051] Sequence comparison can be performed over the entire length of the two sequences being compared, or over a fragment of the two sequences. Typically, comparison will be performed over the full length of the two sequences being compared. However, sequence identity can be performed over a region of, for example, 20, 50, 100 or more consecutive amino acid residues.

[0052] "Sequence identity" as known in the art refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., a reference sequence and a given sequence that is compared to the reference sequence.Sequence identity is determined by comparing a given sequence to a reference sequence after optimally aligning the sequences to produce the highest degree of sequence similarity as determined by the match between strings of such sequences.Upon such alignment, sequence identity is ascertained position by position, e.g., a sequence is "identical" at a particular position if the nucleotide or amino acid residue at that position is identical.Then, the total number of such positions that are identical is divided by the total number of nucleotides or residues in the reference sequence to obtain the sequence identity percentage. Sequence identity may be determined using methods such as, but not limited to, Computational Molecular Biology, Lesk, AN, ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073, the teachings of which are incorporated herein by reference. (1988).Preferred methods for determining sequence identity are designed to give the largest match between the sequences tested. Methods for determining sequence identity are codified in publicly available computer programs that determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference. (1990)). These programs optimally align sequences using default gap weights to provide the highest level of sequence identity between a given sequence and a reference sequence. As one illustration, by a polynucleotide having a nucleotide sequence having at least, e.g., 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% "sequence identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence, except that the given polynucleotide sequence may contain up to 5, up to 4, up to 3, up to 2, up to 1, or up to 0 point mutations per each 100 nucleotides of the reference nucleotide sequence.In other words, in a polynucleotide having a nucleotide sequence with at least 95%, for example at least 96%, 97%, 98%, 99%, or 100% sequence identity to a reference nucleotide sequence, up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, or up to 0% of the nucleotides of the reference sequence can be deleted, replaced with another nucleotide, or up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, or up to 0% of the total nucleotides of the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, either individually among the nucleotides in the reference sequence or in one or more consecutive groups within the reference sequence. Similarly, by a polypeptide having a given amino acid sequence having at least, e.g., 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence, except that the given polypeptide sequence may contain up to 5, up to 4, up to 3, up to 2, up to 1, or up to 0 amino acid changes per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence with at least 95%, for example at least 96%, 97%, 98%, 99%, or 100% sequence identity with a reference amino acid sequence, up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, or up to 0% of the amino acid residues of the reference sequence can be deleted or replaced with another amino acid, or up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, or up to 0% of the total number of amino acid residues of the reference sequence can be inserted into the reference sequence. These changes in the reference sequence can occur at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually among the residues in the reference sequence or in one or more consecutive groups within the reference sequence. Preferably, the positions of the residues that are not identical differ by conservative amino acid substitutions.However, when determining sequence identity, conservative substitutions are not included as matches.

[0053] As used herein, the term "immune response" includes natural immune response, T cell-mediated immune response, and / or B cell-mediated immune response. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity, and B cell responses, such as antibody production. In addition, the term "immune response" includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen-presenting cells (e.g., professional antigen-presenting cells, such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen-presenting cells, such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes (e.g., neutrophils).

[0054] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intraperitoneal (ip), or intradermal (id) injection or infusion techniques.

[0055] In the medical field, the term "prevent" includes any activity that reduces the burden of mortality or morbidity due to a disease. Prevention can occur at the primary, secondary, and tertiary prevention levels. While primary prevention avoids the onset of a disease, secondary and tertiary levels of prevention include activities aimed at preventing the progression of the disease and the appearance of symptoms, as well as reducing the negative effects of an already established disease by restoring function and reducing disease-related complications.

[0056] A "variant" of the polypeptide according to the invention may be (i) one in which one or more of the amino acid residues are replaced with a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), which may or may not be encoded by the genetic code, (ii) one in which one or more modified amino acid residues are present, e.g., a residue modified by attachment of a substituent group, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the invention, (iv) a fragment of the polypeptide, and / or (v) one in which the polypeptide is fused to another polypeptide, e.g., a leader or secretion sequence, or a sequence used for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). Fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally or chemically modified. Such variants are deemed to be within the scope of one of skill in the art from the teachings herein.

[0057] Within the meaning of the present invention, the term "co-administration" is used to refer to the administration of a composition according to the invention and another therapeutic agent simultaneously in one composition, or simultaneously in different compositions, or sequentially (preferably within 24 hours).

[0058] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being fully explained in the literature. See, among others, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins eds.(1985); Transcription and Translation (BD Hames & SJ Higgins, eds. (1984); Animal Cell Culture (RI Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.), Current Protocols in See Molecular Biology, John Wiley & Sons, Inc. (1994).

[0059] Peptide Compositions of the Present Invention Modifications of the amino acid structure of CP1 have led to the discovery of additional peptides capable of modulating complement activation, such as C1q activity. Modifications such as pegylation have been shown to enhance the potency of the parent molecule in in vitro assays of classical complement pathway activation / inhibition, myeloperoxidase (MPO) inhibition, antioxidant activity, and inhibition of NET activity. It has previously been shown that PEGylation enhances peptide solubility and potent inhibition of biological activity compared to TIFF2024522988000005.tif4128. Peptides with a C-terminal monodisperse 24mer PEGylated moiety were found to be highly soluble and have potent inhibition of the complement system. TIFF2024522988000006.tif4151. A sarcosine substitution scan of SEQ ID NO:2 revealed a peptide that is water soluble without PEGylation by replacing the isoleucine at position 8 with sarcosine: It has been found that this results in TIFF2024522988000007.tif4128 (as described in U.S. Patent No. 10,005,818).

[0060] As used herein, the term "peptide" refers to a naturally occurring amino acid sequence or a peptidomimetic, peptide analog, and / or synthetic derivative of about 15 amino acids based on SEQ ID NO:2 and / or 3 (including, but not limited to, pegylated peptides). Additionally, a peptide may be less than about 15 amino acid residues, such as about 10 to about 15 amino acid residues, such as about 5 to about 10 amino acid residues. For example, peptide residues of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 amino acids may be peptides within the context of the present invention as well. A peptide may also be more than 15 amino acids, such as 16, 17, 18, 19, and 20 or more amino acids.

[0061] The disclosed peptides are generally amino acid sequences that are constrained (i.e., have certain elements of structure, such as, for example, the presence of amino acids that initiate a β-turn or β-pleated sheet, or are cyclized, for example, by the presence of disulfide-bonded Cys residues) or unconstrained (i.e., linear) of greater than, about, or less than about 15 amino acid residues.

[0062] Substitutes for an amino acid in a peptide sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Amino acids containing aromatic ring structures include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. For example, one or more amino acid residues in a sequence can be substituted with another amino acid of similar polarity that acts as a functional equivalent, resulting in a silent change.

[0063] Conservative changes generally do not lead to much change in the structure and function of the resulting protein. Non-conservative changes are more likely to change the structure, activity or function of the resulting protein. For example, the peptides of the present disclosure include one or more of the following conservative amino acid substitutions: aliphatic amino acids, such as alanine, valine, leucine and isoleucine, are replaced with other aliphatic amino acids; serine is replaced with threonine; threonine is replaced with serine; acidic residues, such as aspartic acid and glutamic acid, are replaced with other acidic residues; residues with amide groups, such as asparagine and glutamine, are replaced with other residues with amide groups; basic residues, such as lysine and arginine, are replaced with other basic residues; and aromatic residues, such as phenylalanine and tyrosine, are replaced with other aromatic residues.

[0064] Particularly preferred amino acid substitutions include: (a) Ala to Glu or vice versa, so that the negative charge can be reduced; (b) Lys to Arg or vice versa, such that a positive charge can be maintained; (c) Ala to Arg or vice versa, so that the positive charge can be reduced; (d) Glu to Asp or vice versa, such that a negative charge can be maintained; (e) Ser to Thr or vice versa, such that a free -OH can be maintained; (f) Gln to Asn or vice versa, such that free NH2 can be maintained; (g) Ile for Leu or Val or vice versa, as roughly equivalent hydrophobic amino acids; (h) Phe for Tyr or vice versa, as roughly equivalent aromatic amino acids; and (i) Ala to Cys or vice versa, such that disulfide bonds are affected.

[0065] Substitutions for amino acids within the peptide sequence can be selected from any amino acid, including, but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine, N-formyl-L-methionine, sarcosine, or other N-methylated amino acids. In some embodiments, sarcosine is substituted for an amino acid within the peptide sequence.

[0066] In one aspect, the present invention discloses a synthetic peptide derived from a human astrovirus coat protein, the peptide comprising the amino acid sequence of SEQ ID NO:2 and / or 3 and modifications.

[0067] Table 1: List of peptides of the present invention TIFF2024522988000008.tif54166

[0068] In other embodiments, the synthetic peptides can alter cytokine expression, including, but not limited to, in models of acute lung injury (ALI). In some embodiments, the present invention provides methods of altering cytokine expression, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0069] In other embodiments, the synthetic peptides can inhibit or alter neutrophil binding and / or adhesion. In some embodiments, the present invention provides methods of inhibiting or altering neutrophil binding and / or adhesion comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0070] In other embodiments, the synthetic peptides can enhance neutrophil survival. In some embodiments, the present invention provides methods for enhancing neutrophil survival comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0071] In other embodiments, the synthetic peptides are capable of binding in vivo to cell surface receptors, such as, for example, but not limited to, integrins and / or ICAMs. In some embodiments, methods are provided for inhibiting or altering neutrophil binding to cell surface receptors comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0072] The disclosed peptides can selectively modulate C1q and MBL activation without affecting alternative pathway activity and are therefore ideal for the prevention and treatment of diseases mediated by dysregulated activation of the classical and lectin pathways. Specific blockade of the classical and lectin pathways is especially needed since both of these pathways are involved in ischemia-reperfusion-induced injury in many animal models [Castellano et al., “Therapeutic targeting of classical and lectin pathways of complement protects from ischemia-reperfusion-induced renal damage.” Am J Pathol. 2010; 176(4):1648-59; Lee et al., “Early complement factors in the local tissue immunocomplex generated during intestinal ischemia / reperfusion injury.” Mol. Immunol. 2010 February; 47(5):972-81; Tjernberg, et al., “Acute antibody-mediated complement activation mediates lysis of pancreatic islets cells and may cause tissue loss in clinical islet transplantation.” Transplantation. 2008 Apr. 27; 85(8):1193-9; Zhang et al. “The role of natural IgM in myocardial ischemia-reperfusion injury.” J Mol Cell Cardiol. 2006 July; 41(1):62-7). The alternative pathway is essential for immune surveillance against invading pathogens, and humans with defects in the alternative pathway suffer from severe bacterial infections.By binding to and inactivating C1q and MBL, the peptides can effectively modulate activation of the classical and lectin pathways while leaving the alternative pathway intact.

[0073] As used herein, the term "modulate" refers to (i) controlling, reducing, inhibiting, or regulating the biological function of an enzyme, protein, peptide, factor, by-product, or derivative thereof, either individually or in a complex; (ii) reducing the amount of a biological protein, peptide, or derivative thereof, either in vivo or in vitro; or (iii) interrupting a biological chain of events, cascade, or pathway that is known to involve a series of related biological or chemical reactions. Thus, the term "modulate" can be used to describe, for example, reducing the amount of a single component of the complement cascade, reducing the rate or total amount of formation of a component or a complex of components, or reducing the overall activity of a complex process or series of biological reactions, resulting in cell lysis, formation of a convertase enzyme, formation of a complement-derived membrane attack complex, inflammation, or inflammatory disease, etc. In in vitro assays, the term "modulate" can refer to a measurable change or reduction in some biological or chemical event, although one of skill in the art will recognize that a measurable change or reduction need not be absolute in order to be "modulatory."

[0074] In some embodiments, the present invention relates to therapeutically active peptides that have the effect of modulating the complement system and preventing, treating, and / or ameliorating toxic side effects of checkpoint inhibitors, such as intestinal necrosis or damage.

[0075] In one aspect, the present invention relates to therapeutically active peptides that have the effect of preventing, treating, and / or alleviating intestinal necrosis or damage, such as necrosis or damage caused by severe inflammatory responses, intestinal infarction (i.e., ischemia-reperfusion injury of intestinal tissue), autoimmune inflammatory bowel disease (IBD) and related therapies, and chemotherapy-induced or toxin-induced intestinal necrosis.

[0076] Pharmaceutical Compositions of the Present Invention The present disclosure provides a pharmaceutical composition capable of regulating the complement system, comprising at least one peptide as described above and at least one pharma- ceutically acceptable carrier, diluent, stabilizer, or excipient. The pharma- ceutically acceptable carrier, excipient, or stabilizer is non-toxic to a recipient at the dosage and concentration used. They may be solid, semi-solid, or liquid. The pharmaceutical composition of the present invention may be in the form of a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, or syrup.

[0077] The pharmaceutical composition of the present invention is prepared by mixing the peptide with appropriate purity with a pharma- ceutically acceptable carrier, diluent, or excipient. Examples of formulations and methods for preparing such formulations are well known in the art. The pharmaceutical composition of the present invention is useful as a prophylactic and therapeutic agent for various disorders and diseases as described above. In one embodiment, the composition comprises a therapeutically effective amount of the peptide. In another embodiment, the composition comprises at least one other active ingredient that is effective for regulating the complement system. In another embodiment, the composition comprises at least one other active ingredient that is effective for treating at least one disease associated with the complement system. In another embodiment, the composition comprises at least one other active ingredient that is effective for treating at least one disease not associated with the complement system. As used herein, the term "therapeutically effective amount" refers to the total amount of each active ingredient that is sufficient to show a benefit to the subject.

[0078] The therapeutically effective amount of peptide varies depending on several factors, such as the condition to be treated, the severity of the condition, administration time, administration route, the excretion rate of the peptide used, the duration of treatment, the concomitant therapy involved, and the age, sex, weight and condition of the subject.Those skilled in the art can determine the therapeutically effective amount.Therefore, those skilled in the art may need to titrate the dosage and modify the administration route to obtain the maximum therapeutic effect.

[0079] Effective daily doses are generally in the range of about 0.001 to about 200 milligrams per kilogram of body weight (mg / kg), including about 5 to about 160 mg / kg, about 10 to about 160 mg / kg, about 40 mg / kg to about 160 mg / kg, and about 40 mg / kg to about 100 mg / kg. This dose can be achieved with a dosing regimen of 1 to 6 times daily. Alternatively, optimal treatment can be achieved with sustained release formulations using less frequent dosing regimens. In some embodiments, a therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 10 mg / kg to about 160 mg / kg. In some embodiments, a therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 20 mg / kg to about 160 mg / kg. In some embodiments, a therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 40 mg / kg to about 160 mg / kg. In some embodiments, a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered in at least one dose, a first dose comprising from about 1 mg / kg to about 160 mg / kg of SEQ ID NO:2 and / or 3. In some embodiments, a second dose comprising a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered, the second dose comprising from about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, e.g., from about 10 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3. In some embodiments, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered in two doses, a first dose comprising from about 1 mg / kg to about 160 mg / kg of SEQ ID NO:2 and / or 3, e.g., from about 10 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, and a second dose comprising from about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, e.g., from about 1 mg / kg to about 40 mg / kg of SEQ ID NO:2 and / or 3. In some embodiments, the second dose is administered 30 seconds to 10 hours after the first dose is administered, e.g., about 8 hours after administration of the first dose.In some embodiments, a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered in multiple doses over a period of about 1 week to about 2 weeks, each dose containing about 1 mg / kg to about 160 mg / kg of SEQ ID NO:2 and / or 3, e.g., about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, administered every 4 to 10 hours, e.g., about every 8 hours.

[0080] In some embodiments, at least one loading dose of about 10 mg / kg to about 160 mg / kg of SEQ ID NO:2 and / or 3, e.g., about 10 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, is administered, followed by at least one maintenance dose of about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3, e.g., about 1 mg / kg to about 40 mg / kg of SEQ ID NO:2 and / or 3. In some embodiments, the first maintenance dose is administered 4-10 hours after the last loading dose. In some embodiments, the maintenance doses are administered every 4-10 hours for a period of about 1 week to about 2 weeks. In some embodiments, the first maintenance dose is administered 8 hours after the last loading dose, and the maintenance doses are administered every 8 hours for a period of about 1 week to about 2 weeks.

[0081] In another aspect, the invention is a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NO:2 and / or 3 and at least one pharma- ceutically acceptable carrier, diluent, or excipient.

[0082] The composition of the present invention may include carriers and / or excipients. Although it is possible to use the peptide of the present invention directly for therapy, it may be preferable to administer it in a pharmaceutical formulation, for example in admixture with suitable pharmaceutical excipients and / or carriers selected with respect to the intended route of administration and standard pharmaceutical practice. The excipients and / or carriers must be "acceptable" in the sense of being compatible with the other components of the formulation and not harmful to their recipient. Acceptable excipients and carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (AR Gennaro edit. 2005). The choice of pharmaceutical excipients and carriers may be selected with respect to the intended route of administration and standard pharmaceutical practice. Oral formulations easily accommodate further admixtures, such as, for example, milk, yogurt, and infant formula. Oral solid dosage forms can also be used, and can include, for example, capsules, tablets, caplets, pills, troches, lozenges, powders, and granules.Non-limiting examples of suitable excipients include, for example, diluents, buffers (e.g., sodium bicarbonate), preservatives, stabilizers, binders, compression agents, lubricants, dispersion enhancers, disintegrants, antioxidants, flavoring agents, sweeteners, and coloring agents.Those skilled in the art can fully prepare suitable solutions.

[0083] In one embodiment of any of the compositions of the present invention, the composition is formulated for delivery by routes such as oral, topical, rectal, mucosal, sublingual, nasal, naso / oro gastric gavage, parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal, and intratracheal administration.In one embodiment of any of the compositions of the present invention, the composition is in the form of a liquid, foam, cream, spray, powder, or gel.In one embodiment of any of the compositions of the present invention, the composition includes a buffering agent (e.g., sodium bicarbonate).

[0084] The administration of peptides and compositions in the method of the present invention can be achieved by any method known in the art.Non-limiting examples of useful routes of delivery include oral, rectal, fecal (by enema), and naso-oral gastric gavage, as well as parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal, and intratracheal administration.Active agents may be systemic after administration, or may be localized by using regional administration, intramural administration, or by using implants that act to retain the active dose at the site of implantation.

[0085] The useful dosage of the compounds and formulations of the present invention may vary widely depending on the nature of the disease, the medical history of the patient, the frequency of dosing, the mode of administration, clearance of the agent from the host, and the like. The initial dose may be higher, followed by a lower maintenance dose. The dose may be administered less frequently, such as weekly or biweekly, to maintain an effective dosage level, or may be divided into smaller doses and administered daily, semiweekly, and the like. It is contemplated that a range of doses may be effective to achieve a therapeutic effect. While it is possible to use the compounds of the present invention as such for therapy, it may be preferable to administer them in a pharmaceutical formulation, e.g., in admixture with suitable pharmaceutical excipients, diluents, or carriers selected with regard to the intended route of administration and standard pharmaceutical practice. The excipients, diluents, and / or carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (AR Gennaro edit. 2005). The choice of pharmaceutical excipients, diluents, and carriers can be selected with respect to the intended route of administration and standard pharmaceutical practice.

[0086] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous suspensions which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0087] The solutions or suspensions can contain any of the following components in any combination: sterile diluents, such as, by way of example and not limitation, water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citric acid, and phosphate; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.

[0088] When the agent shows insufficient solubility, methods can be used to solubilize the agent.Such methods are known to those skilled in the art and include, but are not limited to, the use of cosolvents such as dimethylsulfoxide (DMSO), the use of surfactants such as TWEEN® 80, or dissolving in aqueous sodium bicarbonate.In formulating effective pharmaceutical compositions, pharma-ceutically acceptable derivatives of the agent can also be used.

[0089] The composition can include, together with the active agent, for example, but not limited to: diluents, such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; lubricants, such as magnesium stearate, calcium stearate, and talc; and binders, such as starch, natural gums, such as acacia gum, gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose, and derivatives thereof, povidone, crospovidone, and other such binders known to those skilled in the art. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, or mixing the active agent and any pharmaceutical auxiliary as defined above in a carrier, such as, for example, but not limited to, water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc., thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain small amounts of non-toxic auxiliary substances, such as wetting agents, emulsifiers, or solubilizers, pH buffering agents, and the like, such as, for example and without limitation, acetic acid, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.The actual method of preparing such dosage forms is known or will be apparent to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975).The composition or formulation to be administered will in any event contain a quantity of active agent sufficient to alleviate the symptoms of the subject to be treated.

[0090] The active agents or pharma- ceutically acceptable derivatives can be prepared with carriers that protect the agent against rapid elimination from the body, such as time-release formulations or coatings. The compositions can include other active agents to obtain desired combinations of properties.

[0091] Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly, or intravenously, and is also contemplated herein. Injections can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for liquid solution or suspension before injection, or as emulsions. Suitable excipients include, by way of example and without limitation, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered can also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0092] The lyophilized powder can be reconstituted for administration of solutions, emulsions, and other mixtures, or can be formulated as a solid or gel. Sterile lyophilized powders are prepared by dissolving the agent provided herein or a pharma- ceutically acceptable derivative thereof in a suitable solvent. The solvent can include excipients that improve stability or other pharmacological components of the powder or the reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent can also include a buffer, such as citrate, sodium, or potassium phosphate, or other such buffers known to those skilled in the art, typically at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. Generally, the resulting solution can be apportioned into vials for lyophilization. Each vial can contain, by way of example and without limitation, a single dosage (10-1000 mg, e.g., 100-500 mg) or multiple dosages of agent. The lyophilized powder can be stored under appropriate conditions, e.g., at about 4° C. to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.

[0093] Combination therapy A further aspect of the present invention provides a method for regulating the complement system, comprising administering to a subject a pharmaceutical composition of the present invention. The pharmaceutical compositions of the present invention can be administered as the only active pharmaceutical agent, but they can also be used in combination with one or more therapeutic or prophylactic agents effective for regulating the complement system. In this aspect, the method of the present invention comprises administering the pharmaceutical composition of the present invention before, simultaneously with, and / or after one or more additional therapeutic or prophylactic agents effective for regulating the complement system.

[0094] The pharmaceutical composition of the present invention can be administered with an additional agent in a combination therapy, either together or separately, or by combining the pharmaceutical composition and the additional agent into one composition. The dosage is administered and adjusted to achieve maximum regulation of the complement system. For example, both the pharmaceutical composition and the additional agent are usually present at dosage levels between about 10% and about 150%, more preferably between about 10% and about 80%, of the dosage normally administered in a monotherapy regimen.

[0095] In any embodiment, the pharmaceutical composition of the present invention is administered in combination with a checkpoint inhibitor, either simultaneously or sequentially in any order.For example, but not limited to, the pharmaceutical composition can be administered after a checkpoint inhibitor and / or can be administered as a prophylactic to a subject who has been treated with a checkpoint inhibitor and has experienced intestinal inflammation, damage, or necrosis as a result of the checkpoint inhibitor.In any embodiment, the pharmaceutical composition of the present invention can be administered to a subject who is currently being treated with a checkpoint inhibitor, who has been treated with a checkpoint inhibitor, and / or who will be treated with a checkpoint inhibitor.Non-limiting examples of checkpoint inhibitors include CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors, such as pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo), and atezolizumab (Tecentriq).

[0096] In any embodiment, the pharmaceutical compositions of the present invention can prevent, treat, and / or mitigate toxic side effects of checkpoint inhibitors, such as intestinal necrosis and / or damage.

[0097] In any embodiment, the pharmaceutical compositions of the present invention can prevent, treat, and / or alleviate intestinal necrosis and / or damage, such as necrosis or damage caused by severe inflammatory responses, intestinal infarction (i.e., ischemia-reperfusion injury of intestinal tissue), autoimmune inflammatory bowel disease (IBD), and related therapies, as well as chemotherapy-induced or toxin-induced intestinal necrosis.

[0098] How to use In one aspect, the present invention provides a method for preventing, treating, and / or ameliorating toxic side effects of checkpoint inhibitors comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0099] In one aspect, the present invention provides a method of preventing, treating, and / or alleviating intestinal necrosis and / or damage, such as necrosis or damage caused by severe inflammatory responses, intestinal infarction (i.e., ischemia-reperfusion injury of intestinal tissue), autoimmune inflammatory bowel disease (IBD), and related therapies, and chemotherapy-induced or toxin-induced intestinal necrosis, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0100] In one aspect, the present invention provides a method of preventing, treating, and / or alleviating intestinal necrosis and / or damage in a subject who is being, has been treated, and / or will be treated with at least one checkpoint inhibitor, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3.

[0101] In any embodiment of any of the foregoing methods, the composition further comprises at least one pharma- ceutically acceptable carrier, diluent, stabilizer, or excipient. In one embodiment of any of the foregoing methods, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 10 mg / kg to about 160 mg / kg. In one embodiment of any of the foregoing methods, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 20 mg / kg to about 160 mg / kg. In one embodiment of any of the foregoing methods, the therapeutically effective amount of SEQ ID NO:2 and / or 3 is about 40 mg / kg to about 160 mg / kg. In one embodiment of any of the foregoing methods, the composition is formulated for subcutaneous, intravenous, intraperitoneal, or intramuscular administration. In one embodiment, the composition further comprises a pharma- ceutically acceptable carrier and / or excipient. EXAMPLES

[0102] The present invention is also described and illustrated through the following examples. However, the use of these and other examples anywhere in this specification is merely illustrative and in no way limits the scope and meaning of the invention or any exemplified term. Similarly, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention may become apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in its spirit or scope. Therefore, the present invention should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0103] Example 1: Use of PA-dPEG24 to prevent, treat, and / or alleviate intestinal necrosis Intestinal necrosis is a potentially life-threatening medical condition that can result from a variety of clinical etiologies. Necrosis of intestinal tissue leads to luminal damage and leakage of intestinal bacteria, as well as elicitation of an aggressive immune response involving the complement system and neutrophils. PA-dPEG24 (RLS-0071) is a peptide inhibitor of the classical and lectin pathways, as well as myeloperoxidase activity and NETosis, the major effectors of neutrophils. Therefore, we decided to evaluate the extent to which immune modulation via inhibition of the complement system and neutrophil effectors could affect survival in the context of intestinal necrosis and leakage of intestinal contents. Adolescent male Long-Evans rats were subjected to cecal ligation and puncture (CLP), an established rat model of intestinal necrosis, and a single dose of 40 mg / kg RLS-0071 was given to one cohort 30 minutes after surgery, while the control group was not treated. The survival of the rats was then evaluated up to 5 days after surgery. Surprisingly, animals treated with RLS-0071 showed 80% survival compared to 50% in the untreated group. To evaluate whether the unexpected increase in survival could be due to reduced inflammatory response, markers of NETosis, free DNA in plasma, and the proinflammatory cytokine IL-6 were evaluated. For animals treated with RLS-0071, a reduction in the blood levels of free DNA and the inflammatory cytokine IL-6 was observed. These results indicate that a single dose of RLS-0071 can increase survival in intestinal necrosis, possibly by reducing certain inflammatory responses.

[0104] material and method Animal testing A previously established CLP model

[10] was utilized with modifications. Male Long-Evans rats (250–300 grams) were induced with 5% isoflurane in oxygen and anesthesia was maintained with 1.5–2% isoflurane in oxygen delivered via a nose cone. Adequate anesthesia was confirmed by toe pinch. The surgical area (lower abdominal quadrant) was shaved and disinfected with 10% iodine followed by 70% ethanol for a total of three times. Buprenorphine-SR (1 mg / kg) was given subcutaneously before surgery for pain management. For the surgical procedure, the animals were placed in a supine position with the head facing away from the surgeon. After placement of a sterile drape, a longitudinal midline incision (approximately 3–4 cm) was made with a sterile scalpel. After the initial incision, small scissors were used to enter the peritoneal cavity. The linea alba of the abdominal musculature was dissected and the fascial and peritoneal layers were incised intermuscularly. The cecum was located and exteriorized, leaving the remnant of the intestine in the peritoneal cavity. The cecal mesentery was dissected, taking care to avoid damage to the cecal branch of the ileocecal artery and to avoid hemorrhagic complications. The cecum was ligated at 75% using a sterile 3-0 non-absorbable suture. The cecum was perforated twice by penetrating puncture using a sterile 18-gauge needle, midway between the ligated part and the tip of the cecum, in the mesenteric-to-antementeric direction. The cecum was repositioned in the abdominal cavity. The peritoneum was closed using a 4-0 non-absorbable suture and the skin was closed with sterile metal wound clips. The animals were given 5 mL of pre-warmed (37°C) normal saline subcutaneously and allowed to recover. For RLS-0071 treated animals (n=9), 30 minutes after surgery, rats were given a single dose of 40 mg / kg RLS-0071 intravenously through an indwelling jugular catheter. Amino acid sequence: The peptide, consisting of TIFF2024522988000009.tif4128, had a monodisperse 24-mer polyethylene glycol (PEG) tail and was manufactured by PolyPeptide Group (San Diego, CA) to a purity of ≥95% as verified by HPLC and mass spectrometry. Lyophilized RLS-0071 was solubilized in 0.05 M histidine buffer and pH adjusted to 6.5. The control group (n=6) received no compound. Animals were monitored every 30 minutes for at least 2 hours after surgery. Once vivacious, alert, and responsive, animals were returned to their home cages. Animals were quantitatively assessed for morbidity twice daily for the duration of the study (Shrum, B., Anantha, RV, Xu, SX et al. robust scoring system to evaluate sepsis severity in an animal model. BMC Res Notes 7, 233 (2014). https: / / doi.org / 10.1186 / 1756-0500-7-233). Scoring criteria included appearance, level of consciousness, activity, response to stimuli, eyes open vs. closed, respiratory rate, and quality of breathing. Each category was ranked from 0 (best) to 4 (worst). Animals were humanely euthanized if their cumulative score exceeded 21 or their quality of breathing exceeded 3. At the end of the 5th day, surviving animals were euthanized by carbon dioxide asphyxiation and cervical dislocation. Animals continued to be monitored a minimum of twice daily.

[0105] Blood samples were collected before surgery (prebleed) and 24 hours after surgery. Plasma was isolated from the blood and analyzed for free DNA concentration and cytokine levels as described below.

[0106] Measurement of plasma DNA Free DNA was measured by PicoGreen in rat plasma samples as previously described [8]. Briefly, plasma samples were diluted in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 (TE) buffer, and 50 uL of each sample was added to wells along with 50 uL of a 1:200 dilution of PicoGreen (Life Technologies, Carlsbad, CA, USA) and incubated for 10 min at room temperature protected from light. A DNA standard curve was prepared in TE buffer. Fluorescence was then read at an excitation wavelength of 485 nm and an emission wavelength of 520 nm using a BioTek microplate reader. All free DNA measurements were performed in triplicate.

[0107] Cytokine Analysis Rat IL-6 ELISA was purchased from R&D Systems. Plasma samples from experimental animals were subjected to analysis according to the manufacturer's instructions. Briefly, diluted rat plasma samples were added overnight to wells previously coated with capture IL-6 antibody. Plates were blocked with 1% BSA for 60 minutes at room temperature. 100uL of plasma samples (1:4 dilution) or standards were added to the plate and incubated at room temperature for 2 hours, and any unbound components were then removed by washing. Next, 100μL of detection IL-6 antibody (1:60 dilution) was added, incubated at room temperature for 2 hours, and washed. Next, 100μL of streptavidin-horseradish peroxidase (HRP) conjugate (1:40 dilution) was added and incubated at room temperature for 20 minutes in the dark. After washing, 100μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added and left at room temperature for approximately 5 minutes. The reaction was stopped by adding 100 μL of sulfuric acid, and the absorbance was measured at 450 nm using a BioTek microplate reader. Sample concentrations were calculated using an IL-6 standard curve generated with 1:2 serial dilutions.

[0108] statistical analysis Data are expressed as the mean and standard error of the mean.

[0109] result RLS-0071 reduces mortality in a CLP model To test whether modulation of the complement system and neutrophil immune mechanisms increases mortality from bacterial sepsis in the setting of intestinal necrosis, RLS-0071 was tested in the CLP model. In the CLP model, ligation of the cecum causes infarction of the cecum punch, which then undergoes necrosis. A previously developed CLP model in Long-Evans rats reported that induction of moderate necrosis, which results in approximately 40% survival, requires ligation of the cecum at half the distance between the distal pole and base of the cecum, followed by penetrating the ligated cecum with an 18-gauge needle

[11] . Our experimental protocol utilized 75% ligation of the cecum, followed by two punctures of the cecum with an 18-gauge needle, resulting in 20% survival. Surprisingly, as shown in Figure 1, rats that underwent CLP and received a single 40 mg / kg dose of RLS-0071 delivered intravenously 30 minutes after surgery had a 50% survival rate, a 2.5-fold increase, compared to the non-rescue treatment group. Thus, rather than modulation of the complement system and neutrophil effectors causing increased mortality from an uncontrollable infection, administration of RLS-0071 appeared to increase survival in the setting of intestinal necrosis.

[0110] RLS-0071 inhibits the accumulation of free DNA in the blood The finding that RLS-0071 could increase survival in the setting of intestinal necrosis raised the possibility that inhibition of inflammation in rats might affect rat survival. We then assayed two important aspects of inflammation in the blood of CLP animals. Neutrophil extracellular traps (NETs), released from activated neutrophils, have previously been shown to play a pathogenic role in a variety of autoimmune, metabolic, and inflammatory diseases

[12] and have been hypothesized to contribute to immune thrombosis and disseminated intravascular coagulation (DIC) in sepsis [4]. NETs have been observed in mouse models of virus-induced acute lung injury. In the second aspect, the presence of free DNA in the bloodstream is a biomarker of NETs in the blood of human patients with acute lung injury [13, 14] and COVID-19 patients

[15] . To ascertain whether free DNA is present in the blood of septic rats, the levels of free DNA were measured by PicoGreen fluorescence 24 hours after surgery. Free DNA levels were increased in animals subjected to the CLP procedure compared to blood obtained from animals prior to surgery (pre-bleed). Animals receiving RLS-0071 had reduced levels of free DNA compared to untreated animals. Additionally, animals were tested for IL-1B and found to be negative. As shown in Figure 2, this reduction in free DNA in animals treated with RLS-0071 suggests that RLS-0071 reduces NET formation in this model.

[0111] RLS-0071 reduces levels of the inflammatory cytokine IL-6 in the blood In cases of intestinal necrosis, significant amounts of proinflammatory cytokines are produced in response to infection. This so-called "cytokine storm" has been well documented for sepsis, and this aggressive inflammatory response is associated with severe outcomes, e.g., end-organ damage and sometimes death [4]. To ascertain the effect of RLS-0071 on inflammatory cytokine levels in this model, blood IL-6 levels were measured. IL-6 is a potent proinflammatory cytokine released primarily by macrophages and plays a major role in many inflammatory diseases, including inflammatory bowel disease

[16] . Blood obtained from animals prior to surgery (prebleeding) did not have detectable levels of IL-6. Animals subjected to the CLP procedure showed increased blood levels of cytokines 24 hours after surgery. In contrast, as shown in Figure 3, animals receiving RLS-0071 showed reduced IL-6 compared to rescue non-intervention animals, suggesting that RLS-0071 can reduce the production of inflammatory cytokines in this model. Additionally, animals were tested for IL-1B and found to be negative.

[0112] Consideration We performed experiments to determine whether the immunomodulatory molecule RLS-0071 could affect survival from bacterial sepsis in the context of intestinal necrosis. Surprisingly, RLS-0071 was able to increase survival in an established model of intestinal necrosis caused by cecal ligation. As previously reported, the cecal ligation and puncture (CLP) rat model has been available for over 40 years and is considered the gold standard model of intestinal necrosis and sepsis

[11] . In this model, the cecum is ligated below the ileocecal valve, followed by needle puncture of the cecum. Upon necrosis and perforation of the cecum, bacteria, toxins, and other contaminating microorganisms are released into the peritoneal cavity, resulting in bacterial peritonitis. These mixed intestinal bacteria are then transported into the blood compartment, causing bacterial sepsis. Enteropathogenic bacterial sepsis commonly leads to hypotension, disseminated intravascular coagulation (DIC), multiple organ failure, and potentially death.

[0113] RLS-0071 has been shown to inhibit classical complement activation in in vitro, in vivo, and ex vivo studies, as well as NET formation via inhibition of myeloperoxidase in in vitro and ex vivo studies [6-8]. Given its immunomodulatory activity on the complement system and neutrophil effectors, we hypothesized that RLS-0071 may exacerbate bacterial sepsis and increase mortality in CLP animal models. Our results showed the surprising finding that RLS-0071 delivered as a single dose 30 minutes after surgery increased survival by 2.5-fold. This result suggested that RLS-0071 may increase survival by reducing the fatal aspect of the inflammatory response to intestinal necrosis. CLP animals treated with RLS-0071 showed reduced levels of free DNA, which serves as a biomarker of NETosis, and reduced inflammatory cytokine IL-6. The ability of RLS-0071 to increase survival and reduce inflammation in the CLP model has potential utility as a clinical therapeutic for a variety of disease processes associated with intestinal necrosis, such as intestinal infarction (i.e., ischemia-reperfusion injury of intestinal tissue), autoimmune inflammatory bowel disease (IBD) and associated medications, chemotherapy-induced or toxin-induced intestinal necrosis, as well as intestinal necrosis or damage resulting from severe inflammatory responses.

[0114] Example 2: Multiple doses of RLS-0071 and RLS-0088 reduce mortality in a CLP model We next evaluated whether a multiple dose regimen of RLS-0071 would have an effect on survival. RLS-0071 was administered to animals as in Example 1 at 0.5, 24, 48, and 72 hours after surgery. Rats receiving RLS-0071 showed a significant increase in survival (p=0.032) (Figure 4). A second generation EPICC peptide, RLS-0088, was then tested in the CLP model to determine whether it would have a similar effect on improving survival. Using the same multiple dose experimental approach as shown in Example 1, rats were subjected to CLP and 40 mg / kg of RLS-0088 was administered to the rats at 0.5, 24, 48, and 72 hours after surgery. Rats receiving RLS-0088 showed an increase in survival (Figure 5).

[0115] Example 3: Administration of pharmaceutical formulations To treat a disease or condition, a pharmaceutical composition containing a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered to a subject in need thereof. Administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

[0116] To regulate the complement system in a subject, a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered to a subject in need thereof. Administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

[0117] To alter cytokine expression in a subject, a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered to a subject in need thereof. Administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

[0118] To prevent, treat, and / or mitigate toxic side effects of checkpoint inhibitors, such as intestinal necrosis or damage, a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered to a subject in need thereof. Administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

[0119] To prevent, treat, and / or alleviate intestinal necrosis and / or damage, such as necrosis or damage caused by severe inflammatory reactions, intestinal infarction (i.e., ischemia-reperfusion injury of intestinal tissue), autoimmune inflammatory bowel disease (IBD), and related drug therapies, and chemotherapy-induced or toxin-induced intestinal necrosis, a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered to a subject in need thereof. Administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

[0120] To prevent, treat, and / or alleviate intestinal necrosis and / or damage in a subject who is being, has been, and / or will be treated with at least one checkpoint inhibitor, a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NO: 2 and / or 3 is administered to a subject in need thereof. Administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

[0121] Although several possible embodiments are disclosed above, the embodiments of the present invention are not so limited. These exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the invention, but instead have been selected and described to explain the principles of the invention so that others skilled in the art may practice the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0122] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

[0123] References TIFF2024522988000010.tif228164TIFF2024522988000011.tif131164

Claims

1. A pharmaceutical comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3 for use in a method for altering cytokine expression in a subject in need thereof.

2. A pharmaceutical comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3 for use in a method for preventing, treating, and / or mitigating toxic side effects of a checkpoint inhibitor in a subject in need thereof.

3. A pharmaceutical comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3 for use in a method for preventing, treating, and / or ameliorating intestinal necrosis and / or damage in a subject in need thereof.

4. A pharmaceutical comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and / or 3 for use in a method for preventing, treating, and / or ameliorating intestinal necrosis and / or damage in a subject who is being treated, has been treated, or will be treated with at least one checkpoint inhibitor.

5. The pharmaceutical composition of any one of claims 1 to 4, further comprising at least one pharma- ceutically acceptable carrier, diluent, stabilizer, or excipient.

6. The method of any one of claims 1 to 4, wherein the therapeutically effective amount of SEQ ID NO: 2 and / or 3 is from about 10 mg / kg to about 160 mg / kg.

7. The method of any one of claims 1 to 4, wherein the therapeutically effective amount of SEQ ID NO: 2 and / or 3 is from about 20 mg / kg to about 160 mg / kg.

8. The method of any one of claims 1 to 4, wherein the therapeutically effective amount of SEQ ID NO: 2 and / or 3 is from about 40 mg / kg to about 160 mg / kg.

9. The pharmaceutical composition of any one of claims 1 to 4, wherein the therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered in at least one dose, wherein a first dose comprises from about 1 mg / kg to about 160 mg / kg of SEQ ID NO:2 and / or 3.

10. The pharmaceutical composition of claim 9, wherein a second dose comprising a therapeutically effective amount of SEQ ID NO:2 and / or 3 is administered, the second dose comprising from about 1 mg / kg to about 120 mg / kg of SEQ ID NO:2 and / or 3.

11. The therapeutically effective amount of SEQ ID NO: 2 and / or 3 is administered in two doses, the first dose comprising SEQ ID NO: 2 and / or 3 at about 1 mg / kg to about 160 mg / kg, and the second dose comprising SEQ ID NO: 2 and / or 3 at about 1 mg / kg to about 120 mg / kg, the pharmaceutical according to any one of claims 1 to 4.

12. The pharmaceutical according to claim 11, wherein the second dose is administered 30 seconds to 10 hours after the first dose is administered.

13. The therapeutically effective amount of SEQ ID NO: 2 and / or 3 is administered in multiple doses over a period of about 1 week to about 2 weeks, each dose comprising SEQ ID NO: 2 and / or 3 at about 1 mg / kg to about 160 mg / kg and being administered every 4 to 10 hours, the pharmaceutical according to any one of claims 1 to 4.

14. The pharmaceutical according to claim 13, wherein each dose is administered every 8 hours.

15. The therapeutically effective amount of SEQ ID NO: 2 and / or 3 is administered in at least one loading dose of SEQ ID NO: 2 and / or 3 at about 10 mg / kg to about 160 mg / kg, followed by at least one maintenance dose of SEQ ID NO: 2 and / or 3 at about 1 mg / kg to about 120 mg / kg, the first maintenance dose being administered 4 to 10 hours after the last loading dose, and the maintenance dose being administered every 4 to 10 hours over a period of about 1 week to about 2 weeks, the pharmaceutical according to any one of claims 1 to 4.

16. The pharmaceutical according to claim 15, wherein the first maintenance dose is administered 8 hours after the last loading dose and the maintenance dose is administered every 8 hours over a period of about 1 week to about 2 weeks.

17. The pharmaceutical according to any one of claims 1 to 4, wherein the subject is also being treated with, has previously been treated with, or will be treated with a checkpoint inhibitor.

18. The pharmaceutical according to claim 17, wherein the checkpoint inhibitor is selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor, such as pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo), and atezolizumab (Tecentriq).