Compositions comprising trem-1 inhibitors and uses thereof

Abstract

Provided herein are purified peptide inhibitors of triggering receptor expressed on myeloid cells 1 (TREM-1) that are suitable for administration as therapeutics to treat diseases or disorders. Also provided herein are manufacturing processes to produce peptide inhibitors of TREM-1 and compositions comprising such inhibitors that have desirable purity, concentration, and stability characteristics.

Description

COMPOSITIONS COMPRISING TREM-1 INHIBITORS AND USES THEREOFFIELD

[0001] The disclosure relates to compositions comprising a purified peptide inhibitor of triggering receptor expressed on myeloid cells- 1 (TREM-1), processes for producing such compositions, and uses of such compositions to treat diseases or disorders.BACKGROUND

[0002] Triggering receptor expressed on myeloid cells- 1 (TREM-1) is a transmembrane receptor expressed on innate immune cells and part of the family of TREM receptors. Activation of TREM-1 triggers the release of pro-inflammatory cytokines and chemokines and contributes to the dysregulated immune response in sepsis (Siskind et al., 2022, Front. Immunol. 13:907387). TREM-like transcript 1 (TLT-1) also belongs to the TREM family and is specific to platelets and megakaryocytes, where it plays a role in hemostasis / thrombosis (Washington et al., 2004, Blood 104(4):1042-1047). A TLT-1- derived peptide (LR12) has been shown to exhibit anti-inflammatory properties by inhibiting TREM-1 signaling (Derive etal., 2012, J Immunol. 188:5585-5592). Administration ofLR12 in an adult minipig model of induced sepsis reduced cardiovascular failure and improved survival in animals after induction of fecal peritonitis (Derive et al., 2013, Shock 39(2): 176-182).

[0003] The Applicant found that LR12 tends to dimerize into a dimer that is biologically inactive. The presence of said LR12 dimer has potential negative effects on the dosage of the active LR12 monomer with a risk to reduce the therapeutic efficacy of an LR12 therapeutic administration scheme. To the Applicant’s knowledge, the problem of LR12 dimerization and its effect on the treatment efficacy has not been disclosed in the art. Given that LR12 ispreferably administered via parenteral administration route, there is a need to supply pure LR12 compositions in order to ensure the therapeutic efficacy.

[0004] Furthermore, in order to administer sufficient therapeutic amounts of LR12 parenterally, the LR12 peptide needs to be solubilized in a liquid pharmaceutical vehicle such as saline water. The benefits of the appropriate use of fluids in intensive care units (ICUs) and hospitals are well described in the art, and there is growing knowledge regarding the potential risks of volume overload and / or osmotic shock on organ failure and mortality, in particular in vulnerable subjects such as the subjects suffering from septic shock or systemic inflammatory response syndrome. Therefore, there is a need to supply LR12 compositions that can be reconstituted into a liquid parenteral formulation wherein the LR12 presents an optimal solubility by enabling the minimal possible amount of excipients. Otherwise expressed, there is a need for LR12 compositions that can be reconstituted into a liquid parenteral formulation having a high concentration of LR12, allowing the administration to the patient of a volume of liquid parenteral formulation which is the smallest as possible, while having a high purity of LR12, a high stability of LR12 and an optimal osmolality.

[0005] Lastly, the Applicant observed that LR12 dimers may still be formed during the shelf-life of the LR12 formulations and / or within its liquid formulation during the intravenous administration.

[0006] Consequently, there is a need for improved LR12 pharmaceutical compositions that have desirable purity, concentration, and stability characteristics for therapeutic administration while respecting the clinical prerequisites with regard to the fluid administration and osmotic pressure compliance, particularly for subjects vulnerable to volume overload and / or osmotic shock.

[0007] Advantageously, the pharmaceutical composition of the present disclosure allows to overcome the above shortcomings thereby optimizing the practicability of LR12 administration in a hospital setting, the drug product being efficiently solubilized, with asuitable stability, from the production to the administration of the pharmaceutical composition, while being compliant with regard to osmolality prerequisites.SUMMARY

[0008] The present application relates to a pharmaceutical composition comprising a purified substance of LR12 peptide of amino acid sequence SEQ ID NO: 1, in association with at least one solubility enhancing agent, preferably arginine; and sodium citrate buffer; wherein the purified LR12 peptide substance comprises at least 85% w / w of peptide content, in weight of the total weight of the purified LR12 peptide substance; and wherein the pharmaceutical composition comprises less than 5% w / w of LR12 dimer in weight relative to the peptide content.

[0009] The present application also relates to a pharmaceutical composition comprising: a purified substance of LR12 peptide of amino acid sequence SEQ ID NO: 1; at least one solubility enhancing agent, preferably the at least one solubility enhancing agent is at least one amino acid selected from arginine, lysine and histidine, more preferably the at least one solubility enhancing agent is at least one amino acid selected from arginine and lysine, even more preferably the at least one solubility enhancing agent is arginine; and a citrate buffer, preferably sodium citrate buffer; wherein the pharmaceutical composition comprises less than 5% w / w of LR12 dimer in weight relative to the peptide content; and wherein the amount of the at least one solubility enhancing agent ranges from 20% to 30% in weight relative to the dry weight of the purified LR12 peptide substance.

[0010] In some embodiments, the purified LR12 peptide substance comprises at least 85% w / w of LR12 peptide content, in weight of the total weight of the purified LR12 peptide substance.

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

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

[0013] In some embodiments, the pharmaceutical composition is a powder composition, preferably a lyophilized powder composition. Advantageously, the powder humidity content of the pharmaceutical composition is of equal or less than 5.0 % w / w, preferably equal or less than 3.0 % w / w. In some embodiments, the pharmaceutical composition is an aqueous solution further comprising an aqueous medium, preferably selected from water and saline water solution, preferably a saline water solution having 0.9% sodium chloride in weight relative to the total weight of the saline water solution; and / or wherein the pharmaceutical composition has a pH ranging from 5.0 to 6.0, preferably about 5.5.

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

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

[0016] The present application also relates to a purified substance of LR12 peptide having or consisting of an amino acid sequence as set forth in SEQ ID NO: 1, comprising at least 85% w / w of peptide content in weight of the total weight of the purified substance of LR12; comprising less than 2.7% of LR12 dimer in weight relative to the total weight of the peptide content.

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

[0018] The present application also relates to the pharmaceutical composition or purified LR12 peptide substance as described herein for use as a medicament. The present application further relates to the pharmaceutical composition or purified LR12 peptide substance as described herein for use in the treatment of a disease in a subject in need thereof, wherein said disease is selected from inflammatory disease or disorder, preferably selected from septic shock, sepsis, severe sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), hemorrhagic shock, ischemia-reperfusion and pancreatitis; cardiovascular disease, preferably selected from myocardial infarction, cerebral infarction, acute myocardial infarction, ischemia, coronary heart disease, acute coronary syndrome, stroke, aneurysm, stable angina pectoris, effort angina pectoris, cardiomyopathy, hypertensive heart disease, chronic heart failure, acute heart failure, cor pulmonale, cardiac dysrhythmias, inflammatory heart disease (such as endocarditis and myocarditis), peripheral arterial disease, SIRS-associated myocardial and / or vascular dysfunction, and atherosclerosis; and infectious diseases such as COVID- 19.

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

[0020] The present application also relates to a process for preparing a purified LR12 peptide substance as described herein, comprising the steps of: i. synthesizing the LR12 peptide on solid phase, leading to a crude LR12 composition; ii. providing a first peptide solution comprising the crude LR12 composition; iii. purifying the first peptide solution by ion exchange column chromatography (IEC), leading to a first pre-purified substance of LR12; iv. further purifying the pre-purified substance of LR12 by reversed-phase high performance liquid chromatography (HPLC), thereby leading to HPLC pooled fractions comprising a second pre-purified substance of LR12; v. preparing an arginine solution between 5 and 25 mM to be added to the pooled fraction during step vi.; vi. concentrating the pooled fractions using membrane diafiltration or ultrafiltration, typically on a 150 to 300 Dalton membrane, to a concentrated solution of at least 120 g / L; and vii. filtering and lyophilizing the concentrated solution obtained in step vi) to obtain the purified substance of LR12 peptide.

[0021] In some embodiments, the HPLC pooled fractions comprising the second prepurified substance of LR12 are cooled to a temperature of 15°C or less and / or are bubbled with nitrogen.

[0022] The present application further relates to a process for preparing a pharmaceutical composition as described herein, comprising the steps of: a) supplying a purified substance of LR12 peptide as described herein, preferably by carrying out the steps i) to vii) according to the process as described above;b) mixing the purified substance of LR12 peptide with a citrate buffer, preferably sodium citrate buffer, and with at least one solubility enhancing agent selected from arginine, lysine and histidine, preferably selected from arginine and lysine, more preferably being arginine, preferably: the amount of the at least one solubility enhancing agent, preferably arginine, ranging from 20.0% to 30.0%, preferably from 22.0% to 28.0%, more preferably from 22.3% to 27.6%, even more preferably is about 24.5%, in weight relative to the dry weight of the purified substance of LR12 peptide, and the amount of citrate buffer, preferably sodium citrate buffer, ranging from 3.0% to 7.0%, preferably from 4.0% to 6.0%, more preferably from 4.5% to 5.5%, even more preferably is about 4.8%, in weight relative to the dry weight of the purified substance of LR12 peptide; thereby leading to a bulk solution, c) optionally adjusting the pH of the bulk solution, typically with HC1, to a pH 5.5, d) lyophilizing the bulk solution to obtain a lyophilized powder composition of the pharmaceutical composition, and e) optionally adding an aqueous medium, preferably selected from water and saline water solution, preferably saline water solution having 0.9% sodium chloride in weight relative to the total weight of the saline water solution; to obtain an aqueous solution of the pharmaceutical composition.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a graph showing the results from flow cytometric quantification (DCFDA reduction) of neutrophils ROS production after 2 hours stimulation with LPS, LPS + lOpg / mL LR12, LPS + 50pg / mL LR12, LPS + lOpg / mL LR12-dimer (dimer), LPS + 50pg / mL LR12-dimer (dimer). *p<0.005; **p< 0.01 (T test).DEFINITIONS

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

[0025] The terms “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Similarly, the expressions “at least one” and “one or more” are interchangeable.

[0026] “About” preceding a value is used to indicate that the value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used herein, the terms “about” and “approximately” may be used as equivalents.

[0027] “APT’ is an active pharmaceutical ingredient, i.e., LR12 in the present disclosure. An API is an agent that has a treatment effect; it may be a chemical or a biological ingredient.

[0028] “At least” means “greater than or equal to” and is thus different from “strictly greater than” which does not comprise “equal to”.

[0029] “Buffer” relates to a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid, allowing the pH of a pharmaceutical composition to be maintained constant when a small amount of strong acid or base is added thereto. “Buffering agent” refers to the specific chemical that is present in both an acidic and a basic forms in a buffer, allowing the pH of a pharmaceutical composition to be maintained constant.

[0030] “Comprising” or “comprise” is to be construed in an open, inclusive sense, not limited to the features following this term.

[0031] “Consisting of’ or “consist” is to be construed in a close, non-inclusive sense, limited to the features following this term.

[0032] “Dose” refers to the amount of API administered at one time.

[0033] “Drug substance” or “LR12 drug substance”, in connection with LR12, refers to a purified LR12 peptide substance (also referred as a purified composition of LR12 peptide). The purified LR12 peptide substance as described herein, also referred to as drug substance, is a powder. This powder is obtained by the lyophilization of a solution, as explained in the process for preparing a purified LR12 peptide substance described in the present application. Advantageously, the humidity content of the purified LR12 peptide substance is of equal or less than 10.0 % w / w, preferably equal or less than 5.0 % w / w.

[0034] “Drug product”, in connection with LR12, refers to a pharmaceutical composition comprising a purified LR12 peptide substance. The drug product may either be in the form of a powder (e.g., storage form) or in the form of a liquid (e.g., in-use form, above-mentioned as “liquid parenteral formulation”). The drug product which is in the form of a powder is obtained by dissolving the purified LR12 peptide substance as described herein and carrying out the other steps a) to d) of the process for preparing a pharmaceutical composition described in the present application, implying a step of lyophilization. Advantageously, the humidity content of the drug product which is in the form of a powder is of equal or less than 5.0 % w / w, preferably equal or less than 3.0 % w / w. The drug product which is in the form of a liquid is obtained by adding an aqueous medium to the drug product which is in the form of a powder, i.e. by carrying out step e) of the process for preparing a pharmaceutical composition described in the present application.To summarize, the manufacturing chain is as follows: process for preparing a purified LR12 peptide substance described in the present application > obtention of the purified LR12peptide substance as described herein (powder) > steps a) to d) of the process for preparing a pharmaceutical composition described in the present application > obtention of the drug product in the form of a powder (e.g., storage form) > step e) of the process for preparing a pharmaceutical composition described in the present application > obtention of the drug product in the form of a liquid (e.g., in-use form).

[0035] “Excipient” refers to any inactive ingredient, which is required for the formulation of an API in a suitable dosage form.

[0036] “From X to Y” refers to the range of values between X and Y, the limits X and Y being included in said range.

[0037] Numerical values are subject to measurement errors which place limits on their accuracy, as known by the skilled person. Thus, in the present application, the general convention in the scientific and technical literature is applied: the last decimal place of a numerical value indicates its degree of accuracy. Where no other error margins are given, the maximum margin is ascertained by applying the rounding-off convention 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 for example by 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 (of the purified LR12 peptide substance).

[0040] “LR12” or “LR12 peptide” refers to the TLT-1 derived peptide of formula (I) consisting of the amino acid sequence as set forth in SEQ ID NO: 1 (LQEEDAGEYGCM).Formula (I)

[0041] “Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to an excipient or carrier that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, such as a human. It includes any and all solvents, such as, for example, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. A pharmaceutically acceptable excipient or carrier refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the regulatory offices such as the EMA (European Medicines Agency) or FDA (US Food and Drug Administration).

[0042] “Pharmaceutical composition” refers to the combination of at least one API and at least one pharmaceutically acceptable excipient.

[0043] “Powder” refers to a set of dry and more or less fine solid (micro)particles. Advantageously, the humidity content of any powder as described herein is of equal or less than 10.0 % w / w.

[0044] “Room temperature” refers to a temperature ranging from 20°C to 25°C, preferably a temperature of 20°C.

[0045] “Rpm” or “revolutions per minute” refers to the number of turns in one minute.

[0046] “Subject” refers to a warm-blooded animal, more preferably a mammal. The term “mammal” refers here to any mammal, including humans. In some embodiments, a subject may be a “patient”, who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of the targeted disease or condition.

[0047] “Treatment” refers to a therapeutic (or curative) treatment, to a prophylactic (or preventative) treatment, or to both a therapeutic (or curative) treatment and a prophylactic (or preventative) treatment, wherein the object is to prevent, reduce, slow down (lessen), or cure one or more of the symptom(s) or manifestation(s) of the targeted pathologic condition or disorder, such as an inflammatory disease or disorder, in particular septic shock, sepsis or SIRS, a cardiovascular disease, disorder or condition, or an infectious disease. Those in need of treatment include those already suffering from an inflammatory disease or disorder, a cardiovascular disease, disorder or condition, or an infectious disease, as well as those prone to develop an inflammatory disease or disorder, a cardiovascular disease, disorder or condition, or an infectious disease, or those in whom an inflammatory disease or disorder, a cardiovascular disease, disorder or condition, or an infectious disease is to be prevented.DETAILED DESCRIPTION

[0048] The present disclosure relates to compositions comprising a peptide inhibitor of TREM-1 that are optimized for therapeutic administration to treat diseases or disorders (e.g., septic shock, sepsis or SIRS). The LR12 peptide inhibitor of TREM-1 (also known as nangibotide; LQEEDAGEYGCM - SEQ ID NO: 1) contains a cysteine that leads to formation of disulfide bridges between molecules of LR12, creating dimerized LR12. The LR12-dimer is composed of 2 LR12 peptide monomers covalently bound by a disulfide bond through the cysteine at position 11. The LR12 dimer, i.e., the compound of formula II, was found to be inactive in a preclinical in vitro pharmacology model and that can negatively impact the LR12 treatment efficacy.

[0049] Given the biological inactivity of the LR12 dimer, the present invention relates to a purified LR12 peptide substance and the pharmaceutical composition (drug product) comprising thereof that are advantageous in terms of therapeutic efficacy over LR12 peptide substances comprising more than about 5% of LR12 dimer in weight relative to the total weight of the peptide content of the LR12 substance. In other terms, the purified LR12 peptide substance and the pharmaceutical composition (drug product) comprising thereof are advantageous in terms of chemical stability. According to a particularly advantageous aspect, such chemical stability is maintained throughout the shelf-life of the purified LR12 peptide substance and the pharmaceutical composition (drug product) comprising thereof. Additionally, such chemical stability is maintained even if the pharmaceutical composition comprising LR12 peptide is solubilized into a liquid formulation and more importantly it is maintained during the intravenous administration of the liquid formulation.

[0050] In view of the above, according to some advantageous embodiments, the present purified LR12 peptide substance and the present pharmaceutical composition comprising the purified LR12 peptide substance confer the same therapeutic effects, (by supplying the sameamount of the LR12 bioactive monomer) at an inferior amount of drug compared to the drug substances and products of the prior art.Purified LR12 peptide substance

[0051] Thus, according to a first aspect, the present application relates to a purified composition of LR12 peptide, also referred to as purified LR12 peptide substance (or sometimes as LR12 drug substance). It is understood that such substance comprises a peptide content (or fraction) wherein peptides such as LR12 or peptide impurities, different than LR12, are comprised. Typically, the peptide fraction of the purified LR12 peptide substance described herein is at least about 85% in weight relative to the total weight of the purified LR12 peptide substance. The peptide fraction of a substance can be determined by any means known in the art as defined above, preferably by elemental analysis. In one preferred embodiment, the peptide fraction of the LR12 drug substance is determined by elemental analysis comprising a dry combustion step at a temperature ranging from 1000°C to 1200°C in the presence of oxygen followed by thermal conductivity detection, typically using an elemental analyzer for simultaneous determination of C, H, N and S. During the dry combustion step, the total content of nitrogen in the drug substance is quantitatively converted to N2 and can be subsequently determined by measuring the thermal conductivity after separation of the other gaseous components (CO2, H2O and SO2).

[0052] The purified LR12 peptide substance as described herein comprises less than about 5%, about 4.5%, about 4%, about 3.5%, about 3%, about 2.7%, or about 2.5% of LR12 dimer in 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 substance comprises at least about 85% w / w, preferably at least about 86% of peptide content in weight of the total weight of the purified substance of LR12; comprising less than about 5%, preferably less than about 3%, more preferably less than about 2.7% of LR12 dimer in weight relative to the total weight of the peptide content.

[0054] In some embodiments, the purified LR12 peptide substance comprises at least 85% w / w of peptide content in weight of the total weight of the purified substance of LR12; comprising less than 2.7% of LR12 dimer in weight relative to the total weight of the peptide content.

[0055] Otherwise expressed, the purified LR12 peptide substance as described herein is a composition comprising:- at least about 85% w / w of peptide content in weight relative to the total weight of the purified LR12 peptide substance; and- less than about 5% w / w, less than about 4.5% w / w, less than about 4% w / w, less than about 3.5%, less than about 3% w / w, less than about 2.7% w / w, or less than about 2.5% w / w of LR12 dimer in weight relative to the total weight of the peptide content of the purified LR12 peptide substance.

[0056] In some embodiments, the purified LR12 peptide substance comprises at least about 85% w / w of peptide content in weight of the total weight of the purified substance of LR12; comprising more than or equal to about 95 % of LR12 (monomer) in weight relative to the total weight of the peptide content.

[0057] Provided herein is a purified peptide composition comprising a plurality of peptides comprising the amino acid sequence of SEQ ID NO: 1, wherein less than about 2.7% of the peptides are dimerized at the cysteine residue in SEQ ID NO: 1.

[0058] Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises no more than about 2.5%, about 2%, about 1.5%, about 1.2%, about 1%, about 0.5% or about 0.3% Des-Gln-LR12 in weight relative to the total weight of the peptide content. Des-Gln-LR12 is a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (LEEDAGEYGCM) and its structure corresponds to the hereinbelow presented formula III. Typically, the purified LR12 peptide substance as described herein comprises less or equal to about 1.2%, preferably less or equal to 1.0%, more preferably lessor equal to 0.5% Des-Gln-LR12 in weight relative to the total weight of the peptide content of the purified LR12 peptide substance.

[0059] Provided herein is a purified LR12 peptide substance, comprising no more than about 2.5%, about 2%, about 1.5%, about 1.2%, about 1%, about 0.75%, about 0.5%, or about 0.3% Met(O)12-LR12 in weight relative to the total weight of the peptide content. Met(O)12-LR12 is an oxidized LR12 product of formula IV presented hereinbelow. 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 into methionine sulfoxide also referred to as Met(O) or MetO. Typically, the purified LR12 peptide substance as described herein comprises less or equal to 1.0%, preferably less or equal to about 0.75%, more preferably less or equal to about 0.5% Met(O)12-LR12 in weight relative to the total weight of the peptide content of the purified LR12 peptide substance.

[0060] In some embodiments, the purified LR12 peptide substance comprises less or equal to 1.2% of Des-Gln-LR12 and / or less or equal to 0.5% of Met(O)12-LR12 and / or less or equal to 0.5% of other single impurity, in weight relative to the total weight of the peptide content of the purified LR12 peptide substance.

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

[0062] Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises no more than about 5%, about 4.5%, about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, or about 0.75% acetate in weight relative to the total weight of the purified LR12 peptide substance. Provided herein is a purified LR12 peptide substance comprising LR12, wherein the purified LR12 peptide substance comprises no more than about 1% acetate.

[0063] Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises less than about 750 ppm, about 700 ppm, about 650 ppm, about 600 ppm, about 550 ppm, about 500 ppm, or about 450 ppm acetonitrile. Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises less than about 410 ppm acetonitrile.

[0064] Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises less than about 7000 ppm, about 6500 ppm, about 6000 ppm, about 5500 ppm, about 5000, or about 4500 ppm methyl-t-butyl ether. Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises less than about 5000 ppm methyl-t-butyl ether.

[0065] Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises less than about 7000 ppm, about 6500 ppm, about 6000 ppm, about 5500 ppm, about 5000, or about 4500 ppm n-heptane. Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises less than about 5000 ppm n-heptane.

[0066] Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance comprises less than about 500 ppm acetonitrile, less than about 5000 ppm methyl-t-butyl ether, and less than about 5000 ppm n-heptane.

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

[0068] Provided herein is a purified LR12 peptide substance, wherein the purified LR12 peptide substance has the following characteristics:- has less than about 2.7% cysteine dimerization of LR12 in weight relative to the total weight of the peptide content;- comprises no more than about 1.2%, preferably no more than about 1.0%, more preferably no more than about 0.5% of Des-Gln-LR12 in weight relative to the total weight of the peptide content;- comprises no more than about 1.0%, preferably no more than about 0.75%, more preferably no more than about 0.5% of Met(O)12-LR12 in weight relative to the total weight of the peptide content;- comprises no more than about 0.5% other single impurity;- comprises no more than 5% of total peptide impurities (i.e., peptides with undesired sequences) in weight relative to the total weight of the peptide content.;- comprises no more than about 1 % acetate, in weight relative to the total weight of the purified LR12 peptide substance;- comprises less than about 500 ppm acetonitrile in weight relative to the total weight of the purified LR12 peptide substance;- comprises less than about 5000 ppm methyl-t-butyl ether; and- comprises less than about 5000 ppm n-Heptane.

[0069] In some embodiments, the purified substance of LR12 peptide has the following characteristics:- comprises at least about 85% w / w of peptide content in weight of the total weight of the purified substance of LR12; preferably comprising more than or equal to about 95 % of LR12 (monomer) in weight relative to the total weight of the peptide content;- has less than about 2.7% cysteine dimerization of LR12 in weight relative to the total weight of the peptide content;- comprises no more than about 1.2%, preferably no more than about 1.0%, more preferably no more than about 0.5% of Des-Gln-LR12 in weight relative to the total weight of the peptide content;- comprises no more than about 1.0%, preferably no more than about 0.75%, more preferably no more than about 0.5% of Met(O)12-LR12 in weight relative to the total weight of the peptide content;- comprises no more than about 1 % acetate, in weight relative to the total weight of the purified LR12 peptide substance;- comprises less than about 500 ppm acetonitrile in weight relative to the total weight of the purified LR12 peptide substance;- comprises less than about 5000 ppm methyl-t-butyl ether in weight relative to the total weight of the purified LR12 peptide substance; and- comprises less than about 5000 ppm n-Heptane in weight relative to the total weight of the purified LR12 peptide substance.

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

[0071] Otherwise expressed, the purified LR12 peptide substance as described herein is a composition comprising:- at least about 85% w / w of peptide content in weight relative to the total weight of the purified LR12 peptide substance;- less than about 5% w / w, less than about 4.5% w / w, less than about 4% w / w, less than about 3.5% w / w, less than about 3% w / w, less than about 2.7% w / w, or less than about 2.5% w / w of LR12 dimer in weight relative to the total weight of the peptide content of the purified LR12 peptide substance; and- optionally from 2% w / w to 5% w / w of arginine in weight relative to the total weight of the purified LR12 peptide substance.

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

[0073] The purified LR12 peptide substance as described herein allows the obtention of a drug product comprising a high dose of LR12 peptide (“high dose formulation” as mentioned herein), which allows a reduced volume of administration of the in-use form of this drug product to the patient.Pharmaceutical composition comprising the purified LR12 peptide substance (drug product)

[0074] In some embodiments, the maximal dimer content in the purified LR12 peptide substance is preferably equal to or lower than 2.7% and, subsequently, the dimer content in the drug product is equal to or lower than 5.0%.

[0075] Briefly, to reduce the formation of disulfide bridges, the pH during the formulation process was reduced to below 6.0. However, LR12 contains 4 acidic, negatively charged amino acids, namely glutamate (Glu) and aspartate (Asp) and no basic amino acids.Consequently, LR12 displays a theoretical pl (isoelectric point) of 3.57 and accordingly, is barely soluble below pH 5.0. To solve this problem, solubility aids were screened at pH 5.5 and 6.5. Tensioactive (Tween® 20), polyol (mannitol), sugars (sucrose and trehalose), zwitterion (betaine), chaotropic (urea), redox (glutathione) and counter ion (ammonium acetate and citrate) molecules failed to improve the solubility of LR12.

[0076] Unexpectedly, solubility at 40 mg / mL could only be reached by adding the positively charged solubility enhancing agents such as amino acids lysine or arginine or histidine. Without willing to be bound by a theory, these amino acids may improve LR12 solubility by neutralizing the negative charges in the LR12 sequence. The administration of LR12 in the compositions provided herein at a dose of 1 to 3 mg / kg / h delivers only a small fraction of the daily amount of arginine administered to patients under parenteral nutrition.

[0077] Provided herein is a pharmaceutical composition (=drug product) comprising the purified LR12 peptide substance disclosed herein, in association with one or more pharmaceutically acceptable solubility enhancing agent(s) and at least one pharmaceutically acceptable buffer composition. The pharmaceutical compositions provided herein have desirable concentration and stability characteristics for clinical administration while enabling the minimal possible amount of excipients.

[0078] Preferably, the amount of the at least one solubility enhancing agent, preferably of arginine, lysine or histidine, more preferably of arginine or lysine, even more preferably of arginine, ranges from 20% to 30% in weight relative to the dry weight of the purified LR12 peptide substance.

[0079] The at least one solubility enhancing agent 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. The at least one pharmaceutically acceptable buffer composition is preferably a citrate buffer, i.e., a mixture of a citrate, typically sodium citrate, with citric acid. In some embodiments, citrate buffer compositions are readily available. In some embodiments, citrate buffer can be1 prepared by mixing a citrate salt such as sodium citrate with hydrochloric acid, thereby leading to a buffer of citrate / citric acid.

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

[0081] In some embodiments, the pharmaceutical composition comprises purified LR12 peptide substance, in association with at least one solubility enhancing agent, preferably arginine; and a citrate buffer, preferably sodium citrate buffer; wherein the purified LR12 peptide substance comprises at least 85% w / w of peptide content in weight of the total weight of the purified LR12 peptide substance; and wherein the composition comprises less than 5% w / w, of LR12 dimer in weight relative to the peptide content.

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

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

[0084] In some embodiments, the pharmaceutical composition comprises the purified LR12 peptide substance in an amount ranging from 70.0% to 80.0%, preferably from 75.2% to 79.0%, more preferably about 77.3% in weight relative to the total dry weight of the pharmaceutical composition, and: the amount of the at least one solubility enhancing agent, preferably of arginine, lysine or histidine, more preferably of arginine or lysine, even more preferably of arginine, ranges from 20.0% to 30.0%, preferably from 22.0% to 28.0%, more preferably from 22.3% to 27.6%, even more preferably is about 24.5%, in weight relative to the dry weight of the purified LR12 peptide substance, and / or the amount of the at least one pharmaceutically acceptable buffer, preferably of the citrate buffer, more preferably of sodium citrate buffer, ranges from 3.0% to 7.0%, preferably from 4.0% to 6.0%, more preferably from 4.5% to 5.5%, even more preferably is about 4.8%, in weight relative to the dry weight of the purified LR12 peptide substance.

[0085] In some embodiments, the pharmaceutical composition comprises the purified LR12 peptide substance in an amount ranging from 70.0% to 80.0%, preferably from 73.0% to 77.0%, more preferably about 75.2% in weight relative to the total dry weight of the pharmaceutical composition, and: the amount of the at least one solubility enhancing agent, preferably of arginine lysine or histidine, more preferably of arginine or lysine, even more preferably of arginine, ranges from 25.0% to 30.0%, preferably from 26.0% to 28.0%, more preferably from 27.0% to 28.0%, even more preferably is about 27.6%, in weight relative to the LR12 amount, and / or the amount of the at least one pharmaceutically acceptable buffer, preferably of the citrate buffer, more preferably of sodium citrate buffer, ranges from 3.0% to 7.0%,preferably from 4.0% to 6.0%, more preferably from 5.0% to 6.0%, even more preferably is about 5.4%, in weight relative to the LR12 amount.

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

[0087] In some embodiments, the pharmaceutical composition as described herein has a pH ranging from 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, the pH is measured using standard techniques known to the person skilled in the art, for example after re-solubilizing the powder in a solvent, preferably in water.

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

[0089] According to some advantageous embodiments, the dimer content in pharmaceutical composition does not significantly evolve in its in-use form, i.e., the pharmaceutical composition in liquid form (aqueous solution) described herein above. In some embodiments, the maximal dimer content in the in-use form of the pharmaceutical composition is equal to or lower than 10.0% in weight relative to the total weight of the peptide content of the in-use form of the pharmaceutical composition. A dimer content of equal to or less than 10.0% in weight relative to the total weight of the peptide content of the in-use form of the pharmaceutical composition allows a sufficient therapeutic efficacy of the pharmaceutical composition.

[0090] The invention also relates to a pharmaceutical composition comprising a purified LR12 peptide substance as described herein, in association 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 ranges from 20% to 30% in weight relative to the dry weight of the purified LR12 peptide substance; wherein the pharmaceutical composition comprises less than 10.0% w / w of LR12 dimer in weight relative to the peptide content; and wherein the pharmaceutical composition is an aqueous solution further comprising an aqueous medium, preferably selected from water and saline water solution, preferably in saline water solution having 0.9% sodium chloride in weight relative to the saline water solution.

[0091] In some embodiments, the aqueous solution comprises: the purified LR12 peptide substance in an amount ranging from 70.0% to 80.0%, preferably from 75.2% to 79.0%, more preferably about 77.3% in weight relative to the total dry weight of the pharmaceutical composition; at least one pharmaceutically acceptable buffer, preferably a citrate buffer, more preferably sodium citrate buffer, in an amount ranging from 2.0% to 6.0%, preferably from 3.0% to 5.0%, more preferably about 3.7% in weight relative to the total dry weight of the pharmaceutical composition, and at least one solubility enhancing agent, preferably arginine, lysine or histidine, more preferably arginine or lysine, even more preferably arginine, in an amount ranging from 15.0% to 25.0%, preferably from 18.0% to 22.0%, more preferably about 19.0% in weight relative to the total dry weight of the pharmaceutical composition.

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

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

[0094] In some embodiments, the pharmaceutical composition comprises at least one solubility enhancing agent, preferably arginine, lysine or histidine, more preferably arginine or lysine, even more preferably arginine at a concentration ranging from about 100 mM to about 250 mM.

[0095] In some embodiments, the pharmaceutical composition comprises no more than about 2.5%, about 2%, about 1.5%, about 1.2%, about 1%, about 0.5% or about 0.3% Des- Gln-LR12, in weight relative to the total weight of the peptide content. In some embodiments, the pharmaceutical composition comprises less or equal than about 1.2%, preferably less or equal to 1.0%, more preferably less or equal to 0.5% Des-Gln-LR12in weight relative to the total weight of the peptide content of the pharmaceutical composition.

[0096] In some embodiments, the pharmaceutical composition comprises no more than about 2.5%, about 2%, about 1.5%, about 1.2%, about 1%, about 0.75%, about 0.5%, or about 0.3% Met(O)12-LR12, in weight relative to the total weight of the peptide content. In some embodiments, the pharmaceutical composition comprises less or equal to 1.0%, preferably less or equal to about 0.75%, more preferably less or equal to about 0.5% Met(O)12-LR12, in weight relative to the total weight of the peptide content of the pharmaceutical composition.

[0097] In some embodiments, the pharmaceutical composition is lyophilized. Provided herein is a pharmaceutical composition that has a 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 whenreconstituted in aqueous solution. Provided herein is a pharmaceutical composition that has a solubility of at least about 175 mg / ml when reconstituted in aqueous solution.

[0098] In some embodiments, the pharmaceutical composition comprises from 800 mg to 2500 mg of LR12 peptide, preferably from 1200 mg to 2000 mg of LR12 peptide. Otherwise expressed, in some embodiments, the pharmaceutical composition comprises from 800 mg to 2500 mg of LR12 peptide per unit dosage, preferably from 1200 mg to 2000 mg of LR12 peptide per unit dosage. Examples of unit dosage include vials.

[0099] In some embodiments, the pharmaceutical composition comprises about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, about 2000 mg, about 2050 mg, about 2100 mg, about 2150 mg, about 2200 mg, about 2250 mg, about 2300 mg, about 2350 mg, about 2400 mg, about 2450 mg, or about 2500 mg, of LR12 peptide. Otherwise expressed, in some embodiments, the pharmaceutical composition comprises about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, about 2000 mg, about 2050 mg, about 2100 mg, about 2150 mg, about 2200 mg, about 2250 mg, about 2300 mg, about 2350 mg, about 2400 mg, about 2450 mg, or about 2500 mg, of LR12 peptide per unit dosage. Examples of unit dosage include vials.

[0100] Provided herein is a vial containing a lyophilized pharmaceutical composition or a purified LR12 peptide substance disclosed herein. In some embodiments, a vial contains from about 400 to about 1200 mg of a lyophilized pharmaceutical composition or a purified LR12 peptide substance, preferably of LR12 peptide. In some embodiments, a vial contains fromabout 400 to about 1200 mg of a lyophilized pharmaceutical composition or of a LR12 peptide, preferably of LR12 peptide disclosed herein reconstituted in 10 ml of aqueous solution. In view of the present disclosure, the skilled artisan can calculate the amount of LR12 Active Pharmaceutical Ingredient comprised in the purified LR12 peptide substance.

[0101] Provided herein is a vial containing a pharmaceutical composition or a purified LR12 peptide substance disclosed herein. In some embodiments, a vial containing a powder pharmaceutical composition or a purified LR12 peptide substance disclosed herein contains from about 800 mg to about 2500 mg of LR12 peptide, preferably from about 1200 mg to about 2000 mg of LR12 peptide. In some embodiments, a vial containing a liquid pharmaceutical composition disclosed herein contains from about 800 mg to about 2000 mg of LR12 peptide, preferably from about 1200 mg to about 2000 mg of LR12 peptide, reconstituted in 10 ml of aqueous solution.

[0102] Provided herein is a syringe containing a pharmaceutical composition or a purified LR12 peptide substance disclosed herein. In some embodiments, a syringe contains from about 400 to about 1200 mg of LR12 peptide disclosed herein diluted in 50 ml of aqueous solution. In some embodiments, a syringe contains from about 800 to about 2500 mg of LR12 peptide disclosed herein, preferably 1200 to about 2000 mg of LR12 peptide disclosed herein, diluted in 50 ml of aqueous solution.

[0103] Provided herein is a pharmaceutically acceptable solution comprising from about 400 to about 1200 mg of a LR12 peptide dissolved in about 40 to about 60 ml of aqueous solution.

[0104] Provided herein is a pharmaceutically acceptable solution having osmolality as close as possible to 300 mOsm / kg (and maximum 500 mOsm / kg).Process of manufacturing purified LR12 peptide substance and compositions comprising thereof

[0105] The manufacturing processes disclosed herein allows to further reduce the formation of cysteine dimers while maintaining solubility.

[0106] Provided herein is a method or process for purifying LR12 (i.e., for preparing a purified LR12 peptide substance), the process comprising: i. preparing crude LR12 in solid phase; ii. providing a first peptide solution comprising a crude LR12; iii. loading the first peptide solution onto a column with ion exchange chromatography (IEC) to purify fractions; iv. loading the fractions onto a column with reversed-phase chromatography (RPC) to produce pooled fractions; v. preparing a solution containing at least one solubility enhancing agent, preferably arginine between 5 mM and 25 mM to be added to the pooled fractions during step vi.; and vi. concentrating and purifying the pooled fractions by ultrafiltration / diafiltration, typically using a 150 to 300 Dalton membrane, to a concentrated solution of at least 40g / L.

[0107] In some embodiments, the process for preparing a purified substance of LR12 peptide comprises: i. synthesizing the LR12 peptide on solid phase, leading to a crude LR12 composition; ii. providing a first peptide solution comprising the crude LR12 composition; iii. purifying the first peptide solution by ion exchange column chromatography (IEC), leading to a first pre-purified substance of LR12;iv. further purifying the pre- purified substance of LR12 by reversed-phase high performance liquid chromatography (HPLC), thereby leading to HPLC pooled fractions comprising a second pre-purified substance of LR12; v. preparing a solution containing at least one solubility enhancing agent, preferably arginine between 5 and 25 mM to be added to the pooled fractions during step vi.; vi. concentrating the pooled fractions using membrane diafiltration or ultrafiltration, typically on a 150 to 300 Dalton membrane, to a concentrated solution of at least 120 g / L; and vii. filtering and lyophilizing the concentrated solution obtained in step vi) to obtain the purified LR12 peptide substance.

[0108] In some embodiments, concentrating the pooled fractions comprises 2 steps of concentration.

[0109] In some embodiments, the HPLC pooled fractions comprising the second prepurified substance of LR12 are cooled to a temperature of 15°C or less and / or are bubbled with nitrogen.

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

[0111] In some embodiments of the methods provided herein, the 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, the 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, LR12 dimer formation is reduced by using buffers at pH between 5 and 6 for IEC, RPC and diafiltration, and adjusting the pH between 5 and 6 of the pooled fractions from RPC; cooling the solutions below 15°C; and bubbling the solutions with nitrogen.

[0113] According to an advantageous aspect of the invention, a control of the manufacturing timing with the disclosed drug product formulation allow a reproducible control of the dimer content in the final product.

[0114] The improved manufacturing processes provided herein controls timing between steps to allow an acceptable and reproducible control of the dimer content in the final drug product. To optimize the practicability of administration in a hospital setting, the drug product is highly concentrated, with a suitable stability.

[0115] Thus, provided herein are methods of manufacturing the TREM-1 peptide inhibitor substances and compositions (e.g., substances and compositions comprising LR12) described herein. The manufacturing methods decrease the formation of LR12 dimers. The timing between solubilization of the active pharmaceutical ingredient, addition of excipients (pH adjustment, arginine), filling of vials and temperature adjustment in the freeze-dryer directly influences dimer formation and consequently, dimer content of the drug product. A tight control of the manufacturing timing with the formulation provided herein allows an acceptable and reproducible control of the dimer content in the final product.

[0116] The methods provided herein control the pH to limit dimer formation during the manufacturing process and after reconstitution of the product; and achieve a desired target concentration (e.g., 24 mg / mL). To optimize the practicability of administration in a hospital setting, the drug product is highly concentrated, with a suitable stability.

[0117] To facilitate the manipulation of the LR12 drug product at clinical sites, the number of vials to be reconstituted per dose is reduced. Therefore, a high dose (HD) formulation of LR12 (nangibotide) is provided herein. Using the excipients arginine or lysine or histidine,and sodium citrate, the concentration of LR12 drug substance (i.e., purified LR12 peptide substance) in the solution to be lyophilized is increased by 5 fold, up to 200 mg of LR12 / mL, with no effect on the formation of LR12 dimer. A formulation at a concentration of 120 mg of LR12 / mL in the solution to be lyophilized was developed. This formulation is called LR12 high dose (HD) drug product (1200 mg / vial).

[0118] Provided herein is a process for preparing a pharmaceutical composition as described herein, comprising the steps of: a) supplying a purified LR12 peptide substance as described herein, preferably by carrying out the steps i) to vii) according to the process as described above; b) mixing the purified substance of LR12 peptide with at least one solubility enhancing agent, preferably arginine or lysine or histidine, more preferably arginine or lysine, even more preferably arginine, and at least one pharmaceutically acceptable buffer, preferably a citrate buffer, even more preferably sodium citrate buffer, preferably: the amount of the at least one solubility enhancing agent, preferably of arginine or lysine or histidine, more preferably arginine or lysine, even more preferably of arginine, ranges from 20.0% to 30.0%, preferably from 22.0% to 28.0%, more preferably from 22.3% to 27.6%, even more preferably is about 24.5%, in weight relative to the dry weight of the purified LR12 peptide substance, and / or the amount of the at least one pharmaceutically acceptable buffer, preferably of the citrate buffer, more preferably of sodium citrate buffer, ranges from 3.0% to 7.0%, preferably from 4.0% to 6.0%, more preferably from 4.5% to 5.5%, even more preferably is about 4.8%, in weight relative to the dry weight of the purified LR12 peptide substance; thereby leading to bulk solution, c) optionally adjusting the pH of the bulk solution, typically with HC1, to a pH 5.5, d) lyophilizing the bulk solution to obtain a lyophilized powder composition of the pharmaceutical composition, ande) optionally adding an aqueous medium, preferably selected from water and saline water solution, preferably a saline water solution having 0.9% sodium chloride in weight relative to the saline water solution; to obtain an aqueous solution of the pharmaceutical composition.

[0119] Advantageously, step e) is carried out extemporaneously.

[0120] Provided herein is a method of preparing the LR12 drug, wherein- filling is performed in a vial of 20 mL (which is more appropriate to the filling volume (10 mL) before lyophilization) instead of the 50 mL vial used previously;- a saline solution is used (preferably NaCl at 0.9% w / w for both reconstitution and dilution); and- a syringe (50 mL) and an automatic syringe pump are used to dispense the defined dose of 1 mg / kg / h administered by continuous intravenous infusion.

[0121] Provided herein is a method of preparing the LR12 drug product (i.e., the in-use form, also referred to as liquid parenteral formulation), wherein- filling is performed in a vial of 20 mL (which is more appropriate to the filling volume (10 mL) before lyophilization) instead of the 50 mL vial used previously;- a saline solution is used (preferably NaCl at 0.9% w / w for both reconstitution and dilution); and- a syringe (50 mL) and an automatic syringe pump are filled with the LR12 adapted for administration of a dose of 1 mg / kg / h by continuous intravenous infusion.

[0122] These steps allow reduction of the total amount of liquid to be administered per day (e.g., 2 syringes (50mL) / day per subject in intensive care unit).Peptide inhibitors and compositions for their therapeutic use

[0123] Another aspect of the present disclosure is a purified substance of LR12 peptide as described herein, or a pharmaceutical composition as described herein, for use as a medicament.

[0124] A further aspect of the present disclosure is the purified substance of LR12 peptide as described herein, or the pharmaceutical composition as described herein, for use in the treatment of a disease selected from an inflammatory disease or disorder, a cardiovascular disease, disorder or condition, and an infectious disease in a subject in need thereof.

[0125] In some embodiments, the disease to be 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, 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 and pancreatitis.

[0127] In some embodiments, the inflammatory disease or disorder is an acute inflammatory disease or disorder. Examples of acute inflammatory disorders include, without being 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. In some embodiments, the inflammatory disease or disorder is septic shock.

[0129] SIRS is characterized by systemic inflammation and widespread tissue injury. Clinically, SIRS is defined as fulfilling at least two of the following four criteria: fever > 38.0°C or hypothermia < 36.0°C; tachycardia > 90 beats / minute;tachypnea > 20 breaths / minute; and leukocytosis > 12x109 / L or leucopenia < 4xlO9 / L (Bone et al., Chest. 1992 Jun; 101 (6): 1644-55). SIRS may occur as a response to a nonspecific insult of either infectious or non-infectious origin. Examples of insults of non- infectious origin include, without being limited to, trauma, thermal injury, pancreatitis, autoimmune disorders and surgery. Examples of insults of infectious origin include bacterial infections (e.g., respiratory infections, abdominal infections 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 a dysregulated response of the human subject to infection (Singer et al., JAMA. 2016 Feb 23;315(8): 801 -10). Patients with sepsis can be clinically identified as patients suffering from a documented or suspected infection and from an organ dysfunction. Organ dysfunction in a human subject may be identified using an organ dysfunction score, notably upon admission of the human subject in ICU or emergency unit. Examples of organ dysfunction scores include, without being limited to, the SOFA (Sequential Organ Failure Assessment) score, the qSOFA (quick SOFA) score, the MODS (Multiple Organ Dysfunction Score), the P-MODS (Pediatric Multiple Organ Dysfunction Score) and the LODS (Logistic Organ Dysfunction System).

[0131] Septic shock is defined as a subset of sepsis, in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone. Thus, sepsis encompasses septic shock. Patients with septic shock can be clinically identified as patients suffering from sepsis and having (i) persisting hypotension requiring vasopressors to maintain their mean arterial pressure >65 mm Hg despite adequate volume resuscitation and (ii) a serum lactate level >2 mmol / L (18mg / dL) (Sepsis-3 definition as described in Singer et al., JAMA. 2016 Feb 23 ;315(8): 801 -10).

[0132] In some embodiments, the infection inducing the dysregulated response of the subject suffering from septic shock, sepsis, or SIRS, is a bacterial, fungal, parasitic, or viralinfection. Examples of infections include, without being limited to, respiratory infections, abdominal infections, and urinary tract infections (Ulis).

[0133] In some embodiments, the disease to be 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 pectoris, effort angina pectoris, cardiomyopathy, hypertensive heart disease, chronic heart failure, acute heart failure, cor pulmonale, cardiac dysrhythmias, inflammatory heart diseases (such as endocarditis and myocarditis), vasculitis, peripheral arterial disease, SIRS-associated 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 pectoris, effort angina pectoris, cardiomyopathy, hypertensive heart disease, chronic heart failure, acute heart failure, cor pulmonale, cardiac dysrhythmias, inflammatory heart disease, peripheral arterial disease, SIRS-associated myocardial and / or vascular dysfunction, and atherosclerosis.

[0135] In some embodiments, the disease, disorder or condition to be 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 infectious diseases caused by bacteria, viruses, fungi, and parasites, respectively. Examples of bacterial diseases include Escherichia coli infections. Examples of viral diseases include SARSCoV-2 infections (also referred to as coronavirus disease 2019 or COVID-19). In some embodiments, the infection disease is COVID-19.

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

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

[0138] In some embodiments, the purified substance of LR12 peptide or the pharmaceutical composition as described herein are for administration at a dose (or rate) ranging from about 0.1 mg / kg / h to about 3 mg / kg / h (mg of LR12 per kg body weight of the subject per hour), preferably from about 0.3 mg / kg / h to about 3 mg / kg / h. In some embodiments, the pharmaceutical composition or purified LR12 peptide substance is for intravenous administration at a rate ranging from 0.3 to 3.0 mg / kg / hr (mg of LR12 per kilo body weight per hour).

[0139] Indeed, the high LR12 peptide content in the pharmaceutical composition (drug product) according to the invention allows a high rate of administration, for example ranging from 0.3 to 3.0 mg / kg / hr (mg of LR12 per kg bodyweight of the subject per hour). This high rate of administration improves patient comfort by limiting administration time, and reduces the risk of volume overload.

[0140] In some embodiments, the purified substance of LR12 peptide or the pharmaceutical composition as described herein are for administration at a dose (or rate) ranging from about 0.1 mg / kg / h to about 1 mg / kg / h (mg per kg body weight of the subject per hour), preferably from about 0.3 mg / kg / h to about 1 mg / kg / h.

[0141] In some embodiments, the purified substance of LR12 peptide or the pharmaceutical composition as described herein are for administration at a dose (or rate) 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 the pharmaceutical composition as described herein are for administration, preferably continuous administration, for at least 24, 48, 72, 96, or 120 hours. In some embodiments, the purified LR12 peptide substance or the pharmaceutical composition as described herein are for administration, preferably continuous administration, for up to 7, 6, 5, 4, or 3 days. In some embodiments, the purified LR12 peptide substance or the pharmaceutical composition as described herein are for administration, preferably continuous administration, for up to 5 days.

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

[0144] In some embodiments, the subject suffers from a shock or hypotensive shock or distributive shock, which refer to a diminished or insufficient perfusion that impairs organ function and is generally associated with decreased arterial blood pressure. Accordingly, in some embodiments, the subject received or is receiving fluid therapy, i.e., a therapy aiming at restoring and / or maintaining the volume and composition of the body fluids to normal, in particular with respect to the water-electrolyte balance. Fluid therapy thus aims at correcting and / or preventing volume and / or electrolyte deficit. In some embodiments, the subject is vulnerable to hypertonic solutions and / or to volume overload.

[0145] In some embodiments, a subject suffering from an inflammatory disease or disorder, in particular septic shock, sepsis, or SIRS is successfully "treated", if, after being administered a purified substance of LR12 peptide or pharmaceutical composition as described herein, the subject shows at least one of the following: o a reversal of a hypotensive shock, wherein a hypotensive shock reversal is defined as the absence of any vasopressor therapy during at least 24 hours (i.e., not requiring to restart a vasopressor therapy in the 24 hours following the end of a vasopressor therapy); o a decrease of a severity score used to assess the severity of the disease and / or the prognosis of the subject upon admission in ICU or emergency unit, such as the APACHE II (Acute Physiology And Chronic Health Evaluation II) score, the APACHE (Acute Physiology And Chronic Health Evaluation III) III score, the APACHE IV (Acute Physiology And Chronic Health Evaluation IV) score, the SAPS II (Simplified Acute Physiology Score II) score or the SAPS III (Simplified Acute Physiology Score III) score; o a decrease of an organ dysfunction score used to assess the presence of organ dysfunction in the subject upon admission in ICU or emergency unit, such as the SOFA score, the qSOFA score, the MODS, the P-MODS or the LODS; o a decrease of the SOFA score and / or of the qSOFA score, preferably with reference to the SOFA score and / or the qSOFA score assessed upon admission in ICU or emergency unit or before the start of the administration of a purified LR12 peptide substance or pharmaceutical composition as described herein; o a decrease in the requirement for cardiovascular support, for example a decrease in the use of vasopressor therapy; o a decrease in the requirement for respiratory support, for example a decrease in the use of invasive mechanical ventilation (IMV); o a decrease in the requirement for renal support, for example a decrease in the use of continuous or discontinuous renal replacement therapy also referred to as RRT (e.g., dialysis);o a decrease in the risk of reinfection or an absence of reinfection; o a decrease in the risk of rehospitalization or an absence of rehospitalization; o an increase in the chance of survival; o a decrease in the risk of sepsis-related death; o a decrease in the risk of the all-cause death; o a decrease in the risk of post-sepsis or post-shock morbidity; o an increase in the quality of life, in particular in the post-sepsis or post-shock quality of life, that may be assessed for example through an evaluation of survival and quality-adjusted life years (QALYs), estimated from the EQ5D. For example, health-related quality of life (HRQoL) scores from the EQ 5D 5L may be calculated and converted to utility scores.The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0146] Also provided herein are methods of using the TREM-1 peptide inhibitor substances and compositions (e.g., substances and compositions comprising LR12) described herein to treat a disease or a disorder, in particular an inflammatory disease or disorder, a cardiovascular disease, disorder or condition, or an infectious disease. Provided herein are methods of using the TREM-1 peptide inhibitor substances and compositions described herein to treat septic shock in a subject in need thereof. Provided herein are methods of using the TREM-1 peptide inhibitor substances and compositions described herein to treat sepsis in a subject in need thereof.

[0147] Provided herein is a method of treating an inflammatory disease or disorder, a cardiovascular disease, disorder or condition, or an infectious disease, as described herein in a subject in need thereof, said method comprising administering to the subject a purified substance of LR12 peptide as described herein, or a pharmaceutical composition as described herein.

[0148] Provided herein is a method of treating septic shock or sepsis in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable composition described herein intravenously at a rate of about 0.3 mg / kg / hr - about 3.0 mg / kg / hr (mg of LR12 per kg body weight of the subject per hour). Provided herein is a method of ameliorating a symptom of septic shock or sepsis in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable composition described herein intravenously at a rate of about 0.3 - about 3.0 mg / kg / hr. In some embodiments, the rate is about 1.0 mg / kg / hr (mg of LR12 per kg body weight of the subject per hour).

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

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

[0151] Also provided herein is the use of a purified substance of LR12 peptide as described herein, or a pharmaceutical composition as described herein, in the manufacture of a medicament for the treatment of a disease as described herein in a subject in need thereof.

[0152] Further provided herein is a pharmaceutical composition as described herein for the treatment or for use in the treatment of a disease as described herein in a subject in need thereof.

[0153] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define a term that contradicts that term’s definition in the application, thedefinition that appears in this disclosure controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.EXAMPLES

[0154] The present disclosure is further illustrated by the following examples. The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure. It will be understood by those of skill in the art that numerous and various modifications can be made to yield essentially similar results without departing from the spirit of the present disclosureExample 1: Absence of biological activity of the LR12 dimerMaterials and Methods

[0155] Neutrophils produce reactive oxygen species (ROS) in presence of LPS (lipopolysaccharides) following activation of the TREM-1 pathway and are thus used in the present example in order to assess TREM-1 activation and the biological activity of the LR12 dimer on the TREM-1 pathway. ROS production induced by TREM-1 activation and its modulation by LR12 can be quantified by flow cytometry. Any decrease in ROS production by LR12 (LPS+LR12 conditions) translates into a decrease in mean fluorescence intensity (MFI) as compared to the ROS production observed following the TREM-1 activation in presence of LPS (LPS condition).

[0156] Primary human neutrophils were isolated from the peripheral blood of healthy donors by immunomagnetic negative cell sorting with EasySep™ Human Monocyte / Neutrophil Isolation Kits (StemCell, Canada) following the manufacturer’s instructions. Purity was assessed by flow cytometry. Cells were suspended in RPMI 1640medium containing GlutaMAX and supplemented with 10% FCS, 25 mMHEPES, 100 U / ml penicillin and streptomycin before stimulation. Human primary neutrophils were incubated in resting conditions (also referred to as control conditions or Ctrl 2), or with lOOng / mL LPS from E. coli serotype 0127:B8 (Sigma-Aldrich), with or without LR12 or LR12-dimer (dimer) at the indicated concentrations.

[0157] ROS production was quantified by using a fluorogenic substrate (DCFDA or 2',7'-dichlorofluorescein diacetate), a chemically reduced form of fluorescein used as an indicator of the presence of ROS in cells. Upon cleavage of the acetate groups by intracellular esterases and oxidation, the nonfluorescent DCFDA is converted to the highly fluorescent 2', 7'-di chlorofluorescein (DCF). 2.5xl05isolated human neutrophils were incubated 2 hours at 37°C - 5% CO2 with 5pM of DCFDA, with or without lOOng / mL LPS, and with or without LR12 or LR12-dimer (dimer) at the indicated concentrations. Data were acquired using flow cytometry. Results are expressed as mean fluorescence intensity (MFI).Results

[0158] As shown on Figure 1, LPS induced a significant increase of ROS production by human primary neutrophils after 2 hours stimulation. LR12 monomer was able to decrease in a dose-dependent manner the LPS-induced release of ROS. Surprisingly, LR12 dimer did not display any TREM-1 inhibiting property. Indeed, LR12-dimer was not able to decrease the production of ROS by LPS-activated neutrophils.

[0159] These results show that, despite the fact that the LR12-dimer is composed of 2 LR12 peptides covalently bound by a disulfide bond through the cysteine at position 11, it is not able to induce any pharmacological inhibition of TREM-1.Example 2: Preparation of the purified LR12 peptide substance (drug substance)

[0160] A stepwise solid-phase synthesis of LR12 peptide (15mol) was performed using Fmoc / tBu chemistry in a 270L SPPS reactor. Sequential acylation reactions are carried out at 20°C ±3°C under stirring at 30 ± 20 rpm using appropriate Na-Fmoc-protected aminoacids, which are carboxyl-activated using Oxyma, DITU and DIC in DMF. Capping step by acylation with acetic anhydride is repeated after each amino acid coupling. Upon completion of the sequential acylation reactions, the peptide-resin is washed with IP A, filtered and dried in vacuo.

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

[0162] The dried crude LR12 peptide is re-solubilized in aqueous ammonia, its pH is adjusted to 6.5-7.0, and let under stirring for 3h at 50-55°C, before removing the spent resin by filtration. Purification of the dissolved crude solution is carried through ion-exchange chromatography (TEC) with load of <30 g / L Column Volume (CV) on a 450 mm diameter column (2.5cm / min), followed by Reversed-phase chromatography (RPC) with load of 15-22 g / L CV on a 450 mm diameter column (4.7cm / min) for which fractions are eluted with NH4O Ac / Acetonitrile (MeCN), collected in water and kept under cold storage (2-8°C). The pooled fractions are concentrated (in 2 steps) and purified by ultrafiltration / diafiltration using 300 Dalton membrane. 15mM Arginine is added after the 1st concentration (approx. 65 g / L). The solution is then further processed to a concentrated solution of 170g / L. Nitrogen inerting of the bulk solutions (cooled below 15°C) is performed to maintain a low-level oxygen content.

[0163] The concentrated LR12 solution is immediately filtered and lyophilized to obtain a purified LR12 peptide substance containing i- 85% peptide content in weight of the total purified LR12 peptide substance weight and ii- 4.7% arginine in weight of the total purified LR12 peptide substance weight. The amount of the LR12 dimer was 0.7% in weight relative to the total weight of the peptide content.Example 3: Preparation of the pharmaceutical composition (drug product)

[0164] The pH during the formulation process of the drug substance according to Example 2 is maintained between 5 and 6. The timing between the solubilization of the drug substance (comprising the active pharmaceutical ingredient, API), the addition of excipients (pH adjustment, arginine), and the filling of vials and the temperature adjustment in the freeze- dryer is set below 24 hours. Nitrogen inerting of the bulk solutions is performed by bubbling nitrogen to the solutions in order to get an oxygen content below 5 ppm. At the time of administration, the pharmaceutical composition (drug product powder in a 20mL vial) is reconstituted with 9.5 mL of NaCl 0.9% w / w and then further diluted in a 50 mL syringe with NaCl 0.9% w / w. The concentration of obtained LR12 solution is 24 mg / mL with an osmolality of 380 mOsm / kg.

[0165] The quantitative composition of the drug product is presented in Table 1.* containing 1200mg API (LR12)Table 1: Composition of the LR12 drug product

[0166] LR12-dimer content measured by analytical reversed phase HPLC-UV and expressed as a percentage in weight relative to the weight of the LR12 peptide for every process step are presented in Table 2 below.Table 2: Content of LR12 dimer, Des-Gln-LR12 and Met(O)12-LR12 % of the purified LR12 peptide substance, of the drug product and of the reconstituted drug product; the % being relative to the peptide content of the LR12 drug substance, drug product and the reconstituted drug product respectively. Comparative Examples 4-9,: Preparation of comparative pharmaceutical compositionsComparative Example 4a (NaCl):

[0167] The purified substance of LR12 peptide (of example 2) is formulated using the following excipients: 50 mM sodium citrate pH 5.5; 50 mMNaCl. The concentration of bulk solution (before freeze drying) is 10 mg / mL (due to solubility of LR12 limit). Comparative Example 4b (NaCl, pH 6.5):

[0168] The purified substance of LR12 peptide (of example 2) is formulated using the following excipients: 50 mM sodium phosphate pH 6.5; 50mM NaCl. The concentration of bulk solution (before freeze drying) is 20 mg / mL (due to solubility of LR12 limit).Comparative Example 5 (mannitol):

[0169] The purified substance of LR12 peptide (of example 2) is formulated using the following excipients: 50 mM sodium citrate pH 5.5; 200 mM mannitol. The concentration of bulk solution (before freeze drying) is 10 mg / mL (due to solubility of LR12 limit).Comparative Example 6 (betaine):

[0170] The purified substance of LR12 peptide (of example 2) is formulated using the following excipients: 50 mM sodium citrate pH 5.5; 150 mM betaine. The concentration of bulk solution (before freeze drying) is 10 mg / mL (due to solubility of LR12 limit).Comparative Example 7a (lysine):

[0171] The purified substance of LR12 peptide (of example 2) is formulated using the following excipients: 5 OmM sodium citrate pH 5.5; 50 mM lysine; 50 mM NaCl. The concentration of bulk solution (before freeze drying) is 40 mg / mL (due to solubility of LR12 limit).Comparative Example 7b (arginine):

[0172] The purified substance of LR12 peptide (of example 2) is formulated using the following excipients: 50mM sodium citrate pH 5.5; 50 mM arginine. The concentration of bulk solution (before freeze drying) is 40 mg / mL (due to solubility of LR12 limit).Comparative Example 8 (sodium citrate; arginine; pH 5.5):

[0173] The purified substance of LR12 peptide (of example 2) is formulated using the following excipients: 25mM sodium citrate pH 5.5; 225 mM arginine; 65 mM HC1. The concentration of bulk solution (before freeze drying) is 150 mg / mL; osmolality 512 mOsm / kg.Series of Comparative Examples 9 (90-420 mM Arginine; pH 5.5):

[0174] Formulations using incremental concentrations of the purified substance of LR12 peptide (of example 2) and incremental concentrations of the following excipients: Arginine 90 to 420 mM, using only HC1 for pH adjustment to 5.5.

[0175] The results regarding the solubility, the LR12 stability and osmotic pressure of the obtained pharmaceutical compositions is presented in Table 3 below.Table 3: Solubility, stability and osmolality of the compared pharmaceutical compositions

[0176] Among all different formulations tested, citrate or phosphate buffers alone only allowed for up to 20 mg / mL LR12 solubility (comparative examples 4a and 4b). The use of excipients such as mannitol or betaine did not lead to increased solubility of LR12 (examples 5 and 6). Both arginine and lysine at 50mM were able to increase the solubility of LR12 up to 40mg / mL, (comparative examples 7a and 7b). Increasing concentrations of arginine from 90 to 420 mM (example 9) were associated with higher solubility of LR12, but were also associated with significant higher dimer increase during formulation, despite the fact that pH was maintained as per in the pharmaceutical composition of the invention.

[0177] The combination of arginine and citrate buffer, according to the invention, allowed thus not only to enhance the LR12 solubility but also to obtain the lowest dimer content products while using the minimum possible amount of excipients and keeping the osmolality of the obtained pharmaceutical composition below 500 mOsm / kg.Comparative Example 11: Comparative process regarding the preparation of LR12 drug substance composition

[0178] The dried crude LR12 peptide (of example 2) is re-solubilized in aqueous ammonia, pH adjusted to 6.5-7.0, and under stirring for 3h at 50-55°C, before removing the spent resin by filtration. Purification of the dissolved crude solution is carried through ion-exchange chromatography (IEC) with load of <30 g / L CV on a 450mm diameter column (2.5cm / min), followed by Reversed-phase chromatography (RPC) at room temperature -25°C) with load of 15-22 g / L CV on a 450mm diameter column (4.7cm / min) for which fractions are eluted with NH4OAc / MeCN, collected in water, without Nitrogen inerting. The pooled fractions are concentrated and purified by ultrafiltration / diafiltration using 300 Dalton membrane to a concentrated solution of 15g / L. The concentrated LR12 solution is filtered and lyophilized to obtain a purified substance of LR12 peptide.

[0179] LR12-dimer contents for every process step are presented in Table 4.Table 4: LR12-dimer content

[0180] The results of the comparative example 11 show that the process steps according to the invention, in particular the N2 inerting and / or the reversed phase, ultrafiltration / diafiltration steps at chilled temperature of less than 15°C presented in example 2 may further inhibit the LR12-dimer formation in the purified substance of LR12 peptide.Examples 12-18: Preparation of pharmaceutical compositions according to the invention and of comparative pharmaceutical compositions

[0181] Material and methods

[0182] Pharmaceutical compositions were prepared using incremental concentrations of LR12 peptide in the purified substance of LR12 peptide (of example 2); incremental amounts of amino acids (arginine or lysine) from 15% to 26% in weight relative to the dry weight of the purified LR12 peptide substance; and incremental amounts of sodium citrate buffer from 5% to 7% in weight relative to the dry weight of the purified substance of LR12 peptide. The obtained pharmaceutical compositions were lyophilized powders (= drug product in powder form) containing from 1200 mg to 2000 mg of LR12 peptide (per vial).

[0183] In order to prepare these pharmaceutical compositions in powder form, the buffer solution was first prepared according to table 5. The purified LR12 peptide substance and at least one amino acid selected from arginine and lysine were added into the prepared buffer solution. The pH during the formulation process was adjusted to get a bulk solution (before the freeze-drying step) around 5.5. To further limit dimer formation during the manufacturingprocess, nitrogen inerting of the bulk solutions was performed in order to get an oxygen content below 5 ppm.

[0184] The prepared bulk solution was visually checked and filtered using 0.2 pm disposable filters. Then, 20 mL vials were filled with the filtered bulk solutions. Filling volumes varied from lOmL to 15mL depending on the LR12 peptide concentration in each bulk solution (from 80 mg / mL to 200 mg / mL). The vials containing the filtered bulk solutions were then lyophilized to get pharmaceutical compositions in the form of powders (= drug product in storage form).

[0185] The pharmaceutical compositions according to the invention are: - LR12 120mg / mL & Arginine (155mM),LR12 120mg / mL & Lysine (190mM),LR12 120mg / mL & Arginine (190mM),LR12 200mg / mL & Arginine (3 OOmM), LR12 200mg / mL & Lysine (300mM).

[0186] The pharmaceutical compositions not according to the invention (comparative) are:LR12 80mg / mL & Arginine (80mM), andLR12 lOOmg / mL & Arginine (120mM).

[0187] The material and methods are summarized in the following Table 5:Table 5: Preparation of the compared pharmaceutical compositions

[0188] Results

[0189] The results are presented in the following Table 6.Table 6: Results obtained with the compared pharmaceutical compositions

[0190] The results show that there are precipitates for the two comparative examples having an amount of the amino acid of less than 20% in weight relative to the dry weight of the purified LR12 peptide substance. Those precipitates, observed in the bulk solution before the filtration, are not suitable for further processing. Thus, it is not possible to obtain a pharmaceutical composition in a powder form (=drug product in a storage form) when the amount of the amino acid is of less than 20% in weight relative to the dry weight of the purified LR12 peptide substance.

[0191] The results show that, upon reconstitution with 9.5 mL of NaCl 0.9% w / w, the pharmaceutical composition (drug product powder in a 20mL vial) comprises (in weight relative to the peptide content) less than 5.0% w / w of LR12 dimer in weight relative to the peptide content, less than 1.2% of Des-Gln-LR12 in weight relative to the peptide content and less than 1.0% of Met(O)12-LR12 in weight relative to the peptide content. The osmolality is confirmed to be less than the acceptable limit of 500 mOsm / kg.

[0192] Therefore, a pharmaceutical composition according to the invention in a powder form, having an amount of arginine, lysine or histidine ranging from 20% to 30% in weight relative to the dry weight of the purified LR12 peptide substance, allows the obtention of a pharmaceutical composition to have less than 5.0% w / w of LR12 dimer in weight relative to the peptide content, high LR12 peptide amounts of 1200 mg and 2000 mg and an osmolality of less than the acceptable limit of 500 mOsm / kg. All these technical advantages contribute to allow the pharmaceutical composition in its in-use form to, at least: be suitable for administration with a high rate of administration, improving the patient comfort and limiting the risk of volume overload; decreasing the ratio between the weight content of excipients to the weight content of LR12 peptide, limiting the daily exposure to excipients, i.e., the total amount of the excipients that would be taken or used in a day basis (in mg / kg of excipients per kilo body weight per hour); be suitable for administration to pediatric population with body weight below 30kg;be tolerated by the patient body (osmolality of less than the acceptable limit of 500 mOsm / kg); and have a sufficient therapeutic effect of the LR12 peptide thanks to high purity and stability of LR12.

[0193] On the contrary, pharmaceutical compositions in a powder form (=drug product in a storage form) were not obtained when the amount of the amino acid was of less than 20% in weight relative to the dry weight of the purified LR12 peptide substance.

Claims

CLAIMS1. A pharmaceutical composition comprising a purified LR12 peptide substance of amino acid sequence SEQ ID NO: 1, in association 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 ranges from 20% to 30% in weight relative to the dry weight of the purified LR12 peptide substance; and wherein the pharmaceutical composition comprises less than 5.0% w / w of LR12 dimer in weight relative to the peptide content.

2. The pharmaceutical composition according to claim 1, wherein said pharmaceutical composition comprises from 800 mg to 2500 mg of LR12 peptide content, preferably from 1200 mg to 2000 mg of LR12 peptide content.

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

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

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

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

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein: the pharmaceutical composition is an aqueous solution further comprising an aqueous medium, preferably selected from water and saline water solution, preferably in saline water solution having 0.9% sodium chloride in weight relative to the saline water solution; and / or the pharmaceutical composition has a pH ranging from 5.0 to 6.0, preferably about 5.5.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein said pharmaceutical composition presents an osmolality of less than 500 mOsm / kg, preferably ranging from 300 to 400 mOsm / kg, more preferably from 320 to 390 mOsm / kg, even more preferably about 380 mOsm / kg.

9. A purified LR12 peptide substance having an amino acid sequence as set forth in SEQ ID NO: 1, comprising at least 85% w / w of peptide content in weight of the total weight of the purified substance of LR12; and comprising less than 2.7% of LR12 dimer in weight relative to the total weight of the peptide content.

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

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

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

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

14. The pharmaceutical composition or purified LR12 peptide substance for use according to claim 12 or 13, wherein said pharmaceutical composition or purified LR12 peptide substance is for intravenous administration at a rate ranging from 0.3 to 3.0 mg / kg / hr (mg of LR12 per kg body weight per hour).

15. A process for preparing a purified LR12 peptide substance according to any one of claims 9 to 11, comprising the steps of: i. synthesizing the LR12 peptide on solid phase, leading to a crude LR12 composition; ii. providing a first peptide solution comprising the crude LR12 composition;iii. purifying the first peptide solution by ion exchange column chromatography (IEC), leading to a first pre-purified substance of LR12; iv. further purifying the pre-purified substance of LR12 by reversed-phase high performance liquid chromatography (HPLC), thereby leading to HPLC pooled fractions comprising a second pre-purified substance of LR12; v. preparing an arginine solution between 5 and 25 mM to be added to the pooled fractions during step vi.; vi. concentrating the pooled fractions using membrane diafiltration or ultrafiltration, typically on a 150 to 300 Dalton membrane, to a concentrated solution of at least 120 g / L; and vii. filtering and lyophilizing the concentrated solution obtained in step vi) to obtain the purified substance of LR12 peptide.

16. The process of claim 15, wherein the HPLC pooled fractions comprising the second pre-purified substance of LR12 are cooled to a temperature of 15°C or less and / or are bubbled with nitrogen.

17. A process for preparing a pharmaceutical composition according to any one of claims 1 to 8, comprising the steps of: a) supplying a purified LR12 peptide substance according to any one of claims 9 to 11, preferably by carrying out the steps i) to vii) according to claim 15 or claim 16; b) mixing the purified LR12 peptide substance with citrate and at least one amino acid selected from arginine, lysine and histidine, preferably: the amount of the at least one amino acid ranging from 20.0% to 30.0%, preferably from 22.0% to 28.0%, more preferably from 22.3% to 27.6%, even more preferably is about 24.5%, in weight relative to the dry weight of the purified LR12 peptide substance of LR12 peptide, and the amount of citrate ranging from 3.0% to 7.0%, preferably from 4.0% to 6.0%, more preferably from 4.5% to 5.5%, even more preferably is about4.8%, in weight relative to the dry weight of the purified LR12 peptide substance of LR12 peptide; thereby leading to bulk solution c) optionally adjusting the pH of the bulk solution, typically with HC1, to a pH 5.5, d) lyophilizing the bulk solution to obtain a lyophilized powder composition of the pharmaceutical composition, and e) optionally adding an aqueous medium, preferably selected from water and saline water solution, preferably saline water solution having 0.9% sodium chloride in weight relative to the saline water solution; to obtain an aqueous solution of the pharmaceutical composition.

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