CN117396215A

CN117396215A - Peptides and methods of use

Info

Publication number: CN117396215A
Application number: CN202280037280.6A
Authority: CN
Inventors: 尼尔·K·克里希纳; 肯吉·昆尼昂; 乌尔里希·蒂内尔
Original assignee: Rierta Life Sciences
Current assignee: Rierta Life Sciences
Priority date: 2021-06-01
Filing date: 2022-05-31
Publication date: 2024-01-12

Abstract

The present invention provides peptides comprising C-terminal PEGylation, which are synthetic modifications of polar-selective (PA) peptides. The invention also 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

Peptides and methods of use

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application Ser. Nos. 63/279,423 and 63/195,401, filed on day 1 and month 6 of 2021, filed on day 11 of 2021. The disclosure of the U.S. provisional application is incorporated herein by reference in its entirety.

Sequence listing

The present application contains a sequence listing that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created at 25 days 5 of 2022, named 251110_000164_sl.txt, 1,196 bytes in size.

Background

Embodiments of the present invention relate generally to synthetic peptides and their use for therapy, and more particularly, to synthetic peptide modifications that can prevent, treat and/or mitigate toxicity, particularly intestinal necrosis or injury, caused by checkpoint inhibitors.

Complement system

The complement system is an important component of the innate immune system, playing a key role as a defense mechanism against invading pathogens, initiating adaptive immune responses, and helping to clear immune complexes and apoptotic cells. The complement system is composed of three distinct pathways: classical pathway, lectin pathway and alternative pathway. C1q and mannose-binding lectin (MBL) are structurally related recognition molecules of the classical and lectin pathways, respectively. IgM or clustered IgG is the primary ligand of C1q, while MBL recognizes polysaccharides such as mannans. The C1q and MBL binding ligands result in sequential activation of C4 and C2, forming classical pathway and lectin pathway C3 convertases, respectively. In contrast, alternative pathway activation does not require recognition molecules, but can amplify C3 activation initiated by either the classical pathway or the lectin pathway. Activation of any of these three pathways results in the formation of inflammatory mediators (C3 a and C5 a) and Membrane Attack Complexes (MACs), leading to cell lysis.

Although the complement system plays a key role in many protective immune functions, complement activation is an important mediator of tissue damage in a wide range of autoimmune and inflammatory disease processes. (Ricklin and Lambris, "Complement-targeted therapeutic (component-targeted therapeutics)", nat Biotechnol 2007;25 (11): 1265-75).

Complement modulators are needed. On the one hand, the complement system is an important host defense against pathogenic organisms. On the other hand, its unchecked activation can cause destructive damage to the host cell. Currently, although many disease processes, including autoimmune diseases such as those associated with morbidity and mortality in systemic lupus erythematosus, myasthenia gravis, and multiple sclerosis are known to be associated with complement dysregulation, only two anticomplementary therapies have recently been approved for use in humans: 1) Ekulizumab (Soliris) ^TM ) And 2) Wu Tuomi Ralset (ultomiris) (Ravulizumab) ^TM ) Two humanized long-acting anti-C5 monoclonal antibodies are used to treat sudden nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). PNH and aHUS are orphan diseases that rarely occur in humans. Currently, no complement regulator is approved for the more common disease processes in which complement activation disorders play a critical role. Complement activation disorders may play a role in chronic and acute disease indications.

Peptides need to be developed to inhibit the classical, lectin and alternative pathways of the complement system, as each of these three pathways has been shown to contribute to many autoimmune and inflammatory disease processes. Specific blockade of the classical and lectin pathways is particularly desirable because both pathways are involved in ischemia reperfusion-induced injury and other diseases in many animal models. People with alternative pathway defects can suffer from serious bacterial infections. Thus, a functional alternative pathway is essential for immune surveillance of invading pathogens.

The PIC1 family of molecules (also referred to herein as the epcc family or epcc peptides) comprises a number of rationally designed peptides based on hybrid astrovirus coat proteins that have a variety of anti-inflammatory functionalities including complement classical pathway inhibition, myeloperoxidase inhibition, neutrophil Extracellular Trap (NET) inhibition, and antioxidant activity. The original compound is a 15 amino acid peptide sequence IALILEPICCQERAA (SEQ ID NO: 1) with a C-terminal monodisperse 24-mer PEGylated portion (IALILEPICCQERAA-dPEG 24; PA-dPEG24; RLS-0071; SEQ ID NO: 2) that enhances its water solubility. PA-dPEG24 is a peptide inhibitor of the classical and lectin pathways, myeloperoxidase activity and NETosis, which are the major effectors of neutrophils [6-8]. Sarcosine substitution scan of SEQ ID NO. 2 shows that substitution of isoleucine at position 8 with sarcosine produces the water-soluble non-PEGylated peptide IALILEP (Sar) CCQERAA (PA-I8 Sar; RLS-0088; SEQ ID NO. 3) (as described in U.S. Pat. No. 10,005,818).

Other characteristics of the PA-dPEG24 molecule and the PA-I8Sar molecule are discussed herein.

Checkpoint therapeutics and related toxicities

Intestinal necrosis is a potentially life threatening medical condition that may be caused by a variety of causes, including bacterial sepsis and Systemic Inflammatory Response Syndrome (SIRS). The etiology of intestinal necrosis includes a variety of disease processes that may lead to vascular damage, such as venous thrombosis, chronic ischemia, mechanical and non-obstructive mesenteric ischemia [1], as well as autoimmune inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, or toxic megacolon caused by Clostridium difficile (C.difficile) colitis. Furthermore, intestinal necrosis caused by severe inflammatory reactions is a treatment-limiting serious adverse event associated with cancer checkpoint inhibitor drugs. [2]

Intestinal necrosis often damages the intestinal lumen, resulting in trans-migration of intestinal bacteria, ultimately resulting in massive leakage of intestinal contents containing bacteria, toxins and other microbial products into the peritoneal and systemic circulation. This process may then lead to bacterial sepsis, hypotension, disseminated intravascular coagulation, multiple system organ failure, and often to death. The main therapeutic interventions for the treatment of intestinal necrosis include antibiotics aimed at killing bacterial pathogens in the peritoneum and blood stream. The use of anti-complement therapies such as eculizumab may lead to impaired immunity in the subject and increase the risk of invasive neisseria meningitidis (n.menningitidis) infection. Furthermore, persistent neutropenia is associated with a high risk of life threatening bacterial sepsis. Thus, there is a concern that modulation of complement system and neutrophil effector function may exacerbate the risk of an overwhelming bacterial infection due to intestinal necrosis.

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 for preventing, treating and/or alleviating the toxic side effects associated with checkpoint inhibitors, in particular intestinal necrosis or injury.

Disclosure of Invention

As described in the background section, there is a great need in the art for techniques to identify peptide-based inhibitors of different pathways of the complement system, and to use this understanding to develop novel therapeutic peptides. The present invention meets this and other needs. Embodiments of the present invention relate generally to synthetic peptides, and more particularly, to synthetic peptides that are pegylated or contain sarcosine substitutions, and their use in methods of modulating the complement system and preventing, treating, and/or alleviating toxic side effects associated with checkpoint therapeutics, particularly intestinal necrosis or injury.

In one aspect, the invention provides synthetic peptides that modulate the complement system and methods of using these peptides. In particular, in some embodiments, the synthetic peptide can bind, modulate, and inactivate C1 and MBL, and thus can effectively inhibit classical and lectin pathway activation at the earliest point of the complement cascade, while leaving the alternative pathway intact. These peptides are of therapeutic value in terms of selectively modulating and inhibiting C1 and MBL activation without affecting alternative pathways, and are useful in the treatment of diseases mediated by deregulation of classical and lectin pathway activation. In other embodiments, the peptide modulates classical pathway activation but does not modulate lectin pathway activation. The peptides are useful in a variety of therapeutic indications.

In any embodiment, the synthetic peptide is capable of preventing, treating, and/or alleviating the toxic side effects of a checkpoint inhibitor.

In any embodiment, the synthetic peptide is capable of preventing, treating and/or reducing intestinal necrosis and/or injury, such as necrosis or injury caused by severe inflammatory reactions, 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.

In any embodiment, the invention is based on the identification and modification of a 15 amino acid peptide from a polar selection (PA) peptide (SEQ ID NO: 1), derivatives of said peptide and methods of use thereof. PA peptide is a promiscuous peptide derived from a human astrovirus protein called CP 1. 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. The PEGylated form of the PIC1 peptide is referred to as PA-dPEG24 or RLS-0071 (SEQ ID NO: 2), has 24 PEG units at the C-terminus of the peptide, and exhibits improved solubility in aqueous solutions. Sarcosine substitution scan of SEQ ID NO. 2 shows that substitution of isoleucine at position 8 with sarcosine produces the water-soluble non-PEGylated peptide IALILEP (Sar) CCQERAA (PA-I8 Sar; RLS-0088; SEQ ID NO. 3) (as described in U.S. Pat. No. 10,005,818).

In one aspect, the invention provides a method of preventing, treating and/or alleviating the 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 NOs 2 and/or 3.

In one aspect, the invention provides a method of preventing, treating and/or reducing intestinal necrosis and/or injury, such as necrosis or injury caused by severe inflammatory reactions, 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.

In one aspect, the invention provides a method of preventing, treating and/or alleviating intestinal necrosis and/or injury in a subject being treated with, having been treated with and/or to be treated with at least one checkpoint inhibitor, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NOs 2 and/or 3.

In one embodiment of any of the above methods, the composition further comprises at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient. In one embodiment of any of the above methods, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is from about 10mg/kg to about 160mg/kg. In one embodiment of any of the above methods, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is from about 20mg/kg to about 160mg/kg. In one embodiment of any of the above methods, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is from about 40mg/kg to about 160mg/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 1mg/kg to about 160mg/kg 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 about 1mg/kg to about 120mg/kg SEQ ID NO:2 and/or 3, including about 10mg/kg to about 120mg/kg 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 about 1mg/kg to about 160mg/kg SEQ ID NO 2, including about 10mg/kg to about 120mg/kg SEQ ID NO 2 and/or 3, and the second dose comprising about 1mg/kg to about 120mg/kg SEQ ID NO 2 and/or 3, including about 1mg/kg to about 40mg/kg SEQ ID NO 2 and/or 3. In any embodiment, the second dose is administered 30 seconds to 10 hours after administration of the first dose, including about eight hours after administration of the first dose. In any embodiment, a therapeutically effective amount of SEQ ID NO 2 and/or 3 is administered in a plurality of doses over a period of about one week to about two weeks, each dose comprising about 1mg/kg to about 160mg/kg SEQ ID NO 2 and/or 3, including about 1mg/kg to about 120mg/kg SEQ ID NO 2 and/or 3, administered every 4 to 10 hours, including about every eight hours.

In any embodiment, at least one loading dose of about 10mg/kg to about 160mg/kg SEQ ID NO:2 and/or 3, including about 10mg/kg to about 120mg/kg SEQ ID NO:2 and/or 3, is administered followed by at least one maintenance dose of about 1mg/kg to about 120mg/kg SEQ ID NO:2 and/or 3, including about 1mg/kg to about 40mg/kg SEQ ID NO:2 and/or 3. In any embodiment, the first maintenance dose is administered 4 to 10 hours after the last loading dose. In any embodiment, the maintenance dose is administered every 4 to 10 hours over a period of about one week to about two weeks. In any embodiment, 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 one week to about two weeks.

In any one of the above methods, the composition is formulated for subcutaneous, intravenous, intraperitoneal, or intramuscular administration. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier and/or excipient.

These and other objects, features and advantages of the present invention will become more fully apparent upon review of the following specification, appended description, claims and drawings.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. The present patent or application contains at least one color drawing. The patent office will provide copies of this patent or patent application publication with color drawings at the request and after payment of the necessary fee.

FIG. 1 shows that PA-dPEG24 (also referred to herein as RLS-0071) increases survival of rats after cecal ligation. Evaluation of Kaplan-Meier survival curves. The red line represents the results for untreated animals after 75% CLP (n=6), while the red curve represents the results after animals received a single dose of 40mg/kg RLS-0071 (n=9).

FIG. 2 shows that RLS-0071 reduces free DNA levels in blood. Plasma was isolated from CLP-administered (n=4) and non-administered (n=3) single dose RLS-0071 animals prior to surgery (pre-blood collection) (n=7) and 24 hours post-surgery. Plasma samples were incubated with PicoGreen. Fluorescence was read in a microplate reader at 485nm excitation wavelength and 520nm emission wavelength. Data are mean and mean standard error.

FIG. 3 shows that RLS-0071 reduces IL-6 levels in blood. Plasma was isolated from CLP-administered (n=3) and non-administered (n=5) single dose RLS-0071 animals prior to surgery (pre-blood collection) (n=8) and 24 hours post-surgery. Plasma samples were analyzed in an IL-6ELISA according to the manufacturer's instructions. Data are mean and mean standard error.

FIG. 4 shows that multiple doses of RLS-0071 improved survival of rats after cecal ligation as assessed by Kaplan-Meier survival curves. The black line represents the results for animals that did not receive treatment after 75% CLP (n=12), while the green line represents the results after animals received 40mg/kg RLS-0071 dosing at 0.5 hours, 24 hours, 28 hours and 72 hours post-surgery (n=12).

FIG. 5 shows that multiple doses of RLS-0088 increased survival in rats after cecal ligation as assessed by Kaplan-Meier survival curves. The red line represents the results for animals receiving only normal saline after 75% CLP (n=3), while the green line represents the results after animals received 40mg/kg RLS-0071 dose at 0.5 hours, 24 hours, 28 hours and 72 hours post-surgery (n=5). SID = once daily administration.

Detailed Description

As described in the background section, there is a great need in the art for techniques to identify peptide-based inhibitors of different pathways of the complement system, and to use this understanding to develop novel therapeutic peptides. The present invention meets this and other needs. Embodiments of the present invention relate generally to synthetic peptides, and more particularly, to synthetic peptides that are pegylated or contain sarcosine substitutions, and their use in methods of modulating the complement system and alleviating toxic side effects associated with checkpoint therapeutics, particularly intestinal necrosis or injury.

In order to facilitate an understanding of the principles and features of various embodiments of the invention, different illustrative embodiments are explained below. While exemplary embodiments of the invention have been explained in detail, it should be understood that other embodiments are contemplated. Accordingly, it is not intended that the scope of the invention be limited to the details of construction and arrangement of the components set forth in the following description or illustrated in the examples. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Furthermore, in describing exemplary embodiments, specific terminology will be resorted to for the sake of clarity.

It must also be noted that, as used in the specification and claims, unless the context clearly indicates otherwise, no particular number of a reference includes a plurality of references. For example, references to components are also intended to include compositions of multiple components. References to compositions containing ingredients are intended to include ingredients other than those specified. In other words, the terms "said" and "the" do not denote a limitation of quantity, but rather denote the presence of "at least one of the referenced item.

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

Furthermore, in describing exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term encompasses its broadest meaning as understood by those skilled in the art and includes all technical equivalents that operate in a similar manner to accomplish a similar purpose. It is understood that embodiments of the disclosed technology may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description. References to "one embodiment," "an embodiment," "example embodiment," "some embodiments," "certain embodiments," "various embodiments," etc., indicate that the embodiment of the disclosed technology so described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Furthermore, the repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, but it may.

As used herein, the term "about" should be taken to mean two numbers specified as endpoints of any range. Any reference to a range should be construed as providing support for any subset within that range. Ranges may be expressed herein as from "about" or "approximately" or "substantially" one particular value, and/or to "about" or "approximately" or "substantially" another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and/or to the other particular value. Furthermore, the term "about" means within an acceptable error range for the particular value 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, according to practice in the art, "about" may mean within an acceptable standard deviation. Alternatively, "about" may mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, more preferably up to ±1% of a given value. Alternatively, particularly in terms of biological systems or processes, the term may mean within an order of magnitude, preferably within a factor of 2 of the value. In describing particular values in the present application and claims, unless otherwise indicated, the term "about" is implicit and in this case is intended to be within the acceptable error range of the particular value.

Throughout this disclosure, various aspects of the invention may be provided 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. Accordingly, the description of a range should be considered to explicitly disclose all possible sub-ranges and individual values within the range. For example, descriptions of ranges such as 1 to 6 should be considered as explicitly disclosing sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

Also, as used herein, "substantially free of" something, or "substantially pure" and similar descriptions may include "at least substantially free of" something, or "at least substantially pure" and "completely free of" something, or "completely pure".

"comprising" or "containing" or "including" means that at least the compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, materials, particles, method steps have the same function as the listed compounds, materials, particles, method steps.

Throughout the specification, various components having particular values or parameters may be identified, however, these terms are provided as exemplary embodiments. Indeed, the exemplary embodiments are not limiting of the various aspects and concepts of the invention as many equivalent parameters, sizes, ranges, and/or values may be implemented. The terms "first," "second," and the like, "primary," "secondary," and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

It should be noted that terms like "specifically," "preferably," "typically," "generally," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present invention. It should also be noted that terms such as "substantially" and "about" are utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation.

The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Rather, unless otherwise indicated, 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 "50mm" is intended to mean "about 50mm".

It should also be understood that reference to one or more method steps does not exclude the presence of other method steps or intermediate method steps between those explicitly identified steps. Also, it should be understood that reference to one or more components in a composition does not preclude the presence of other components than the specifically identified components.

The materials described below, which make up the various elements of the invention, are intended to be illustrative, not limiting. Many suitable materials that will perform the same or similar functions as the materials described herein are intended to be within the scope of the present invention. For example, such other materials not described herein may include, but are not limited to, materials developed after the development time of the present invention. Any dimensions listed in the various figures are for illustration purposes only and are not intended to be limiting. Other dimensions and proportions are contemplated and are intended to be included within the scope of the present invention.

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

As used herein, the term "combination" of a synthetic peptide and at least a second pharmaceutically active ingredient according to the claimed invention means at least two, but any desired combination of compounds may be delivered simultaneously or sequentially (e.g., over a 24 hour period). It is contemplated that the compositions and methods of the present invention, when used to treat various diseases, may be used with other therapeutic methods/agents suitable for the same or similar diseases. Such other therapeutic methods/agents may be co-administered (simultaneously or sequentially) to produce additive or synergistic effects. Due to additive or synergistic effects, the appropriate therapeutically effective dose of each agent can be reduced.

A "disease" is a state of health of a subject, wherein the subject is unable to maintain homeostasis, and wherein the subject's health will continue to deteriorate if the disease is not ameliorated. In contrast, a "disorder" of a subject refers to a state of health in which the subject is able to maintain homeostasis, but the subject's state of health is less good than the state of health in the absence of the disorder. Even if untreated, the condition does not necessarily result in a further decrease in the health of the subject.

The term "treating" a state, disorder or condition includes: (1) Preventing or delaying the occurrence of at least one clinical or subclinical symptom of the state, disorder or condition in a subject who may be suffering from or susceptible to the state, disorder or condition but has not experienced or displayed a clinical or subclinical symptom of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, alleviating or delaying the progression of the disease or its recurrence (in the case of maintenance therapy) or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., ameliorating the state, disorder or condition or at least one clinical or sub-clinical symptom thereof. The benefit to the treated subject is either statistically significant or at least perceptible to the patient or physician.

As used herein, the term "treatment" means treatment and/or prevention. Therapeutic effects are obtained by inhibiting, alleviating or eradicating the disease state.

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

The phrase "pharmaceutically acceptable" when used in connection with the compositions of the present invention means that the molecular entities and other ingredients of such compositions are physiologically tolerable and do not typically produce adverse reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

The term "pharmaceutical carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or carrier with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Preferably, water or aqueous physiological saline solution, aqueous dextrose and aqueous glycerol are employed as carriers, particularly for injectable solutions. Alternatively, the pharmaceutical carrier may be a solid dosage form carrier including, but not limited to, one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavoring agent, and a coloring agent. Suitable pharmaceutical carriers are described in Remington pharmaceutical (Remington's Pharmaceutical Sciences) of e.w. martin.

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 as the unmodified form in vivo and/or in vitro.

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

There are several different computer programs available for determining the degree of identity between two sequences. For example, comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm. In a preferred embodiment, the percentage identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (j. Mol. Biol. (48): 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the Accelrys GCG software package (available at www.accelrys.com/products/GCG), using the Blosum 62 matrix or 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 produce slightly different results, but the overall percentage identity of the two sequences will not change significantly using different algorithms.

Sequence comparison may be performed over the entire length of the two sequences being compared or over a fragment of the two sequences. Typically, the comparison is made over the entire length of the two sequences being compared. However, sequence identity may be over a region of, for example, twenty, fifty, one hundred or more consecutive amino acid residues.

"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 to be compared to the reference sequence. Sequence identity is determined by comparing a given sequence after it has been optimally aligned with a reference sequence to yield the highest degree of sequence similarity, as determined by matching between strings of such sequences. After such alignment, sequence identity is determined from position to position, e.g., if the nucleotide or amino acid residues at a particular position are identical, the sequence is "identical" at that position. The total number of such positional identities is then divided by the total number of nucleotides or residues in the reference sequence to yield% sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to the methods described in the following documents: computing molecular biology (Computational Molecular Biology), lesk, a.n. code Oxford University Press, new York (1988); biological calculation: informatics and genome project (Biocomputing: informatics and Genome Projects), smith, d.w. editions, academic Press, new York (1993); computer analysis of sequence data, first Part (Computer Analysis of Sequence Data, part I), griffin, a.m. and Griffin, h.g. editions, humana Press, new Jersey (1994); sequence analysis in molecular biology (Sequence Analysis in Molecular Biology), von Heinge, g., academic Press (1987); sequence analysis primers (Sequence Analysis Primer), grisskov, M.and Devereux, J.code, M.Stockton Press, new York (1991); and Carilo, H.and Lipman, D., SIAM J.applied Math.,48:1073 (1988), the teachings of which are incorporated herein by reference. The preferred method of determining sequence identity aims at obtaining the maximum match between the sequences being sequenced. Methods of determining sequence identity are compiled in publicly available computer programs that determine sequence identity between given sequences. Examples of such procedures include, but are not limited to, GCG package (Devereux, J. Et al, nucleic Acids Research,12 (1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S.F. et al, J.molecular. Biol.,215:403-410 (1990)). BLASTX programs are publicly available from NCBI and other sources (BLAST Manual, altschul, S.et al, NCVI NLM NIH Bethesda, md.20894; altschul, S.F. et al, J.molecular. Biol.,215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs use default gap weights to optimally align sequences to produce the highest degree of sequence identity between a given sequence and a reference sequence. As one example, a nucleotide sequence of a polynucleotide having at least, for example, 95%, e.g., at least 96%, 97%, 98%, 99%, or 100% "sequence identity" to a reference nucleotide sequence means that the nucleotide sequence of the given polynucleotide is identical to the reference sequence, but the given polynucleotide sequence may include up to 5, 4, 3, 2, 1, or 0 point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having at least 95%, e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity with respect to the reference nucleotide sequence, up to 5%, 4%, 3%, 2%, 1% or 0% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5%, 4%, 3%, 2%, 1% or 0% of the number of nucleotides in the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 'or 3' end positions of the reference nucleotide sequence or at any of those end positions, either interspersed alone between nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Similarly, a given amino acid sequence of a polypeptide having at least, for example, 95%, e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity to a reference amino acid sequence means that the given amino acid sequence of the polypeptide is identical to the reference sequence, but for every 100 amino acids of the reference amino acid sequence, the given polypeptide sequence may include up to 5, 4, 3, 2, 1 or 0 amino acid changes. In other words, to obtain a given polypeptide sequence having at least 95%, e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity to a reference amino acid sequence, up to 5%, 4%, 3%, 2%, 1% or 0% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or up to 5%, 4%, 3%, 2%, 1% or 0% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or at any of those terminal positions, either interspersed alone between residues in the reference sequence or interspersed among one or more consecutive groups within the reference sequence. Preferably, the different residue positions differ by conservative amino acid substitutions. However, conservative substitutions are not included in the match when determining sequence identity.

As used herein, the term "immune response" includes an innate immune response, a T cell-mediated immune response, and/or a 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 affected by T cell activation, such as antibody production (humoral response) and cytokine-reactive cell (e.g., macrophage) activation. Immune cells involved in immune responses 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; bone marrow cells such as macrophages, eosinophils, mast cells, basophils and granulocytes (e.g., neutrophils).

"parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intraperitoneal (i.p.), or intradermal (i.d.) injection, or infusion techniques.

In the context of the medical field, the term "prevention" encompasses any activity that reduces the mortality or morbidity burden of the disease. Prevention can be performed at primary, secondary and tertiary prevention levels. Primary prevention can avoid disease occurrence, while secondary and tertiary prevention levels encompass activities aimed at preventing disease progression and symptoms appearance, as well as reducing the negative impact of established disease by restoring function and reducing disease-related complications.

A "variant" of a polypeptide according to the invention may be (i) a variant in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and the substituted amino acid residue may or may not be an amino acid residue encoded by the genetic code, (ii) a variant in which one or more modified amino acid residues are present, e.g. residues modified by ligation substituents, (iii) a variant in which the polypeptide is an alternative splice variant of a polypeptide of the invention, (iv) a fragment of a polypeptide, and/or (v) a variant in which the polypeptide is fused to another polypeptide such as a leader or secretory sequence or a sequence for purification (e.g. His tag) or detection (e.g. Sv5 epitope tag). Such fragments include polypeptides produced by proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally or chemically modified. Such variations are considered to be within the ability of those skilled in the art in light of the teachings herein.

Within the meaning of the present invention, the term "co-administration" is used to mean that the composition according to the invention and the further therapeutic agent are administered simultaneously in one composition, or simultaneously in different compositions, or sequentially (preferably over a period of 24 hours).

Conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art can be employed in accordance with the present invention. Such techniques are well elucidated in the literature. See, e.g., sambrook, fritsch and manitis, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (1989) Cold Spring Harbor Laboratory Press, cold Spring Harbor, new York (herein "Sambrook et al, 1989"); cloning of DNA: a practical method (DNA Cloning: A Practical Approach), volumes I and II (D.N.Glover, 1985); oligonucleotide synthesis (Oligonucleotide Synthesis) (m.j.gait, 1984); nucleic acid hybridization (Nucleic Acid Hybridization) (code B.D.Hames and S.J.Higgins (1985)), transcription and translation (Transcription and Translation) (code B.D.Hames and S.J.Higgins (1984)), animal cell culture (Animal Cell Culture) (code R.I.Fresnel (1986)), immobilized cells and enzymes (Immobilized Cells and Enzymes) (IRL Press (1986)), B.Perbal, instructions of molecular cloning (A Practical Guide To Molecular Cloning) (1984), F.M.Ausubel et al (code), latest protocols for molecular biology (Current Protocols in Molecular Biology), john Wiley & Sons, inc. (1994), and the like.

Peptide compositions of the invention

Modification of the amino acid structure of CP1 led to the discovery of other peptides capable of modulating complement activation such as C1q activity. It has been previously demonstrated that modifications such as PEGylation enhance the solubility of peptides and effective inhibition of biological activity compared to the parent molecule (IALILEPICCQERAA; SEQ ID NO: 1) in vitro assays of classical complement pathway activation/inhibition, myeloperoxidase (MPO) inhibition, antioxidant activity and NET activity inhibition. Peptides with C-terminal monodisperse 24-mer PEGylated moieties were found to be highly soluble and have a strong inhibitory effect on the complement system (IALILEPICCQERAA-dPEG 24; SEQ ID NO:2; PA-DPEG24; PA-dPEG24; RLS-0071). Sarcosine substitution scan of SEQ ID NO. 2 shows that substitution of isoleucine at position 8 with sarcosine produces the water-soluble non-PEGylated peptide IALILEP (Sar) CCQERAA (PA-I8 Sar; RLS-0088; SEQ ID NO. 3) (as described in U.S. Pat. No. 10,005,818).

As used herein, the term "peptide" refers to a potentially naturally occurring amino acid sequence of about 15 amino acids based on SEQ ID nos. 2 and/or 3, or a peptidomimetic, peptide analogue and/or synthetic derivative (including, for example, but not limited to, pegylated peptides). In addition, the 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 are equally possible peptides within the scope of the invention. Peptides may also be more than 15 amino acids, for example 16, 17, 18, 19 and 20 or more amino acids.

The disclosed peptides are typically constrained (i.e., having a structural element such as an amino acid present that initiates a beta turn or beta sheet, or cyclized such as by the presence of a disulfide-bonded Cys residue) or unconstrained (i.e., linear) amino acid sequences of greater than about 15 amino acid residues, or less than about 15 amino acid residues.

Substitutions of amino acids within the peptide sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Amino acids containing an aromatic ring structure 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 within the sequence may be substituted with another amino acid of similar polarity as a functional equivalent, resulting in a silent change.

Conservative changes typically result in less structural and functional changes in the resulting protein. Non-conservative changes are more likely to alter the structure, activity, or function of the resulting protein. For example, the peptides of the present disclosure comprise one or more of the following conservative amino acid substitutions: an aliphatic amino acid such as alanine, valine, leucine and isoleucine is replaced with another aliphatic amino acid; serine to threonine; threonine to serine; replacement of an acidic residue such as aspartic acid and glutamic acid with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; basic residues such as lysine and arginine are exchanged for another basic residue; and the replacement of an aromatic residue such as phenylalanine and tyrosine with another aromatic residue.

Particularly preferred amino acid substitutions include:

a) Ala replaces Glu or vice versa, which reduces negative charge;

b) Lys replaces Arg or vice versa, which can maintain a positive charge;

c) Ala replaces Arg or vice versa, which reduces the positive charge;

d) Glu replaces Asp or vice versa, which can maintain a negative charge;

e) Ser replaces Thr or vice versa, so that free-OH can be maintained;

f) Gln replaces Asn or vice versa, so that free NH2 can be maintained;

g) Ile replaces Leu or Val or vice versa as a substantially equivalent hydrophobic amino acid;

h) Phe replaces Tyr or vice versa as a substantially equivalent aromatic amino acid; and

i) Ala replaces Cys or vice versa, thus affecting disulfide bonds.

Substitutions of amino acids within the peptide sequence may 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, the sarcosine replaces an amino acid within a peptide sequence.

In one embodiment, the invention discloses synthetic peptides derived from human astrovirus coat proteins, said peptides comprising the amino acid sequence and modifications of SEQ ID NO. 2 and/or 3.

Table 1. List of peptides of the invention.

SEQ ID NO.	Sequence(s)	Description of the invention
			1	IALILEPICCQERAA	PA(PIC1)
2	IALILEPICCQERAA-PEG24	PA-dPEG24；RLS-0071
			3	IALILEP(Sar)CCQERAA	PA-I8Sar；RLS-0088

In other embodiments, the synthetic peptide is capable of altering cytokine expression, including but not limited to Acute Lung Injury (ALI) models. In some embodiments, the invention provides a method of altering cytokine expression, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NOs 2 and/or 3.

In other embodiments, the synthetic peptide is capable of inhibiting or altering neutrophil binding and/or adhesion. In some embodiments, the invention provides a method of inhibiting or altering neutrophil binding and/or adhesion, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NOs 2 and/or 3.

In other embodiments, the synthetic peptide is capable of increasing neutrophil survival. In some embodiments, the invention provides a method of increasing neutrophil survival, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NOs 2 and/or 3.

In other embodiments, the synthetic peptide may bind to a cell surface receptor in vivo, such as, but not limited to, integrin and/or ICAM. In some embodiments, the methods provide a method of inhibiting or altering neutrophil binding to a cell surface receptor, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NOs 2 and/or 3.

The disclosed peptides can selectively modulate C1q and MBL activation without affecting alternative pathway activity and are therefore ideal choices for the prevention and treatment of diseases mediated by deregulated activation of the classical pathway and the lectin pathway. Specific blocking of the classical and lectin pathways is particularly desirable, as both pathways are involved in ischemia reperfusion-induced injury in many animal models. [ Castillano et al, "treatment targeting complement classical and lectin pathways can prevent ischemia reperfusion-induced kidney injury (Therapeutic targeting of classical and lectin pathways of complement protects from ischemia-reperfusion-induced renal damage)," Am J pathl.2010; 176 (4) 1648-59; lee et al, "early complement factors in the local tissue immune complex generated during intestinal ischemia/reperfusion injury (Early complement factors in the local tissue immunocomplex generated during intestinal ischemia/reperfusion injury)," mol.immunol.2010 month 2; 47 (5) 972-81; tjernberg et al, "Acute antibody mediated complement activation mediated islet cell lysis and may lead to tissue loss of clinical islet transplantation (act anti-body-mediated complement activation mediates lysis of pancreatic islets cells and may cause tissue loss in clinical islet transplantation)," transformation.2008, 4 months, 27 days; 85 1193-9; zhang et al, "role of natural IgM in myocardial ischemia reperfusion injury (The role of natural IgM in myocardial ischemia-reperfusion injury)," J Mol Cell cardiol.2006, 7; 41 (1):62-7). Alternative pathways are essential for immune surveillance of invading pathogens, whereas persons with defects in alternative pathways may suffer from serious bacterial infections. By binding and inactivating C1q and MBL, the peptide can effectively modulate classical and lectin pathway activation while leaving the alternative pathway intact.

As used herein, the term "modulate" refers to i) controlling, reducing, inhibiting or modulating a biological function of an enzyme, protein, peptide, factor, by-product or derivative thereof, alone or in complex form; ii) reducing the amount of a biological protein, peptide or derivative thereof in vivo or in vitro; or iii) interrupt an event biological chain, cascade or pathway known to comprise a series of related biological or chemical reactions. Thus, the term "modulate" may be used, for example, to describe reducing the number of individual components of the complement cascade, reducing the rate or total amount of component or component complex formation, or reducing the overall activity of a complex process or series of biological reactions, which results in such consequences as cell lysis, invertase formation, complement-derived membrane attack complex formation, inflammation or inflammatory disease, as compared to a control sample. In an in vitro assay, the term "modulation" may refer to a measurable change or decrease in some biological or chemical event, but it will be understood by those of ordinary skill in the art that the measurable change or decrease need not all be "modulated".

In any embodiment, the invention relates to therapeutically active peptides having the effect of modulating the complement system and preventing, treating and/or alleviating the toxic side effects of checkpoint inhibitors such as intestinal necrosis or injury.

In any embodiment, the present invention relates to therapeutically active peptides having the effect of preventing, treating and/or reducing intestinal necrosis or injury, such as necrosis or injury caused by severe inflammatory reactions, 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.

Pharmaceutical compositions of the invention

The present disclosure provides pharmaceutical compositions capable of modulating the complement system comprising at least one peptide as described above and at least one pharmaceutically acceptable carrier, diluent, stabilizer or excipient. The pharmaceutically acceptable carrier, excipient or stabilizer is non-toxic to the recipient at the dosage and concentration employed. They may be solid, semi-solid or liquid. The pharmaceutical compositions of the present invention may be in the form of tablets, pills, powders, troches, sachets, cachets, elixirs, suspensions, emulsions, solutions or syrups.

The pharmaceutical compositions of the invention are prepared by mixing a peptide of suitable purity with a pharmaceutically acceptable carrier, diluent or excipient. Examples of formulations and methods of making such formulations are well known in the art. As described above, the pharmaceutical composition of the present invention is useful as a prophylactic and therapeutic agent for various conditions and diseases. In one embodiment, the composition comprises a therapeutically effective amount of a peptide. In another embodiment, the composition comprises at least one additional active ingredient that is effective in modulating the complement system. In another embodiment, the composition comprises at least one additional active ingredient effective in treating at least one disease associated with the complement system. In another embodiment, the composition comprises at least one additional active ingredient effective in 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 the various active ingredients sufficient to exhibit a benefit to the subject.

The therapeutically effective amount of the peptide will vary depending on several factors, such as the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the peptide employed, the duration of the treatment, the co-therapy involved, and the age, sex, weight and condition of the subject, etc. One of ordinary skill in the art can determine a therapeutically effective amount. Thus, one of ordinary skill in the art may need to titrate the dose and modify the route of administration to obtain maximum therapeutic effect.

Effective daily dosages typically range from about 0.001 to about 200 milligrams per kilogram (mg/kg) of body weight, including from about 5 to about 160mg/kg, from about 10 to about 160mg/kg, from about 40mg/kg to about 160mg/kg, and from about 40mg/kg to about 100mg/kg. The dose may be achieved by a 1-6 daily dosing regimen. Alternatively, optimal treatment may be achieved by a slow release formulation at a less frequent dosing regimen. In some embodiments, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is from about 10mg/kg to about 160mg/kg. In some embodiments, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is from about 20mg/kg to about 160mg/kg. In some embodiments, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is about 40mg/kg to about 160mg/kg. In some embodiments, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is administered in at least one dose, the first dose comprising about 1mg/kg to about 160mg/kg 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 about 1mg/kg to about 120mg/kg SEQ ID NO:2 and/or 3, including about 10mg/kg to about 120mg/kg 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, the first dose comprising about 1mg/kg to about 160mg/kg SEQ ID NO 2 and/or 3, including about 10mg/kg to about 120mg/kg SEQ ID NO 2 and/or 3, and the second dose comprising about 1mg/kg to about 120mg/kg SEQ ID NO 2 and/or 3, including about 1mg/kg to about 40mg/kg SEQ ID NO 2 and/or 3. In some embodiments, the second dose is administered 30 seconds to 10 hours after administration of the first dose, including about eight hours after administration of the first dose. In some embodiments, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is administered in a plurality of doses over a period of about one week to about two weeks, each dose comprising about 1mg/kg to about 160mg/kg SEQ ID NO 2 and/or 3, including about 1mg/kg to about 120mg/kg SEQ ID NO 2 and/or 3, administered every 4 to 10 hours, including about every eight hours.

In some embodiments, at least one loading dose of about 10mg/kg to about 160mg/kg SEQ ID NO:2 and/or 3, including about 10mg/kg to about 120mg/kg SEQ ID NO:2 and/or 3, is administered followed by at least one maintenance dose of about 1mg/kg to about 120mg/kg SEQ ID NO:2 and/or 3, including about 1mg/kg to about 40mg/kg SEQ ID NO:2 and/or 3. In some embodiments, the first maintenance dose is administered 4 to 10 hours after the last loading dose. In some embodiments, the maintenance dose is administered every 4 to 10 hours over a period of about one week to about two weeks. In some embodiments, 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 one week to about two weeks.

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 pharmaceutically acceptable carrier, diluent or excipient.

The compositions of the present invention may comprise a carrier and/or excipient. Although the peptide of the invention may be used as such for treatment, it may be preferred that it is administered in the form of a pharmaceutical formulation, for example, in the form of a mixture with suitable pharmaceutical excipients and/or carriers selected according to the intended route of administration and standard pharmaceutical practice. The excipient and/or carrier 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 and carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in the following documents: remington pharmaceutical science and practice (Remington: the Science and Practice of Pharmacy). Lippincott Williams & Wilkins (A.R. Gennaro, 2005). The choice of pharmaceutical excipients and carriers can be selected according to the intended route of administration and standard pharmaceutical practice. Oral formulations are readily adaptable to other mixtures, such as milk, yogurt and infant formulas. Solid dosage forms for oral administration may also be used and may 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, compactors, lubricants, dispersion enhancing agents, disintegrants, antioxidants, flavoring agents, sweeteners, and colorants. Those skilled in the art will be well able to prepare suitable solutions.

In one embodiment of any of the compositions of the present invention, the composition is formulated for delivery by such routes as oral, topical, rectal, mucosal, sublingual, nasal, nasogastric/orogastric lavage, 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 comprises a buffer (e.g., sodium bicarbonate).

In the methods of the invention, administration of the peptides and compositions can be accomplished by any method known in the art. Non-limiting examples of useful delivery routes include oral, rectal, fecal (by enema) and gastric lavage by nasogastric/orogastric, as well as parenteral, intraperitoneal, intradermal, transdermal, intrathecal, nasal and intratracheal administration. The active agent may be systemic after administration or may be localized by use of regional administration, intrawall administration, or use of an implant that retains the active agent at the implantation site.

The useful dosage of the compounds and formulations of the present invention can vary widely depending on the nature of the disease, the patient's medical history, the frequency of administration, the mode of administration, the rate of clearance of the agent by the host, and the like. The initial dose may be larger, followed by a smaller maintenance dose. The frequency of administration of the dose may be as low as once per week or every two weeks, or divided into smaller doses and administered daily, every half week, etc., to maintain an effective dose level. It is contemplated that a variety of doses may be effective to achieve a therapeutic effect. Although the compound of the present invention may be used as such for treatment, it may be preferred that it is administered in the form of a pharmaceutical formulation, for example, in the form of a mixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient, diluent and/or carrier 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 the following documents: remington pharmaceutical science and practice (Remington: the Science and Practice of Pharmacy). Lippincott Williams & Wilkins (A.R. Gennaro, 2005). The choice of pharmaceutical excipients, diluents and carriers can be selected according to the intended route of administration and standard pharmaceutical practice.

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 sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

The solution or suspension may comprise any combination of any of the following components: sterile diluents, including for example, but not limited to, water for injection, physiological saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid (EDTA); buffers such as acetate, citrate and phosphate; and tonicity adjusting agents such as sodium chloride or dextrose.

In the case where the agent exhibits insufficient solubility, a method of solubilizing the agent may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents such as Dimethylsulfoxide (DMSO), the use of surfactants such as 80, or dissolved in aqueous sodium bicarbonate. Pharmaceutically acceptable derivatives of the agents may also be used to formulate effective pharmaceutical compositions.

The composition may contain an active agent and an agent such as, but not limited to: diluents such as lactose, sucrose, dicalcium phosphate or carboxymethyl cellulose; lubricants, such as magnesium stearate, calcium stearate, and talc; and binders such as starch, natural gums such as gum arabic gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose and its derivatives, povidone, crospovidone and other such binders known to those skilled in the art. For example, liquid pharmaceutically administrable compositions may be prepared by dissolving, dispersing or otherwise mixing the active agent as defined above and optionally a pharmaceutical adjuvant in a carrier such as, but not limited to, water, physiological saline, aqueous dextrose, glycerol, ethylene glycol, ethanol, or the like, to form a solution or suspension. If desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying or solubilizing agents, pH buffering agents and the like, for example, but not limited to, acetate salts, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate and other such agents. The actual methods of making such dosage forms are known or will be apparent to those skilled in the art (e.g., remington's Pharmaceutical Sciences, mack Publishing Company, easton, pa., 15 th edition, 1975). In any event, the composition or formulation to be administered will contain an amount of active agent sufficient to alleviate the symptoms of the subject being treated.

The active agent or pharmaceutically acceptable derivative may be formulated with a carrier, such as a time release formulation or coating, that prevents rapid elimination of the agent from the body. The composition may include other active agents to achieve the desired combination of properties.

Parenteral administration, typically characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, for example, but are not limited to, water, physiological saline, dextrose, glycerol, or ethanol. In addition, the pharmaceutical compositions to be administered may also contain minor 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, as desired.

The lyophilized powder may be reconstituted into solutions, emulsions, and other mixtures or formulated as a solid or gel for administration. Sterile lyophilized powders are prepared by dissolving the agents provided herein, or pharmaceutically acceptable derivatives thereof, in a suitable solvent. The solvent may contain excipients that improve stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffers known to those skilled in the art, typically at a pH of about neutral. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to yield the desired formulation. Typically, the resulting solution may be dispensed into vials for lyophilization. Each vial may contain, for example, but not limited to, a single dose (10-1000 mg, such as 100-500 mg) or multiple doses of the agent. The lyophilized powder may be stored under suitable conditions, such as about 4 ℃ to room temperature. Reconstitution of the lyophilized powder with water for injection results in a formulation for parenteral administration.

Combination therapy

Another embodiment of the invention provides a method of modulating the complement system comprising administering to a subject a pharmaceutical composition of the invention. Although the pharmaceutical compositions of the present invention may be administered as the sole active agent, they may also be used in combination with one or more therapeutic or prophylactic agents that are effective in modulating the complement system. In this regard, the methods of the invention comprise administering the pharmaceutical compositions of the invention prior to, concurrently with, and/or after one or more other therapeutic or prophylactic agents that are effective in modulating the complement system.

The pharmaceutical composition of the present invention may be administered in combination therapy with other agents, either co-administered or separately administered, or by combining the pharmaceutical composition and the other agents into one composition. The dosage is administered and adjusted to achieve maximum modulation of the complement system. For example, the pharmaceutical composition and the other agent are typically present at a dosage level of about 10% to about 150%, more preferably about 10% to about 80%, of the dosage typically administered in a single drug treatment regimen.

In any embodiment, the pharmaceutical composition of the 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 may be administered after the checkpoint inhibitor, and/or may be administered as a prophylactic agent to a subject who has been treated with the checkpoint inhibitor and has undergone inflammation, injury, or necrosis of the intestine due to the checkpoint inhibitor. In any embodiment, the pharmaceutical composition of the invention may be administered to a subject currently being treated with, having been treated with, and/or to 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), ipilimab (Yervoy), nivolumab (Opdivo), and atrazomib (tecontriq).

In any embodiment, the pharmaceutical composition of the invention is capable of preventing, treating and/or alleviating the toxic side effects of checkpoint inhibitors, such as intestinal necrosis and/or injury.

In any embodiment, the pharmaceutical compositions of the invention are capable of preventing, treating and/or reducing intestinal necrosis and/or injury, such as necrosis or injury caused by severe inflammatory reactions, 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.

Application method

In one aspect, the invention provides a method of preventing, treating and/or alleviating intestinal necrosis and/or injury in a subject being treated with, having been treated with or to be treated with at least one checkpoint inhibitor, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NOs 2 and/or 3.

In any of the above methods, the composition further comprises at least one pharmaceutically acceptable carrier, diluent, stabilizer, or excipient. In one embodiment of any of the above methods, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is from about 10mg/kg to about 160mg/kg. In one embodiment of any of the above methods, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is from about 20mg/kg to about 160mg/kg. In one embodiment of any of the above methods, the therapeutically effective amount of SEQ ID NO 2 and/or 3 is from about 40mg/kg to about 160mg/kg. In one embodiment of any of the above methods, the composition is formulated for subcutaneous, intravenous, intraperitoneal, or intramuscular administration. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier and/or excipient.

Examples

The invention is also described and illustrated by the following examples. However, these and other embodiments used in any of the present specification are merely illustrative and in no way limit the scope and meaning of the invention or any exemplary terms. Also, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading the present specification, and such variations may be made without departing from the spirit or scope of the invention. The invention is therefore to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Example 1: prevention, treatment and/or alleviation of intestinal necrosis using PA-dPEG24

Intestinal necrosis is a potentially life threatening medical condition that can be caused by a variety of clinical causes. Intestinal tissue necrosis causes damage to the lumen and leakage of intestinal bacteria, and contributes to invasive immune responses, including complement system and neutrophils. PA-dPEG24 (RLS-0071) is a peptide inhibitor of the classical and lectin pathways, the major effectors of neutrophil myeloperoxidase activity and NETosis. Thus, the inventors decided to evaluate the extent of the effect of immunomodulation on survival by inhibition of complement system and neutrophil effectors in the case of intestinal necrosis and leakage of intestinal contents. Cecal Ligation Punctures (CLP) were performed on pubertal male Long-Evans rats, a established model of enteric necrosis rats, one group received a single dose of 40mg/kg RLS-0071 thirty minutes after surgery, while the control group received no treatment. Rats were then assessed for survival until 5 days post-surgery. Surprisingly, animals treated with RLS-0071 showed 80% survival compared to 50% in untreated groups. To assess whether the unexpected increase in survival was likely due to a moderation of the inflammatory response, the inventors evaluated NETosis markers, free DNA in plasma, and pro-inflammatory cytokine IL-6. For animals treated with RLS-0071, a decrease in blood levels of free DNA and inflammatory cytokine IL-6 was observed. These results indicate that a single dose of RLS-0071 may increase the survival rate of intestinal necrosis by reducing certain inflammatory responses.

Materials and methods

Animal experiment

The CLP model [10] established previously is utilized and modified. Male Long-Evans rats (250-300 g) were induced with 5% isoflurane and oxygen and anesthesia was maintained by nasal cone delivery of 1.5-2% isoflurane and oxygen. Adequate anesthesia was confirmed by pinching the toe. The surgical area (lower abdominal quadrant) was shaved, washed with 10% iodine, followed by 70% ethanol for a total of three times. buprenorphine-SR (1 mg/kg) was administered subcutaneously preoperatively for pain management. For surgical procedures, the animal is in a supine position with the head away from the operator. After placement of sterile gauze, a longitudinal midline incision (about 3-4 cm) was made with a sterile scalpel. After initial incision, small scissors are used to access the peritoneal cavity. The white line of abdominal musculature was dissected to dissect the fascia and peritoneal layers intermuscularly. The cecum is positioned and removed, leaving the remainder of the intestine in the peritoneal cavity. The mesentery of the cecum was carefully dissected to avoid damaging the cecal branches of the ileocecum and to avoid bleeding complications. The cecum was ligated 75% using sterile 3-0 non-absorbable suture. The cecum was perforated twice in the mesenteric-trans-mesenteric direction with a sterile 18 gauge needle intermediate the ligation and cecal tip. The cecum is repositioned into the abdominal cavity. The peritoneum was closed with a 4-0 non-absorbable suture and the skin was closed with a sterile metal wound clip. Animals received 5mL of pre-warmed (37 ℃) standard saline subcutaneously for recovery. For RLS-0071 treated animals (n=9), rats received a single dose of 40mg/kg RLS-0071 intravenously via jugular indwelling catheter 30 minutes post-surgery. A peptide consisting of the amino acid sequence IALILEPICCQERAA (SEQ ID NO: 2) and the tail of a monodisperse 24-mer polyethylene glycol (PEG) was manufactured from PolyPeptide Group (San Diego, calif.), and was verified to be 95% pure by HPLC and mass spectrometry analysis. Lyophilized RLS-0071 was dissolved in 0.05M histidine buffer and pH was adjusted to 6.5. The control group (n=6) received no compound. Animals were monitored every 30 minutes for at least two hours post-surgery. Once smart, alert, responsive, the animal is returned to its home cage. Animals were quantitatively assessed for morbidity twice daily for the duration of the study. (Shrum, b., anantha, r.v., xu, s.x., etc.), a powerful scoring system (A robust scoring system to evaluate sepsis severity in an animal model) for assessing sepsis severity in animal models, BMC Res Notes 7,233 (2014), https:// doi.org/10.1186/1756-0500-7-233). Scoring criteria include appearance, level of consciousness, activity, response to stimulus, open and closed eyes, respiration rate, and respiration quality. The respective categories are ranked from 0 (best) to 4 (worst). Animals were humanly euthanized if their cumulative score was greater than 21 or if their respiratory quality was greater than 3. At the end of 5 days, surviving animals were euthanized by carbon dioxide asphyxiation and cervical dislocation. The animals were then monitored at least twice daily.

Blood samples were collected pre-operatively (pre-harvest) and 24 hours post-operatively. Plasma was isolated from blood and analyzed for free DNA concentration and cytokine levels as described below.

Plasma DNA measurement

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

Cytokine analysis

Rat IL-6ELISA was purchased from R & D Systems. Plasma samples of experimental animals were processed according to the manufacturer's instructions. Briefly, diluted rat plasma samples were added to wells pre-coated with the captured IL-6 antibodies overnight. Plates were blocked with 1% BSA for 60 min at room temperature. 100uL of plasma sample (1:4 dilution) or standard was added to the plate and incubated for 2 hours at room temperature, followed by washing to remove any unbound components. Next, 100. Mu.L of detection IL-6 antibody (1:60 dilution) was added, incubated for 2 hours at room temperature and washed. Next, 100. Mu.L of streptavidin-horseradish peroxidase (HRP) conjugate (1:40 dilution) was added and incubated in the dark at room temperature for 20 minutes. After washing, 100. Mu.L of 3,3', 5' -Tetramethylbenzidine (TMB) substrate was added at room temperature for about 5 minutes. The reaction was terminated by adding 100. Mu.L of sulfuric acid, and absorbance was measured at 450nm using a BioTek microplate reader. Sample concentrations were calculated using IL-6 standard curves made with 1:2 serial dilutions.

Statistical analysis

Data are expressed as mean and mean standard error.

Results

RLS-0071 reduces mortality in CLP model

To test whether modulation of the complement system and neutrophil immune mechanisms would increase death due to bacterial sepsis in the case of intestinal necrosis, the inventors tested RLS-0071 in CLP model. In CLP model, cecal ligation resulted in cecal puncture infarct, followed by necrosis. CLP model in Long-Evans rats previously developed reported that induction of moderate necrosis resulted in about 40% survival, requiring ligation of the cecum at half the distance between the distal cecum pole and the base of the cecum, followed by puncture of the ligated cecum with an 18-gauge needle [11]. The inventors' experimental protocol utilized 75% cecal ligation, followed by puncture of the cecum with two 18 gauge needles, with a survival rate of 20%. Surprisingly, the survival rate of rats receiving CLP and delivered intravenously at 30 minutes post-surgery a single dose of 40mg/kg RLS-0071 was 50% increased by a factor of 2.5 compared to the non-rescue treatment group, as shown in figure 1. Thus, rather than saying that modulation of the complement system and neutrophil effectors leads to increased death due to overwhelming infections, administration of RLS-0071 appears to increase survival in the case of intestinal necrosis.

RLS-0071 inhibited free DNA accumulation in blood

RLS-0071 was found to increase survival in the case of intestinal necrosis, suggesting the possibility of inhibiting inflammation in rats if it affects its survival. The inventors then determined two key aspects of inflammation in CLP animal blood. The release of Neutrophil Extracellular Traps (NET) from activated neutrophils has previously been shown to play pathogenic roles in a variety of autoimmune, metabolic and inflammatory diseases [12] and is postulated to contribute to immune thrombosis and Disseminated Intravascular Coagulation (DIC) in sepsis [4]. NET was observed in the virus-induced mouse acute lung injury model. In a second aspect, the presence of free DNA in the blood stream is a NET biomarker in the blood of human acute lung injury patients [13,14] and of COVID-19 patients [15 ]. To determine whether free DNA was present in the blood of sepsis rats, the inventors measured the level of free DNA by PicoGreen fluorescence 24 hours after surgery. The free DNA levels of CLP-operated animals were increased compared to blood taken from animals prior to surgery (prior to blood collection). The free DNA level was reduced in animals receiving RLS-0071 compared to untreated animals. In addition, animals were tested for IL-1B and found to be negative. This free DNA reduction in animals treated with RLS-0071 indicated that RLS-0071 reduced NET formation in this model, as shown in figure 2.

RLS-0071 reduces inflammatory cytokine IL-6 levels in blood

In the case of intestinal necrosis, a large amount of pro-inflammatory cytokines are produced in response to infection. This so-called "cytokine storm" has been well proven for sepsis and this invasive inflammatory response is associated with serious consequences including damage to the final organ, sometimes leading to death [4]. To determine the effect of RLS-0071 on inflammatory cytokine levels in this model, blood IL-6 levels were measured. IL-6 is a powerful pro-inflammatory cytokine, released mainly by macrophages, and plays a major role in many inflammatory diseases, including inflammatory bowel disease [16]. Preoperatively (pre-harvest) blood from animals is free of detectable levels of IL-6. Animals subjected to CLP surgery showed elevated blood cytokine levels 24 hours post-surgery. In contrast, animals receiving RLS-0071 showed a decrease in IL-6 compared to animals not receiving rescue intervention, indicating that RLS-0071 may reduce inflammatory cytokine production in this model, as shown in figure 3. In addition, animals were tested for IL-1B and found to be negative.

Discussion of the invention

The inventors have conducted experiments to determine whether the immunomodulatory molecule RLS-0071 can affect the survival rate of bacterial sepsis in the case of intestinal necrosis. Surprisingly, RLS-0071 was able to increase the survival of the model of intestinal necrosis established by cecal ligation. As reported previously, the Cecal Ligation Puncture (CLP) rat model has been used for over 40 years, and is considered the gold standard for the intestinal necrosis and sepsis model [11]. In this model, the cecum was ligated under the ileocecal valve and then the cecum was punctured with a needle. After cecal necrosis and perforation, bacteria, toxins and other microbial contaminants are released into the peritoneal cavity, resulting in bacterial peritonitis. These mixed intestinal bacteria are then transported to the blood chamber, resulting in bacterial sepsis. Enteropathogenic bacterial sepsis often contributes to hypotension, disseminated Intravascular Coagulation (DIC), multiple system organ failure, and possible death.

RLS-0071 has been shown to inhibit classical complement activation in vitro, in vivo and ex vivo studies, and to inhibit NET formation by inhibiting myeloperoxidase in vitro and ex vivo studies [6-8]. Considering the immunomodulatory activity on the complement system and on neutrophil effectors, the inventors hypothesize that RLS-0071 aggravates bacterial sepsis in CLP animal models and increases the mortality. Our results demonstrate a surprising finding that RLS-0071 delivered as a single dose at 30 minutes post-surgery improved survival by a factor of 2.5. This result suggests that RLS-0071 may improve survival by reducing an important aspect of the inflammatory response of intestinal necrosis. CLP animals treated with RLS-0071 showed reduced levels of free DNA as NETosis biomarker and reduced inflammatory cytokine IL-6. The ability of RLS-0071 to increase survival and reduce inflammation in CLP models has potential for use as a clinical treatment for various disease processes associated with intestinal necrosis such as intestinal infarction (i.e., intestinal tissue ischemia reperfusion injury), autoimmune Inflammatory Bowel Disease (IBD) and related drugs, chemotherapy-induced or toxin-induced intestinal necrosis, and intestinal necrosis or injury caused by severe inflammatory reactions.

Example 2: multiple doses of RLS-0071 and RLS-0088 reduced mortality in the CLP model

The inventors next evaluated whether the multi-dose regimen of RLS-0071 would have an impact on survival. RLS-0071 was administered to animals at 0.5 hours, 24 hours, 48 hours and 72 hours post-surgery as in example 1. Rats receiving RLS-0071 showed a significant increase in survival (p=0.032) (fig. 4). The second generation EPICC peptide, RLS-0088, was then tested in the CLP model to determine if it had a similar effect of improving survival. Using the same multi-dose experimental procedure as described in example 1, rats received CLP and were administered 40mg/kg RLS-0088 at 0.5, 24, 48 and 72 hours post-operatively. Rats receiving RLS-0088 showed increased survival (figure 5).

Example 3: administration of pharmaceutical formulations

Administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NOs 2 and/or 3 to treat a disease or disorder. The administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

Administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NOs 2 and/or 3 to modulate the complement system of said subject. The administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

Administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NOs 2 and/or 3 to alter cytokine expression in said subject. The administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

Administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NOs 2 and/or 3 to prevent, treat and/or mitigate toxic side effects of checkpoint inhibitors, such as intestinal necrosis or injury. The administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

A pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NOs 2 and/or 3 is administered to a subject in need thereof to prevent, treat and/or reduce intestinal necrosis and/or injury, such as necrosis or injury caused by severe inflammatory reactions, intestinal infarction (i.e., ischemia reperfusion injury of intestinal tissue), autoimmune Inflammatory Bowel Disease (IBD) and related drugs, and chemotherapy-induced or toxin-induced intestinal necrosis. The administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

Administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of SEQ ID NOs 2 and/or 3 to prevent, treat and/or reduce intestinal necrosis and/or injury in a subject being treated with, having been treated with and/or to be treated with at least one checkpoint inhibitor. The administration may be by any suitable route (e.g., injection, infusion, implantation, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration).

************

Although several possible embodiments are disclosed above, embodiments of the invention are not so limited. These exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the present invention, but are selected and described in order to explain the principles of the present invention so that those skilled in the art may practice the present 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 claims.

All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if actually present in the present specification.

Reference to the literature

1.Erik A.Campbell,Michael Silberman intestinal necrosis (Bowell necrosis). Treasure Island (FL): statPearls Publishing;2021 month 1.

2.Dahiya DS,Wani F,Guidi JC,Kichloo A gastrointestinal adverse effects of immunotherapeutic agent: systematic overview (Gastrointestinal Adverse Effects of Immunotherapeutic Agents: A Systematic Review.) the journal of Gastroenterology Res.2020, month 12; 13 (6) 227-232.Doi:10.14740/gr1340.2020, 12-month 23-day electronic publication. PMID:33447301.

3.Karasu E,Nilsson B,J, lambris JD, huber-Lang M. Multiple injury and Sepsis Induced Multiple-organ dysfunction (Targeting Complement Pathways in Polytrauma-and Sepsis-Induced Multiple-Organ Dysfunction) Front immunol.2019, 3, 21; 10:543.doi:10.3389/fimmu.2019.00543. ECallIng 2019.PMID:30949180

4.Chen Z,Zhang H,Qu M,Nan K,Cao H,Cata JP,Chen W,Miao C New role of neutrophil extracellular traps in sepsis and sepsis-related thrombosis (The Emerging Role of Neutrophil Extracellular Traps in Sepsis and Sepsis-Associated Thrombosis). Front Cell Infect Microbiol.2021, 3, 17; 653228.Doi:10.3389/fcimb.2021.653228.

5.Chakraborty RK,Burns B systemic inflammatory response syndrome (Systemic Inflammatory Response Syndrome) 2021, 3/1. StatPearls [ Internet ]. Treasure Island (FL): statPearls Publishing;2021 month 1.PMID 31613449.

6.Sharp JA,Hair PS,Pallera HK,Kumar PS,Mauriello CT,Nyalwidhe JO and the like, peptide inhibitors of complement C1 (RLS-0071) rapidly inhibit complement activation (Peptide Inhibitor of Complement C (RLS-0071) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats) after intravascular injection in rats PLoS One 2015;10 (7) e0132446.

7.Hair PS,Sass LA,Krishna NK,Cunnion KM peptide inhibitors of complement C1 (RLS-0071) inhibit myeloperoxidase activity in cystic fibrosis sputum (Inhibition of Myeloperoxidase Activity in Cystic Fibrosis Sputum by Peptide Inhibitor of Complement C (RLS-0071)). PLoS One2017;12 (1) e0170203.

8.Hair PS,Enos AI,Krishna NK,Cunnion KM peptide inhibitors of complement C1 inhibit the immune complex complement activation and neutrophil extracellular trap formation (Inhibition of Immune Complex Complement Activation and Neutrophil Extracellular Trap Formation by Peptide Inhibitor of Complement C1.) Front Immunol 2018, month 3, 26; 9:558.

9.Gregory Rivera M,Hair PS,Cunnion KM,Krishna NK peptide inhibitors of complement C1 (RLS-0071) exhibit antioxidant activity by Single Electron Transfer (SET) and Hydrogen Atom Transfer (HAT) (Peptide Inhibitor of Complement C (RLS-0071) demonstrates antioxidant activity via Single Electron Transport (SET) and Hydrogen Atom Transfer (HAT)). PLoS One 2018, 3 months 2; 13 (3) e0193931.

10.Hair PS,Gregory Rivera M,Enos AI,Pearsall SE,Sharp JA,Yousefieh N et al, peptide inhibitors of complement C1 (RLS-0071) inhibit pathogenic bacterial growth (Peptide Inhibitor of Complement C (RLS-0071) Inhibits Growth of Pathogenic Bacteria). Int J Pept Res Therap 2017. Ori/10.1007/s 10989-017-9651-z

11.Rittirsch D,Huber-Lang MS, flierl MA, ward PA. immunization against experimental sepsis by cecal ligation and puncture (Immunodesign of experimental sepsis by cecal ligation and puncture). Nat protoc.2009;4 (1) 31-6.Doi:10.1038/nprot.2008.214.PMID 19131954.

Review of Neutrophil Extracellular Traps (NET) in mutua V and Gershwin LJ. disease: potential anti-NET therapies (A review of Neutrophil Extracellular Traps (NETs) in treatment: potential anti-NET therapeutics). Clin Rev Allergy Immunol 2020;1:1-18.

13.Caudrillier A,Kessenbrock K,Gilliss BM,Nguyen JX,Marques MB,Monestier M, et al, platelets induce neutrophil extracellular traps in transfusion-associated acute lung injury (Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury). J Clin Invest 2012;122:2661-2671.

14.Thomas GM,Carbo C,Curtis BR,Martinod K,Mazo IB,Schatzberg D, et al, extracellular DNA traps are associated with pathogenesis of human and mouse trani (Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice). Blood 2012;119:6335-6343.

15.Skendros P,Mitsios A,Chrysanthopoulou A,Mastellos DC,Metallidis S,Rafailidis P, et al, complement and tissue factor enriched neutrophil extracellular traps are key drivers of the formation of the COVID-19 immune thrombus (Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombis). J Clin Invest 2020;130 (11):6151-6157.

16.Kamieniak A,Krawiec P,E. Interleukin 6: biological significance and effect in inflammatory bowel disease (Intereukin 6:biological signific)ance and role in inflammatory bowel diseases)./>Adv Clin Exp Med.2021, 4 th month 28 th day doi:10.17219/acem/130356.PMID:33908198./ >

Sequence listing

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Claims

1. A method of altering cytokine expression, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NOs 2 and/or 3.

2. A method of preventing, treating and/or alleviating the toxic side effects of a checkpoint inhibitor in a subject in need thereof, the method comprising administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and/or 3.

3. A method of preventing, treating and/or reducing intestinal necrosis and/or intestinal injury in a subject in need thereof, the method comprising administering to the subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NO:2 and/or 3.

4. A method of preventing, treating and/or reducing intestinal necrosis and/or injury in a subject being treated with, having been treated with or to be treated with at least one checkpoint inhibitor, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a synthetic peptide comprising SEQ ID NOs 2 and/or 3.

5. The method of any one of claims 1 to 4, wherein the composition further comprises at least one pharmaceutically acceptable carrier, diluent, stabilizer or excipient.

6. The method of any one of claims 1 to 5, wherein the therapeutically effective amount of SEQ ID NOs 2 and/or 3 is from about 10mg/kg to about 160mg/kg.

7. The method of any one of claims 1 to 5, wherein the therapeutically effective amount of SEQ ID NOs 2 and/or 3 is from about 20mg/kg to about 160mg/kg.

8. The method of any one of claims 1 to 5, wherein the therapeutically effective amount of SEQ ID NOs 2 and/or 3 is from about 40mg/kg to about 160mg/kg.

9. The method of any one of claims 1 to 5, wherein the therapeutically effective amount of SEQ ID NOs 2 and/or 3 is administered in at least one dose, the first dose comprising from about 1mg/kg to about 160mg/kg of SEQ ID NOs 2 and/or 3.

10. The method of claim 9, wherein a second dose comprising a therapeutically effective amount of SEQ ID No. 2 and/or 3 is administered, said second dose comprising about 1mg/kg to about 120mg/kg of SEQ ID No. 2 and/or 3.

11. The method of any one of claims 1 to 5, wherein the therapeutically effective amount of SEQ ID NOs 2 and/or 3 is administered in two doses, a first dose comprising about 1mg/kg to about 160mg/kg of SEQ ID NOs 2 and/or 3 and a second dose comprising about 1mg/kg to about 120mg/kg of SEQ ID NOs 2 and/or 3.

12. The method of claim 11, wherein the second dose is administered 30 seconds to 10 hours after administration of the first dose.

13. The method of any one of claims 1 to 5, wherein the therapeutically effective amount of SEQ ID NOs 2 and/or 3 is administered in a plurality of doses over a period of about one week to about two weeks, each dose comprising about 1mg/kg to about 160mg/kg of SEQ ID NOs 2 and/or 3 and administered once every 4 to 10 hours.

14. The method of claim 13, wherein each dose is administered every 8 hours.

15. The method of any one of claims 1 to 5, wherein the therapeutically effective amount of SEQ ID NO. 2 and/or 3 is administered at least one loading dose of about 10mg/kg to about 160mg/kg SEQ ID NO. 2 and/or 3, followed by at least one maintenance dose of about 1mg/kg to about 120mg/kg SEQ ID NO. 2 and/or 3,

wherein the first maintenance dose is administered 4 to 10 hours after the last loading dose, and

wherein the maintenance dose is administered every 4 to 10 hours over a period of about one week to about two weeks.

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

17. The method of any one of claims 1 to 16, wherein the subject is also being treated with, has been previously treated with, or is about to be treated with a checkpoint inhibitor.

18. The method of claim 17, wherein the checkpoint inhibitor is selected from CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors, such as pembrolizumab (Keytruda), ipilimab (Yervoy), nivolumab (Opdivo), and atumumab (tecentiq).