Compositions containing TREM-1 inhibitors and their use

Abstract

This specification provides purified peptide inhibitors of trigger receptors expressed on the surface of myeloid cell 1 (TREM-1) suitable for administration as therapeutic agents for treating diseases or disorders. This specification also provides manufacturing processes for producing TREM-1 peptide inhibitors and compositions comprising such inhibitors having desirable purity, concentration, and stability properties.

Description

Technical Field

[0001] The present disclosure relates to compositions comprising purified peptide inhibitors of trigger receptors expressed on the surface of triggering receptor expressed on myeloid cells-1 (TREM-1), processes for manufacturing such compositions, and the use of such compositions for treating a disease or disorder.

Background Art

[0002] The trigger receptor expressed on the surface of triggering receptor expressed on myeloid cells-1 (TREM-1) is a transmembrane receptor expressed on the surface of innate immune cells and is a member of the TREM receptor family. Activation of TREM-1 induces the release of pro-inflammatory cytokines and chemokines and contributes to dysregulation of the immune response in sepsis (Siskind et al., 2022, Front. Immunol. 13:907387). Trigger-like transcript 1 (TLT-1) also belongs to the TREM family, is specific for platelets and megakaryocytes, and is involved in hemostasis / thrombosis (Washington et al., 2004, Blood 104(4):1042-1047). A peptide (LR12) derived from TLT-1 has been shown to exhibit anti-inflammatory properties by inhibiting TREM-1 signaling (Derive et al., 2012, J Immunol. 188:5585-5592). Administration of LR12 in an adult minipig model of induced sepsis reduced cardiovascular failure and improved the survival rate of animals after induction of fecal peritonitis (Derive et al., 2013, Shock 39(2):176-182).

[0003] The present applicant has discovered that LR12 tends to dimerize into a biologically inactive dimer. The presence of the LR12 dimer potentially adversely affects the dosage of active LR12 monomer and has the risk of reducing the therapeutic effect of the LR12 treatment regimen. To the knowledge of the present applicant, the problem of LR12 dimerization and its influence on the therapeutic effect have not been disclosed in the art. Considering that LR12 is preferably administered by a parenteral administration route, it is necessary to supply a pure LR12 composition to ensure the therapeutic effect.

[0004] Furthermore, in order to administer sufficient therapeutic doses of LR12 parenterally, the LR12 peptide needs to be solubilized in a liquid pharmaceutical medium such as saline. The benefits of appropriate use of fluids in intensive care units (ICUs) and hospitals are well documented in the art, and knowledge is growing regarding the potential risks of fluid overload and / or osmotic shock to organ failure and mortality, particularly in vulnerable subjects such as those suffering from septic shock or systemic inflammatory response syndrome. Therefore, there is a need to supply LR12 compositions that can be reconstituted into liquid parenteral formulations exhibiting optimal solubility by enabling the minimum possible amount of excipients. In other words, there is a need for LR12 compositions that can be reconstituted into liquid parenteral formulations with high concentrations of LR12, enabling the administration of the smallest possible amount of liquid parenteral formulation to patients while maintaining high purity, high stability, and optimal osmolality.

[0005] Finally, the applicant has noticed that LR12 dimers may still form in the liquid formulation of the LR12 formulation during its effective period and / or during intravenous administration.

[0006] Therefore, there is a need for improved LR12 pharmaceutical compositions that possess desirable purity, concentration, and stability characteristics for therapeutic administration, while respecting clinical requirements regarding fluid administration and osmotic compliance, particularly for subjects vulnerable to fluid overload and / or osmotic shock.

[0007] Advantageously, the pharmaceutical composition of this disclosure overcomes the aforementioned drawbacks, thereby optimizing the feasibility of LR12 administration in a hospital setting. The pharmaceutical product is efficiently solubilized and exhibits suitable stability from the manufacture to administration of the pharmaceutical composition while complying with the required osmolality. [Overview of the Initiative]

[0008] This application relates to a pharmaceutical composition comprising a purified LR12 peptide having the amino acid sequence of SEQ ID NO: 1, mixed with at least one solubility enhancer, preferably arginine and sodium citrate buffer, wherein the purified LR12 peptide substance contains at least 85% w / w of peptide by weight relative to the total weight of the purified LR12 peptide substance, and the pharmaceutical composition comprises less than 5% w / w of LR12 dimers by weight relative to the peptide content.

[0009] This application is, • Purified LR12 peptide having the amino acid sequence of SEQ ID NO: 1, Preferably, at least one amino acid selected from arginine, lysine, and histidine, more preferably at least one amino acid selected from arginine and lysine, and even more preferably at least one solubility enhancer which is arginine. • Citrate buffer, preferably sodium citrate buffer A pharmaceutical composition containing, This also relates to a pharmaceutical composition containing less than 5% w / w of LR12 dimers by weight relative to the peptide content, and in which the amount of at least one solubility enhancer is in the range of 20% to 30% by weight relative to the dry weight of the purified LR12 peptide substance.

[0010] In some embodiments, the purified LR12 peptide material contains at least 85% w / w of LR12 peptide by weight relative to the total weight of the purified LR12 peptide material.

[0011] In some embodiments, the pharmaceutical composition contains 800 mg to 2500 mg of LR12 peptide, preferably 1200 mg to 2000 mg of LR12 peptide.

[0012] In some embodiments, the pharmaceutical composition contains purified LR12 peptide in an amount of 70.0% to 80.0%, preferably in the range of 75.2% to 79.0%, and more preferably about 77.3%, relative to the total dry weight of the pharmaceutical composition. In some embodiments, the pharmaceutical composition contains, The amount of at least one solubility enhancer, preferably arginine, is in the range of 20.0% to 30.0% by weight relative to the dry weight of the purified LR12 peptide, preferably 22.0% to 28.0%, more preferably 22.3% to 27.6%, and even more preferably about 24.5%, and / or The amount of citrate buffer, preferably sodium citrate buffer, is in the range of 3.0% to 7.0%, preferably 4.0% to 6.0%, more preferably 4.5% to 5.5%, and even more preferably about 4.8%, relative to the dry weight of the purified LR12 peptide.

[0013] In some embodiments, the pharmaceutical composition is a powder composition, preferably a freeze-dried powder composition. Advantageously, the powder moisture content of the pharmaceutical composition is 5.0% w / w or less, preferably 3.0% w / w or less. In some embodiments, the pharmaceutical composition is preferably an aqueous medium selected from water and physiological saline, preferably an aqueous solution further comprising physiological saline having 0.9% sodium chloride by weight relative to the total weight of physiological saline, and / or the pharmaceutical composition has a pH in the range of 5.0 to 6.0, preferably about 5.5.

[0014] In some embodiments, the pharmaceutical composition has a pH in the range of 5.0 to 6.0, preferably about 5.5.

[0015] In some embodiments, the pharmaceutical composition exhibits an osmolality of less than 500 mOsm / kg (mOsm / kg and mOsmol / kg can be used interchangeably), preferably in the range of 300 to 400 mOsm / kg, more preferably in the range of 320 to 390 mOsm / kg, and even more preferably in the range of about 380 mOsm / kg.

[0016] This application also relates to a purified LR12 peptide having or consisting of the amino acid sequence described in Sequence ID No. 1, which contains at least 85% w / w of peptide content by weight relative to the total weight of the purified LR12 product, and less than 2.7% by weight of LR12 dimers relative to the total weight of the peptide content.

[0017] In some embodiments, the purified LR12 peptide contains 1.2% or less by weight of Des-Gln-LR12 and / or 0.5% or less of Met(O)12-LR12 relative to the total weight of the peptide content. In some embodiments, the purified LR12 peptide contains 1% or less by weight of acetate, 500 ppm or less of acetonitrile, less than 5000 ppm of methyl-t-butyl ether and / or less than 5000 ppm of n-heptane relative to the peptide substance.

[0018] This application also relates to the pharmaceutical compositions or purified LR12 peptide substances described herein for use as a medicine. This application further relates to the pharmaceutical compositions or purified LR12 peptide substances described herein for use in the treatment of diseases in subjects requiring such treatment, wherein the diseases are preferably inflammatory diseases or disorders selected from septic shock, sepsis, severe sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), hemorrhagic shock, ischemia-reperfusion injury and pancreatitis, preferably cardiovascular diseases selected from myocardial infarction, cerebral infarction, acute myocardial infarction, ischemia, coronary heart disease, acute coronary syndrome, stroke, aneurysm, stable angina pectoris, exertional angina pectoris, cardiomyopathy, hypertensive heart disease, chronic heart failure, acute heart failure, cor pulmonale, cardiac arrhythmias, inflammatory heart diseases (such as endocarditis and myocarditis), peripheral artery disease, SIRS-related myocardial and / or vascular dysfunction and atherosclerosis, as well as infectious diseases such as COVID-19.

[0019] In some embodiments, the pharmaceutical composition or purified LR12 peptide substance is intended for intravenous administration at a rate ranging from 0.3 to 3.0 mg / kg / hr (mg of LR12 per kg of body weight of the subject per hour).

[0020] This application is a process for preparing the purified LR12 peptide substance described herein, i. Synthesizing the LR12 peptide on a solid phase to obtain a crude LR12 composition, ii. Obtaining a first peptide solution containing the crude LR12 composition, iii. Purifying the first peptide solution by ion exchange column chromatography (IEC) to obtain a first pre-purified substance of LR12, iv. Further purifying the pre-purified substance of LR12 by reverse phase high performance liquid chromatography (HPLC) to obtain an HPLC pooled fraction containing a second pre-purified substance of LR12, v. Preparing a 5 - 25 mM arginine solution for addition to the pooled fraction during step vi, vi. Concentrating the pooled fraction to at least a 120 g / L concentrated solution using membrane diafiltration or ultrafiltration, typically with a membrane of 150 - 300 Daltons, and vii. Filtering and lyophilizing the concentrated solution obtained in step vi) to obtain a purified substance of the LR12 peptide also relates to a process comprising.

[0021] In some embodiments, the HPLC pooled fraction containing the second pre-purified substance of LR12 is cooled to a temperature of 15°C or lower and / or bubbled with nitrogen.

[0022] This application further relates to a process for preparing a pharmaceutical composition described herein, a) Supplying a purified substance of the LR12 peptide described herein by performing steps i) - vii) preferably according to the process described above, b) A purified substance of the LR12 peptide, a citrate buffer, preferably a sodium citrate buffer, and at least one solubility enhancer selected from arginine, lysine, and histidine, preferably selected from arginine and lysine, more preferably arginine · At least one solubility enhancer, preferably arginine, in an amount ranging from 20.0% to 30.0%, preferably from 22.0% to 28.0%, more preferably from 22.3% to 27.6%, and even more preferably about 24.5% by weight based on the dry weight of the purified substance of the LR12 peptide, and · A citrate buffer, preferably sodium citrate buffer, in an amount ranging from 3.0% to 7.0%, preferably from 4.0% to 6.0%, more preferably from 4.5% to 5.5%, and even more preferably about 4.8% by weight based on the dry weight of the purified substance of the LR12 peptide Mixing them to obtain a bulk solution, c) Optionally, adjusting the pH of the bulk solution typically to pH 5.5 with HCl, d) Lyophilizing the bulk solution to obtain a lyophilized powder composition of the pharmaceutical composition, and e) Optionally, adding a physiological saline having 0.9% sodium chloride by weight based on the total weight of an aqueous medium preferably selected from water and physiological saline, preferably physiological saline, to obtain an aqueous solution of the pharmaceutical composition relates to a process comprising.

Brief Description of Drawings

[0023] [Figure 1] Graph showing the results from quantification of neutrophil ROS production by flow cytometry (reduction by DCFDA) after 2-hour stimulation with LPS, LPS + 10 μg / mL of LR12, LPS + 50 μg / mL of LR12, LPS + 10 μg / mL of LR12 dimer (dimer), LPS + 50 μg / mL of LR12 dimer (dimer). *p < 0.005, **p < 0.01 (t-test).

[0024] Definitions In the present disclosure, the following terms have the following meanings.

[0025] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects of the article. For example, "one element" means one or more elements. Similarly, the expressions "at least one" and "one or more" are interchangeable.

[0026] The term "approximately" preceding a value is used to indicate that the value includes inherent variations in the error of the instrument or method used to determine the value, or variations that exist between the samples being measured. Unless otherwise specified or evident from the context, the term "approximately" means within plus or minus 10% of the reported number (except when such a number exceeds 100% of the possible value or falls below 0%). When used in conjunction with a range of values ​​or a set of values, the term "approximately" applies to each of the values ​​listed in the range or set of values, unless otherwise specified. The terms "approximately" and "about" as used herein may be used synonymously.

[0027] "API" is a pharmaceutical active ingredient, i.e., LR12 in this disclosure. The API is a therapeutic agent, which may be a chemical or biological component.

[0028] "At least" means "greater than or equal to," and is therefore different from "strictly greater than" (which does not include "equal to").

[0029] A "buffer solution" refers to a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid, which can maintain a constant pH when a small amount of strong acid or strong base is added to a pharmaceutical composition. A "buffering agent" refers to a specific chemical substance that exists in both acidic and basic forms in a buffer solution and can maintain a constant pH of a pharmaceutical composition.

[0030] "Comprising" or "comprise" is meant to be interpreted in a non-restrictive and comprehensive sense, and is not limited to the characteristics that follow this term.

[0031] "Consisting of" or "consisting of" should be interpreted in a limited and non-inclusive sense, and should be limited to the characteristics that follow the term.

[0032] "Dosage" refers to the amount of API administered at one time.

[0033] With respect to LR12, "API" or "LR12 API" refers to the purified LR12 peptide substance (also known as the purified LR12 peptide composition). The purified LR12 peptide substance described herein, also known as the API, is a powder. This powder is obtained by lyophilization of a solution, as described in the process for preparing the purified LR12 peptide substance described in this application. Advantageously, the water content of the purified LR12 peptide substance is 10.0% w / w or less, preferably 5.0% w / w or less.

[0034] With respect to LR12, “pharmaceutical product” refers to a pharmaceutical composition comprising a purified LR12 peptide substance. The pharmaceutical product may be in either powder form (e.g., storage form) or liquid form (e.g., form of use as referred above as “liquid parenteral formulation”). The pharmaceutical product in powder form is obtained by dissolving the purified LR12 peptide substance described herein and performing the other steps a) to d) of the process for preparing the pharmaceutical composition described herein (meaning the lyophilization step). Advantageously, the moisture content of the pharmaceutical product in powder form is 5.0% w / w or less, preferably 3.0% w / w or less. The pharmaceutical product in liquid form is obtained by adding an aqueous medium to the pharmaceutical product in powder form, i.e., by performing step e) of the process for preparing the pharmaceutical composition described herein. In summary, the series of manufacturing steps is as follows: process for preparing the purified LR12 peptide substance described in this application > step of obtaining the purified LR12 peptide substance (powder) described herein > steps a) to d) of the process for preparing the pharmaceutical composition described in this application > step of obtaining the pharmaceutical product in powder form (e.g., storage form) > step e) of the process for preparing the pharmaceutical composition described in this application > step of obtaining the pharmaceutical product in liquid form (e.g., form for use).

[0035] "Excipients" refer to any inactive components necessary for the formulation of the API in a suitable dosage form.

[0036] "X~Y" refers to the range of values ​​between X and Y, where the upper and lower limits of X and Y are included within the aforementioned range.

[0037] Numerical values ​​are subject to measurement errors that limit their accuracy, as is known to those skilled in the art. Therefore, this application applies the general convention in scientific and technical literature, namely, that the last decimal place of a numerical value indicates its degree of accuracy. Unless other tolerances are specified, the maximum tolerance is determined by rounding to the last decimal place.

[0038] "Peptide content" refers to the peptide fraction of the purified LR12 peptide substance. The peptide content can be determined by any means known in the art, such as elemental analysis, quantitative amino acid analysis, high-performance liquid chromatography (HPLC), or the Kjeldahl method, preferably by elemental analysis.

[0039] "Peptide purity" refers to the LR12 fraction of the peptide fraction (purified LR12 peptide substance).

[0040] "LR12" or "LR12 peptide" refers to the TLT-1 derived peptide of formula (I), which consists of the amino acid sequence (LQEEDAGEYGCM) described in Sequence ID No. 1. [ka]

[0041] A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient or carrier that does not cause adverse reactions, allergic reactions, or other adverse reactions when administered to mammals such as humans. This includes all solvents, such as dispersions, coatings, antimicrobial agents, antifungal agents, isotonic agents, and absorption retarders. A pharmaceutically acceptable excipient or carrier refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating agent, or formulation aid. For administration to humans, the formulation must meet the sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities such as the EMA (European Medicines Agency) or the FDA (U.S. Food and Drug Administration).

[0042] "Pharmaceutical composition" refers to a combination of at least one API and at least one pharmaceutically acceptable excipient.

[0043] "Powder" refers to an aggregate of fine solid (micro) particles, varying in degree of dryness. Advantageously, any powder described herein has a moisture content of 10.0% w / w or less.

[0044] "Room temperature" refers to a temperature in the range of 20°C to 25°C, preferably 20°C.

[0045] "rpm" or "revolutions per minute" refers to the number of rotations per minute.

[0046] The term “subject” refers to a warm-blooded animal, more preferably a mammal. The term “mammal” here refers to any mammal, including humans. In some embodiments, the subject may be a “patient” who is waiting for or receiving medical care, or who has been, is currently, or will be the subject of medical treatment in the past or in the future, or who is being observed for the onset of a target disease or condition.

[0047] "Treatment" refers to therapeutic (or curative) treatment or prophylactic treatment, or both therapeutic (or curative) and prophylactic treatment, aimed at preventing, reducing, slowing (delaying progression), or curing one or more symptoms or manifestations of a targeted pathological condition or disorder, particularly septic shock, sepsis or SIRS, cardiovascular disease, disorder or condition, or infection. Those requiring treatment include individuals already suffering from an inflammatory disease or disorder, cardiovascular disease, disorder or condition, or infection, as well as those who are susceptible to developing an inflammatory disease or disorder, cardiovascular disease, disorder or condition, or infection, or those who should be prevented from developing an inflammatory disease or disorder, cardiovascular disease, disorder or condition, or infection. [Modes for carrying out the invention]

[0048] This disclosure relates to compositions comprising a TREM-1 peptide inhibitor optimized for therapeutic administration to treat a disease or disorder (e.g., septic shock, sepsis, or SIRS). The TREM-1 LR12 peptide inhibitor (also known as nangibotide; LQEEDAGEYGCM-SEQ ID NO: 1) contains cysteine, which results in the formation of a disulfide bond between LR12 molecules, thereby forming a dimerized LR12. The LR12 dimer consists of two LR12 peptide monomers covalently linked by a disulfide bond via cysteine ​​at position 11. The LR12 dimer, i.e., the compound of formula II, has been shown to be inactive in preclinical in vitro pharmacological models and may adversely affect the therapeutic effect of LR12. [ka]

[0049] Considering the biological inactivity of LR12 dimers, the present invention relates to a purified LR12 peptide substance and a pharmaceutical composition (pharmaceutical product) containing the same, which is more advantageous in terms of therapeutic efficacy than an LR12 peptide substance containing more than 5% by weight of LR12 dimers relative to the total weight of the peptide content of the LR12 substance. In other words, the purified LR12 peptide substance and the pharmaceutical composition (pharmaceutical product) containing the same are advantageous in terms of chemical stability. In a particularly advantageous embodiment, such chemical stability is maintained throughout the entire shelf life of the purified LR12 peptide substance and the pharmaceutical composition (pharmaceutical product) containing the same. Furthermore, such chemical stability is maintained even when the pharmaceutical composition containing the LR12 peptide is solubilized in a liquid formulation, and more importantly, during intravenous administration of the liquid formulation.

[0050] In view of the above, according to several advantageous embodiments, purified LR12 peptide substances and pharmaceutical compositions comprising purified LR12 peptide substances provide the same therapeutic effect with a smaller amount of drug (by supplying the same amount of LR12 bioactive monomers) compared to the prior art active pharmaceutical ingredients and products.

[0051] Purified LR12 peptide substance Accordingly, according to the first aspect, this application relates to a purified composition of LR12 peptide, also known as purified LR12 peptide material (or sometimes referred to as LR12 active pharmaceutical ingredient). Naturally, such material includes a peptide content (or fraction) containing peptides such as LR12 or peptide impurities other than LR12. Typically, the peptide fraction of the purified LR12 peptide material described herein is at least about 85% by weight of the total weight of the purified LR12 peptide material. The peptide fraction of the material can be determined by any means known in the art as defined above, preferably by elemental analysis. In a preferred embodiment, the peptide fraction of the LR12 active pharmaceutical ingredient is determined by elemental analysis, typically including a dry combustion step at a temperature in the range of 1000°C to 1200°C in the presence of oxygen, followed by detection of thermal conductivity, using an elemental analyzer for the simultaneous determination of C, H, N, and S. During the dry combustion step, the total nitrogen content in the active pharmaceutical ingredient can be quantitatively converted to N2 and then determined by measuring the thermal conductivity after separation of other gaseous components (CO2, H2O, and SO2).

[0052] The purified LR12 peptide substances described herein contain LR12 dimers in an amount of about 5%, about 4.5%, about 4%, about 3.5%, about 3%, about 2.7%, or less than about 2.5% by weight relative to the total weight of the peptide content of the purified LR12 peptide substance.

[0053] According to some embodiments, the purified LR12 peptide material contains a peptide content of at least about 85% w / w, preferably at least about 86%, relative to the total weight of the purified LR12 material, and contains LR12 dimers of less than about 5%, preferably less than about 3%, and more preferably less than 2.7%, relative to the total weight of the peptide content.

[0054] In some embodiments, the purified LR12 peptide material contains at least 85% w / w of peptide by weight relative to the total weight of the purified LR12 material, and contains less than 2.7% by weight of LR12 dimers relative to the total weight of the peptide content.

[0055] In other words, the purified LR12 peptide substance described herein is • A peptide content of at least approximately 85% w / w by weight relative to the total weight of the purified LR12 peptide substance, • LR12 dimers containing less than approximately 5% w / w, less than approximately 4.5% w / w, less than approximately 4% w / w, less than approximately 3.5%, less than approximately 3% w / w, less than approximately 2.7% w / w, or less than approximately 2.5% w / w of peptide content relative to the total weight of the purified LR12 peptide substance. It is a composition containing [the specified ingredient].

[0056] In some embodiments, the purified LR12 peptide material contains at least about 85% w / w of peptide by weight relative to the total weight of the purified LR12 material, and contains about 95% or more of LR12 (monomers) by weight relative to the total weight of the peptide content.

[0057] This specification provides a purified peptide composition comprising several peptides containing the amino acid sequence of SEQ ID NO: 1, wherein less than approximately 2.7% of the peptide is dimerized at the cysteine ​​residue of SEQ ID NO: 1.

[0058] This specification provides purified LR12 peptide materials containing Des-Gln-LR12 in an amount of approximately 2.5%, approximately 2%, approximately 1.5%, approximately 1.2%, approximately 1%, approximately 0.5%, or approximately 0.3% or less by weight relative to the total weight of the peptide content. Des-Gln-LR12 is a peptide consisting of the amino acid sequence (LEEDAGEYGCM) of SEQ ID NO: 2, and its structure corresponds to Formula III shown below. Typically, the purified LR12 peptide materials described herein contain Des-Gln-LR12 in an amount of approximately 1.2% or less, preferably 1.0% or less, and more preferably 0.5% or less by weight relative to the total weight of the peptide content of the purified LR12 peptide material. [ka]

[0059] This specification provides purified LR12 peptide substances containing Met(O)12-LR12 in an amount of about 2.5%, about 2%, about 1.5%, about 1.2%, about 1%, about 0.75%, about 0.5%, or less by weight relative to the total weight of the peptide content. Met(O)12-LR12 is the oxidized LR12 product of formula IV shown below. Met(O)12-LR12 is a peptide consisting of the amino acid sequence of SEQ ID NO: 3 (LEEDAGEYGCM) in which the methionine at position 12 is oxidized to a methionine sulfoxide also known as Met(O) or MetO. Typically, the purified LR12 peptide substances described herein contain 1.0% or less, preferably about 0.75% or less, and more preferably about 0.5% or less by weight relative to the total weight of the peptide content of the purified LR12 peptide substance. [ka]

[0060] In some embodiments, the purified LR12 peptide material contains 1.2% or less by weight of Des-Gln-LR12 and / or 0.5% or less of Met(O)12-LR12 and / or 0.5% or less of other single impurities relative to the total weight of the peptide content of the purified LR12 peptide material.

[0061] In some embodiments, the purified LR12 peptide material contains Des-Gln-LR12 in an amount of 1.2% or less by weight, preferably 1.0% or less, more preferably 0.5% or less, relative to the total weight of the peptide content of the purified LR12 peptide material, and / or Met(O)12-LR12 in an amount of 1.0% or less by weight, preferably 0.5% or less, relative to the total weight of the peptide content of the purified LR12 peptide material.

[0062] This specification provides purified LR12 peptide substances containing acetate in amounts of approximately 5%, approximately 4.5%, approximately 4%, approximately 3.5%, approximately 3%, approximately 2.5%, approximately 2%, approximately 1.5%, approximately 1%, or approximately 0.75% or less by weight relative to the total weight of the purified LR12 peptide substance. This specification also provides purified LR12 peptide substances containing LR12 with acetate in amounts of approximately 1% or less.

[0063] This specification provides purified LR12 peptide substances containing approximately 750 ppm, approximately 700 ppm, approximately 650 ppm, approximately 600 ppm, approximately 550 ppm, approximately 500 ppm, or less than approximately 450 ppm of acetonitrile. This specification also provides purified LR12 peptide substances containing less than approximately 410 ppm of acetonitrile.

[0064] This specification provides purified LR12 peptide substances containing approximately 7000 ppm, approximately 6500 ppm, approximately 6000 ppm, approximately 5500 ppm, and less than approximately 5000 or approximately 4500 ppm of methyl-t-butyl ether. This specification also provides purified LR12 peptide substances containing less than approximately 5000 ppm of methyl-t-butyl ether.

[0065] This specification provides purified LR12 peptide substances containing n-heptane in amounts of approximately 7000 ppm, approximately 6500 ppm, approximately 6000 ppm, approximately 5500 ppm, approximately 5000 ppm, or less than approximately 4500 ppm. This specification also provides purified LR12 peptide substances containing n-heptane in amounts of less than approximately 5000 ppm.

[0066] This specification provides a purified LR12 peptide substance containing less than 500 ppm of acetonitrile, less than 5000 ppm of methyl-t-butyl ether, and less than 5000 ppm of n-heptane.

[0067] In some embodiments, the purified LR12 peptide substance comprises less than 1% by weight of acetate relative to the total weight of the purified LR12 peptide substance, less than 500 ppm by weight of acetonitrile relative to the total weight of the purified LR12 peptide substance, less than 5000 ppm of methyl-t-butyl ether and / or less than 5000 ppm of n-heptane.

[0068] In this specification, • Contains less than 2.7% cysteine ​​dimerization of LR12 by weight relative to the total weight of the peptide content. • Contains Des-Gln-LR12 in an amount of approximately 1.2% or less by weight relative to the total weight of the peptide content, preferably approximately 1.0% or less, and more preferably approximately 0.5% or less. • Contains Met(O)12-LR12 in an amount of about 1.0% or less by weight relative to the total weight of the peptide content, preferably about 0.75% or less, and more preferably about 0.5% or less. • Contains approximately 0.5% or less of other single impurities. • Contains 5% or less total peptide impurities (i.e., peptides with undesirable sequences) by weight relative to the total weight of the peptide content. • Contains less than 1% acetate by weight relative to the total weight of the purified LR12 peptide substance. • Contains less than 500 ppm of acetonitrile by weight relative to the total weight of the purified LR12 peptide substance. • Contains less than approximately 5000 ppm of methyl-t-butyl ether, • Contains n-heptane at less than approximately 5000 ppm A purified LR12 peptide substance having the following characteristics is provided.

[0069] In some embodiments, the purified LR12 peptide is • Contains at least about 85% w / w of peptides by weight relative to the total weight of the purified LR12, preferably containing about 95% or more of LR12 (monomers) by weight relative to the total weight of the peptide content. • Contains less than 2.7% cysteine ​​dimerization of LR12 by weight relative to the total weight of the peptide content. • Contains Des-Gln-LR12 in an amount of approximately 1.2% or less by weight relative to the total weight of the peptide content, preferably approximately 1.0% or less, and more preferably approximately 0.5% or less. • Contains Met(O)12-LR12 in an amount of about 1.0% or less by weight relative to the total weight of the peptide content, preferably about 0.75% or less, and more preferably about 0.5% or less. • Contains less than 1% acetate by weight relative to the total weight of the purified LR12 peptide substance. • Contains less than 500 ppm of acetonitrile by weight relative to the total weight of the purified LR12 peptide substance. • Contains less than approximately 5000 ppm of methyl-t-butyl ether by weight relative to the total weight of the purified LR12 peptide substance, • Contains less than 5000 ppm of n-heptane by weight relative to the total weight of the purified LR12 peptide substance. It possesses the following characteristics.

[0070] According to some embodiments, the purified LR12 peptide substance described above further contains arginine in an amount ranging from 2 to 5% w / w by weight relative to the total weight of the purified LR12 peptide substance.

[0071] In other words, the purified LR12 peptide substance described herein is • Peptide content of at least approximately 85% w / w by weight relative to the total weight of the purified LR12 peptide substance. • LR12 dimers with a peptide content of less than approximately 5% w / w, less than approximately 4.5% w / w, less than approximately 4% w / w, less than approximately 3.5% w / w, less than approximately 3% w / w, less than approximately 2.7% w / w, or less than approximately 2.5% w / w relative to the total weight of the purified LR12 peptide substance, and Optionally, 2% w / w to 5% w / w arginine by weight relative to the total weight of the purified LR12 peptide substance. It is a composition containing [the specified ingredient].

[0072] The purified LR12 peptide substance described herein is a powder, preferably a lyophilized powder.

[0073] The purified LR12 peptide substance described herein can be used to obtain a pharmaceutical product containing a high dose of LR12 peptide (referred to herein as the “high-dose formulation”) which can reduce the dosage of this pharmaceutical product in the form used for patient administration.

[0074] Pharmaceutical composition (pharmaceutical product) containing purified LR12 peptide substance In some embodiments, the maximum dimer content in the purified LR12 peptide substance is preferably 2.7% or less, and thereafter the dimer content in the pharmaceutical product becomes 5.0% or less.

[0075] In short, the pH during the formulation process was lowered to below 6.0 to reduce the formation of disulfide crosslinks. However, LR12 contains four acidic, negatively charged amino acids, namely glutamic acid (Glu) and aspartic acid (Asp), and no basic amino acids. Therefore, LR12 exhibits a theoretical pI (isoelectric point) of 3.57 and is therefore almost insoluble below pH 5.0. To solve this problem, solubilizers were selected for pH 5.5 and 6.5. Surfactants (Tween® 20), polyols (mannitol), sugars (sucrose and trehalose), zwitterions (betaine), chaotropic compounds (urea), redox compounds (glutathione), and counterions (ammonium acetate and citrate) molecules failed to improve the solubility of LR12.

[0076] Unexpectedly, a solubility of 40 mg / mL could only be achieved by adding positively charged solubility enhancers such as the amino acids lysine, arginine, or histidine. While we do not wish to be bound by theory, these amino acids may improve the solubility of LR12 by neutralizing the negative charge of the LR12 sequence. Administration of LR12 in the compositions provided herein at doses of 1–3 mg / kg / h delivers only a small fraction of the daily dose of arginine administered to patients receiving parenteral nutrition.

[0077] This specification provides pharmaceutical compositions (=pharmaceutical products) comprising a purified LR12 peptide substance disclosed herein, mixed with one or more pharmaceutically acceptable solubility enhancers and at least one pharmaceutically acceptable buffer composition. The pharmaceutical compositions provided herein have desirable concentrations and stability properties for clinical administration while allowing for the smallest possible amount of excipients.

[0078] Preferably, the amount of at least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, and even more preferably arginine, is in the range of 20% to 30% by weight relative to the dry weight of the purified LR12 peptide substance.

[0079] At least one solubility enhancer may be selected from the group comprising or consisting of arginine, lysine, histidine, or a combination thereof, preferably from the group comprising or consisting of arginine, lysine, or a combination thereof. At least one pharmaceutically acceptable buffer composition is preferably a citrate buffer, i.e., a citrate, typically a mixture of sodium citrate and citric acid. In some embodiments, citrate buffer compositions are readily available. In some embodiments, a citrate buffer can be prepared by mixing a citrate, such as sodium citrate, with hydrochloric acid to obtain a citrate / citric acid buffer.

[0080] Accordingly, in some embodiments, the pharmaceutical composition comprises a purified LR12 peptide substance having at least 85% w / w peptide content as described above, mixed with at least one solubility enhancer, preferably selected from arginine, lysine, and histidine, more preferably selected from arginine and lysine, and even more preferably arginine, and at least one buffer composition, preferably citrate buffer, more preferably sodium citrate buffer, and the pharmaceutical composition comprises less than 5% w / w of LR12 dimers by weight relative to the peptide content.

[0081] In some embodiments, the pharmaceutical composition comprises a purified LR12 peptide substance mixed with at least one solubility enhancer, preferably arginine and a citrate buffer, preferably a sodium citrate buffer, wherein the purified LR12 peptide substance contains at least 85% w / w of peptide by weight relative to the total weight of the purified LR12 peptide substance, and the composition contains less than 5% w / w of LR12 dimers by weight relative to the peptide content.

[0082] In some embodiments, the pharmaceutical composition contains purified LR12 peptide material in an amount of 70.0% to 80.0%, preferably 75.2% to 79.0%, and more preferably about 77.3%, relative to the total dry weight of the pharmaceutical composition.

[0083] In some embodiments, in this pharmaceutical composition, • At least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, and even more preferably the amount of arginine being in the range of 20.0% to 30.0% by weight relative to the dry weight of the purified LR12 peptide substance, preferably 22.0% to 28.0%, more preferably 22.3% to 27.6%, and even more preferably about 24.5%, and / or The amount of at least one pharmaceutically acceptable buffer, preferably a citrate buffer, more preferably a sodium citrate buffer, is in the range of 3.0% to 7.0%, preferably 4.0% to 6.0%, more preferably 4.5% to 5.5%, and even more preferably about 4.8%, relative to the dry weight of the purified LR12 peptide substance.

[0084] In some embodiments, the pharmaceutical composition contains purified LR12 peptide material in an amount of 70.0% to 80.0%, preferably in the range of 75.2% to 79.0%, more preferably about 77.3%, relative to the total dry weight of the pharmaceutical composition, and • At least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, and even more preferably the amount of arginine being in the range of 20.0% to 30.0% by weight relative to the dry weight of the purified LR12 peptide substance, preferably 22.0% to 28.0%, more preferably 22.3% to 27.6%, and even more preferably about 24.5%, and / or The amount of at least one pharmaceutically acceptable buffer, preferably a citrate buffer, more preferably a sodium citrate buffer, is in the range of 3.0% to 7.0%, preferably 4.0% to 6.0%, more preferably 4.5% to 5.5%, and even more preferably about 4.8%, relative to the dry weight of the purified LR12 peptide substance.

[0085] In some embodiments, the pharmaceutical composition contains purified LR12 peptide material in an amount of 70.0% to 80.0%, preferably in the range of 73.0% to 77.0%, more preferably about 75.2%, relative to the total dry weight of the pharmaceutical composition, and • At least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, and even more preferably the amount of arginine being in the range of 25.0% to 30.0% by weight relative to the amount of LR12, preferably 26.0% to 28.0%, more preferably 27.0% to 28.0%, and even more preferably about 27.6%, and / or The amount of at least one pharmaceutically acceptable buffer, preferably a citrate buffer, more preferably a sodium citrate buffer, is in the range of 3.0% to 7.0% by weight relative to the amount of LR12, preferably 4.0% to 6.0%, more preferably 5.0% to 6.0%, and even more preferably about 5.4%.

[0086] In some embodiments, the pharmaceutical composition is a powder composition, preferably a freeze-dried powder composition. In some embodiments, the pharmaceutical composition is preferably an aqueous medium selected from water and physiological saline, preferably an aqueous solution further comprising physiological saline having 0.9% sodium chloride by weight relative to physiological saline.

[0087] In some embodiments, the pharmaceutical compositions described herein have a pH in the range of 5.0 to 6.0, preferably about 5.5. The pH of the pharmaceutical composition is valid whether the composition is a powder or a liquid (i.e., a solution). In the case of a powder, for example, the pH is measured using standard techniques known to those skilled in the art after redissolving the powder in a solvent, preferably water.

[0088] In some embodiments, the pharmaceutical composition is preferably an aqueous medium selected from water and physiological saline, preferably an aqueous solution further comprising physiological saline having 0.9% sodium chloride by weight relative to physiological saline, and the pharmaceutical composition has a pH in the range of 5.0 to 6.0, preferably about 5.5.

[0089] According to several advantageous embodiments, the dimer content in the pharmaceutical composition does not change significantly in its form of use, i.e., the liquid form (aqueous solution) of the pharmaceutical composition described herein. In some embodiments, the maximum dimer content in the form of use of the pharmaceutical composition is 10.0% or less by weight relative to the total weight of the peptide content in the form of use of the pharmaceutical composition. A dimer content of 10.0% or less by weight relative to the total weight of the peptide content in the form of use of the pharmaceutical composition enables sufficient therapeutic effect of the pharmaceutical composition.

[0090] The present invention also relates to a pharmaceutical composition comprising a purified LR12 peptide substance described herein, mixed with a citrate buffer and at least one amino acid selected from arginine, lysine, and histidine, wherein the amount of at least one amino acid is in the range of 20% to 30% by weight relative to the dry weight of the purified LR12 peptide substance, the pharmaceutical composition comprises less than 10.0% w / w of LR12 dimers by weight relative to the peptide content, and the pharmaceutical composition is preferably an aqueous medium selected from water and physiological saline, preferably an aqueous solution further comprising physiological saline having 0.9% sodium chloride by weight relative to physiological saline.

[0091] In some embodiments, the aqueous solution is • Purified LR12 peptide substance in an amount of 70.0% to 80.0%, preferably in the range of 75.2% to 79.0%, more preferably about 77.3%, relative to the total dry weight of the pharmaceutical composition. • At least one pharmaceutically acceptable buffer, preferably citrate buffer, more preferably sodium citrate buffer, in an amount of 2.0% to 6.0%, preferably in the range of 3.0% to 5.0%, more preferably about 3.7%, relative to the total dry weight of the pharmaceutical composition, and • At least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, and even more preferably arginine, in an amount of 15.0% to 25.0%, preferably 18.0% to 22.0%, and more preferably about 19.0%, relative to the total dry weight of the pharmaceutical composition, is included. Includes.

[0092] In some embodiments, the pharmaceutical composition, preferably in liquid form (aqueous solution), exhibits an osmolality of less than 500 mOsm / kg, preferably 300-400 mOsm / kg, more preferably 320-390 mOsm / kg, and even more preferably about 380 mOsm / kg.

[0093] In some embodiments, the pharmaceutical composition described herein comprises at least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, and even more preferably arginine, in a concentration that increases the solubility of the composition to at least 120 mg / mL when dissolved in an aqueous solution. In some embodiments, the pharmaceutical composition comprises at least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, and even more preferably arginine, and the purified LR12 peptide substance has less than 2.7% LR12 dimers.

[0094] In some embodiments, the pharmaceutical composition contains at least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, and even more preferably arginine, at a concentration in the range of about 100 mM to about 250 mM.

[0095] In some embodiments, the pharmaceutical composition contains Des-Gln-LR12 in an amount of about 2.5%, about 2%, about 1.5%, about 1.2%, about 1%, about 0.5%, or about 0.3% or less by weight relative to the total weight of the peptide content. In some embodiments, the pharmaceutical composition contains Des-Gln-LR12 in an amount of about 1.2% or less, preferably 1.0% or less, and more preferably 0.5% or less by weight relative to the total weight of the peptide content of the pharmaceutical composition.

[0096] In some embodiments, the pharmaceutical composition contains Met(O)12-LR12 in an amount of about 2.5%, about 2%, about 1.5%, about 1.2%, about 1%, about 0.75%, about 0.5%, or about 0.3% or less by weight relative to the total weight of the peptide content. In some embodiments, the pharmaceutical composition contains Met(O)12-LR12 in an amount of 1.0% or less, preferably about 0.75% or less, and more preferably about 0.5% or less by weight relative to the total weight of the peptide content of the pharmaceutical composition.

[0097] In some embodiments, the pharmaceutical composition is lyophilized. This specification provides pharmaceutical compositions having solubility of at least about 250 mg / mL, about 225 mg / mL, about 200 mg / mL, about 175 mg / mL, or about 150 mg / mL when reconstituted in an aqueous solution. This specification also provides pharmaceutical compositions having solubility of at least about 175 mg / mL when reconstituted in an aqueous solution.

[0098] In some embodiments, the pharmaceutical composition contains 800 mg to 2500 mg of LR12 peptide, preferably 1200 mg to 2000 mg of LR12 peptide. In other words, in some embodiments, the pharmaceutical composition contains 800 mg to 2500 mg of LR12 peptide per unit dose, preferably 1200 mg to 2000 mg of LR12 peptide per unit dose. An example of a unit dose is a vial.

[0099] In some embodiments, this pharmaceutical composition is available in doses of approximately 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, and 16 Contains 50 mg, approximately 1700 mg, approximately 1750 mg, approximately 1800 mg, approximately 1850 mg, approximately 1900 mg, approximately 1950 mg, approximately 2000 mg, approximately 2050 mg, approximately 2100 mg, approximately 2150 mg, approximately 2200 mg, approximately 2250 mg, approximately 2300 mg, approximately 2350 mg, approximately 2400 mg, approximately 2450 mg, or approximately 2500 mg of LR12 peptide. In other words, in some embodiments, the pharmaceutical composition is administered in doses of approximately 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, and 16 Contains LR12 peptide in amounts of 00 mg, approximately 1650 mg, approximately 1700 mg, approximately 1750 mg, approximately 1800 mg, approximately 1850 mg, approximately 1900 mg, approximately 1950 mg, approximately 2000 mg, approximately 2050 mg, approximately 2100 mg, approximately 2150 mg, approximately 2200 mg, approximately 2250 mg, approximately 2300 mg, approximately 2350 mg, approximately 2400 mg, approximately 2450 mg, or approximately 2500 mg. Vials are an example of unit doses.

[0100] This specification provides vials containing a lyophilized pharmaceutical composition or purified LR12 peptide material disclosed herein. In some embodiments, the vial contains about 400 to about 1200 mg of the lyophilized pharmaceutical composition or purified LR12 peptide material, preferably LR12 peptide. In some embodiments, the vial contains about 400 to about 1200 mg of the lyophilized pharmaceutical composition or LR12 peptide, preferably LR12 peptide reconstituted with 10 mL of aqueous solution as disclosed herein. In view of this disclosure, a person skilled in the art can calculate the amount of LR12 pharmacokinetic active ingredient to be included in the purified LR12 peptide material.

[0101] This specification provides vials containing the pharmaceutical compositions or purified LR12 peptide substances disclosed herein. In some embodiments, a vial containing the powdered pharmaceutical compositions or purified LR12 peptide substances disclosed herein contains about 800 mg to about 2500 mg of LR12 peptide, preferably about 1200 mg to about 2000 mg of LR12 peptide. In some embodiments, a vial containing the liquid pharmaceutical compositions disclosed herein contains about 800 mg to about 2000 mg of LR12 peptide, preferably about 1200 mg to about 2000 mg of LR12 peptide reconstituted with 10 mL of aqueous solution.

[0102] This specification provides syringes containing the pharmaceutical compositions or purified LR12 peptide substances disclosed herein. In some embodiments, the syringe contains about 400 to about 1200 mg of the LR12 peptide disclosed herein, diluted with 50 mL of aqueous solution. In some embodiments, the syringe contains about 800 to about 2500 mg of the LR12 peptide disclosed herein, preferably 1200 to about 2000 mg of the LR12 peptide disclosed herein, diluted with 50 mL of aqueous solution.

[0103] This specification provides pharmaceutically acceptable solutions containing approximately 400 to 1200 mg of LR12 peptide dissolved in approximately 40 to 60 mL of aqueous solution.

[0104] This specification provides pharmaceutically acceptable solutions having osmolality concentrations as close as possible to 300 mOsm / kg (maximum 500 mOsm / kg).

[0105] Process for producing purified LR12 peptide substance and compositions containing the same The manufacturing process disclosed herein can further reduce the formation of cysteine ​​dimers while maintaining solubility.

[0106] This specification describes a method or process for purifying LR12 (i.e., preparing a purified LR12 peptide substance), wherein the process is: i. Steps for preparing crude LR12 in a solid phase, ii. A step to obtain a first peptide solution containing crude LR12, iii. A step of purifying the fraction by packing the first peptide solution into a column equipped with ion exchange chromatography (IEC), iv. A step of filling the fraction into a column equipped with reverse-phase chromatography (RPC) to produce a pooled fraction. v. A step of preparing a solution containing 5 mM to 25 mM of at least one solubility enhancer, preferably arginine, to be added to the pool fraction during step vi, and vi. A process of concentrating and purifying the pooled fraction to a concentrated solution of at least 40 g / L by ultrafiltration / diafiltration, typically using a membrane with a density of 150–300 Daltons. A method or process including this is provided.

[0107] In some embodiments, the process for preparing a purified LR12 peptide is as follows: i. A step to synthesize LR12 peptide on a solid phase and obtain a crude LR12 composition. ii. A step to obtain a first peptide solution containing a crude LR12 composition, iii. A step to purify the first peptide solution by ion exchange column chromatography (IEC) to obtain a first pre-purified product of LR12. iv. A step of further purifying the pre-purified LR12 by reverse-phase high-performance liquid chromatography (HPLC) to obtain an HPLC pool fraction containing a second pre-purified LR12. v. A step of preparing a solution containing 5 to 25 mM of at least one solubility enhancer, preferably arginine, to be added to the pool fraction during step vi. vi. A step of concentrating the pooled fraction to a concentrated solution of at least 120 g / L using membrane dialysis filtration or ultrafiltration, typically with a membrane of 150-300 Daltons, and vii. A step to obtain purified LR12 peptide material by filtering and freeze-drying the concentrated solution obtained in step vi. Includes.

[0108] In some embodiments, the process of concentrating the pool fraction includes a two-step concentration process.

[0109] In some embodiments, the HPLC pool fraction containing a second pre-purified LR12 is cooled to a temperature of 15°C or below and / or bubbling with nitrogen.

[0110] According to some embodiments, the purified LR12 peptide substance obtained in step vii contains at least one solubility enhancer, preferably arginine, in an amount ranging from 2 to 5% w / w by weight relative to the total weight of the purified LR12 peptide substance.

[0111] In some embodiments of the methods provided herein, a concentrated LR12 solution is filtered and lyophilized to obtain a purified LR12 peptide substance having a peptide purity of at least 94%, 95%, 96%, or 97%. In some embodiments of the methods provided herein, a concentrated LR12 solution is filtered and lyophilized to obtain a purified LR12 peptide substance having a peptide purity of at least 95%.

[0112] In some embodiments of the methods disclosed herein, a buffer with a pH of 5-6 is used for IEC, RPC, and diafiltration, and the formation of LR12 dimers is reduced by adjusting the pH of the pooled fraction from the RPC to 5-6, the solution is cooled to below 15°C, and the solution is bubbled with nitrogen.

[0113] By controlling the manufacturing timing using the disclosed pharmaceutical product formulations, it becomes possible to reproducibly control the dimer content in the final product.

[0114] The improved manufacturing process provided in this invention allows for acceptable and reproducible control of the dimer content in the final pharmaceutical product by controlling the timing between steps. To optimize the feasibility of administration in a hospital setting, the pharmaceutical product is highly concentrated and has suitable stability.

[0115] Accordingly, this specification provides a method for producing the TREM-1 peptide inhibitors and compositions described herein (e.g., substances and compositions containing LR12). This production method reduces the formation of LR12 dimers. The timing between solubilization of the active pharmaceutical ingredient, addition of excipients (pH adjustment, arginine), vial filling, and freeze-dryer temperature adjustment directly affects dimer formation and, consequently, the dimer content of the pharmaceutical product. Strict control of the production timing using the formulations provided herein enables acceptable and reproducible control of the dimer content in the final product.

[0116] The method provided herein controls pH to limit dimer formation during the manufacturing process and after product reconstitution, thereby achieving the desired target concentration (e.g., 24 mg / mL). To optimize the feasibility of administration in a hospital setting, the pharmaceutical product is highly concentrated and possesses suitable stability.

[0117] To facilitate the handling of LR12 pharmaceutical products in clinical settings, the number of vials reconstituted per administration is reduced. Therefore, this specification provides a high-dose (HD) formulation of LR12 (nangibotide). Using excipients such as arginine, lysine, or histidine and sodium citrate, the concentration of the LR12 active pharmaceutical ingredient (i.e., purified LR12 peptide substance) in the lyophilized solution is increased up to fivefold, reaching 200 mg of LR12 / mL without affecting the formation of LR12 dimers. A formulation with a concentration of 120 mg of LR12 / mL in the lyophilized solution has been developed. This formulation is referred to as the LR12 high-dose (HD) pharmaceutical product (1200 mg / vial).

[0118] This specification describes a process for preparing the pharmaceutical compositions described herein, a) Preferably, the process of supplying the purified LR12 peptide substance described herein by performing steps i) to vii) in accordance with the process described above. b) Purified LR12 peptide substance, at least one solubility enhancer, preferably arginine, lysine, or histidine, more preferably arginine or lysine, even more preferably arginine, and at least one pharmaceutically acceptable buffer, preferably citrate buffer, even more preferably sodium citrate buffer, preferably · At least one solubility enhancer in an amount of 20.0% to 30.0%, preferably 22.0% to 28.0%, more preferably 22.3% to 27.6%, and even more preferably about 24.5%, relative to the dry weight of the purified LR12 peptide substance, preferably arginine, lysine, or histidine, more preferably arginine, or lysine, even more preferably arginine, and / or • At least one pharmaceutically acceptable buffer, preferably citrate buffer, more preferably sodium citrate buffer, in an amount of 3.0% to 7.0%, preferably 4.0% to 6.0%, more preferably 4.5% to 5.5%, and even more preferably about 4.8% by weight relative to the dry weight of the purified LR12 peptide substance, wherein the buffer is preferably citrate buffer. The process of mixing with and thereby obtaining a bulk solution, c) Optionally, adjust the pH of the bulk solution to pH 5.5, typically with HCl. d) A step of freeze-drying the bulk solution to obtain a freeze-dried powder composition of the pharmaceutical composition, and e) Optionally, an aqueous medium selected from water and physiological saline, preferably physiological saline containing 0.9% sodium chloride by weight relative to physiological saline, is added to obtain an aqueous solution of the pharmaceutical composition. A process including this is provided.

[0119] Advantageously, step e) is executed immediately.

[0120] In this specification, • Instead of the previously used 50 mL vials, fill them into 20 mL vials (which are more suitable for the pre-freeze filling volume (10 mL)). • Use physiological saline (preferably 0.9% w / w NaCl for both reconstitution and dilution), and Dispense the prescribed dose of 1 mg / kg / h, which is administered by continuous intravenous infusion, using a syringe (50 mL) and an automatic injector pump. A method for preparing LR12 drugs is provided.

[0121] In this specification, • Instead of the previously used 50 mL vials, fill them into 20 mL vials (which are more suitable for the pre-freeze filling volume (10 mL)). • Use physiological saline (preferably 0.9% w / w NaCl for both reconstitution and dilution), and Fill the syringe (50 mL) and automatic injector pump with LR12 suitable for administration at a dose of 1 mg / kg / h by continuous intravenous infusion. A method for preparing an LR12 pharmaceutical product (i.e., the form for use, also called a liquid parenteral formulation) is provided.

[0122] These processes can reduce the total amount of fluid administered per day (for example, two syringes (50 mL) per patient in the intensive care unit per day).

[0123] Peptide inhibitors and compositions for their therapeutic use Another aspect of this disclosure is a purified substance of the LR12 peptide described herein or a pharmaceutical composition described herein for use as a medicine.

[0124] Further aspects of this disclosure are purified LR12 peptides or pharmaceutical compositions described herein for use in the treatment of diseases selected from inflammatory diseases or disorders, cardiovascular diseases, disorders or conditions and infections in subjects requiring such treatment.

[0125] In some embodiments, the disease being treated is an inflammatory disease or disorder. Examples of inflammatory diseases or disorders include septic shock, sepsis, severe sepsis, systemic inflammatory response syndrome (SIRS), sepsis-associated organ dysfunction, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), acute kidney injury (AKI), ischemia-reperfusion injury, pancreatitis, pneumonia, endotoxemia, and hemorrhagic shock.

[0126] In some embodiments, the inflammatory disease or disorder is selected from septic shock, sepsis, severe sepsis, SIRS, ARDS, hemorrhagic shock, ischemia-reperfusion injury, and pancreatitis.

[0127] In some embodiments, the inflammatory disease or disorder is an acute inflammatory disease or disorder. Examples of acute inflammatory diseases include, but are not limited to, septic shock, sepsis, SIRS, ARDS, AKI, pancreatitis, and hemorrhagic shock.

[0128] In some embodiments, the inflammatory disease or disorder is septic shock, sepsis, or SIRS.

[0129] SIRS is characterized by systemic inflammation and extensive tissue damage. Clinically, SIRS is defined by the following four criteria: fever greater than 38.0°C or hypothermia less than 36.0°C, tachycardia greater than 90 beats / min, tachypnea greater than 20 breaths / min, and 12 × 10⁻¹⁰ 9 Leukocytosis exceeding / L or 4×10 9 SIRS is defined as meeting at least two of the following criteria: leukopenia of less than 1 / L (Bone et al., Chest. June 1992; 101(6):1644-55). SIRS may occur as a reaction to infectious or non-infectious nonspecific injuries. Examples of non-infectious injuries include, but are not limited to, trauma, burns, pancreatitis, autoimmune disorders, and surgery. Examples of infectious injuries include bacterial infections (e.g., respiratory, abdominal, and urinary tract infections (UTIs)), fungal infections (e.g., respiratory infections), and viral infections (e.g., respiratory infections).

[0130] Sepsis is defined as a life-threatening organ dysfunction caused by dysregulation of the human subject's response to infection (Singer et al., JAMA. February 23, 2016; 315(8):801-10). Patients with sepsis can be clinically identified as those with confirmed or suspected infection and those suffering from organ dysfunction. Organ dysfunction in human subjects can be identified using organ dysfunction scores, particularly when human subjects are admitted to the ICU or emergency center. Examples of organ dysfunction scores, but not limited to, include the SOFA (Sequential Organ Failure Assessment) score, qSOFA (Rapid SOFA) score, MODS (Multiple Organ Dysfunction Score), P-MODS (Pediatric Multiple Organ Dysfunction Score), and LODS (Logistic Organ Dysfunction System).

[0131] Septic shock is defined as a subtype of sepsis, and particularly severe cardiovascular, cellular, and metabolic abnormalities in septic shock carry a higher risk of death than sepsis alone. Therefore, sepsis encompasses septic shock. Patients with septic shock can be clinically identified as those who have sepsis and have (i) persistent hypotension requiring vasopressors to maintain mean arterial pressure above 65 mmHg despite adequate fluid resuscitation, and (ii) serum lactate levels greater than 2 mmol / L (18 mg / dL) (as defined in Singer et al., JAMA. February 23, 2016; 315(8):801-10).

[0132] In some embodiments, the infection causing dysregulation of the response in a subject suffering from septic shock, sepsis, or SIRS is a bacterial, fungal, parasitic, or viral infection. Examples of infections, but not limited to, include respiratory infections, abdominal infections, and urinary tract infections (UTIs).

[0133] In some embodiments, the disease being treated is a cardiovascular disease, disorder, or condition. Examples of cardiovascular diseases, disorders, or conditions include myocardial infarction, acute myocardial infarction, cerebral infarction, ischemia, coronary heart disease, acute coronary syndrome, stroke, aneurysm, stable angina, exertional angina, cardiomyopathy, hypertensive heart disease, chronic heart failure, acute heart failure, cor pulmonale, cardiac arrhythmias, inflammatory heart diseases (such as endocarditis and myocarditis), vasculitis, peripheral artery disease, SIRS-related myocardial and / or vascular dysfunction, and atherosclerosis.

[0134] In some embodiments, the cardiovascular disease, disorder, or condition is selected from myocardial infarction, cerebral infarction, acute myocardial infarction, ischemia, coronary heart disease, acute coronary syndrome, stroke, aneurysm, stable angina, exertional angina, cardiomyopathy, hypertensive heart disease, chronic heart failure, acute heart failure, cor pulmonale, cardiac arrhythmia, inflammatory heart disease, peripheral artery disease, SIRS-related myocardial and / or vascular dysfunction, and atherosclerosis.

[0135] In some embodiments, the disease, disorder, or condition being treated is an infectious disease. As used herein, the term “infectious disease” refers to a pathological condition or disorder resulting from an infection. Examples of infectious diseases include bacterial diseases (or bacterial infections), viral diseases (or viral infections), fungal diseases (or fungal infections), and parasitic diseases (or parasitic infections), which are infections caused by bacteria, viruses, fungi, and parasites, respectively. An example of a bacterial disease is Escherichia coli infection. An example of a viral disease is SARS-CoV-2 infection (also known as coronavirus disease 2019 or COVID-19). In some embodiments, the infectious disease is COVID-19.

[0136] In some embodiments, the diseases to be treated are selected from cardiovascular diseases, including septic shock, sepsis, severe sepsis, SIRS, ARDS, hemorrhagic shock, ischemia-reperfusion injury and pancreatitis, preferably myocardial infarction, cerebral infarction, acute myocardial infarction, ischemia, coronary heart disease, acute coronary syndrome, stroke, aneurysm, stable angina, exertional angina, cardiomyopathy, hypertensive heart disease, chronic heart failure, acute heart failure, cor pulmonale, cardiac arrhythmias, inflammatory heart diseases (such as endocarditis and myocarditis), peripheral artery disease, SIRS-related myocardial and / or vascular dysfunction and atherosclerosis, as well as infectious diseases such as COVID-19.

[0137] Preferably, the purified LR12 peptide or pharmaceutical composition described herein is for administration by infusion or injection, preferably intravenous infusion or intravenous injection. In some embodiments, the purified LR12 peptide or pharmaceutical composition described herein is for administration by continuous infusion, preferably continuous intravenous infusion.

[0138] In some embodiments, the purified LR12 peptide or pharmaceutical composition described herein is intended for administration at doses (or rates) in the range of about 0.1 mg / kg / h to about 3 mg / kg / h (mg of LR12 per kg of body weight per hour), preferably about 0.3 mg / kg / h to about 3 mg / kg / h. In some embodiments, the pharmaceutical composition or purified LR12 peptide substance is intended for intravenous administration at rates in the range of 0.3 to 3.0 mg / kg / hr (mg of LR12 per kg of body weight per hour).

[0139] In fact, the high LR12 peptide content in the pharmaceutical composition (pharmaceutical product) according to the present invention enables rapid administration in the range of, for example, 0.3 to 3.0 mg / kg / hr (mg of LR12 per kg of the subject's body weight per hour). This rapid administration improves patient comfort by limiting the administration time and reduces the risk of fluid overload.

[0140] In some embodiments, the purified LR12 peptide or pharmaceutical composition described herein is intended for administration at doses (or rates) ranging from about 0.1 mg / kg / h to about 1 mg / kg / h (mg per kg of body weight per hour), preferably from about 0.3 mg / kg / h to about 1 mg / kg / h.

[0141] In some embodiments, the purified LR12 peptide or pharmaceutical composition described herein is intended for administration at doses (or rates) of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / kg / h.

[0142] In some embodiments, the purified LR12 peptide substance or pharmaceutical composition described herein is for administration of at least 24, 48, 72, 96, or 120 hours, preferably for continuous administration. In some embodiments, the purified LR12 peptide substance or pharmaceutical composition described herein is for administration of up to 7, 6, 5, 4, or 3 days, preferably for continuous administration. In some embodiments, the purified LR12 peptide substance or pharmaceutical composition described herein is for administration of up to 5 days, preferably for continuous administration.

[0143] In some embodiments, the subject is a mouse, rat, guinea pig, rabbit, dog, pig, or non-human primate. In some embodiments, the subject is a human. In some embodiments, the subject is an adult human. In some embodiments, the subject is an adolescent human. In some embodiments, the subject is a pediatric human.

[0144] In some embodiments, the subject is suffering from shock, hypotensive shock, or distributive shock (which impair organ function and generally refer to decreased or insufficient perfusion accompanied by a decrease in arterial blood pressure). Accordingly, in some embodiments, the subject has previously received or is currently receiving fluid therapy, i.e., treatment aimed at restoring and / or maintaining normal fluid volume and composition, particularly with respect to water and electrolyte balance. Accordingly, fluid therapy aims to correct and / or prevent fluid volume and / or electrolyte deficiencies. In some embodiments, the subject is vulnerable to hypertonic fluids and / or fluid overload.

[0145] In some embodiments, subjects suffering from inflammatory diseases or disorders, particularly septic shock, sepsis, or SIRS, after being administered the purified LR12 peptide or pharmaceutical composition described herein, • Recovery from hypotensive shock, defined as not receiving any vasopressor therapy for at least 24 hours (i.e., not needing to resume vasopressor therapy within 24 hours of the end of vasopressor therapy). • Reduction in severity scores used to assess the severity and / or prognosis of the disease upon admission to the ICU or emergency center, such as the APACHE II (Acute Physiology and Chronic Health Evaluation) score, APACHE III (Acute Physiology and Chronic Health Evaluation) score, APACHE IV (Acute Physiology and Chronic Health Evaluation) score, SAPS II (Simplified Acute Physiology Score) score, or SAPS III (Simplified Acute Physiology Score) score. • Decrease in organ dysfunction scores, such as SOFA score, qSOFA score, MODS, P-MODS, or LODS, which are used to assess the presence of organ dysfunction in subjects upon admission to the ICU or emergency center. Preferably, a decrease in the SOFA score and / or qSOFA score based on the SOFA score and / or qSOFA score assessed when admitted to the ICU or emergency center or before the initiation of administration of the purified LR12 peptide substance or pharmaceutical composition described herein. • Decreased need for cardiovascular support, for example, reduced use of vasopressor therapy. • Reduced need for respiratory support, for example, reduced use of invasive ventilation (IMV). • A decrease in the need for renal support, such as a decrease in the use of continuous or non-continuous renal replacement therapy, including RRT (e.g., dialysis). • Reduced risk of reinfection or no reinfection. • Reduced risk of readmission or no readmission. • Increased probability of survival, • Reduced risk of sepsis-related deaths, • Reduction of the risk of death from any cause • Reduced risk of post-sepsis or post-shock morbidity. • Improvements in quality of life, particularly after sepsis or shock, which can be assessed by evaluating the estimated survival years and quality-adjusted life years (QALYs) from EQ5D (for example, a health-related quality of life (HRQoL) score may be calculated from EQ-5D-5L and converted to a utility value). A treatment is considered successful if at least one of the following is observed. The above parameters for evaluating treatment success and disease improvement can be easily measured using routine procedures familiar to physicians.

[0146] This specification also provides methods for treating diseases or disorders, particularly inflammatory diseases or disorders, cardiovascular diseases, disorders or conditions or infections, using the TREM-1 peptide inhibitors and compositions described herein (e.g., substances and compositions containing LR12). This specification also provides methods for treating septic shock in subjects requiring the use of the TREM-1 peptide inhibitors and compositions described herein. This specification also provides methods for treating sepsis in subjects requiring the use of the TREM-1 peptide inhibitors and compositions described herein.

[0147] This specification provides a method for treating an inflammatory disease or disorder, cardiovascular disease, disorder or condition or infection described herein in a subject requiring such treatment, the method comprising administering to the subject a purified LR12 peptide or a pharmaceutical composition described herein.

[0148] This specification provides a method for treating septic shock or sepsis in a subject requiring such treatment, comprising intravenously administering a pharmaceutically acceptable composition described herein to the subject at a rate of about 0.3 mg / kg / hr to about 3.0 mg / kg / hr (mg of LR12 per kg of the subject's body weight per hour). This specification also provides a method for relieving symptoms of septic shock or sepsis in a subject requiring such treatment, comprising intravenously administering a pharmaceutically acceptable composition described herein to the subject at a rate of about 0.3 to about 3.0 mg / kg / hr. In some embodiments, the rate is about 1.0 mg / kg / hr (mg of LR12 per kg of the subject's body weight per hour).

[0149] Symptoms of septic shock or sepsis that are relieved by the methods or treatments described herein include sepsis-induced cardiovascular dysfunction, organ failure, inflammation, fever, and hypothermia.

[0150] In some embodiments, the subject has septic shock or sepsis induced by a bacterial, fungal, parasitic, or viral infection.

[0151] This specification also provides the use of purified LR12 peptides or pharmaceutical compositions described herein in the manufacture of drugs for the treatment of diseases described herein in subjects requiring such use.

[0152] This specification further provides pharmaceutical compositions described herein for the treatment or use in the treatment of diseases described herein in subjects requiring such treatment.

[0153] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. However, all documents or parts of documents cited herein, including patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for any purpose. If one or more incorporated documents or parts of documents define a term that conflicts with the definitions of terms in this application, the definitions provided in this disclosure shall prevail. However, no reference herein to any reference, article, publication, patent, patent gazette, or patent application shall be construed, and should not be construed, as an acknowledgment or any form of suggestion that they constitute valid prior art in any country of the world or form part of common knowledge. [Examples]

[0154] The present disclosure is further illustrated by the following embodiments, which are included for illustrative purposes only and are not intended to limit the scope of the present disclosure. Those skilled in the art will understand that numerous and varied modifications can be made without departing from the spirit of the present disclosure to obtain essentially similar results.

[0155] Example 1: Lack of biological activity of LR12 dimer material and method Since neutrophils generate reactive oxygen species (ROS) in the presence of LPS (lipopolysaccharide) after activation of the TREM-1 pathway, this example uses neutrophils to evaluate TREM-1 activation and the biological activity of LR12 dimers on the TREM-1 pathway. ROS generation induced by TREM-1 activation and its regulation by LR12 can be quantified by flow cytometry. Any reduction in ROS generation by LR12 (LPS + LR12 conditions) is converted to mean fluorescence intensity (MFI) compared to the ROS generation observed after TREM-1 activation in the presence of LPS (LPS conditions).

[0156] Primary human neutrophils were isolated from the peripheral blood of healthy donors by immunomagnetic negative cell sorting using EasySep® Human Monocyte / Neutrophil Isolation Kits (StemCell, Canada) according to the manufacturer's instructions. Purity was assessed by flow cytometry. Cells were suspended in RPMI1640 medium containing GlutaMAX supplemented with 10% FCS, 25 mM HEPES, 100 U / mL penicillin, and streptomycin before stimulation. Primary human neutrophils were incubated under quiescent conditions (also known as control conditions or ctrl 2) or with 100 ng / mL of LPS derived from E. coli serotype 0127:B8 (Sigma-Aldrich), with and without LR12 or LR12 dimers (dimers) at the indicated concentrations.

[0157] ROS generation was quantified using a fluorescent substrate (DCFDA or 2',7'-dichlorofluorescein diacetate), a chemically reduced form of fluorescein used as an indicator of intracellular ROS presence. Non-fluorescent DCFDA is converted to highly fluorescent 2',7'-dichlorofluorescein (DCF) by cleavage and oxidation of the acetate group by intracellular esterases. 2.5 × 10⁻⁶ 5 Isolated human neutrophils were incubated with 5 μM DCFDA at 37°C and 5% CO2 for 2 hours, with and without 100 ng / mL LPS, and with and without the indicated concentrations of LR12 or LR12 dimers (dimers). Data were obtained using flow cytometry. Results are expressed as mean fluorescence intensity (MFI).

[0158] result As shown in Figure 1, LPS induced a significant increase in ROS production by human primary neutrophils after 2 hours of stimulation. LR12 monomers were able to dose-dependently reduce LPS-induced ROS release. Surprisingly, LR12 dimers did not exhibit TREM-1 inhibitory activity. In fact, LR12 dimers were unable to reduce ROS production by LPS-activated neutrophils.

[0159] These results indicate that no pharmacological inhibition of TREM-1 can be induced, despite the fact that the LR12 dimer consists of two LR12 peptides covalently linked by a disulfide bond via a cysteine ​​at position 11.

[0160] Example 2: Preparation of purified LR12 peptide substance (active pharmaceutical ingredient) Stepwise solid-phase synthesis of LR12 peptide (15 mol) was performed using Fmoc / tBu chemistry in a 270 L SPPS reactor. A series of acylation reactions were carried out in DMF at 20°C ± 3°C with stirring at 30 ± 20 rpm using appropriate Nα-Fmoc-protected amino acids that were carboxylated with Oxyma, DITU, and DIC. After each amino acid coupling, the capping step was repeated by acylation with acetic anhydride. After the completion of the series of acylation reactions, the peptide resin was washed with IPA, filtered, and vacuum-dried.

[0161] The resin-bound peptide is added to the cleavage reactor at a rate such that the product temperature does not exceed 25°C. Cleavage of the peptidyl resin and deprotection of the side chains are performed by treatment with a prepared TFA / DTT / TIS / H2O solution at 20°C ± 5°C for 3-4 hours. After cooling the cleavage solution to 0°C ± 5°C, precipitation is carried out with MBTE and n-hexane. The precipitate is filtered, washed with MBTE, and vacuum dried.

[0162] The dried crude LR12 peptide is resolubilized in aqueous ammonia, its pH is adjusted to 6.5-7.0, and after stirring at 50-55°C for 3 hours, the used resin is removed by filtration. The dissolved crude solution is purified by ion exchange chromatography (IEC) on a 450 mm diameter column (2.5 cm / min) with a column volume (CV) of 30 g / L or less, followed by reverse-phase chromatography (RPC) on a 450 mm diameter column (4.7 cm / min) with a CV of 15-22 g / L. These fractions are eluted with NH4OAc / acetonitrile (MeCN), recovered in water, and stored under refrigeration (2-8°C). The pooled fraction is concentrated (in two stages) and purified by ultrafiltration / dialysis filtration using a 300 Dalton membrane. After the first concentration (approximately 65 g / L), 15 mM arginine is added. Next, this solution is further processed to obtain a concentrated solution of 170 g / L. The bulk solution is nitrogen-inactivated (by cooling to below 15°C) to maintain a low oxygen content.

[0163] The concentrated LR12 solution was immediately filtered and freeze-dried to obtain a purified LR12 peptide substance containing i) 85% peptide by weight relative to the total weight of the purified LR12 peptide substance, and ii) 4.7% arginine by weight relative to the total weight of the purified LR12 peptide substance. The amount of LR12 dimer was 0.7% by weight relative to the total weight of the peptide content.

[0164] Example 3: Preparation of the pharmaceutical composition (pharmaceutical product) The pH of the active pharmaceutical ingredient (API) in Example 2 is maintained at 5-6 during the formulation process. The timing between solubilization of the API (containing the active pharmaceutical ingredient (API)), addition of excipients (pH adjuster, arginine), filling of the vial, and temperature adjustment of the freeze-dryer is set to less than 24 hours. Nitrogen inactivation of the bulk solution is performed by bubbling nitrogen into the solution to reduce the oxygen content to less than 5 ppm. At the time of administration, the pharmaceutical composition (pharmaceutical product powder in a 20 mL vial) is reconstituted with 9.5 mL of NaCl (0.9% w / w), and then further diluted with NaCl (0.9% w / w) in a 50 mL syringe. The resulting LR12 solution has a concentration of 24 mg / mL and an osmolality of 380 mOsm / kg.

[0165] The quantitative composition of this pharmaceutical product is shown in Table 1. [Table 1]

[0166] The LR12 dimer content, measured by analytical reverse-phase HPLC-UV and expressed as a weight percentage of the LR12 peptide weight in each process step, is shown in Table 2 below. [Table 2]

[0167] Comparative Examples 4-9: Preparation of Comparative Pharmaceutical Compositions Comparative Example 4a (NaCl) The purified LR12 peptide (Example 2) was formulated using the following excipients: 50 mM sodium citrate (pH 5.5) and 50 mM NaCl. The concentration of the bulk solution (before lyophilization) was 10 mg / mL (due to the solubility limit of LR12).

[0168] Comparative example 4b (NaCl, pH 6.5): The purified LR12 peptide (Example 2) was formulated using the following excipients: 50 mM sodium phosphate (pH 6.5) and 50 mM NaCl. The concentration of the bulk solution (before lyophilization) was 20 mg / mL (due to the solubility limit of LR12).

[0169] Comparative Example 5 (Mannitol): The purified LR12 peptide (Example 2) was formulated using the following excipients: 50 mM sodium citrate (pH 5.5) and 200 mM mannitol. The concentration of the bulk solution (before lyophilization) was 10 mg / mL (due to the solubility limit of LR12).

[0170] Comparative Example 6 (Betaine): The purified LR12 peptide (Example 2) was formulated using the following excipients: 50 mM sodium citrate (pH 5.5) and 150 mM betaine. The concentration of the bulk solution (before lyophilization) was 10 mg / mL (due to the solubility limit of LR12).

[0171] Comparative Example 7a (Lysine): The purified LR12 peptide (Example 2) was formulated using the following excipients: 50 mM sodium citrate (pH 5.5), 50 mM lysine, and 50 mM NaCl. The concentration of the bulk solution (before lyophilization) was 40 mg / mL (due to the solubility limit of LR12).

[0172] Comparative Example 7b (Arginine): The purified LR12 peptide (Example 2) was formulated using the following excipients: 50 mM sodium citrate (pH 5.5) and 50 mM arginine. The concentration of the bulk solution (before lyophilization) was 40 mg / mL (due to the solubility limit of LR12).

[0173] Comparative Example 8 (Sodium citrate, arginine, pH 5.5): The purified LR12 peptide (Example 2) was formulated using the following excipients: 25 mM sodium citrate (pH 5.5), 225 mM arginine, and 65 mM HCl. The bulk solution (before lyophilization) had a concentration of 150 mg / mL and an osmolality of 512 mOsm / kg.

[0174] A series of comparative examples 9 (90-420 mM arginine, pH 5.5): A formulation using purified LR12 peptide at gradually increasing concentrations (Example 2) and the following excipients at gradually increasing concentrations: arginine 90-420 mM, with HCl used only to adjust the pH to 5.5.

[0175] The results regarding the solubility, LR12 stability, and osmotic pressure of the obtained pharmaceutical compositions are shown in Table 3 below. [Table 3]

[0176] Of all the different formulations tested, citrate or phosphate buffer alone yielded a maximum LR12 solubility of only 20 mg / mL (Comparative Examples 4a and 4b). The use of excipients such as mannitol or betaine did not increase the solubility of LR12 (Examples 5 and 6). The solubility of LR12 could be increased to 40 mg / mL with both 50 mM arginine and lysine (Comparative Examples 7a and 7b). When the ruginine concentration was increased from 90 mM to 420 mM (Example 9), not only did the solubility of LR12 increase, but the increase in dimers in the formulation also significantly increased, even though the pH was maintained in the same manner as in the pharmaceutical composition of the present invention.

[0177] Therefore, the combination of arginine and citrate buffer according to the present invention not only increases the solubility of LR12, but also allows for the use of the smallest possible amount of excipients and the acquisition of a product with the lowest possible dimer content while maintaining the osmotic pressure of the resulting pharmaceutical composition at less than 500 mOsm / kg.

[0178] Comparative Example 11: Comparative Process for the Preparation of LR12 Active Pharmaceutical Ingredient Composition The dried crude LR12 peptide (Example 2) was resolubilized in aqueous ammonia, the pH was adjusted to 6.5-7.0, and the mixture was stirred at 50-55°C for 3 hours. The used resin was then removed by filtration. The dissolved crude solution was purified by ion exchange chromatography (IEC) on a 450 mm diameter column (2.5 cm / min) with a CV load of ≤30 g / L, followed by reverse-phase chromatography (RPC) at room temperature (-25°C) on a 450 mm diameter column (4.7 cm / min) with a CV load of 15-22 g / L. The fraction was eluted with NH4OAc / MeCN and recovered in water without nitrogen inactivation. The pooled fraction was concentrated and purified by ultrafiltration / dialysis filtration using a 300 Dalton membrane to obtain a concentrated solution of 15 g / L. The concentrated LR12 solution was filtered and freeze-dried to obtain the purified LR12 peptide.

[0179] The LR12 dimer content for all process steps is shown in Table 4. [Table 4]

[0180] The results of Comparative Example 11 indicate that the process steps according to the present invention at a cooling temperature of less than 15°C as shown in Example 2, particularly the N2 inactivation and / or reverse-phase, ultrafiltration / dialysis filtration steps, may further inhibit the formation of LR12 dimers in the purified LR12 peptide.

[0181] Examples 12-18: Preparation of pharmaceutical compositions and comparative pharmaceutical compositions according to the present invention material and method

[0182] A pharmaceutical composition was prepared using a purified LR12 peptide (Example 2) with gradually increasing concentrations of LR12 peptide, gradually increasing amounts of amino acids (arginine or lysine) at 15% to 26% by weight relative to the dry weight of the purified LR12 peptide, and gradually increasing amounts of sodium citrate buffer at 5% to 7% by weight relative to the dry weight of the purified LR12 peptide. The resulting pharmaceutical composition was a lyophilized powder (pharmaceutical product in powder form) containing 1200 mg to 2000 mg of LR12 peptide (per vial).

[0183] To prepare these pharmaceutical compositions in powder form, buffers were first prepared according to Table 5. Purified LR12 peptide material and at least one amino acid selected from arginine and lysine were added to the prepared buffer. The pH during the formulation process was adjusted to obtain a bulk solution (before the freeze-drying step) of approximately 5.5. To further limit dimer formation during the manufacturing process, nitrogen inactivation of the bulk solution was performed to obtain an oxygen content of less than 5 ppm.

[0184] The prepared bulk solution was visually inspected and filtered using a 0.2 μm disposable filter. The filtered bulk solution was then filled into 20 mL vials. The filling volume varied from 10 mL to 15 mL depending on the LR12 peptide concentration in each bulk solution (80 mg / mL to 200 mg / mL). The vials containing the filtered bulk solution were then freeze-dried to obtain a pharmaceutical composition (a pharmaceutical product in storage form) in powder form.

[0185] The pharmaceutical composition according to the present invention is • LR12 (120 mg / mL) & Arginine (155 mM) LR12 (120 mg / mL) & Lysine (190 mM) • LR12 (120 mg / mL) & Arginine (190 mM) • LR12 (200 mg / mL) & Arginine (300 mM) LR12 (200 mg / mL) & Lysine (300 mM) That is the case.

[0186] Pharmaceutical compositions not related to the present invention (comparative pharmaceutical compositions) are: ·LR12 (80 mg / mL) & Arginine (80 mM), and • LR12 (100 mg / mL) & Arginine (120 mM) That is the case.

[0187] The materials and methods are summarized in Table 5 below. [Table 5]

[0188] result

[0189] These results are shown in Table 6 below. [Table 6]

[0190] These results indicate the presence of precipitates in two comparative examples where the amount of amino acids was less than 20% by weight relative to the dry weight of the purified LR12 peptide material. These precipitates, observed in the bulk solution before filtration, are not suitable for further processing. Therefore, it is not possible to obtain a pharmaceutical composition in powder form (= pharmaceutical product in storage form) when the amount of amino acids is less than 20% by weight relative to the dry weight of the purified LR12 peptide material.

[0191] These results indicate that, when reconstituted with 9.5 mL of NaCl (0.9% w / w), the pharmaceutical composition (pharmaceutical product powder in a 20 mL vial) contains less than 5.0% w / w of LR12 dimers, less than 1.2% by weight of Des-Gln-LR12, and less than 1.0% by weight of Met(O)12-LR12 (by weight relative to peptide content). The osmolality has been confirmed to be below the acceptable limit of 500 mOsm / kg.

[0192] Therefore, a pharmaceutical composition according to the present invention in powder form having arginine, lysine, or histidine in an amount ranging from 20% to 30% by weight relative to the dry weight of the purified LR12 peptide substance can be obtained to have LR12 dimers of less than 5.0% w / w by weight relative to the peptide content, high amounts of LR12 peptide of 1200 mg and 2000 mg, and an osmolality below the permissible limit of 500 mOsm / kg. All these technical advantages are that the pharmaceutical composition in its form of use is at least • Suitable for rapid administration, improving patient comfort and limiting the risk of fluid overload. • Reduce the ratio of the weight content of the excipient to the weight content of the LR12 peptide, thereby limiting the daily exposure to the excipient, i.e., the total amount of the excipient ingested or used on a daily basis (mg / kg of excipient per kg of body weight per hour). • Suitable for administration to pediatric populations weighing less than 30 kg. • Tolerable to the patient's body (osmolar concentration below the permissible limit of 500 mOsm / kg), and • Due to the high purity and stability of LR12, the LR12 peptide has sufficient therapeutic effect. The goal is to make this possible.

[0193] Conversely, when the amount of amino acids was less than 20% by weight relative to the dry weight of the purified LR12 peptide substance, a pharmaceutical composition in powder form (i.e., storage form of the pharmaceutical product) could not be obtained.

Claims

1. A pharmaceutical composition comprising a purified LR12 peptide substance having the amino acid sequence of SEQ ID NO: 1, mixed with a citrate buffer and at least one amino acid selected from arginine, lysine, and histidine, wherein the amount of the at least one amino acid is in the range of 20% to 30% by weight relative to the dry weight of the purified LR12 peptide substance, and The pharmaceutical composition is a pharmaceutical composition comprising LR12 dimers in an amount of less than 5.0% w / w by weight relative to the peptide content.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition contains 800 mg to 2500 mg of LR12 peptide, preferably 1200 mg to 2000 mg of LR12 peptide.

3. The pharmaceutical composition according to claim 1 or 2, comprising the purified LR12 peptide substance in an amount of 70.0% to 80.0%, preferably 75.2% to 79.0%, and more preferably about 77.3%, relative to the total dry weight of the pharmaceutical composition.

4. The pharmaceutical composition according to claim 3, wherein the purified LR12 peptide substance contains at least 85% w / w of LR12 peptide by weight relative to the total weight of the purified LR12 peptide substance.

5. - The amount of the at least one amino acid is in the range of 20.0% to 30.0% by weight relative to the dry weight of the purified LR12 peptide substance, preferably 22.0% to 28.0%, more preferably 22.3% to 27.6%, and even more preferably about 24.5%, and / or The amount of sodium citrate buffer is 3.0% to 7.0% by weight relative to the dry weight of the purified LR12 peptide substance, preferably 4.0% to 6.0%, more preferably 4.5% to 5.5%, and even more preferably about 4.8%. A pharmaceutical composition according to any one of claims 1 to 4.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is a powder composition, preferably a freeze-dried powder composition.

7. - The pharmaceutical composition is preferably an aqueous medium selected from water and physiological saline, preferably an aqueous solution further containing physiological saline having 0.9% sodium chloride by weight relative to physiological saline, and / or The pharmaceutical composition has a pH in the range of 5.0 to 6.0, preferably about 5.

5. A pharmaceutical composition according to any one of claims 1 to 5.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition exhibits an osmolality of less than 500 mOsm / kg, preferably 300 to 400 mOsm / kg, more preferably in the range of 320 to 390 mOsm / kg, and even more preferably about 380 mOsm / kg.

9. A purified LR12 peptide substance having the amino acid sequence described in Sequence ID No. 1, comprising at least 85% w / w of peptide content by weight relative to the total weight of the purified LR12 substance, and less than 2.7% by weight of LR12 dimers relative to the total weight of the peptide content.

10. The purified LR12 peptide substance according to claim 9, comprising 1.2% or less by weight of Des-Gln-LR12 and / or 0.5% or less by weight of Met(O)12-LR12 relative to the total weight of the peptide content.

11. The purified LR12 peptide substance according to claim 9 or 10, comprising 1% or less by weight of acetate, 500 ppm or less of acetonitrile, less than 5000 ppm of methyl-t-butyl ether and / or less than 5000 ppm of n-heptane, relative to the purified LR12 peptide substance.

12. A pharmaceutical composition according to any one of claims 1 to 8 for use as a medicine, or a purified LR12 peptide substance according to any one of claims 9 to 11.

13. A pharmaceutical composition according to any one of claims 1 to 8 or a purified LR12 peptide substance according to any one of claims 9 to 11 for use in the treatment of a disease in a subject requiring such treatment, wherein the disease is preferably an inflammatory disease or disorder selected from septic shock, sepsis, severe sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), hemorrhagic shock, ischemia-reperfusion injury and pancreatitis, preferably a cardiovascular disease selected from myocardial infarction, cerebral infarction, acute myocardial infarction, ischemia, coronary heart disease, acute coronary syndrome, stroke, aneurysm, stable angina pectoris, exertional angina pectoris, cardiomyopathy, hypertensive heart disease, chronic heart failure, acute heart failure, cor pulmonale, cardiac arrhythmia, inflammatory heart disease (such as endocarditis and myocarditis), peripheral artery disease, cardiovascular disease selected from SIRS-related myocardial and / or vascular dysfunction and atherosclerosis, and infectious diseases such as COVID-19.

14. A pharmaceutical composition or purified LR12 peptide substance for use according to claim 12 or 13, for intravenous administration at a rate in the range of 0.3 to 3.0 mg / kg / hr (mg of LR12 per kg of body weight per hour).

15. i. A step of synthesizing the LR12 peptide on a solid phase to obtain a crude LR12 composition. ii. A step of obtaining a first peptide solution containing the crude LR12 composition, iii. A step of purifying the first peptide solution by ion exchange column chromatography (IEC) to obtain a first pre-purified product of LR12. iv. A step of further purifying the pre-purified LR12 by reverse-phase high-performance liquid chromatography (HPLC) to obtain an HPLC pool fraction containing a second pre-purified LR12. v. A step of preparing a 5-25 mM arginine solution to be added to the pool fraction during step vi. vi. A step of concentrating the pooled fraction to a concentrated solution of at least 120 g / L using membrane dialysis filtration or ultrafiltration, typically with a membrane of 150 to 300 Daltons, and vii. A step to obtain a purified LR12 peptide by filtering and freeze-drying the concentrated solution obtained in step vi). A process for preparing the purified LR12 peptide substance according to any one of claims 9 to 11, comprising:

16. The process according to claim 15, wherein the HPLC pool fraction containing the second pre-purified material of LR12 is cooled to a temperature of 15°C or lower and / or bubbled with nitrogen.

17. a) Preferably a step of supplying the purified LR12 peptide substance according to any one of claims 9 to 11 by performing steps i) to vii) according to claim 15 or claim 16, b) The purified LR12 peptide substance is a citrate and at least one amino acid selected from arginine, lysine, and histidine, preferably - At least one amino acid in an amount of 20.0% to 30.0%, preferably 22.0% to 28.0%, more preferably 22.3% to 27.6%, and even more preferably about 24.5%, relative to the dry weight of the purified LR12 peptide substance, and - A citrate in an amount of 3.0% to 7.0%, preferably 4.0% to 6.0%, more preferably 4.5% to 5.5%, and even more preferably about 4.8%, relative to the dry weight of the purified LR12 peptide substance. The process of mixing with and thereby obtaining a bulk solution, c) Optionally, the step of adjusting the pH of the bulk solution to pH 5.5, typically with HCl. d) A step of freeze-drying the bulk solution to obtain a freeze-dried powder composition of the pharmaceutical composition, and e) Optionally, an aqueous medium selected from water and physiological saline, preferably physiological saline containing 0.9% sodium chloride by weight relative to physiological saline, is added to obtain an aqueous solution of the pharmaceutical composition. A process for preparing the pharmaceutical composition according to any one of claims 1 to 8, including the following:

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