Stable aqueous formulation of anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment

Abstract

The subject of the present invention is an aqueous pharmaceutical formulation of an antibody. In particular, the present invention relates to a stable aqueous antibody formulation comprising arginine, trehalose, a surfactant and histidine, and its pharmaceutical preparation and use. The present invention is exemplified by an aqueous formulation of an anti-ADM antibody or an anti-ADM antibody fragment.

Description

[Technical field]

[0001] The present invention relates to the field of pharmaceutical formulations of antibodies. In particular, the subject of the present invention relates to a stable liquid antibody formulation comprising arginine, trehalose, a surfactant and histidine, and its pharmaceutical preparation and use. [Background technology]

[0002] Antibody preparations used for therapeutic or prophylactic purposes require stabilizers to prevent loss of protein activity or structural integrity due to denaturation, oxidation, or aggregation effects over a period of storage and transport prior to use. These problems are exacerbated by the presence of high levels of antibodies, which are often desirable for treating disease. A major objective in the development of antibody formulations is to preserve the antibody, its solubility, stability, and antigen-binding ability. It is particularly important to avoid particle crowding in the solution, which requires sterile filtration before use for intravenous or subcutaneous administration and limits the route of administration. The formulation of antibody preparations requires careful selection of these factors, among others, to avoid protein denaturation and loss of antigen-binding activity. Thus, there is a need for stable aqueous antibody formulations that support stable high concentrations of bioactive antibodies in solution and are suitable for parenteral administration, including intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection.

[0003] Moreover, various diseases or illnesses may have common, partially non-specific symptoms that may range from unpleasant to intolerable to the individual suffering from them. Individuals who experience more than one symptom often need to take several drugs to experience relief from these symptoms. There is a continuous need for new forms of treatment or prevention of symptoms associated with many different underlying diseases or illnesses. In particular, it would be useful to provide a medicine or drug that can be used to treat or prevent more than one symptom associated with an underlying disease or illness.

[0004] The peptide adrenomedullin (ADM) was first reported in 1993 (Kitamura K. et al. 1993. Biochemical and Biophysical Research Communications Vol. 192(2):553-560) as a new antihypertensive peptide containing 52 amino acids, which was isolated from human pheochromocytoma.

[0005] In the same year, a cDNA encoding a precursor peptide containing 185 amino acids and the complete amino acid sequence of this precursor peptide were also reported.

[0006] In particular, the precursor peptide, which contains a signal sequence of 21 amino acids at the N-terminus, is called "pre-pro-adrenomedullin" (pre-proADM). In this specification, all amino acid positions specified generally relate to pre-proADM, which contains 185 amino acids. The peptide adrenomedullin (ADM) is a peptide containing 52 amino acids (SEQ ID NO: 13) and including amino acids 95-146 of pre-proADM, which are formed therefrom by proteolytic cleavage. To date, only a few fragments of the peptide fragments formed upon cleavage of pre-proADM have been more precisely characterized, in particular the physiologically active peptide adrenomedullin (ADM) and "PAMP", i.e. the peptide containing the 20 amino acids (22-41) following the 21 amino acids of the signal peptide in pre-proADM. The discovery and characterization of ADM in 1993 triggered an intensive research activity, the results of which have been summarized in various review articles, of which in the present context we refer in particular to the articles published in the special issue of the journal "Peptides" on ADM (Editorial, Takahashi K. 2001. Peptides 22:1691) and (Eto T. 2001. Peptides 22:1693-1711). Further reviews can be found in (Hinson et al. 2000. Endocrine Reviews 21(2):138-167). Previous scientific studies have shown, inter alia, that ADM can be considered as a multifunctional regulatory peptide. It is released into the blood circulation in an inactive form extended by glycine (Kitamura K. et al. 1998. Biochem. Biophys. Res. Commun. 244(2):551-555). There are also binding proteins that are specific for ADM and that presumably regulate the effects of ADM as well (Pio R. et al. 2001. The Journal of Biological Chemistry 276(15):12292-12300). The most important physiological action of ADM and PAMP studied to date is their effect on blood pressure.

[0007] ADM is a potent vasodilator, and the antihypertensive effect can be associated with a specific peptide segment in the C-terminal portion of ADM. Moreover, the further physiologically active peptide PAMP formed from pre-proADM was found to have a similar antihypertensive effect, even though it appears to have a different mechanism of action from ADM.

[0008] Furthermore, concentrations of ADM that can be measured in the blood circulation and other biological fluids have been found to be significantly higher in several pathological conditions than those found in healthy control humans. Thus, ADM levels are significantly increased to different degrees in patients with congestive heart failure, myocardial infarction, renal disease, hypertensive disorders, diabetes mellitus, acute phase of shock, and sepsis and septic shock. PAMP concentrations are also increased in some of these pathological conditions, although plasma levels are decreased relative to ADM (Eto, T., supra).

[0009] Furthermore, it is known that abnormally high concentrations of ADM are observed in sepsis, with the highest concentrations observed in septic shock (see (Eto, T., supra) and (Hirata et al. Journal of Clinical Endocrinology and Metabolism 1996. 81(4):1449-1453; Ehlenz K. et al. 1997. Exp Clin Endocrinol Diabetes 105:156-162; Tomoda Y. et al. 2001. Peptides 22:1783-1794; Ueda S. et al. 1999. Am. J. Respir. Crit. Care Med. 160:132-136; and Wang P. Peptides 2001. 22:1835-1840).

[0010] WO 2004 / 097423 describes the use of antibodies against adrenomedullin for the diagnosis, prognosis and treatment of cardiovascular disorders. Treatment of diseases by blocking ADM receptors has also been described in the art (e.g. WO 2006 / 027147, PCT / EP2005 / 012844), and the diseases may be sepsis, septic shock, cardiovascular diseases, infectious diseases, skin diseases, endocrine diseases, metabolic diseases, gastrointestinal diseases, cancer, inflammation, blood diseases, respiratory diseases, musculoskeletal diseases, neurological diseases, urological diseases.

[0011] It has been reported that ADM improves cardiac function and blood supply in the liver, spleen, kidneys, and small intestine during the early stage of sepsis. ADM neutralizing antibodies neutralize the above effects during the early stage of sepsis (Wang, P., "Adrenomedullin and cardiovascular responses in sepsis", Peptides, Vol. 22, pp. 1835-1840 (2001)).

[0012] For other diseases, blocking ADM may be beneficial to some extent. However, since a certain amount of ADM may be required for some physiological functions, it may also be harmful if ADM is completely neutralized. Many reports have emphasized that administration of ADM may be beneficial in certain diseases. In contrast, other reports have reported that ADM is life-threatening when administered in certain conditions.

[0013] Administration of ADM in combination with ADM binding protein-1 has been described in the art for the treatment of sepsis and septic shock. Treatment of septic animals with ADM and ADM binding protein-1 is believed to prevent the transition to the later stages of sepsis. It should be noted that in living organisms, ADM binding protein (complement factor H) is present in high concentrations in the blood circulation of the organism (Pio et al.: Identification, characterization, and physiological actions of factor H as an Adrenomedullin binding Protein present in Human Plasma; Microscopy Res. and Technique, 55:23-27 (2002) and Martinez et al.; Mapping of the Adrenomedullin-Binding domains in Human Complement factor H; Hypertens Res Vol. 26, Suppl (2003), S56-59).

[0014] The efficacy of non-neutralizing antibodies targeted against the N-terminus of ADM was investigated in survival studies in CLP-induced sepsis in mice. Pretreatment with non-neutralizing antibodies resulted in reduced catecholamine infusion rates, renal dysfunction, and ultimately improved survival (Struck et al. 2013. Intensive Care Med Exp 1(1):22; Wagner et al. 2013. Intensive Care Med Exp 1(1):21).

[0015] Due to these positive results, a humanized version of the N-terminal anti-ADM antibody, called adrecizumab, is being developed for further clinical development. The beneficial effects of adrecizumab on vascular barrier function and survival were recently demonstrated in a preclinical model of systemic inflammation and sepsis (Geven et al. 2018. Shock 50(6):648-654). In this study, pretreatment with adrecizumab attenuated renal vascular leakage in endotoxemic rats as well as mice with CLP-induced sepsis, which was consistent with increased renal expression of the protective peptide Ang-1 and decreased expression of the deleterious peptide vascular endothelial growth factor. Pretreatment with adrecizumab also improved 7-day survival in CLP-induced sepsis in mice from 10 to 50% for single dose administration and from 0 to 40% for repeated dose administration. Of particular interest is the proposed mechanism of action of adrecizumab. Both animal and human data demonstrate a strong dose-dependent increase in circulating ADM following administration of this antibody. Based on the pharmacokinetic data and the lack of an increase in MR-proADM (an inactive peptide fragment derived from the same prohormone as ADM), the higher circulating ADM levels cannot be explained by increased production.

[0016] A mechanistic explanation for this increase could be that since ADM is small enough to cross the endothelial barrier, but antibodies are not, excess antibodies in the blood circulation could dump ADM from the interstitium into the blood circulation (Geven et al. 2018.Shock.50(2):132-140; and Voors et al (J.Eur J Heart Fail.2019 Feb;21(2):163-171)). Furthermore, binding of antibodies to ADM results in an extension of the half-life of ADM. Even when NT-ADM antibodies partially inhibit ADM-mediated signaling, a large increase in blood circulating ADM would result in an overall "net" increase in ADM activity in the blood compartment, exerting beneficial effects on ECs (mainly barrier stabilization), whereas the deleterious effects of ADM on VSMCs in the interstitium (vasodilation) are reduced.

[0017] WO 2013 / 072510 describes non-neutralizing anti-ADM antibodies for use in the treatment of a severe chronic or acute disease or condition in a patient, to reduce the risk of mortality in said patient.

[0018] WO 2013 / 072511 describes non-neutralizing anti-ADM antibodies for use in the treatment of a chronic or acute disease or condition in a patient, for the prevention or amelioration of organ dysfunction or failure.

[0019] WO 2013 / 072512 describes the half-life (t 1 / 2 They describe a non-neutralizing anti-ADM antibody that is an ADM stabilizing antibody that extends the half retention time of ADM and blocks the biological activity of ADM by less than 80%.

[0020] WO 2013 / 072513 shows that in patients with chronic or acute diseases or conditions requiring stabilization of blood circulation, anti-adrenomedullin (ADM) antibodies or anti-adrenomedullin antibody fragments or anti-ADM non-Ig scaffolds stabilize the patient's blood circulation and reduce the patient's need for vasopressors, such as catecholamines.

[0021] WO 2013 / 072514 shows that anti-adrenomedullin (ADM) antibodies or anti-adrenomedullin antibody fragments or anti-ADM non-Ig scaffolds can be effectively used to regulate fluid balance in patients with chronic or acute diseases or conditions, particularly in patients suffering from fluid imbalance in the ICU (intensive care unit).

[0022] WO 2017 / 182561 describes a method for determining the total amount of active DPP3 in a patient sample for the diagnosis of diseases associated with necrotic processes. It further describes a method for the treatment of necrosis-associated diseases with antibodies against DPP3.

[0023] The inventors have now found that treatment with anti-adrenomedullin (ADM) antibodies or anti-adrenomedullin antibody fragments may be particularly effective in treating patients suffering from diseases associated with impaired vascular integrity.

[0024] As a result, there is a need to provide a stable aqueous formulation for anti-adrenomedullin (ADM) antibodies. [Brief description of the drawings]

[0025] [Figure 1A] Relative areas [%] of the main peak (A), HMWS (B) and LMWS (C) determined by SEC-HPLC for unstressed and stressed anti-ADM antibody (HAM8101) samples. Graphs show the average of two replicates, each measured twice. [Figure 1B] Relative areas [%] of the main peak (A), HMWS (B) and LMWS (C) determined by SEC-HPLC for unstressed and stressed anti-ADM antibody (HAM8101) samples. Graphs show the average of two replicates, each measured twice. [Figure 1C] Relative areas [%] of the main peak (A), HMWS (B) and LMWS (C) determined by SEC-HPLC for unstressed and stressed anti-ADM antibody (HAM8101) samples. Graphs show the average of two replicates, each measured twice. [Figure 2A] Relative areas [%] of the main peak (A), HMWS (B) and LMWS (C) determined by SEC-HPLC for an anti-ADM antibody (HAM8101) sample: untreated (t=0), freeze / thawed (5 cycles F / T at -80°C / 25°C) and mechanically stressed (overhead rotation and orbital shaking). Graphs show the average of two replicates, each measured twice. [Figure 2B]Relative areas [%] of the main peak (A), HMWS (B) and LMWS (C) determined by SEC-HPLC for an anti-ADM antibody (HAM8101) sample: untreated (t=0), freeze / thawed (5 cycles F / T at -80°C / 25°C) and mechanically stressed (overhead rotation and orbital shaking). Graphs show the average of two replicates, each measured twice. [Figure 2C] Relative areas [%] of the main peak (A), HMWS (B) and LMWS (C) determined by SEC-HPLC for an anti-ADM antibody (HAM8101) sample: untreated (t=0), freeze / thawed (5 cycles F / T at -80°C / 25°C) and mechanically stressed (overhead rotation and orbital shaking). Graphs show the average of two replicates, each measured twice. [Figure 3A] Relative areas [%] of the main peak (A), acidic peak (B), and basic peak (C) of unstressed and stressed anti-ADM antibody (HAM8101) samples as determined by CEX-HPLC. The graph shows the average of two replicates, each measured twice. [Figure 3B] Relative areas [%] of the main peak (A), acidic peak (B), and basic peak (C) of unstressed and stressed anti-ADM antibody (HAM8101) samples as determined by CEX-HPLC. The graph shows the average of two replicates, each measured twice. [Figure 3C] Relative areas [%] of the main peak (A), acidic peak (B), and basic peak (C) of unstressed and stressed anti-ADM antibody (HAM8101) samples as determined by CEX-HPLC. The graph shows the average of two replicates, each measured twice. [Figure 4A] Relative areas [%] of the main peak (A), acidic peak (B) and basic peak (C) determined by CEX-HPLC for anti-ADM antibody (Ham80101) samples: untreated (t=0), freeze / thaw (5 cycles F / T at -80°C / 25°C) and mechanical stress (overhead rotation and orbital shaking). Graphs show the average of two replicates, each measured twice. [Figure 4B]Relative areas [%] of the main peak (A), acidic peak (B) and basic peak (C) determined by CEX-HPLC for anti-ADM antibody (Ham80101) samples: untreated (t=0), freeze / thaw (5 cycles F / T at -80°C / 25°C) and mechanical stress (overhead rotation and orbital shaking). Graphs show the average of two replicates, each measured twice. [Figure 4C] Relative areas [%] of the main peak (A), acidic peak (B) and basic peak (C) determined by CEX-HPLC for anti-ADM antibody (Ham80101) samples: untreated (t=0), freeze / thaw (5 cycles F / T at -80°C / 25°C) and mechanical stress (overhead rotation and orbital shaking). Graphs show the average of two replicates, each measured twice. [Figure 5-1] 1 shows the results of DLS measurements of unstressed and stressed anti-ADM antibody (HAM8101) samples. [Figure 5-2] 1 shows the results of DLS measurements of unstressed and stressed anti-ADM antibody (HAM8101) samples. [Figure 6A] Results for accelerated aging A), mechanical stress (B) and non-reducing conditions CE SDS after freezing and thawing show the mean and SD of two replicates, each measured in duplicate. [Figure 6B] Results for accelerated aging A), mechanical stress (B) and non-reducing conditions CE SDS after freezing and thawing show the mean and SD of two replicates, each measured in duplicate. [Figure 7A] Results for accelerated aging A), mechanical stress (B) and reducing conditions after freezing and thawing CE SDS show the mean and SD of two replicates, each measured in duplicate. [Figure 7B] Results for accelerated aging A), mechanical stress (B) and reducing conditions after freezing and thawing CE SDS show the mean and SD of two replicates, each measured in duplicate. [Figure 8-1] 1 shows DLS results for formulations 1 to 5 after storage at 40° C. and after mechanical stress. [Figure 8-2]1 shows DLS results for formulations 1 to 5 after storage at 40° C. and after mechanical stress. [Figure 8-3] 1 shows DLS results for formulations 1 to 5 after storage at 40° C. and after mechanical stress. [Figure 9-1] 1 shows DLS results for formulations 6 to 11 after storage at 40° C. and after mechanical stress. [Figure 9-2] 1 shows DLS results for formulations 6 to 11 after storage at 40° C. and after mechanical stress. [Figure 9-3] 1 shows DLS results for formulations 6 to 11 after storage at 40° C. and after mechanical stress. [Figure 10-1] 1 shows DLS results for formulations 11 to 15 after storage at 40° C. and after mechanical stress. [Figure 10-2] 1 shows DLS results for formulations 11 to 15 after storage at 40° C. and after mechanical stress. [Figure 10-3] 1 shows DLS results for formulations 11 to 15 after storage at 40° C. and after mechanical stress. [Figure 11-1] 1 shows DLS results for formulations 16 to 21 after storage at 40° C. and after mechanical stress. [Figure 11-2] 1 shows DLS results for formulations 16 to 21 after storage at 40° C. and after mechanical stress. [Figure 11-3] 1 shows DLS results for formulations 16 to 21 after storage at 40° C. and after mechanical stress. [Figure 12A] SEC-HPLC relative area [%] monomer of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 2 weeks (A), after mechanical stress and freezing and thawing (B). Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 12B] SEC-HPLC relative area [%] monomer of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 2 weeks (A), after mechanical stress and freezing and thawing (B). Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 13A]SEC-HPLC relative area [%] HMWS of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 2 weeks (A), and after mechanical stress and freezing and thawing (B). Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 13B] SEC-HPLC relative area [%] HMWS of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 2 weeks (A), and after mechanical stress and freezing and thawing (B). Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 14A] SEC-HPLC relative area [%]LMWS of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 2 weeks (A), after mechanical stress and freezing and thawing (B). Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 14B] SEC-HPLC relative area [%]LMWS of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 2 weeks (A), after mechanical stress and freezing and thawing (B). Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 15A] Particle content of less than 5 μm (A), 5 to 10 μm (B), 10 μm or more (C), and 25 μm or more (D) after storage at 40°C for 2 weeks. [Figure 15B] Particle content of less than 5 μm (A), 5 to 10 μm (B), 10 μm or more (C), and 25 μm or more (D) after storage at 40°C for 2 weeks. [Figure 15C] Particle content of less than 5 μm (A), 5 to 10 μm (B), 10 μm or more (C), and 25 μm or more (D) after storage at 40°C for 2 weeks. [Figure 15D] Particle content of less than 5 μm (A), 5 to 10 μm (B), 10 μm or more (C), and 25 μm or more (D) after storage at 40°C for 2 weeks. [Figure 16A]Content of particles subvisible to the naked eye below 5 μm (A), between 5 and 10 μm (B), above 10 μm (C), and above 25 μm (D) after mechanical stress. [Figure 16B] Content of particles subvisible to the naked eye below 5 μm (A), between 5 and 10 μm (B), above 10 μm (C), and above 25 μm (D) after mechanical stress. [Figure 16C] Content of particles subvisible to the naked eye below 5 μm (A), between 5 and 10 μm (B), above 10 μm (C), and above 25 μm (D) after mechanical stress. [Figure 16D] Content of particles subvisible to the naked eye below 5 μm (A), between 5 and 10 μm (B), above 10 μm (C), and above 25 μm (D) after mechanical stress. [Figure 17A] SEC-HPLC relative area [%] monomer (A), HMWS (B) and LMWS (C) content of formulated anti-ADM antibody (HAM8101) stored at 40° C.±2° C. / 75% rH±5% rH for up to 4 weeks. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 17B] SEC-HPLC relative area [%] monomer (A), HMWS (B) and LMWS (C) content of formulated anti-ADM antibody (HAM8101) stored at 40° C.±2° C. / 75% rH±5% rH for up to 4 weeks. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 17C] SEC-HPLC relative area [%] monomer (A), HMWS (B) and LMWS (C) content of formulated anti-ADM antibody (HAM8101) stored at 40° C.±2° C. / 75% rH±5% rH for up to 4 weeks. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 18A] SEC-HPLC relative area [%] monomer (A), HMWS (B) and LMWS (C) content of formulated anti-ADM antibody (HAM8101) after mechanical stress and five freeze and thaw cycles. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 18B]SEC-HPLC relative area [%] monomer (A), HMWS (B) and LMWS (C) content of formulated anti-ADM antibody (HAM8101) after mechanical stress and five freeze and thaw cycles. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 18C] SEC-HPLC relative area [%] monomer (A), HMWS (B) and LMWS (C) content of formulated anti-ADM antibody (HAM8101) after mechanical stress and five freeze and thaw cycles. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 19] Relative monomer content after external analysis by analytical ultracentrifugation. [Figure 20A-1] DLS results after storage at 40° C. (A) and after mechanical stress (B). [Figure 20A-2] DLS results after storage at 40° C. (A) and after mechanical stress (B). [Figure 20B-1] DLS results after storage at 40° C. (A) and after mechanical stress (B). [Figure 20B-2] DLS results after storage at 40° C. (A) and after mechanical stress (B). [Figure 21A] Particle content after thermal stress, less than 5 μm (A), 5-10 μm (B), 10 μm or more (C), and 25 μm or more (D) after storage at 40°C for 2 weeks. [Figure 21B] Particle content after thermal stress, less than 5 μm (A), 5-10 μm (B), 10 μm or more (C), and 25 μm or more (D) after storage at 40°C for 2 weeks. [Figure 21C] Particle content after thermal stress, less than 5 μm (A), 5-10 μm (B), 10 μm or more (C), and 25 μm or more (D) after storage at 40°C for 2 weeks. [Figure 21D] Particle content after thermal stress, less than 5 μm (A), 5-10 μm (B), 10 μm or more (C), and 25 μm or more (D) after storage at 40°C for 2 weeks. [Figure 22A]Particles after mechanical stress, particle content of less than 5 μm (A), 5-10 μm (B), 10 μm or more (C), and 25 μm or more (D) after mechanical stress. [Figure 22B] Particles after mechanical stress, particle content of less than 5 μm (A), 5-10 μm (B), 10 μm or more (C), and 25 μm or more (D) after mechanical stress. [Figure 22C] Particles after mechanical stress, particle content of less than 5 μm (A), 5-10 μm (B), 10 μm or more (C), and 25 μm or more (D) after mechanical stress. [Figure 22D] Particles after mechanical stress, particle content of less than 5 μm (A), 5-10 μm (B), 10 μm or more (C), and 25 μm or more (D) after mechanical stress. [Figure 23A] Relative area [%] of main (A), acidic (B) and basic (C) species from CEX-HPLC of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 3.5 weeks. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 23B] Relative area [%] of main (A), acidic (B) and basic (C) species from CEX-HPLC of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 3.5 weeks. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 23C] Relative area [%] of main (A), acidic (B) and basic (C) species from CEX-HPLC of formulated anti-ADM antibody (HAM8101) stored at 40°C ± 2°C / 75% rH ± 5% rH for up to 3.5 weeks. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 24A] Relative area [%] of main (A), acidic (B) and basic (C) species from CEX-HPLC of formulated anti-ADM antibody (HAM8101) after mechanical stress and five freeze and thaw cycles. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 24B]Relative area [%] of main (A), acidic (B) and basic (C) species from CEX-HPLC of formulated anti-ADM antibody (HAM8101) after mechanical stress and five freeze and thaw cycles. Graphs show the mean and SD of two replicates, each measured in duplicate. [Figure 24C] Relative area [%] of main (A), acidic (B) and basic (C) species from CEX-HPLC of formulated anti-ADM antibody (HAM8101) after mechanical stress and five freeze and thaw cycles. Graphs show the mean and SD of two replicates, each measured in duplicate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] While the present invention has been described with reference to specific embodiments, this description is not intended to be construed in a limiting sense.

[0027] As used in this specification and the appended claims, the singular forms "a" and "an" include the respective plural forms unless the context clearly indicates otherwise.

[0028] In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a variation of ±20%, preferably ±15%, more preferably ±10%, even more preferably ±5% from the indicated numerical value.

[0029] It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". Hereinafter, when a group is defined as comprising at least a certain number of embodiments, this means that the group preferably also consists only of these embodiments.

[0030] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0031] Below, embodiments of the present invention are provided. Generally, the embodiments include: It should be noted that it may be combined with any other embodiment of the same category (product, process, use, method).

[0032] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the antibody or fragment binding to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1), the antibody or antibody fragment being present at a concentration of 1 mg / mL to 100 mg / mL, 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL, most preferably 20 mg / mL, the pharmaceutical aqueous formulation comprising: Arginine in the range of 1g / L to 100g / L, and Trehalose in the range of 1g / L to 100g / L, and a surfactant selected from polysorbates and poloxamers in the range of 0.01 g / L to 5 g / L; and ●Further containing histidine in the range of 0.1g / L to 6.4g / L, The formulation is an aqueous medicinal formulation, the pH of which is in the range of 4.0 to 8.0.

[0033] A preferred embodiment of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the antibody or fragment binding to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1), the antibody or antibody fragment being present at a concentration of 1 mg / mL to 100 mg / mL, preferably 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL, and most preferably 20 mg / mL, the pharmaceutical aqueous formulation comprising: Arginine or its salts in the range of 1 g / L to 100 g / L based on the content of arginine zwitterion, and Trehalose in the range of 1g / L to 100g / L, and a surfactant selected from polysorbates such as ethoxylated sorbitan esterified with fatty acids, including polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, and from poloxamers such as copolymers based on ethylene oxide and propylene oxide in the range of 0.01 g / L to 5 g / L; and ●Further containing histidine in the range of 0.1g / L to 6.4g / L, The formulation is an aqueous medicinal formulation, the pH of which is in the range of 4.0 to 8.0.

[0034] In certain embodiments of the invention, the ADM antibody or ADM antibody fragment is It may be administered at a dose of at least 0.5 mg / kg body weight, specifically at least 1.0 mg / kg body weight, more specifically 1.0 to 20.0 mg / kg body weight, for example 1.0 to 10 mg / kg, 2.0 to 10 mg / kg body weight, 2.0 to 8.0 mg / kg body weight, or 3.0 to 5.0 mg / kg body weight.

[0035] In certain embodiments of the invention, the ADM antibody or ADM antibody fragment is It is present at a concentration of at least 1 mg / mL to 100 mg / mL, preferably 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL, and most preferably 20 mg / mL.

[0036] In a further embodiment of the invention, the ADM antibody or ADM antibody fragment is present in a total amount of at least 5 mg, specifically at least 10 mg, more specifically 10 mg to 1000 mg, more specifically 50 mg to 700 mg, more specifically 100 mg to 500 mg, and most specifically 200 to 480 mg, for a 10 mL vial. This total amount of ADM antibody or ADM antibody fragment can be used to prepare a ready-to-use solution by diluting the total amount of ADM antibody or ADM antibody fragment to the desired concentration in a suitable buffer, including, for example, phosphate buffered saline, water, and emulsions, such as oil / water emulsions. In one embodiment of the invention, the ADM antibody or ADM antibody fragment is present in a total amount of at least 5 mg, specifically at least 10 mg, more specifically 10 mg to 1000 mg, more specifically 50 mg to 700 mg, more specifically 100 mg to 500 mg, and most specifically 200 to 480 mg, for a 10 mL vial, and may be diluted to 10 mL to 200 mL, preferably 20 mL to 180 mL, more preferably 30 mL to 150 mL, and most preferably 50 mL to 100 mL in a suitable buffer, including phosphate buffered saline, water, and emulsions such as oil / water emulsions.

[0037] In another embodiment of the invention, the ADM antibody or ADM antibody fragment is present in a ready-to-use solution at a concentration of at least 1 mg / mL to 100 mg / mL, preferably 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL, and most preferably 20 mg / mL, and is diluted to 10 mL to 200 mL, preferably 20 mL to 180 mL, more preferably 30 mL to 150 mL, and most preferably 50 mL to 100 mL in a suitable buffer, including phosphate buffered saline, water, and emulsions such as oil / water emulsions.

[0038] A person skilled in the art can then know from the instructions how to prepare a ready-to-use solution from the provided pharmaceutical aqueous formulation, which can then be applied to a patient by known routes of administration.

[0039] In another embodiment, the pharmaceutical aqueous formulation is further diluted in an infusion solution and applied to the patient via infusion. In another embodiment, a physician skilled in the art prepares a ready-to-use solution from the pharmaceutical aqueous formulation according to the patient's needs and then applies the ready-to-use solution directly to the patient. In one embodiment of the present invention, the ADM antibody or ADM antibody fragment may be administered at a dose of at least 0.5 mg / kg body weight, particularly at least 1.0 mg / kg body weight, more particularly 1.0 to 20.0 mg / kg body weight, for example 2.0 to 10 mg / kg body weight, 2.0 to 8.0 mg / kg body weight, or 3.0 to 5.0 mg / kg body weight.

[0040] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-Adrenomedullin (ADM) antibody or an anti-Adrenomedullin antibody fragment, said antibody or fragment being a human monoclonal antibody or fragment thereof that binds to the N-terminal region (aa 1-21) of ADM (SEQ ID NO: 1), the heavy chain having the sequence: CDR1: SEQ ID NO:2 GYTFSRYW CDR2: SEQ ID NO:3 ILPGSGST CDR3: SEQ ID NO:4 Includes TEGYEYDGFDY, The light chain has the sequence: CDR1: SEQ ID NO:5 QSIVYSNGNTY CDR2: SEQ ID NO:6 RVS CDR3: SEQ ID NO:7 A pharmaceutical aqueous formulation containing FQGSHIPYT.

[0041] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-Adrenomedullin (ADM) antibody or an anti-Adrenomedullin antibody fragment, having as a heavy chain the following sequence: SEQ ID NO:8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is >95% identical thereto, The light chain has the following sequence: SEQ ID NO:9 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC or an anti-adrenomedullin antibody against the N-terminus of adrenomedullin, the anti-adrenomedullin antibody comprising a sequence that is >95% identical thereto.

[0042] In a further embodiment of the invention, the pharmaceutical aqueous formulation comprising a human or humanized anti-Adrenomedullin (ADM) antibody or anti-Adrenomedullin antibody fragment comprises the following sequence as the heavy chain: SEQ ID NO:8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is >95%, preferably >98%, preferably >99% identical thereto, The light chain has the following sequence: SEQ ID NO:9 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC or an anti-adrenomedullin antibody against the N-terminus of adrenomedullin comprising a sequence that is >95%, preferably >98%, preferably >99% identical thereto.

[0043] To assess identity between two amino acid sequences, a pairwise alignment is performed, with identity being defined as the percentage of amino acids that are directly matched in the alignment.

[0044] Surfactants as disclosed in the present invention can be used to modify the surface tension of liquid antibody formulations. In certain embodiments, surfactants reduce the surface tension of liquid antibody formulations. In another embodiment, surfactants can contribute to improving the stability of any of the antibodies in the formulation. Surfactants can also reduce the aggregation of the formulated antibody (e.g., during shipping and storage) and / or minimize the formation of particulates in the formulation and / or reduce adsorption (e.g., adsorption to containers). As an example, surfactants can also improve the stability of the antibody during and after freeze / thaw cycles. The surfactant can be, for example, but not limited to, polysorbate, poloxamer, Triton, sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl glycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl taurate, disodium methyl oleyl taurate, dihydroxypropyl PEG5 linoleammonium chloride, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0045] The subject of the present invention is an aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, wherein the surfactant is a poloxamer. In another embodiment, the poloxamer is selected from the group comprising copolymers based on ethylene oxide and propylene oxide, including but not limited to L64, P65, P84, P85, F88, P103, P104, P105, F108, P123 or F127.

[0046] In some embodiments, the surfactant is selected from polysorbates and poloxamers, the polysorbates being derived from ethoxylated sorbitan esterified with fatty acids and selected from the group comprising polyoxyethylene-(20)-sorbitan monolaurate, polyoxyethylene-(4)-sorbitan monolaurate, polyoxyethylene-(20)-sorbitan monopalmitate, polyoxyethylene-(20)-sorbitan monostearate, polyoxyethylene-(4)-sorbitan monostearate, polyoxyethylene-(20)-sorbitan tristearate, polyoxyethylene-(20)-sorbitan monooleate, polyoxyethylene-(5)-sorbitan monooleate, polyoxyethylene-(20)-sorbitan trioleate or polyoxyethylene-(20)-sorbitan monoisostearate, polyoxyethylene derivatives, Tween, PEG3350 and mixtures thereof.

[0047] The subject of the present invention is an aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the formulation being essentially free of NaCl and / or glycine.

[0048] In some embodiments, the aqueous formulation includes, for example, but not limited to, acetate, succinate, gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid, ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate, acetate, malate, imidazole, tris, phosphate, and mixtures thereof. Preferably, the aqueous formulation includes histidine, which may include either L-histidine or D-histidine, a solvated form of histidine, a hydrated form of histidine (e.g., a monohydrate, including L-histidine hydrochloride monohydrate), a salt of histidine (e.g., histidine hydrochloride) or an anhydrous form of histidine, or a mixture thereof.

[0049] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, in which arginine is present in the range of 1 g / L to 100 g / L, preferably 13.1 g / L to 52.2 g / L, more preferably 26.1 g / L. Preferably, the aqueous formulation comprises histidine, which may comprise either L-histidine or D-histidine, a solvated form of histidine, a hydrated form of histidine (e.g., a monohydrate, including L-histidine hydrochloride monohydrate), a salt of histidine (e.g., histidine hydrochloride) or an anhydrous form of histidine, or a mixture thereof.

[0050] In one aspect of the invention, the aqueous formulation comprises a tonicity adjusting agent that protects the antibody or protein in the formulation from freeze-thaw induced aggregation as well as from aggregation during storage. The tonicity adjusting agent can be, for example, but is not limited to, a polyol having a molecular weight less than about 600 kD (e.g., in the range of about 120 to about 400 kD), including a sugar (e.g., reducing and non-reducing sugars or mixtures thereof, sugars, or carbohydrates) containing multiple hydroxyl groups, a sugar alcohol, a sugar acid, or a salt or mixture thereof. Examples of non-reducing sugars include, but are not limited to, sucrose, trehalose, and mixtures thereof. In some embodiments, the polyol is mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, or mixtures thereof. In other embodiments, the polyol can be, for example, but is not limited to, a monosaccharide, a disaccharide, or a polysaccharide, or a mixture of any of the foregoing. The sugar or carbohydrate may be, for example, but is not limited to, fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water soluble glucan, or mixtures thereof. In a preferred embodiment of the present invention, the polyol is trehalose.

[0051] The subject of the present invention is an aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, wherein trehalose is present in an amount ranging from 1 g / L to 100 g / L, preferably from 28.4 g / L to 85.1 g / L, more preferably 56.7 g / L.

[0052] The subject of the present invention is an aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, in which poloxamer is present in the range of 0.01 g / L to 5 g / L, preferably 0.1 g / L to 1.0 g / L, more preferably 0.5 g / L.

[0053] In some embodiments, the aqueous formulation comprises a chelating agent which may be selected from the group consisting of aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycines, 2-(2-amino-2-oxotyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, niacinamide, and desoxycholate, and mixtures thereof. In some embodiments, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycol ether, N,N,N',N'-tetraacetic acid (EGTA), transdiaminocyclohexanetetraacetic acid (DCTA), glutamic acid, and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine), and N-(trishydroxymethylmethyl)10glycine ( Tricine), glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetramine (triene), disodium edetate dihydrate (or disodium EDTA dihydrate or disodium EDTA salt), calcium oxalate, malate, citric acid, citric acid monohydrate, and trisodium citrate dihydrate, 8-hydroxyquinolate, amino acids, histidine, cysteine, methionine, peptides, polypeptides, and proteins, and mixtures thereof. In some embodiments, the chelating agent is selected from the group consisting of salts of EDTA, including dipotassium edetate, disodium edetate, calcium disodium edetate, sodium edetate, trisodium edetate, and potassium edetate, and a suitable salt of deferoxamine (DEF) is deferoxamine mesylate (DFM), or mixtures thereof. The chelating agents used in the present invention may, where possible, be present as the free acid or free base form or salt form of the compound, and as the anhydrous, solvated or hydrated form of the compound or the corresponding salt. In the most preferred embodiment of the present invention, the chelating agent is histidine.

[0054] The subject of the present invention is an aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, wherein histidine is present in the range of 0.1 g / L to 6.4 g / L, preferably 0.8 g / L to 3.2 g / L, more preferably 1.6 g / L.

[0055] The subject of the present invention is an aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the pH of the formulation being in the range of 4.0 to 8.0, preferably 5.0 to 7.0, more preferably 6.0.

[0056] According to the invention, the aqueous formulations are provided at near physiological pH to reduce the risk of pain or anaphylactic-like side effects upon injection, and the aqueous formulations also provide improved antibody stability and resistance to aggregation, oxidation, and fragmentation.

[0057] In some embodiments, the formulation may include a preservative. Preferably, the preservative is selected from the group including phenol, m-cresol, benzyl alcohol, benzalkonium chloride, benzalkonium chloride, phenoxyethanol, and methylparaben.

[0058] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the formulation comprising an anti-adrenomedullin antibody against the N-terminus of adrenomedullin having the following sequence: SEQ ID NO:1, arginine at a concentration of 26.1 g / L, trehalose at a concentration of 56.7 g / L, poloxamer at a concentration of 0.5 g / L, histidine at a concentration of 1.6 g / L, the formulation having a pH of 6.0.

[0059] In another embodiment of the present invention, there is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the formulation comprising an anti-adrenomedullin antibody against the N-terminus of adrenomedullin having the following sequence: SEQ ID NO:1, arginine at a concentration of 1 g / L to 100 g / L, trehalose at a concentration of 1 g / L to 100 g / L, poloxamer at a concentration of 0.01 g / L to 5 g / L, and histidine at a concentration of 0.1 g / L to 6.4 g / L, and the formulation exhibits a pH in the range of 4.0 to 8.0.

[0060] Another aspect of the present invention relates to a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the formulation comprising an anti-adrenomedullin antibody against the N-terminus of adrenomedullin having the following sequence: SEQ ID NO:1, arginine at a concentration of 13.1 g / L to 52.2 g / L, trehalose at a concentration of 28.4 g / L to 85.1 g / L, poloxamer at a concentration of 0.1 g / L to 1 g / L, and histidine at a concentration of 0.8 g / L to 3.2 g / L, and the formulation exhibits a pH of 5.0 to 7.0.

[0061] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the formulation being stable after storage for at least 4 weeks at 2-8° C., and wherein less than 5% w / w, preferably less than 4% w / w, more preferably less than 3% w / w, more preferably less than 2% w / w, more preferably less than 1% w / w, more preferably less than 0.05% molar or most preferably less than 0.01% w / w of the total antibody is aggregated, as measured, for example, by size-exclusion high performance liquid chromatography (SEC-HPLC), and the antibody is present in a concentration of 1 mg / mL to 100 mg / mL, preferably 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL and most preferably 20 mg / mL. Other methods used to characterize the molecular weight distribution of biological macromolecules are, for example, micellar liquid chromatography or ion exchange chromatography.

[0062] A preferred embodiment of the invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, the formulation being stable after storage for at least 4 weeks at 2-8°C, and less than 5% w / w, preferably less than 4% w / w, more preferably less than 3% w / w, more preferably less than 2% w / w, more preferably less than 1% w / w, more preferably less than 0.05% molar or most preferably less than 0.01% w / w of the total antibody is aggregated, as measured, for example, by size-exclusion high performance liquid chromatography (SEC-HPLC), and the antibody may be administered at a dose of 1 mg / kg body weight to 10 mg / kg body weight, more preferably 2 mg / kg body weight to 8 mg / kg body weight, most preferably 3 mg / kg body weight to 5 mg / kg body weight. Other methods used to characterize the molecular weight distribution of biological macromolecules are, for example, micellar liquid chromatography or ion exchange chromatography.

[0063] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, which formulation is stable after storage for at least 4 weeks at 2-8° C. and in which less than 5% w / w, preferably less than 4% w / w, more preferably less than 3% w / w, more preferably less than 2% w / w, more preferably less than 1% w / w, more preferably less than 0.05% molar or most preferably less than 0.01% w / w of the total antibody is aggregated, for example as measured by size-exclusion high performance liquid chromatography (SEC-HPLC), and the antibody is present in a total amount of 10 mg to 1000 mg, more preferably 50 mg to 700 mg, most preferably 100 mg to 500 mg for a 10 mL vial. Other methods used to characterize the molecular weight distribution of biological macromolecules are, for example, micellar liquid chromatography or ion exchange chromatography.

[0064] In one embodiment of the invention, SEC-HPLC is capable of separating protein species with an apparent molecular weight of 150 kDa from other protein species with molecular weights of 300 kDa or greater.

[0065] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-Adrenomedullin (ADM) antibody or an anti-Adrenomedullin antibody fragment, in which the activity of said antibody or antibody fragment in said formulation is stable after stress, which stress can be achieved by storing said formulation at 45° C., preferably at 42° C., more preferably at 38° C., more preferably at 36° C., most preferably at 40° C. and 85% rH, preferably at 80 rH, preferably at 70% rH, most preferably at 75% rH for at least 4 weeks, and / or at 29° C., preferably at 27° C., more preferably at 22° C., more preferably at 20° C., most preferably at 25° C. and / or at 70% rH, preferably at 80% rH, most preferably at 85 ... or 65% rH, preferably 55% rH, more preferably 50% rH and most preferably 60% rH for at least 3 months, and / or by storing the formulation at 9°C, preferably 7°C, more preferably 2°C, more preferably 0°C and most preferably 5°C for at least 6 months, and / or by subjecting the formulation to at least 5 freeze / thaw cycles, and / or by subjecting the formulation to mechanical stresses including orbital shaking and overhead rotation, wherein the stability is determined by evaluating the visual appearance including color, clarity and visible particles of the formulation after the stress.

[0066] The abbreviation "rH" stands for "relative humidity" and refers to a measure of how much water vapor is present in a water-air mixture compared to the maximum amount possible. Moreover, rH is the ratio of the humidity ratio of a particular water-air mixture compared to the saturation humidity ratio at a given temperature (dry bulb). The term relative humidity "rH" is used in accordance with the ICH Q1A guideline "Stability Testing of new Drug Substances and Products". Since relative humidity is temperature dependent, precise relative humidity control requires precise air temperature control. Those skilled in the art will know that humidity is typically measured by a hygrometer, such as a gravimetric hygrometer, a chilled mirror hygrometer, or an electrolytic hygrometer.

[0067] "Stable" according to the present invention means that all important quality attributes of the formulation remain within the specification limits of the quality specifications, in particular that the formulation is stable under stress conditions, e.g. at 45°C, preferably 42°C, more preferably 38°C, more preferably 36°C, most preferably 40°C and / or 29°C, preferably 27°C, more preferably 22°C, more preferably 20°C, most preferably 25°C and / or 70%rH, preferably 65%rH, preferably 55%rH, more preferably 50%rH, most preferably 60%rH and / or 45°C, preferably 42°C, more preferably 38°C, more preferably 36°C, most preferably 40°C and / or 85%rH, preferably 80rH, preferably 70%rH, most preferably 80%rH and / or 85%rH, preferably 8 ...0%rH, most preferably 80%rH and / or 80%rH, most preferably 80%rH and / or 80% It means the absence of any visible particles after applying stress conditions by storing the formulation at 75% rH, preferably by storing the formulation at 29°C, preferably at 27°C, more preferably at 22°C, more preferably at 20°C, most preferably at 25°C and / or 70% rH, preferably at 65% rH, preferably at 55% rH, more preferably at 50% rH, most preferably at 60% rH, or by storing the formulation at 9°C, preferably at 7°C, more preferably at 2°C, more preferably at 0°C, most preferably at 5°C, and / or by subjecting the formulation to at least 5 freeze / thaw cycles and / or by subjecting the formulation to mechanical stresses including orbital shaking and overhead rotation.

[0068] In a further embodiment of the invention, there is provided a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, wherein the activity of the antibody or antibody fragment in the formulation is stable following stress, wherein stress is induced by storing the formulation at 45°C, preferably 42°C, more preferably 38°C, more preferably 36°C, and most preferably 40°C for at least two weeks, and / or by subjecting the formulation to at least five freeze / thaw cycles, and / or by subjecting the formulation to mechanical stress including orbital shaking and overhead rotation, and wherein stability is determined by evaluating the visual appearance, including color, clarity and visible particles, of the formulation following stress.

[0069] In some embodiments of the invention, there is a pharmaceutical aqueous formulation comprising a human or humanized anti-Adrenomedullin (ADM) antibody or anti-Adrenomedullin antibody fragment, wherein the activity of the antibody or antibody fragment in the formulation is stable following stress, wherein stress is induced by storing the formulation at 29° C., preferably 27° C., more preferably 22° C., more preferably 20° C., most preferably 25° C. and / or 70% rH, preferably 65% ​​rH, preferably 55% rH, more preferably 50% rH, most preferably 60% rH for at least 2 months, and / or by subjecting the formulation to at least 5 freeze / thaw cycles, and / or by subjecting the formulation to mechanical stress including orbital shaking and overhead rotation, wherein stability is determined by evaluating the visual appearance, including color, clarity and visible particles, of the formulation following stress.

[0070] Another embodiment of the invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-Adrenomedullin (ADM) antibody or anti-Adrenomedullin antibody fragment, wherein the activity of the antibody or antibody fragment in the formulation is stable following stress, which can be achieved by storing the formulation at 45° C., preferably 42° C., more preferably 38° C., more preferably 36° C., most preferably 40° C. and / or 85% rH, preferably 80 rH, preferably 70% rH, most preferably 75% rH for at least 2 weeks, or at 29° C., preferably 27° C., more preferably 22° C., more preferably 20° C., most preferably 25° C. °C and / or 70% rH, preferably 65% ​​rH, preferably 55% rH, more preferably 50% rH, most preferably 60% rH, or by storing the formulation at 9°C, preferably 7°C, more preferably 2°C, more preferably 0°C, most preferably 5°C, or by subjecting the formulation to at least 5 freeze / thaw cycles and / or by subjecting the formulation to mechanical stresses including orbital shaking and overhead rotation, wherein the stability is determined by evaluating the visual appearance including color, clarity and visible particles of the formulation after the stress. A person skilled in the art would also consider storing the formulation under the aforementioned conditions for less than 2 weeks.

[0071] Another aspect of the invention relates to performing at least five freeze / thaw cycles, where one freeze / thaw cycle comprises first freezing the formulation to −80° C. and then thawing at 1° C. / min to 25° C. In some embodiments, 1 to 50, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, more preferably 2 to 8, and most preferably 5 freeze / thaw cycles are performed.

[0072] In further embodiments, a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, wherein the activity of the antibody or antibody fragment in the formulation is stable following stress, the stress being induced by subjecting the formulation to mechanical stress comprising orbital shaking and overhead rotation. In some embodiments, the stress is applied by overhead rotation at 30 rpm for 24 hours and / or orbital shaking at 400 rpm. In another aspect of the invention, the stress is applied by overhead rotation at 30 rpm for 24 hours, preferably 36 hours, more preferably 48 hours, most preferably 72 hours, and / or orbital shaking at 200 rpm, preferably 400 rpm, more preferably 600 rpm, more preferably 800 rpm, most preferably 1000 rpm.

[0073] Micron-sized protein aggregates and particles (subvisible particles, SVPs) are an important quality attribute of therapeutic protein formulations due to the risk of enhancing immunogenic responses. Thus, quantification of subvisible particles larger than 10 μm and 25 μm is required by pharmacopoeias and is generally performed using light obscuration (LO) techniques. In one embodiment of the invention, stability is determined by evaluating the visual appearance of the formulation after stress, including color, clarity, and visible particles. In some embodiments of the invention, subvisible particles larger than 1 μm, more preferably 5 μm, more preferably 10 μm, more preferably 15 μm, more preferably 20 μm, and most preferably 25 μm are quantified. In another embodiment, particles larger than 25 μm are quantified. In some embodiments, visual and color inspection is performed. Appearance tests of the formulations with regard to color (Ph.Eur. method 2.2.2. Visible particles, according to Ph.Eur. method 2.9.20), transparency and degree of opalescence (Ph.Eur. method 2.2.) are usually analyzed according to Ph.Eur. methods 2.9.20 and 2.2.1, respectively. In one embodiment of the invention, visible particles, appearance, color and transparency are evaluated by eye.

[0074] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, which formulation is stable after storage at 2-8°C for at least 4 weeks, where stable means that the formulation remains free of visible particles.

[0075] The subject of the present invention is an aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, which is stable after storage at 2-8°C for at least 4 weeks, where stable means that the formulation remains free of particles subvisible to the naked eye.

[0076] Those skilled in the art will know that micron-sized protein aggregates and particles (subvisible particles, SVPs) are an important quality attribute of therapeutic protein formulations due to the risk of enhancing immunogenic responses. Therefore, quantification of subvisible particles larger than 10 μm and 25 μm is required by pharmacopoeias and is generally performed using light obscuration (LO) techniques. Currently, the acceptance criteria for SVPs are 6000 NMT≧10 μm / container and 600 NMT≧25 μm / container. SVPs <10 μm need to be monitored and no acceptance criteria have been defined. However, quantification and characterization of particles with sizes less than 10 μm is of increasing interest and, on the other hand, there are regulatory expectations.

[0077] The subject of the present invention is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, wherein the antibody is present at a concentration of 1 mg / mL to 100 mg / mL, preferably 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL, and most preferably 20 mg / mL.

[0078] In some embodiments of the invention, there is a pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, where the antibody or fragment may be administered at a dose of at least 0.5 mg / kg body weight, specifically at least 1.0 mg / kg body weight, more specifically 1.0-20.0 mg / kg body weight, for example 2.0-10 mg / kg body weight, 2.0-8.0 mg / kg body weight, or 2.0-5.0 mg / kg body weight.

[0079] The subject of the present invention is a pharmaceutical lyophilized formulation obtained from a pharmaceutical aqueous formulation. In some embodiments, a formulation is provided that is lyophilized and / or has been subjected to lyophilization. In another aspect of the present invention, a pharmaceutical formulation is provided in a lyophilized form, which is called a "cake" or "powder", is physically stable and is compatible with pharma-ceutically acceptable solvent mixtures. In some embodiments, a composition is provided that is not lyophilized and / or has not been subjected to lyophilization.

[0080] In some embodiments, the aqueous formulations disclosed herein have a shelf life of at least or greater than about 6 months, at least or greater than about 12 months, at least or greater than about 18 months, at least or greater than about 24 months, at least or greater than about 30 months, at least or greater than about 36 months, at least or greater than about 42 months, or at least or greater than about 48 months at 5° C. (e.g., at 5° C., 25° C., or 40° C.). In further embodiments, the formulations of the invention have a shelf life of at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 ​​months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months.

[0081] In some embodiments, the aqueous formulations disclosed herein have a shelf life of at least or greater than about 6 months, at least or greater than about 12 months, at least or greater than about 18 months, at least or greater than about 24 months, at least or greater than about 30 months, at least or greater than about 36 months, at least or greater than about 42 months, or at least or greater than about 48 months at 25° C. (e.g., at 5° C., 25° C., or 40° C.). In further embodiments, the formulations of the invention have a shelf life of at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 ​​months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months.

[0082] In some embodiments, the aqueous formulations disclosed herein have a shelf life of at least or greater than about 6 months, at least or greater than about 12 months, at least or greater than about 18 months, at least or greater than about 24 months, at least or greater than about 30 months, at least or greater than about 36 months, at least or greater than about 42 months, or at least or greater than about 48 months at 40° C. (e.g., at 5° C., 25° C., or 40° C.). In further embodiments, the formulations of the invention have a shelf life of at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 ​​months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months.

[0083] The subject of the present invention is a method for producing a ready-to-use solution, comprising the steps of: a. providing a pharmaceutical aqueous formulation; b. preparing the aqueous pharmaceutical formulation from step a) in a physiologically acceptable solution, optionally by diluting a volume of the pharmaceutical formulation from step a) with a physiologically acceptable solution, and optionally by aliquoting the pharmaceutical formulation from step a), wherein the diluted or prepared aqueous pharmaceutical formulation is suitable for administration in a patient.

[0084] The subject of the present invention is a method for producing a ready-to-use solution, comprising the steps of: a. providing a lyophilized formulation obtained from the aqueous formulation by lyophilization, optionally without the addition of any other bulking reagents; b. Reconstitution of the lyophilisate from step a) in water for injection and / or a physiologically acceptable solution for injection; and c. adjusting the reconstituted formulation from step b) in a physiologically acceptable solution, optionally by diluting a volume of the reconstituted formulation from step b) with a physiologically acceptable solution, and optionally by aliquoting the reconstituted formulation from step b), wherein the diluted or adjusted aqueous pharmaceutical formulation is suitable for administration in a patient.

[0085] A preferred embodiment of the present invention is a method for producing a ready-to-use solution, comprising the steps of: a. providing a pharmaceutical aqueous formulation; b. preparing the aqueous pharmaceutical formulation from step a) in a physiologically acceptable solution, optionally by diluting a volume of the pharmaceutical formulation from step a) with a physiologically acceptable solution, and optionally by aliquoting the pharmaceutical formulation from step a), wherein the diluted or prepared aqueous pharmaceutical formulation is suitable for administration in a patient; The solution is selected from the group consisting of lactated Ringer's solution, glucose solution, electrolyte solution, isotonic sodium chloride solution, and the infusion solution is present in a volume of 10 mL to 200 mL, preferably 20 mL to 180 mL, more preferably 30 mL to 150 mL, and most preferably 50 mL to 100 mL.

[0086] Another preferred embodiment of the present invention is a method for producing a ready-to-use solution, comprising the steps of: a. providing a lyophilized formulation obtained from the aqueous formulation by lyophilization, optionally without the addition of any other bulking reagents; b. Reconstitution of the lyophilisate from step a) in water for injection and / or a physiologically acceptable solution for injection; c. preparing the reconstituted formulation from step b) in a physiologically acceptable solution, optionally by diluting a volume of the reconstituted formulation from step b) with a physiologically acceptable solution, and optionally by aliquoting the reconstituted formulation from step b), wherein the diluted or prepared aqueous pharmaceutical formulation is suitable for administration in a patient; The solution is selected from the group consisting of lactated Ringer's solution, glucose solution, electrolyte solution, isotonic sodium chloride solution, and the infusion solution is present in a volume of 10 mL to 200 mL, preferably 20 mL to 180 mL, more preferably 30 mL to 150 mL, and most preferably 50 mL to 100 mL.

[0087] Routes of antibody administration are well known in the art and may include, for example, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, or by sustained release systems. Sustained release parenteral injections can be divided into several types: oil-based injectable solutions, injectable drug suspensions, polymer-based microspheres, and polymer-based in situ forming. Antibodies may be administered continuously by infusion or bolus injection. Physiologically acceptable solutions are supplementary fluids used, for example, in intravenous therapy, to restore or maintain normal fluid volume and electrolyte balance. Those skilled in the art will know that different physiologically acceptable solutions exist and that different physiologically acceptable solutions may be classified, for example, by their tonicity or purpose. Typically, the tonicity of a physiologically acceptable solution may be either isotonic, hypotonic, or hypertonic. In one embodiment of the present invention, the physiologically acceptable solution may be selected from the group including lactated Ringer's solution, glucose solution, electrolyte solution, isotonic sodium chloride solution. In another embodiment of the present invention, the physiologically acceptable solution is also suitable as a commercially available infusion or injection carrier solution for delivering carbohydrate-free electrolytes, such as isotonic NaCl solution, isotonic glucose solution, lactated Ringer's solution and the like (Rote Liste 2004, List of finished medicinal products of the members of the Federation of the Pharmaceutical Industry eV, Editio Cantor, Aulendorf / Wuertt., main groups 52.1 and 52.2.1), to the desired concentration or dose without physical or chemical incompatibility.

[0088] A subject of the present invention is a device comprising the formulation described above.

[0089] The aqueous pharmaceutical composition according to the present invention can be stored in a device such as a medical container. The medical container according to the present invention is any container suitable for storing the aqueous pharmaceutical composition. A typical medical container according to the present invention can be made of glass or plastic and selected from the group including vials, pre-filled syringes, and cartridges. The medical container can have different sets of closures: rubber stoppers for vials; plungers, needles, and needle shields (or luer tips and caps) for pre-filled syringes, and plungers and seals for cartridges. A person skilled in the art will know how to select a suitable medical container.

[0090] The subject of the present invention is a ready-to-use liquid pharmaceutical formulation obtainable by the method according to the present invention, which contains the human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment in a dose of 1 to 10 mg / kg body weight.

[0091] The subject of the present invention is a ready-to-use aqueous pharmaceutical formulation obtainable by the method according to the present invention, which contains the human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment in a dose of 2 to 8 mg / kg body weight, preferably 2 or 4 or 8 mg / kg body weight.

[0092] In an embodiment of the invention, there is a ready-to-use aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, the antibody or fragment being present at a concentration of at least 0.5 mg / kg body weight, particularly at least 1.0 mg / kg body weight, more particularly 1.0-20.0 mg / kg body weight, for example 2.0-10 mg / kg body weight, 2.0-8.0 mg / kg body weight or 2.0-5.0 mg / kg body weight.

[0093] A subject of the present invention is a pharmaceutical formulation for use in the treatment or prevention of acute diseases or acute conditions selected from the group comprising SIRS, severe infections, sepsis, shock, e.g. septic shock, acute vascular diseases, e.g. heart failure, congestion, in particular diuretic resistant congestion, inflammatory conditions, autoimmune diseases, metabolic diseases, encephalopathy, cardiovascular diseases and drug induced diseases, disease symptoms or diseases characterized by such symptoms, wherein the disease symptoms are selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting and / or migraine.

[0094] The acute disease or condition may be selected from the group including, but not limited to, severe infections such as meningitis, systemic inflammatory response syndrome (SIRS) or sepsis, other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, shock such as septic shock, and organ dysfunction such as renal dysfunction, hepatic dysfunction, burns, surgery, trauma, poisoning, chemotherapy induced damage.

[0095] The pharmaceutical preparation according to the invention is for reducing the risk of death during sepsis and septic shock, ie in the later stages of sepsis.

[0096] Septic shock is a potentially fatal medical condition that occurs when sepsis, an organ dysfunction or injury in response to infection, leads to dangerously low blood pressure and abnormalities in cellular metabolism. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) defines septic shock as a subset of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities are associated with a higher risk of death than sepsis alone. Patients with septic shock can be identified clinically by the need for vasopressors to maintain a mean arterial pressure of 65 mmHg or higher in the absence of hypovolemia, and by serum lactate levels above 2 mmol / L (>18 mg / dL). This combination is associated with a hospital mortality rate of over 40% (Singer et al. 2016. JAMA. 315(8):801-10). The primary infection is most commonly caused by bacteria, but can also be caused by fungi, viruses, or parasites. It can be located in any part of the body, but most commonly in the lungs, brain, urinary tract, skin, or abdominal organs. This can lead to multiple organ dysfunction syndrome (previously known as multiple organ failure) and can be fatal. Often, patients with septic shock are treated in intensive care units. Children, immunocompromised individuals, and the elderly are most commonly affected because their immune systems cannot fight infection as effectively as healthy adults. Mortality from septic shock is approximately 25-50%.

[0097] The following clinical criteria for SIRS define sepsis, severe sepsis, and septic shock:

[0098] 1) Systemic inflammatory host response (SIRS) characterized by at least two of the following symptoms: - The patient is hypotensive (mean arterial pressure is <65mmHg) Elevated serum lactate levels are >4mmol / L Blood sugar > 7.7mmol / L (non-diabetic) -Central venous pressure is not within the range of 8-12 mmHg Urine output is <0.5mL×kg -1 ×time -1 ●Central venous (superior vena cava) oxygen saturation <70% or mixed venous <65% Heart rate is >90 beats per minute Body temperature <36℃ or >38℃ ●Respiration rate>20 / min White blood cell count <4 or >12 x 10 9 / L(white blood cells);>10% immature neutrophils 2) Sepsis At least two of the symptoms mentioned under 1) are followed by a further clinical suspicion of infection: ●Cough / phlegm / chest pain Abdominal pain / bloating / diarrhea Line infection ●Endocarditis ●Difficulty urinating Headache accompanied by stiff neck Cellulitis / wound / joint infection Microbiological positivity for any infection 3) Severe sepsis The patient presents with sepsis and there is clinical suspicion of any organ dysfunction: Systolic blood pressure <90 / mean; <65mmHg ●Lactic acid>2mmol / L Bilirubin > 34μmol / L Urine output <0.5mL / kg / hour in 2 hours Creatinine > 177 μmol / L Platelets < 100 x 10 9 / L ●SpO2>90% unless O2 is given 4) Septic shock 3) At least one sign of end-stage organ dysfunction as described above is present.

[0099] When there is refractory hypotension that does not respond to treatment and intravenous systemic fluid administration alone is insufficient to keep the patient's blood pressure from becoming hypotensive, septic shock is indicated and administration of a pharmaceutical formulation according to the present invention is also provided.

[0100] In one particular embodiment of the invention, a pharmaceutical formulation is provided for use in the treatment of an acute disease or condition, said acute disease or condition may be headache, preferably primary headache, most preferably migraine.

[0101] Thus, the acute disease or condition may be selected from the group including, but not limited to, severe infections such as meningitis, systemic inflammatory response syndrome (SIRS) or sepsis, other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, shock such as septic shock, and organ dysfunction such as renal dysfunction, hepatic dysfunction, burns, surgery, trauma, poisoning, chemotherapy induced damage.

[0102] In one embodiment of the present invention, the patient is not suffering from SIRS, severe infection, sepsis, shock, e.g., septic shock. The severe infection refers, e.g., to meningitis, systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and shock, e.g., septic shock. In this regard, severe sepsis is characterized by the appearance of sepsis in the patient and further the presence of clinical suspicion of any organ dysfunction, which is as follows: Systolic blood pressure <90 / mean; <65mmHg ●Lactic acid>2mmol / L Bilirubin > 34μmol / L Urine output <0.5mL / kg / hour in 2 hours Creatinine > 177 μmol / L Platelets < 100 x 10 9 / L ●SpO2>90% unless O2 is given

[0103] As used herein, the term "nausea" refers to a feeling of uneasiness and discomfort in the upper stomach accompanied by an involuntary urge to vomit (Metz A. 2017. Australian Family Physician Vol. 36(92:688-692). It may precede vomiting, but a person may have nausea without vomiting.

[0104] Over time, it is a debilitating symptom. Nausea is a non-specific symptom, which means there are many possible causes. Some common causes of nausea are motion sickness, dizziness, migraine, fainting, hypoglycemia, gastroenteritis (stomach infection) or food poisoning. Nausea is a side effect of many medicines, including chemotherapy, or morning sickness in early pregnancy. Nausea can also be caused by anxiety, moodiness and depression.

[0105] As used herein, the term "headache" refers to a symptom of pain in any area of ​​the head or neck. It occurs in migraine (sharp or throbbing pain), tension-type headache, and cluster headache (Waldman et al. 2014. J Yoga Phys Ther 2014.4:1). Those with severe headaches are also at increased risk of depression. Headaches can occur as a result of many conditions, whether severe or not. There are many different classification systems for headaches. It is well recognized that causes of headaches can include fatigue, sleep deprivation, stress, effects of medicines, effects of recreational drugs, viral infections, loud noises, colds, head injuries, rapid ingestion of very cold food or drink, and dental or sinus problems.

[0106] Headaches are broadly classified as "primary" or "secondary" (Oleson 2005.Functional Neurology 20(2):61-68). Primary headaches are benign, recurrent headaches that are not caused by an underlying disease or structural problem. For example, migraine is a type of primary headache. Primary headaches can cause significant daily pain and disability, but they are not dangerous. The four categories of primary headaches are migraine, tension-type headache (TTH), cluster headaches and other trigeminal autonomic headaches, and other primary headaches.

[0107] Secondary headaches are of organic, metabolic or drug-induced origin and are caused by underlying diseases such as infections, head injuries, vascular disorders, cerebral hemorrhage or tumors. Secondary headaches can be harmless or dangerous. Migraine is a primary headache disorder characterized by moderate to severe recurrent headaches (for review, see Diener et al. 2012. Nat Rev Neurol. 8(3):162-71). Typically, the headache affects one half of the head, is pulsating in nature, and lasts for 2 to 72 hours. Accompanying symptoms may include nausea, vomiting, and sensitivity to light, sound, or smell. The pain is generally worsened by physical activity. Up to one-third of people have an aura: this is typically a short-lived visual disturbance that signals that a headache is about to occur. Occasionally, the aura may occur with little or no subsequent headache.

[0108] As used herein, the terms "muscle aches" or "muscle pain", also known as "myalgia", refer to symptoms of many diseases and disorders (for review, see Kyriakides et al. 2013. European Journal of Neurology 20:997-1005).

[0109] The most common cause is overuse or overstretching of a muscle or muscle group. Muscle pain without a history of trauma is often due to a viral infection. More prolonged muscle pain may indicate a metabolic myopathy, some malnutrition, or chronic fatigue syndrome.

[0110] As used herein, the term "back pain" refers to a painful sensation in any part of the back. Episodes of back pain can be acute, subacute, or chronic, depending on duration. The pain can be characterized as a dull ache, a shooting or tingling pain, or a burning sensation. The pain can radiate to the arms and hands as well as the legs or feet and can include paresthesias (tingling pain without obvious cause), weakness or numbness in the legs and arms. The anatomical classification of back pain follows the segments of the spine: neck pain (cervical), mid-back pain (thoracic), lower back pain (lower back), or coccygeal pain (tailbone or sacral pain), with the lumbar region being the most common for pain. Pain can originate from muscles, nerves, bones, joints, or other structures within the spinal column (vertebrae), although internal structures such as the gallbladder and pancreas can also cause referred pain in the back (Cohen et al. 2008. BMJ. 33 7.'u2 718).

[0111] As used herein, the term "shivering" (also called "shivering") is a bodily function in warm-blooded animals in response to initial hypothermia or simply by feeling cold. When core body temperature drops, a shivering reflex is triggered to maintain homeostasis. Skeletal muscles begin to vibrate with small movements, creating warmth by expending energy. Shivering can also be a response to fever, as humans may feel cold. During a fever, the hypothalamic temperature set point is elevated. The elevated set point increases body temperature (fever), but also makes the patient feel cold until the new set point is reached. Severe chills accompanied by intense shivering are called rigor. A rigor occurs because the patient's body is shivering in a physiological attempt to raise body temperature to a new set point. Shivering can also appear after surgery, where it is called post-anesthesia shivering.

[0112] As used herein, the term "vomiting", also called emesis and throwing up, among other terms, is the involuntary force of expelling stomach contents through the mouth and sometimes the nose (Metz A. 2017. Australian Family physician Vol. 36(9):688-692). Vomiting can be caused by a wide variety of conditions. It can exist as a specific response to illness, such as gastritis or poisoning, or as a non-specific sequela of disorders ranging from brain tumors and elevated intracranial pressure to overexposure to ionizing radiation.

[0113] As used herein, a disease or disorder condition in a patient in need of treatment and / or prevention of such condition is selected from the group of disease indications including inflammatory conditions, autoimmune diseases, metabolic diseases, encephalopathy, cardiovascular diseases and drug-induced diseases.

[0114] Below, a non-limiting list of symptoms and disease types relevant to the present specification is provided. It should be noted that the treatment or prevention described herein may target more than one type of symptom. It should also be noted that a given medical indication, disease, or disorder may be associated with more than one symptom.

[0115] The symptom "nausea" may be due to an internal abdominal disease, such as an obstructive disorder (e.g., pyloric obstruction, small bowel obstruction, colonic obstruction, superior mesenteric artery syndrome), intestinal infection (e.g., viral or bacterial infection), inflammatory disease (e.g., cholecystitis, pancreatitis, appendicitis, hepatitis), sensorimotor dysfunction (e.g., gastroparesis, intestinal pseudoobstruction, gastroesophageal reflux disease, chronic idiopathic nausea, functional vomiting, cyclic vomiting syndrome, rumination syndrome) or biliary colic; an external abdominal disease, such as a cardiopulmonary disease (e.g., cardiomyopathy, myocardial infarction), an intracranial disease (e.g., pulmonary hypertension, chronic obstruction, chronic pulmonary syndrome ... nausea, vomiting, nausea, vomiting of the brain, vomiting of the brain associated with certain diseases, including nausea, vomiting of the brain associated with certain diseases, ...

[0116] The symptom "back pain" may be associated with inflammatory illness, particularly in the acute phase, which typically lasts from 2 weeks to 3 months, and may be associated with lower back pain, trauma, injury, infection, cancer (especially those known to spread to the spine, such as breast, lung and prostate cancer), etc.

[0117] The condition "myalgia" or "muscle pain" may be associated with injury or trauma, including sprains, hematomas, or overuse, where the muscle has been overworked and used too frequently, which may be due to another injury, chronic strain, myalgia due to rhabdomyolysis, myalgia associated with, for example, viral infections, pressure injuries, myalgia associated with drugs, for example, fibrates and statins, ACE inhibitors, cocaine, some antiretroviral drugs, severe potassium deficiency, fibromyalgia, Ehlers-Danlos syndrome, autoimmune disorders, for example, mixed connective tissue disease, systemic lupus erythematosus, polymyalgia rheumatoid arthritis, myositis, for example, polymyositis, dermatomyositis and inclusion body myositis, multiple sclerosis, myalgic encephalomyelitis (chronic fatigue syndrome), familial including protection against: Mediterranean fever, polyarteritis nodosa, Devic's disease, morphea, sarcoidosis), metabolic diseases (e.g. carnitine palmitoyltransferase II deficiency, Conn's syndrome, adrenal insufficiency, hyperthyroidism, hypothyroidism, diabetes mellitus, hypogonadism, as well as channelopathies, Stickler's syndrome, hypokalemia, hypotonia (low muscle tone), exercise intolerance, mastocytosis, peripheral neuropathy, eosinophilic myalgia syndrome, Barcoo's fever, herpes, hemochromatosis also known as iron overload disorder, delayed onset myalgia, AIDS, HIV infection, tumor-induced osteomalacia, hypovitaminosis D, myocardial infarction.

[0118] The symptom "headache" can be associated with primary headaches. 90% of all headaches are primary headaches. Primary headaches usually first begin when people are 20-40 years old. The most common types of primary headaches are migraine and tension-type headaches. They have different characteristics. Migraine typically presents with pulsating headache, nausea, photophobia (sensitivity to light) and phonophobia (sensitivity to sound). Tension-type headaches usually involve non-pulsating "zonal" pressure on both sides of the head and no other symptoms. Other very rare types of primary headaches include cluster headaches: short episodes (15-180 minutes) of severe pain usually around one eye, accompanied by autonomic symptoms (watering, red eye, stuffy nose) that occur at the same time every day. Cluster headaches can be treated with triptans and prevented with prednisone, ergotamine or lithium, trigeminal or occipital neuralgia is characterized by facial pain, hemicrania continua, i.e., persistent unilateral pain accompanied by episodes of intense pain, primary stabbing headaches are recurrent episodes of stabbing "ice pick" or "jabs and jolts" pain lasting for one second to several minutes without autonomic symptoms (watering, red eyes, nasal congestion), primary cough headaches begin suddenly and last for several minutes after coughing, sneezing, or straining (anything that can increase head pressure). Serious causes (see section on red flags of secondary headaches) must be ruled out, after which a diagnosis of "benign" primary cough headache can be made, and primary exertional headaches are characterized by a throbbing, pulsating pain that begins during or after exercise and lasts from 5 minutes to 24 hours. The mechanism behind these headaches is unclear, possibly because tension causes the veins in the head to dilate, resulting in pain; primary coital headaches are characterized by a dull, bilateral headache that begins during sexual activity and becomes much worse during orgasm; there are hypotensive headaches; secondary headaches may be caused by problems elsewhere in the head or neck.Some of these are not harmful, for example, cervicogenic headache (pain arising from the muscles of the neck), medication overuse headache in those who use excessive painkillers for headaches, meningitis characterized by meningeal inflammation presenting with fever and meningismus, or stiff neck; intracerebral hemorrhage (intracranial bleeding); subarachnoid hemorrhage (acute, severe headache, stiff neck without fever); ruptured aneurysms, arteriovenous malformations, intraparenchymal hemorrhage; temporal arteritis, i.e., an inflammatory disease of the arteries common in the elderly (average age 70 years); polymyalgia rheumatoid arthritis; acute angle-closure glaucoma (increased pressure inside the eye); postictal headache (postictal state) occurring after a convulsion or other type of attack as part of the postictal period. Gastrointestinal disorders, including Helicobacter pylori infection, celiac disease, non-celiac gluten sensitivity, irritable bowel syndrome, inflammatory bowel disease, gastroparesis, and hepatobiliary disease, can cause headaches. Treatment of gastrointestinal disorders can result in remission or improvement of headaches. The term headache is defined as primary or secondary headache. Primary headache is defined as migraine, tension-type headache (TTH), cluster headache and other trigeminal autonomic headaches, as well as other primary headaches. Diagnosis or evaluation of headache is well established in the art. Evaluation can be based on subjective measures such as the patient's characterization of symptoms. For example, migraine can be diagnosed based on the following criteria: 1) episodic attack of headache lasting 4-72 hours; 2) having two of the following symptoms: unilateral pain, palpitations, worsening on movement, and moderate or severe pain; and 3) having one of the following symptoms: nausea or vomiting, and photophobia or phonophobia (Goadsby et al., N.Engl.J.Med.346:257-270,2002).

[0119] The symptom "trembling" may be associated with fever, cold sensitivity, menopause, panic attacks, anxiety, bacterial infections, rickettsial infections, viral infections, drug withdrawal, etc.

[0120] The symptom "vomiting" may be associated with gastritis (inflammation of the stomach wall), gastroenteritis, gastroesophageal reflux disease, celiac disease, non-celiac gluten sensitivity, pyloric stenosis, intestinal obstruction, overeating, acute abdominal and / or peritonitis, ileus, food allergies (often accompanied by hives or swelling); cholecystitis, pancreatitis, appendicitis, hepatitis; food poisoning, allergic reactions to milk proteins, e.g., milk allergy or lactose intolerance; motion sickness, Meniere's disease, concussion, cerebral hemorrhage, migraine, brain tumors, benign intracranial hypertension and hydrocephalus, metabolic disorders, e.g., hypercalcemia, uremic adrenal insufficiency, hypoglycemia, hyperglycemia, drug reactions, alcoholism, opioid uptake, selective serotonin reuptake inhibitors; use of chemotherapy drugs; stomach inflammation caused by various viruses and bacteria, e.g., norovirus, influenza; or psychiatric / behavioral illnesses, such as bulimia nervosa and purging disorder.

[0121] Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to any of the preceding embodiments used in combination with a known medication for nausea, for use in the treatment or prevention of a disease symptom selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, in a subject in need thereof, or for use in the treatment or prevention of a disease characterized by such symptoms, such as migraine.

[0122] Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to any of the preceding embodiments used in combination with a known medication for headache, for use in the treatment or prevention of a disease symptom selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, in a subject in need thereof, or for use in the treatment or prevention of a disease characterized by such symptoms, such as migraine.

[0123] Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to any of the preceding embodiments used in combination with a known medication for migraine headaches, for use in the treatment or prevention of a disease symptom selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, in a subject in need thereof, or for use in the treatment or prevention of a disease characterized by such symptoms, such as migraine headaches.

[0124] Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment or prevention of a disease symptom selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, in a subject in need thereof, according to any of the preceding embodiments, used in combination with a known medication for tremors.

[0125] Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to any of the preceding embodiments used in combination with a known medicament for emesis, for use in the treatment or prevention of a disease symptom selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, in a subject in need thereof, or for use in the treatment or prevention of a disease characterized by such symptoms, such as migraine.

[0126] Another embodiment of the invention relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to any of the preceding embodiments used in combination with a known medication for back pain, for use in the treatment or prevention of a disease symptom selected from the group of nausea, headache, muscle pain, back pain, tremors, vomiting, in a subject in need thereof, or for use in the treatment or prevention of a disease characterized by such symptoms, such as migraine.

[0127] A subject of the present invention is a pharmaceutical formulation for use in the treatment or prophylaxis of an acute disease or condition in a patient for the prevention or reduction of organ dysfunction or for the prevention of organ dysfunction in said patient, the acute disease or condition being selected from the group comprising, for example, severe infections, diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, shock and organ dysfunction, renal dysfunction, hepatic dysfunction, burns, surgery, trauma, poisoning and chemotherapy induced damage, and said disease is not SIRS, sepsis or septic shock.

[0128] "Organ dysfunction" refers to a state or health condition in which an organ does not perform its expected function. "Organ failure" refers to organ dysfunction to the extent that normal homeostasis cannot be maintained without external clinical intervention. The patient population addressed by the present invention may be defined as follows:

[0129] In the following, clinical criteria are mentioned for each organ that is dysfunctional or prone to dysfunction, and thus represent patient groups with a chronic or acute disease or condition according to the invention. The criteria are oriented towards clinical SOFA scores.

[0130] The SOFA system was developed at a consensus conference of the European Society of Intensive Care Medicine in 1994 and further revised in 1996.

[0131] The SOFA is a 6-organ dysfunction / failure score that measures multiple organ dysfunction daily. Each organ is graded from 0 (normal) to 4 (most abnormal), providing a daily score of 0 to 24 points. The purpose of the SOFA is to create a simple, reliable, continuous score for clinical staff.

[0132] Serial assessment of organ dysfunction during the first days of intensive care unit (ICU) or hospitalization is a good indicator of prognosis. Both the mean and maximum SOFA scores are particularly useful predictors of outcome.

[0133] [Table 1] MAP, mean arterial pressure; CNS, central nervous system; SaO2, peripheral arterial oxygen Saturation. a The PaO2 / FIO2 ratio was used preferentially. If not available, the SaO2 / FIO2 ratio was used. b vasoactive Medications (dopamine and norepinephrine μg / kg / min) administered for at least 1 hour.

[0134] SOFA score references 1.Jones AE, Trzeciak S, Kline JA.The Sequential Organ Failure Assessment score for predicting outcome in patients with severe sepsis and evidence of hypoperfusion at the time of emergency department presentation.Crit Care Med.2009 May;37(5):1649-54.

[0135] 2. Ferreira FL, Bota DP, Bross A, Melot C, Vincent JL. Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA.2001 Oct 10;286(14):1754-8.

[0136] 3. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonca A, Bruining H, Reinhart CK, Suter PM, Thijs LG. The SOFA(Sepsis-related Organ Failure Assessment) score to describe organ dysfunction / failure.On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine.Intensive Care Med.1996 Jul;22(7):707-10.

[0137] In certain embodiments, a patient population according to the invention has at least one SOFA score as a lower threshold, of 1 for one of the respiratory, hepatic, coagulation, cardiovascular, CNS, or renal clinical criteria on the day of admission to the hospital or intensive care unit (ICU), and thus is in need of therapeutic intervention according to the invention, and thus in need of prevention or reduction of organ dysfunction or failure.

[0138] In another particular embodiment, a patient population according to the invention has at least two SOFA scores as lower thresholds, 1 for each of the respiratory, and / or hepatic, and / or coagulation, and / or cardiovascular, and / or CNS, and / or renal clinical criteria on the day of admission to the hospital or intensive care unit (ICU), and thus in need of therapeutic intervention according to the invention, and thus in need of prevention or reduction of organ dysfunction or failure.

[0139] In another particular embodiment, the patient population according to the invention has a SOFA score of at least 3 as a lower threshold, being 1 for each of the respiratory, and / or hepatic, and / or coagulation, and / or cardiovascular, and / or CNS, and / or renal clinical criteria on the day of admission to the hospital or intensive care unit (ICU), and thus in need of therapeutic intervention according to the invention, and thus in need of prevention or reduction of organ dysfunction or failure.

[0140] In another particular embodiment, the patient population according to the invention has a SOFA score of at least 4 as a lower threshold, being 1 for each of the respiratory, and / or hepatic, and / or coagulation, and / or cardiovascular, and / or CNS, and / or renal clinical criteria on the day of admission to the hospital or intensive care unit (ICU), and thus in need of therapeutic intervention according to the invention, and thus in need of prevention or reduction of organ dysfunction or failure.

[0141] Patient Group - Renal Dysfunction / Failure In the following, the clinical criteria refer to a patient group with renal impairment / failure. Patients at risk for renal dysfunction / failure: GFR decrease >25%, serum creatinine increase 1.5-fold, or 6-hour urine production <0.5mL / kg / hour Patients with current renal impairment: GFR reduction >50%, doubling of creatinine, or 12-hour urine production <0.5mL / kg / hour ●Patients with renal failure: GFR decline >75%, 3-fold increase in creatinine or creatinine >355μmol / l (with increase >44) (>4mg / dl) or 24-hour urine output <0.3mL / kg / hour Patients with renal function loss: Prolonged acute kidney injury (AKI) or complete loss of renal function for more than 4 weeks End-stage renal disease: Complete loss of kidney function for more than three months

[0142] Patient Group - Liver Dysfunction / Failure The patient group with liver dysfunction / failure is characterized by a lower bilirubin threshold of >1.2 mg / dL, preferably >1.9 mg / dL, more preferably >5.9 mg / dL.

[0143] The pharmaceutical preparations can also be administered prophylactically before the patient shows any signs of organ dysfunction or failure. This can be the case when the patient has a chronic or acute disease or condition, where dysfunction or failure problems can be expected, including, for example, severe infections, such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, other diseases, such as diabetes, cancer, acute and chronic vascular diseases, such as heart failure, myocardial infarction, stroke, atherosclerosis, shock, such as septic shock, and organ dysfunction, such as renal dysfunction, liver dysfunction, burns, surgery, trauma, poisoning. The pharmaceutical preparations can also be administered prophylactically or therapeutically before, during or after chemotherapy. The same applies to surgery, where certain organs may experience ischemic damage, which can lead to organ dysfunction or failure. Prophylactic means before organ damage occurs, and therapeutic means that organ damage has already occurred. The antibodies or fragments or scaffolds according to the invention are particularly useful during sepsis and septic shock, i.e. for reducing the risk of organ dysfunction or failure in the later stages of sepsis.

[0144] A subject of the present invention is a pharmaceutical preparation for use in the treatment or prevention of an acute disease or condition in a patient, the patient suffering from a disease selected from the group comprising SIRS, sepsis, diabetes, cancer, acute vascular diseases such as heart failure, shock, such as septic shock, organ dysfunction, such as renal dysfunction, in order to stabilize the systemic circulation of said patient.

[0145] In a further embodiment of the present invention there is provided a pharmaceutical preparation for use in the treatment of an acute disease or condition of a patient, for stabilizing the blood circulation, in particular the systemic circulation, of said patient. In particular, a subject of the present invention is a pharmaceutical preparation for use in the treatment of an acute disease or condition of a patient, for stabilizing the systemic circulation of said patient, said patient being in need of stabilization of the blood circulation.

[0146] Systemic circulation refers to the portion of the circulatory system where blood leaves the heart to supply the cells of the body and then re-enters the heart. Blood leaves through the left ventricle into the aorta, the largest artery of the body. The aorta connects to smaller arteries, arterioles, and finally capillaries. Waste products and carbon dioxide diffuse from the cells into the blood, and oxygen in the blood diffuses into the cells. The blood then travels to the venous capillaries and then to the large veins, namely the lower inferior vena cava and the upper superior vena cava, through which the blood re-enters the heart at the right atrium.

[0147] Throughout this specification, stabilizing blood circulation means stabilizing systemic circulation. The term systemic circulation does not include the phenomenon of microcirculation. Microcirculation is the delivery of fresh blood to the smallest blood vessels present in the vasculature embedded in organ tissue. This is in contrast to macrocirculation, which transports blood to and from organs. The state of systemic circulation can be measured by parameters such as mean arterial pressure, blood pressure (see above for other parameters). Thus, patients who need to stabilize blood circulation can be those who exhibit a heart rate of >100 beats / min and / or a mean arterial pressure of <65 mmHg. When blood circulation is stabilized by administration of anti-adrenomedullin (ADM) antibodies, or by anti-ADM antibody fragments that bind to adrenomedullin or anti-ADM non-Ig scaffolds that bind to adrenomedullin, this can be measured and characterized by an increase in mean arterial pressure of more than 65 mmHg and / or a decrease in heart rate of less than 100 beats / min.

[0148] It should be emphasized that the provided pharmaceutical preparations are intended by the present invention to be applied to stabilize systemic circulation, and therefore are not necessarily intended as any method of primary or first-line treatment for an acute disease or condition itself, which should be considered as an underlying disease. This means that the present invention does not provide a treatment to heal / cure, for example, cancer, diabetes, meningitis, polytrauma, etc. Thus, the treatment of acute diseases or conditions of patients within the scope of the present invention is related to any kind of systemic circulatory failure, or insufficient systemic circulation of blood as an acute event.

[0149] The subject of the present invention is a pharmaceutical formulation comprising a) a pharmaceutical preparation for use in the treatment of an acute disease or condition in a patient for stabilizing the systemic circulation, the patient being in need of stabilization of the systemic circulation and exhibiting a heart rate of >100 beats / min and / or a mean arterial pressure of <65mmHg, where stabilizing the systemic circulation means increasing the mean arterial pressure to above 65mmHg, or b) A pharmaceutical preparation for prophylactic use in the treatment of an acute disease or condition in a patient to prevent an increase in heart rate of >100 beats per minute and / or a decrease in mean arterial pressure of <65 mmHg.

[0150] In all of the above acute diseases and conditions, there may be a need to stabilize the patient's blood circulation by administration of a pharmaceutical preparation, which may be administered prophylactically in patients with an acute disease or condition to prevent heart rate from increasing to >100 beats / min and / or mean arterial pressure from dropping to <65 mmHg.

[0151] In one embodiment, the pharmaceutical formulation according to the present invention reduces the patient's need for a vasopressor, e.g., a catecholamine. The patient's need for a vasopressor, e.g., a catecholamine, is an indication of the patient's circulatory status. Thus, the pharmaceutical formulation may be administered at the time the patient requires a vasopressor, e.g., a catecholamine.

[0152] In one embodiment of the invention, the patient is one in need of increasing blood pressure.

[0153] The patient who needs stabilization of blood circulation may be a patient with low cardiac output and / or low blood pressure (hypotension). It may be a patient with a heart rate of >100 beats / min. It may be a patient with a mean arterial pressure <65mmHg or <60mmHg. Mean arterial pressure is defined as MAP=(CO×SVR)+CVP, where CO is cardiac output, SVR is systemic vascular resistance, and CVP is central venous pressure, which is usually negligible in this formula. The patient who needs stabilization of blood circulation may also be a patient with a respiratory rate of >20 / min in addition to the above symptoms.

[0154] In certain embodiments of the invention, the patient in need of stabilization of blood circulation may be a patient with low cardiac output and / or low blood pressure (hypotension).

[0155] This may be a patient with a heart rate >90 beats / min. This may be a patient with a mean arterial pressure <65 mmHg or <60 mmHg.

[0156] Some patients with sepsis-induced hypoperfusion may remain hypotensive despite adequate fluid replacement. In these cases, a vasopressor is required to increase MAP. Thus, in one embodiment of the present invention, the patient with chronic or acute disease or acute condition is a patient who requires a vasopressor to increase MAP. Catecholamines, such as dopamine, epinephrine (adrenaline), norepinephrine (noradrenaline), and phenylephrine, have traditionally been used to increase blood pressure, for example, in patients with septic shock. Recently, vasopressin has also been suggested as a potential vasopressor in patients with chronic or acute disease or acute condition who need to stabilize blood circulation.

[0157] A vasopressor such as catecholamine can stabilize the blood circulation of patients with chronic or acute diseases or acute conditions.When the patient's condition (low blood pressure) is very severe, administration of a vasopressor, for example, administration of catecholamine alone, cannot prevent the collapse of blood circulation.For example, additional administration of the pharmaceutical preparation together with administration of catecholamine can help stabilize the blood circulation of patients whose condition is so severe that administration of catecholamine without administration of the pharmaceutical preparation is not sufficient to stabilize the blood circulation of the patient.

[0158] Moreover, vasopressors can have serious side effects. Dopamine stimulates D1 receptors in the local circulation of the kidney, causing vasodilation and increasing blood flow. This is one of the reasons why clinicians have used low doses of dopamine to protect renal function. It has also been suggested that for other vasopressors, increasing blood pressure with certain drugs may be associated with worse outcomes, despite their intuitive appeal as something beneficial.

[0159] A subject of the present invention is therefore a pharmaceutical preparation for use in the treatment of an acute disease or condition in a patient, to completely or partially replace the administration of a vasopressor, which means that the patient according to the invention may be a patient in need of treatment with a vasopressor or a patient undergoing treatment with a vasopressor.

[0160] The pharmaceutical preparation can also be administered prophylactically before the patient shows any signs of severe blood circulation problems.This may be when the patient has a chronic or acute disease or condition, and blood circulation problems may be expected, including severe infections such as meningitis, systemic inflammatory response syndrome (SIRS) or sepsis, other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, shock such as septic shock, and organ dysfunction such as renal dysfunction, hepatic dysfunction, burns, surgery, trauma, poisoning, chemotherapy-induced damage.

[0161] The antibody or fragment or scaffold according to the invention is particularly useful for reducing the risk of death during sepsis and septic shock, i.e. in the later stages of sepsis. The skilled person will recognize that said reduction in risk of death is related to the stabilization of blood circulation according to the invention. The acute disease or condition may be a disease or condition characterized in that the patient needs to stabilize the blood circulation. The need to stabilize the blood circulation is characterized as outlined above, i.e. it may be a patient with a heart rate of preferably >90 beats / min, or even >100 beats / min. It may also be a patient with a mean arterial pressure <65mmHg or <60mmHg. The mean arterial pressure is defined as MAP=(CO×SVR)+CVP, where CO is the cardiac output, SVR is the systemic vascular resistance, and CVP is the central venous pressure, which is usually negligibly small in this formula. The patient in need of stabilization of the blood circulation may also be a patient with a respiratory rate of >20 / min.

[0162] In some embodiments of the invention, there are also pharmaceutical preparations used in combination with another active drug, e.g., used as a primary medicament, and the combination is for use in the treatment or prevention of a chronic or acute disease or acute condition in a patient, to stabilize the patient's blood circulation, in particular the patient's systemic circulation.

[0163] In one embodiment of the invention, the patient having a chronic or acute disease or condition requiring stabilization of blood circulation is characterized in that the patient needs to obtain intravenous fluids.

[0164] Thus, a subject of the present invention in a particular embodiment is said pharmaceutical preparation in combination with an ADM binding protein and / or further active ingredient for use in the treatment or prophylaxis of patients in need of intravenous fluids, meaning that the patients require intravenous fluids to regulate the systemic fluid balance.

[0165] The pharmaceutical formulations having ADM binding proteins and / or further active ingredients may be used in combination with vasopressors, e.g. catecholamines and / or intravenously administered fluids, for use in chronic or acute diseases or acute conditions of patients, to stabilize blood circulation, in particular to stabilize systemic circulation.

[0166] The antibodies, antibody fragments and combinations of the invention can be used to treat or prevent chronic or acute diseases in a patient: for the prevention of organ dysfunction or failure, in particular renal dysfunction or failure, and / or - to stabilize blood circulation, e.g. to reduce the patient's vasopressor requirements, e.g. catecholamine requirements, and / or - To regulate the patient's fluid balance, • It can be used to reduce the risk of death for the patient.

[0167] A subject of the present invention is a pharmaceutical preparation for use in the treatment or prevention of an acute disease or condition in a patient for the regulation of fluid balance, said patient being in need of regulation of fluid balance and suffering from a disease selected from the group comprising systemic inflammatory response syndrome (SIRS), sepsis, diabetes, cancer, heart failure, shock and renal dysfunction.

[0168] The expression "regulating fluid balance" in the context of the present invention covers any correction of an apparent imbalance in the patient's fluid balance due to an underlying chronic or acute disease or condition, which correction is advantageous to re-establish normal blood pressure in the patient. Those skilled in the art are well aware that blood pressure in general, as well as hypertension and hypotension, are closely related to the patient's fluid balance.

[0169] Fluid balance is the balance between the amount of fluid entering and leaving the body to allow metabolic processes to function. Dehydration is defined as the loss of 1% or more of body weight as a result of fluid loss.

[0170] The three components to assess fluid balance and hydration status are clinical assessment, weight and urine output, review of fluid balance charts, and review of blood chemistry.

[0171] All of these are very well known to those skilled in the art (Alison Shepherd, Nursing Tomes 19.07.11 / Vol 107 No 28, pages 12 to 16).

[0172] Thus, in one embodiment, such a patient in need of fluid balance regulation and / or improvement is a human having a weight loss of 1% or more as a result of fluid loss. Fluid balance can be assessed according to Scales and Pilsworth (2008) Nursing Standard 22:47,50-57. For example, normal urine output ranges from 0.5 to 2 mL / kg body weight per hour. The minimum acceptable urine output for a patient with normal renal function is 0.5 mL / kg per hour. All these criteria can be used to assess whether a patient is in need of fluid balance regulation and / or improvement.

[0173] Patients in a state of fluid imbalance can receive intravenous fluids (infusions) as a standard measure of care, especially in an ICU environment. However, it is desirable to reduce or avoid additional fluid administration due to possible complications, such as the development of edema (acral edema). Edema refers to swelling caused by fluid in body tissues. It can occur in the feet and legs, but can involve the whole body, including organs such as the lungs, heart, and eyes. Thus, the pharmaceutical formulation can be administered at the time the patient requires fluid administration. According to the present invention, the patient is one who requires regulation of fluid balance.

[0174] A subject of the present invention is therefore also a pharmaceutical preparation for use in the treatment of an acute disease or condition in a patient, for the regulation of fluid balance, including but not limited to the prevention or reduction of edema.

[0175] Fluid balance / infusion therapy In an acute hospital environment, for example within an ICU, fluid balance is generally closely monitored by clinical staff, as it provides specific information regarding the patient's actual hydration status and thus renal and cardiovascular function.

[0176] However, when the acute fluid loss is greater than the fluid gain, the patient is said to be in negative fluid balance, in which case physiological fluids are often administered intravenously by the physician to replace the loss.

[0177] In contrast, a positive fluid balance, where fluid gain is greater than fluid loss, may provide information about problems with either the kidneys or the cardiovascular system.

[0178] This means particularly, for example in the context of SIRS, sepsis, severe sepsis and septic shock, that blood pressure is also low (commonly referred to as hypotension) and the filtration rate in the kidneys is reduced, thus causing less fluid reabsorption and less urine output.

[0179] The term "fluid therapy" generally refers to the therapeutic administration of fluids (such as saline or water for injection (WFI)) to a patient as a treatment or prophylactic measure. It can be administered via intravenous, intraperitoneal, intraosseous, subcutaneous, and oral routes.

[0180] Fluid therapy is indicated either when there is loss of fluid or when there is a risk of fluid loss due to an underlying disease or condition. The severity of the fluid loss and the compartment to which it is lost will affect the choice of fluid and the rate at which it needs to be administered. If fluid therapy is administered as a treatment, it is necessary to diagnose and treat the underlying disease or condition. Fluid therapy is routinely indicated in cases of hypotension, hypovolemia, metabolic disturbances, reduced oxygen delivery, SIRS, sepsis, severe sepsis, shock, and septic shock.

[0181] However, it should be emphasized that the pharmaceutical preparation, the medicament provided by the present invention, is only intended to be used to regulate fluid balance, and therefore is not for any method of first-line treatment for chronic or acute diseases or conditions itself. This means that the present invention does not provide a treatment to heal / cure damage induced by, for example, meningitis, systemic inflammatory response syndrome (SIRS), or sepsis, or severe sepsis, other diseases, for example, diabetes, cancer, acute and chronic vascular diseases, for example, heart failure, myocardial infarction, stroke, atherosclerosis, shock, for example, septic shock, and organ dysfunction, for example, renal dysfunction, hepatic dysfunction, burns, surgery, trauma, poisoning, or chemotherapy within the scope of the present invention.

[0182] Therefore, the fluid regulation effect of the pharmaceutical preparation supports the first-line treatment of the chronic or acute disease or condition. In the case of chronic or acute disease or condition, such as severe infections, such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, the first-line treatment is, for example, the administration of antibiotics. The pharmaceutical preparation regulates fluid balance and helps to prevent the patient's severe condition from worsening until, for example, the administration of antibiotics is effective. As mentioned above, the pharmaceutical preparation can be administered in a prophylactic or therapeutic manner, which means to prevent fluid imbalance problems or to reduce fluid imbalance when a fluid imbalance problem exists in the patient. Edema is included in the term fluid imbalance problems.

[0183] In one embodiment of the invention, the patient having a chronic or acute disease or condition requiring regulation of fluid balance is characterized in that the patient is required to receive a vasopressor agent, such as a catecholamine.

[0184] Thus, a subject of the present invention in one particular embodiment is said pharmaceutical formulation for use in the treatment of patients in need of hypertensive, eg catecholamine, therapy.

[0185] Patients in need of improving fluid balance may be characterized by capillary leakage and may have a urine output of < / =0.5 to 1 cc / kg per hour.

[0186] The subject of the present invention is a pharmaceutical preparation for use in the treatment or prevention of congestion in a patient, wherein the patient has a disease or condition selected from the group comprising congestive hypertension, swelling or water retention (edema), heart failure, particularly acute heart failure, kidney or liver disease.

[0187] In certain embodiments, the pharmaceutical preparation may be administered to a patient having vascular barrier dysfunction or endothelial dysfunction that can cause congestion.

[0188] Vascular barrier dysfunction or endothelial dysfunction is a systemic pathological condition of the endothelium (the inner layer of blood vessels) and can be broadly defined as an imbalance between vasodilatory and vasoconstrictive substances produced by (or acting on) the endothelium (Deanfield et al. 2005. J Hypertens 23(1):7-17). Normal functions of endothelial cells include mediation of coagulation, platelet adhesion, immune function, and control of the volume and electrolyte content of the intravascular and extravascular spaces. The endothelium is a cell monolayer that underlies the entire cardiovascular system and regulates many processes, including vasotension, thrombosis, angiogenesis, and inflammation. Endothelial cells have been shown to be phenotypically dynamic and can transition between a quiescent state and an activated state in response to various local and systemic stimuli (Colombo et al. 2015. Curr Heart Fail Rep. 12(3):215-222). In recent years, new research has demonstrated that endothelial dysfunction is a major cause of cardiovascular diseases, including hypertension, atherosclerosis, and congestive heart failure (Gutierrez et al. 2013. European Heart Journal 34:3175-3181).

[0189] The endothelium tightly controls the exchange of fluid from the blood circulation to the surrounding tissues, and dysfunction of this barrier results in uncontrolled fluid extravasation that can lead to congestion and / or edema. A common feature of edema (e.g., pulmonary edema) is an increased permeability of water to low molecular weight solutes (Rocker et al. 1987. Thorax 42:620-23).

[0190] Endothelial dysfunction may result from and / or contribute to several disease processes, such as those occurring in hypertension, hypercholesterolemia, diabetes, or septic shock, and is a major pathophysiological mechanism leading to coronary artery disease and other atherosclerotic diseases.

[0191] From preclinical studies in models of sepsis / septic shock, it is known that administration of anti-ADM antibodies induces an increase in plasma bio-ADM concentrations, which coincides with an increase in survival rate (Struck et al. 2013. Intensive Care Med Exp 1(1):22). The mechanism underlying this effect is thought to be as follows. When administered intravenously, pharmaceutical preparations, due to their size, are unable to pass through the endothelial barrier into the interstitium and remain in the blood circulation. In contrast, ADM, as a small peptide, can freely diffuse through the endothelial barrier. Thus, when an antibody is administered in a molar excess far beyond endogenous ADM, it binds to virtually all of the ADM in the plasma, resulting in the transfer of ADM from the interstitium into the blood circulation as a simple consequence of reaching a binding equilibrium. ADM located in the interstitium can bind to vascular smooth muscle cells, inducing relaxation and resulting in vasodilation. This is reduced by the administration of the antibody. Meanwhile, ADM in the plasma binds to endothelial cells, thereby stabilizing or even restoring vascular integrity. Thus, this function is enhanced when plasma ADM levels increase as a result of the administration of an antibody that is a non-neutralizing antibody. Finally, the binding of the antibody to ADM reduces the proteolytic degradation of ADM.

[0192] Surprisingly, the inventors observed in the PROTECT and BIOSTAT studies (see, for example, WO 2018 / 109228) that bio-ADM concentrations increase with the presence and severity of congestion in subjects with heart failure, despite treatment with diuretics. Thus, the increase in bio-ADM in these patients is the body's counterregulation of tissue congestion. However, the natural increase is insufficient to effectively achieve this counterregulation. Tissue congestion also occurs in sepsis. For example, in WO 2018 / 109228, it could be demonstrated that administration of anti-ADM antibodies in a sepsis animal model results in the restoration of impaired vascular integrity. The parallel mechanisms of tissue congestion in both sepsis and heart failure give confidence to those skilled in the art that administration of anti-ADM antibodies will be beneficial in treating congestion in heart failure as well as sepsis / septic shock.

[0193] In certain embodiments of the invention, the pharmaceutical formulation is for use in the intervention and treatment of congestion in a patient according to any embodiment of the invention, wherein the patient is resistant to diuretics or is a non-responder to diuretic therapy.

[0194] Another particular embodiment of the present invention relates to said pharmaceutical formulation for use in the intervention and treatment of congestion in a patient in need thereof, wherein the patient is resistant to diuretics or is a non-responder to diuretic therapy.

[0195] The term "diuretic resistance" is generally defined as the inability to reduce extracellular fluid volume despite adequate diuretic use (Ravnan et al. 2002.CHF 8:80-85). Epstein et al. defined diuretic resistance as the inability of a 160 mg oral furosemide dose administered twice daily to excrete at least 90 mmol of sodium within 72 hours (Epstein et al. 1977.Curr Ther Res.21:656-667).

[0196] Diuretic adaptation and diuretic resistance may be caused by similar mechanisms. Diuretic adaptations can be classified as those that occur during diuresis, those that cause sodium retention in the short term (causing "post-diuretic NaCl retention"), and those that increase sodium retention long term (the "braking phenomenon"). The ways in which the kidney adapts to long term diuretic therapy are as follows: First, nephron segments downstream from the site of diuresis increase NaCl reabsorption during diuretic administration as the NaCl load delivered increases. Second, as the diuretic concentration in the tubules decreases, the renal tubules act to retain Na until the next dose of diuretic is administered. Third, the ability of diuretics to increase renal NaCl excretion decreases over time, an effect that results from both extracellular fluid volume depletion and structural and functional changes in the renal tubules themselves. All of these adaptations increase the rate of NaCl reabsorption, blunting the effectiveness of diuretic therapy. For reviews see Ellison 1999. Semin Nephrol. 19(6):581-97 and De Bruyne 2003. Postgrad Med J 79:268-271.

[0197] Although difficult to quantify, diuretic resistance is believed to occur in one in three patients with congestive HF. Heart failure represents the most common clinical setting in which diuretic resistance is observed. In mild congestive HF, diuretic resistance is not commonly encountered as long as renal function is preserved. However, in patients with moderate and severe congestive HF, diuretic resistance occurs more frequently and often becomes a clinical problem (Brater 1985. Drugs 30: 427-443; Taylor 2000 Cardiol Rev. 8: 104-114).

[0198] The subject of the present invention is a pharmaceutical preparation for use in therapy or prevention, comprising determining the level of a fragment of pre-pro-Adrenomedullin selected from the group including mid-region pro-Adrenomedullin (MR-proADM), C-terminal pro-Adrenomedullin (CT-proADM), Bio-ADM, ADM-gly, or pro-Adrenomedullin N-terminal 20 peptide (PAMP) or fragments thereof in a body fluid obtained from the subject prior to drug administration; comparing said level of a fragment of pre-pro-Adrenomedullin selected from the group comprising MR-proADM, CT-proADM, PAMP, Bio-ADM or ADM-gly with a pre-defined threshold or predetermined level of said fragment, ● For correlation, elevated levels of the fragment of pre-pro-Adrenomedullin selected from the group including MR-proADM, CT-proADM, PAMP, Bio-ADM or ADM-gly or fragments thereof above a certain threshold or above a pre-determined level are pharmaceutical preparations used for patient stratification for therapeutic use according to the present invention.

[0199] As used herein, the term "PAMP" includes both blood circulating forms of PAMP, i.e., biologically inactive C-terminal glycine-extended PAMP (PAMP-Gly) and biologically active C-terminal amidated PAMP (PAMP-amide).

[0200] As used herein, the terms "Bio-ADM" and "ADM-NH2" shall be used synonymously and both refer to circulating bioactive adrenomedullin.

[0201] In another embodiment of the present application, the fragment of pre-pro-Adrenomedullin that can be determined in body fluids is selected from the group comprising: SEQ ID NO: 10 (proadrenomedullin N-20 terminal peptide, PAMP): amino acids 22 to 41 of preproADM ARLDVASEF RKKWNKWALS R SEQ ID NO: 11 (mid-region pro-adrenomedullin, MR-proADM): amino acids 45 to 92 of preproADM ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV SEQ ID NO: 12 (C-terminal pro-adrenomedullin, CT-proADM): amino acids 148 to 185 of preproADM RRR RRSLPEAGP RTLVSSKPQA HGAPAPPSGS APHFL SEQ ID NO: 13 (mature human adrenomedullin (mature ADM); amidated ADM; bio-ADM; ADM-NH2): amino acids 1 to 52 or amino acids 95 to 146 of pro-ADM YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-CONH2 SEQ ID NO: 14 (Adrenomedullin 1-52-Gly (ADM 1-52-G1y): amino acids 95 to 147 of preproADM) YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYG

[0202] In another embodiment of the present application, the fragment of pre-proAdrenomedullin having at least 5 amino acids is selected from the group including MR-proADM (SEQ ID NO: 11), CT-proADM (SEQ ID NO: 12) and / or PAMP (SEQ ID NO: 10).

[0203] In one embodiment of the present application, the level of pre-proADM fragments and / or fragments thereof is determined by using at least one binding agent, which binds to a region contained within the sequence of MR-proADM (SEQ ID NO: 11).

[0204] In another embodiment of the present application, the fragments of pre-proADM and / or the level of said fragments are determined by using at least one binding agent, which binds to a region contained within the sequence of CT-proADM (SEQ ID NO: 12).

[0205] In another embodiment of the present application, the fragments of pre-proADM and / or the level of said fragments are determined by using at least one binding agent, which binds to a region contained within the sequence of PAMP (SEQ ID NO: 10).

[0206] A subject of a particular embodiment of the present application is a method, wherein the fragment may be selected from MR-proADM as set forth in SEQ ID NO: 11, and / or CT-proADM as set forth in SEQ ID NO: 12, and / or PAMP as set forth in SEQ ID NO: 10.

[0207] In a particular embodiment of the diagnostic method, the proADM and / or a fragment thereof having at least 5 amino acids is selected from the group including mature ADM-NH2 (SEQ ID NO: 13), ADM 1-52-Gly (SEQ ID NO: 14), MR-proADM (SEQ ID NO: 11) and CT-proADM (SEQ ID NO: 12).

[0208] In certain embodiments of the diagnostic method, either the level of mature ADM-NH2 (SEQ ID NO: 13) and / or ADM 1-52-Gly (SEQ ID NO: 14) immunoreactivity, or the level of MR-proADM (SEQ ID NO: 11) immunoreactivity, or the level of CT-proADM (SEQ ID NO: 12) immunoreactivity is determined and correlated with the patient's need for treatment or intervention, and the patient is identified as having such a need if the level of mature ADM-NH2 (SEQ ID NO: 13) and / or ADM 1-52-Gly (SEQ ID NO: 14) immunoreactivity, or the level of MR-proADM (SEQ ID NO: 11) immunoreactivity, or the level of CT-proADM (SEQ ID NO: 12) immunoreactivity in the subject's body fluid is above a threshold value.

[0209] In a particular embodiment of the diagnostic method, the level of proADM and / or its fragments is determined by using at least one binding agent selected from the group of a binding agent that binds to a region contained within the following sequence of mature ADM-NH2 (SEQ ID NO: 13) and / or ADM1-52-Gly (SEQ ID NO: 14) and a second binding agent that binds to a region contained within the sequence of mature ADM-NH2 (SEQ ID NO: 13) and / or ADM1-52-Gly (SEQ ID NO: 14).

[0210] In a particular embodiment of the diagnostic method, the level of proADM and / or its fragments is determined by using at least one binding agent selected from the group of a binding agent that binds to a region contained within the sequence of MR-proADM (SEQ ID NO: 11) and a second binding agent that binds to a region contained within the sequence of MR-proADM (SEQ ID NO: 11).

[0211] In a particular embodiment of the diagnostic method, the level of pro-ADM and / or its fragments is determined by using at least one binding agent selected from the group of a binding agent that binds to a region contained within the sequence of CT-proADM (SEQ ID NO: 12) and a second binding agent that binds to a region contained within the sequence of CT-pro-ADM (SEQ ID NO: 12).

[0212] Subject of a particular embodiment of this diagnostic method is a method according to the invention, in which the fragment may be chosen from MR-proADM as set forth in SEQ ID NO:11 or from mature ADM-NH2 as set forth in SEQ ID NO:13.

[0213] In a particular embodiment of the invention the threshold value is within a threshold range for plasma MR-proADM which is 0.5 to 1.5 nmol / L, preferably 0.7 to 1 nmol / L, most preferably a threshold value of 0.8 nmol / L is applied.

[0214] In a specific embodiment of the present invention, the predetermined threshold value for Bio-ADM in the subject's body fluid sample is 40 to 100 pg / mL, more preferably 50 to 90 pg / mL, even more preferably 60 to 80 pg / mL, and most preferably the threshold value is 70 pg / mL.

[0215] In a particular embodiment of the invention the threshold value is within a threshold range for plasma CT-proADM which is between 85 and 350 pmol / L, preferably between 100 and 250 pmol / L, most preferably a threshold value of 150 pmol / L is applied.

[0216] In a particular embodiment of the invention, a threshold value for plasma PAMP-amide of 0.3 to 1.2 pmol / L, preferably 0.4 to 1.0 pmol / L, most preferably a threshold value of 0.8 pmol / L is applied.

[0217] In a particular embodiment of the invention, a threshold value for plasma PAMP-glycine of 0.5 to 2.0 pmol / L, preferably 0.7 to 1.8 pmol / L, most preferably a threshold value of 1.5 pmol / L is applied.

[0218] In a particular embodiment of the invention the threshold value is within a threshold range for plasma ADM-NH2 of 50-100 pg / mL, preferably 60-90 pg / mL, most preferably a threshold of 70 pg / mL is applied.

[0219] In certain embodiments of the invention, the threshold for plasma Bio-ADM is 5 times the median concentration of the normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0220] In a particular embodiment of the invention, the threshold value for plasma MR-proADM is 5 times the median concentration of the normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, most preferably 2 times the median concentration.

[0221] In a particular embodiment of the invention, the threshold value for plasma CT-proADM is 5 times the median concentration of the normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, most preferably 2 times the median concentration.

[0222] In certain embodiments of the invention, the threshold value for plasma ADM-Gly is 5 times the median concentration of the normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0223] In certain embodiments of the invention, the threshold value for plasma PAMP is 5 times the median concentration of the normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0224] The threshold level can be obtained, for example, from a Kaplan-Meier analysis in which the occurrence of the disease correlates with the quartiles of the biomarkers in the population. According to this analysis, subjects with biomarker levels above the 75th percentile are at significantly higher risk of suffering from the disease according to the invention. This result is further supported by a Cox regression analysis fully adjusted for classical risk factors. The highest quartile relative to all other subjects is highly significantly associated with an increased risk of suffering from the disease according to the invention.

[0225] Other preferred cut-off values ​​are, for example, the 90th, 95th or 99th percentile of the normal population.By using a percentile higher than the 75th percentile, the number of identified false positive subjects is reduced, but it may not be possible to identify subjects with medium risk, even though they are still at high risk.Therefore, the cut-off value can be adopted depending on whether it is considered more appropriate to identify the majority of risk subjects at the expense of also identifying "false positives", or whether it is considered more appropriate to identify mainly high risk subjects at the expense of missing some subjects with medium risk.

[0226] The above thresholds may be different for other assays if they are calibrated differently from the assay system used in the present invention. Therefore, the above thresholds shall be applied to such differently calibrated assays, taking into account the difference in calibration. One possibility to quantify the difference in calibration is a method comparison analysis (correlation) between the assay of interest (e.g., bio-ADM assay) and each biomarker assay used in the present invention, by measuring each biomarker (e.g., bio-ADM) in the sample using both methods. Another possibility is to determine the median biomarker level of a representative normal population using the assay of interest, assuming that this test has sufficient analytical sensitivity, and compare the results with the median biomarker levels described in the literature (e.g., Weber et al. 2017.JALM 2(2):222-233), and recalculate the calibration based on the difference obtained by this comparison. Using the calibration used in the present invention, samples from normal (healthy) subjects were measured: median plasma bio-ADM (mature ADM-NH2) was 13.7 pg / mL (interquartile range [IQR] 9.6-18.7 pg / mL) (Weber et al. 2017. JALM 2(2):222-233).

[0227] The subject of this diagnostic method is a method according to the diagnostic method invention, in which the level of Pro-adrenomedullin or a fragment thereof of at least 5 amino acids is determined by using a binding agent for Pro-adrenomedullin or a fragment thereof of at least 5 amino acids.

[0228] The subject of the present invention is a pharmaceutical preparation for use in the treatment or prophylaxis of patients with shock, in particular septic shock, which comprises: - suffers from shock, particularly septic shock, within 8.4 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment; and / or - Admission to an ICU within 8.4 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment; and / or - not receiving any organ support or not receiving organ support within 8.4 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment; The antibody or fragment is a pharmaceutical agent that binds to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1).

[0229] As used herein, the term "ICU admission" refers to a patient admitted for intensive care who has or is likely to have one or several acute, directly life-threatening dysfunctions that require the use of organ support methods. Criteria for admitting patients to intensive care units have been developed and are well documented in the art (Nates et al. (2016), Critical care medicine 44:1553-1602). The term "ICU admission" is intended to encompass admissions under these criteria.

[0230] Types of organ support include: ●Respiratory support therapy, This includes advanced respiratory support therapies such as endotracheal intubation and mechanical ventilation support. Basic respiratory support therapies, such as the use of supplemental oxygen, the use of stimulatory spirometers, and chest percussion nebulization;

[0231] Circulatory support therapies, such as mechanical circulatory support (e.g., use of intra-aortic balloon pumps and ventricular assist devices) and medical treatments, including the use of angiotensin-converting enzymes, beta-blockers, etc.

[0232] Renal support therapies such as hemodialysis and peritoneal dialysis Hemodynamic monitoring or supportive therapy, such as measurement of blood pressure, blood flow and oxygen content of blood, fluid resuscitation or blood transfusion, and the use of vasoactive drugs8, e.g., nitroglycerin, nitric oxide, etc. • Neurological monitoring or support, such as intraventricular catheters.

[0233] Extracorporeal organ support is described in detail, for example, in ICU Management & Practice, Volume 18-Issue 1, 2018.

[0234] In a preferred embodiment, the adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is for use in the treatment of a patient with shock, particularly septic shock, comprising: suffers from shock, particularly septic shock, within 10 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold; and / or - Admission to an ICU within 10 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold; and / or - is not receiving any organ support or is not receiving organ support within 10 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold; the antibody or fragment or scaffold binds to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1); If a patient a) suffers from shock, particularly septic shock, within 10 hours and b) is admitted to an ICU within 10 hours, the initiation point for treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is the shorter of a) and b).

[0235] In another embodiment, if a patient a) has been admitted to an ICU within 10 hours and c) is receiving organ support for 10 hours or less, the initiation time for treatment with an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold is the shorter of a) and c).

[0236] In another embodiment, if the patient b) suffers from shock, in particular septic shock, within 10 hours and c) is receiving organ support within 10 hours, the initiation time for treatment with anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is the shorter of b) and c).

[0237] In another embodiment, if a patient a) suffers from shock, particularly septic shock, within 10 hours, b) the patient is in shock within 10 hours, and c) is receiving organ support for 10 hours or less, the initiation time for treatment with anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is the shortest of a), b) and c).

[0238] In another embodiment, the invention relates to an adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment of a patient with shock, particularly septic shock, comprising: - shock within 10 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold; and / or - Admission to an ICU within 10 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, or anti-ADM non-Ig scaffold; and / or - is not receiving any organ support or is not receiving organ support within 10 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold; The antibody or fragment or scaffold relates to an adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, or an anti-ADM non-Ig scaffold that binds to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1).

[0239] In a preferred embodiment, the patient suffers from shock, in particular septic shock, within 9 hours, preferably 8.4 hours, preferably 8.26 hours (0.344 days), preferably 8 hours, preferably 7 hours, preferably 6 hours, preferably 5.76 hours (0.25 days), preferably 5.75 hours (0.24 days), 5 hours, preferably 4 hours, preferably 3 hours, at the time of initiation of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.

[0240] In a preferred embodiment, the patient suffers from shock, particularly septic shock, within 8.4 hours, preferably 8.26 hours (0.344 days) from the start of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.

[0241] In a preferred embodiment, the patient is admitted to the ICU within 9 hours, preferably 8.4 hours, preferably 8.26 hours (0.344 days), preferably 8 hours, preferably 7 hours, preferably 6 hours, preferably 5.76 hours (0.25 days), preferably 5.75 hours (0.24 days), preferably 5 hours, preferably 4 hours, preferably 3 hours, at the time of initiation of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.

[0242] In a preferred embodiment, the patient is admitted to the ICU within 8.4 hours, preferably within 8.26 hours (0.344 days) of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.

[0243] In a preferred embodiment, the patient is receiving organ support within 9 hours, preferably 8.4 hours, preferably 8.26 hours (0.344 days), preferably 8 hours, preferably 7 hours, preferably 6 hours, preferably 5.76 hours (0.25 days), preferably 5.75 hours (0.24 days), preferably 5 hours, preferably 4 hours, preferably 3 hours, of the start of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.

[0244] In a preferred embodiment, the patient is receiving organ support within 8.4 hours, preferably within 8.26 hours (0.344 days) from the start of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.

[0245] In another embodiment, the anti-adrenomedullin-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is for use in the treatment of a patient suffering from shock, particularly septic shock, wherein the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is Within 10 hours after the onset of shock in the patient; and / or Within 10 hours of the patient's admission to the ICU, and / or - Administered before the patient receives organ support or within 10 hours of receiving organ support; The antibody or fragment or scaffold binds to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO:1).

[0246] In another embodiment, the anti-adrenomedullin-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is for use in the treatment of a patient suffering from shock, particularly septic shock, wherein the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is Within 8.4 hours after the onset of shock in the patient; and / or Within 8.4 hours of the patient's admission to the ICU, and / or - Administered before the patient receives organ support or within 10 hours of receiving organ support; The antibody or fragment or scaffold binds to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO:1).

[0247] In a preferred embodiment, the anti-adrenomedullin-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is for use in the treatment of patients suffering from shock, particularly septic shock, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold comprising: Within 10 hours after the onset of shock in the patient; and / or Within 10 hours of the patient's admission to the ICU, and / or - Administered before the patient receives organ support or within 10 hours of receiving organ support; the antibody or fragment or scaffold binds to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1); ●If an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold can be administered a) within 10 hours after the onset of shock in the patient, and b) within 10 hours after admission of the patient to the ICU, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered at the shorter of a) and b).

[0248] In another embodiment, where an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold can be administered a) within 10 hours after the onset of shock in the patient, and c) before the patient receives organ support or within 10 hours before receiving organ support, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered at the shorter of a) and c).

[0249] In another embodiment, if the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold can be administered b) within 10 hours after the patient is admitted to the ICU, and c) before the patient receives organ support or within 10 hours before receiving organ support, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold can be administered at the shorter of b) and c).

[0250] In another embodiment, where the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold may be administered a) within 10 hours after the onset of shock in the patient, b) within 10 hours after the patient is admitted to an ICU, and c) before the patient receives organ support or within 10 hours of receiving organ support, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold may be administered at the shortest time of a), b), and c).

[0251] In another embodiment, the anti-adrenomedullin-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is for use in the treatment of a patient suffering from shock, particularly septic shock, wherein the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is Within 10 hours after the onset of shock in the patient; and / or Within 10 hours of the patient's admission to the ICU, and / or - Administered before the patient receives organ support or within 10 hours of receiving organ support; The antibody or fragment or scaffold binds to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO:1).

[0252] In a preferred embodiment, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 9 hours, preferably 8.4 hours, preferably 8.26 hours (0.344 days), preferably 8 hours, preferably 7 hours, preferably 6 hours, preferably 5.76 hours (0.25 days), preferably 5.75 hours (0.25 days), preferably 5 hours, preferably 4 hours, preferably 3 hours after the onset of shock and / or sepsis in the patient.

[0253] In a preferred embodiment, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 8.4 hours, preferably 8.26 hours (0.344 days) after the onset of shock and / or sepsis in the patient.

[0254] In a preferred embodiment, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 9 hours, preferably 8.4 hours, preferably 8.26 hours (0.344 days), preferably 8 hours, preferably 7 hours, preferably 6 hours, preferably 5.76 hours (0.25 days), preferably 5.75 hours (0.25 days), preferably 5 hours, preferably 4 hours, preferably 3 hours after admission of the patient to the ICU.

[0255] In a preferred embodiment, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 8.4 hours, preferably 8.26 hours (0.344 days) after admission of the patient to the ICU.

[0256] In a preferred embodiment, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered within 9 hours, preferably 8.4 hours, preferably 8.26 hours (0.344 days), preferably 8 hours, preferably 7 hours, preferably 6 hours, preferably 5.76 hours (0.25 days), preferably 5.75 hours (0.25 days), preferably 5 hours, preferably 4 hours, preferably 3 hours after the patient has received organ support at the start of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.

[0257] In a preferred embodiment, the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is administered 8.4 hours, preferably 8.26 hours (0.344 days) after the patient is receiving organ support, at the start of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold.

[0258] In another particular embodiment of the invention, the shock is selected from the group comprising hypovolemic shock, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic or septic shock.

[0259] In certain embodiments of the invention, the shock is selected from the group comprising: In the case of cardiogenic shock, the patient is suffering from an acute coronary syndrome (e.g., acute myocardial infarction) or has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, myocardial damage, valvular disease, aortic dissection with acute aortic stenosis, traumatic chordae rupture, or massive pulmonary embolism, or In the case of hypovolemic shock, the patient may have a bleeding disorder, including spontaneous bleeding in the setting of gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor encroaching on a large blood vessel) and anticoagulant use, or a non-hemorrhagic disorder, including vomiting, diarrhea, kidney loss, skin loss / anesthesia loss (e.g., burns, heat stroke) or third space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma, or In the case of obstructive shock, the patient may have cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or In the case of distributive shock, the patient has septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.

[0260] In a more preferred embodiment, the shock is septic shock, Covid-19 shock, burn shock or traumatic shock.

[0261] In a further embodiment, the present invention relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment of a patient with shock, in particular septic shock, wherein the patient goes into shock within 10 hours of starting treatment with the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold, and / or the patient is admitted to an ICU within 10 hours of starting treatment, and / or is not receiving any organ support or is not receiving organ support within 10 hours of starting treatment with the anti-Adrenomedullin (ADM) antibody or anti-Adrenomedullin antibody fragment or anti-ADM non-Ig scaffold, and a body fluid sample taken from the patient shows a level of bioADM>70pg / mL, and the body fluid is selected from the group comprising whole blood, plasma, serum.

[0262] A subject of the present invention is a pharmaceutical preparation for use in the treatment or prophylaxis of a patient suffering from shock selected from the group comprising hypovolemic, cardiogenic, obstructive and distributive shock, in particular cardiogenic, septic, Covid-19, burn and traumatic shock.A further embodiment of the present invention is a pharmaceutical preparation for use in the treatment or prophylaxis of a patient suffering from shock due to chemical contamination with acidic, basic or oxidizing agents and chemicals in solid, liquid or gas form.

[0263] As used herein, the term "shock" is characterized by reduced oxygen delivery and / or increased oxygen consumption or inadequate oxygen utilization resulting in cellular and tissue hypoxia. It is a life-threatening condition of blood circulation failure, most commonly manifested as hypotension (systolic blood pressure less than 90 mmHg or MAP less than 65 mmHg). Shock is divided into four main types based on the underlying cause: hypovolemic shock, cardiogenic shock, obstructive shock, and distributive shock (Vincent and De Backer 2014. N. Engl. J. Med. 370(6):583).

[0264] The term cardiogenic shock refers to shock in which a patient may suffer from acute coronary syndrome (e.g., acute myocardial infarction) or has heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, myocardial injury, valvular disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture, or massive pulmonary embolism. Cardiogenic shock (CS) is defined as a state of severe intraorgan hypoperfusion due to reduced cardiac output. In particular, CS forms a spectrum ranging from mild hypoperfusion to severe shock. The established criteria for the diagnosis of CS are (i) systolic blood pressure > ≤ 90 mmHg for 30 min or the need for vasopressors to achieve blood pressure ≥ 90 mmHg; (ii) pulmonary congestion or elevated left ventricular filling pressure; (iii) signs of impaired organ perfusion by at least one of the following criteria: (a) altered mental status; (b) cool, clammy skin; (c) oliguria (< 0.5 mL / kg / hr or < 30 mL / hr); (d) elevated serum lactate (Reynolds and Hochman 2008. Circulation 117:686-697). Acute myocardial infarction (AMI) with subsequent ventricular dysfunction is the most frequent cause of CS, accounting for approximately 80% of cases. Mechanical complications such as ventricular septum (4%) or free wall rupture (2%), and acute severe mitral regurgitation (7%) are less frequent causes of CS after AMI. (Hochman et al. 2000. J Am Coll Cardiol 36:1063-1070). Non-AMI-associated CS may be caused by decompensated valvular disease, acute myocarditis, arrhythmias, etc., and treatment options are heterogeneous. This translates to 40,000-50,000 patients per year in the United States and 60,000-70,000 patients in Europe. Despite advances in treatment, mainly due to early revascularization and subsequent reduced mortality, CS remains the leading cause of death in AMI, with mortality still approaching 40-50% according to recent registry and randomized trials (Goldberg et al. 2009. Circulation 119:1211-1219).

[0265] The term "hypovolemic shock" refers to shock in which the patient may suffer from a bleeding disorder, including spontaneous bleeding in the setting of gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor encroaching on the great vessels) and anticoagulant use, or a non-bleeding disorder, including vomiting, diarrhea, kidney loss, skin loss / anesthesia loss (e.g., burns, heat stroke) or third space loss in the setting of pancreatitis, cirrhosis, ileus, trauma. Hypovolemic shock is characterized by a decrease in intravascular volume and can be divided into two broad subtypes: hemorrhagic and non-hemorrhagic. Common causes of hemorrhagic hypovolemic shock include gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor encroaching on the great vessels) and spontaneous bleeding in the setting of anticoagulant use. Common causes of non-hemorrhagic hypovolemic shock include vomiting, diarrhea, kidney loss, skin / anesthesia loss (e.g., burns, heat stroke), or third space loss in the setting of pancreatitis, cirrhosis, bowel obstruction, and trauma. For review, see Koya and Paul 2018.Shock.StatPearls[Internet].Treasure Island (FL):StatPearls Publishing;2019-2018 Oct 27.

[0266] The term "occlusive shock" refers to shock in which the patient may be suffering from cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis. Obstructive shock is due to a physical blockage of the great vessels or the heart itself. Several conditions can result in this form of shock (e.g., cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis). For a review, see Koya and Paul 2018.Shock.StatPearls[Internet].Treasure Island (FL):StatPearls Publishing;2019-2018 Oct 27.

[0267] The term "distributed shock" refers to shock, where the patient may have septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis. According to the cause, there are four types of distributed shock: neurogenic shock (reduced sympathetic stimulation leading to reduced vascular tone), anaphylactic shock, septic shock and shock due to adrenal crisis. In addition to sepsis, distributed shock can be caused by systemic inflammatory response syndrome (SIRS) due to conditions other than infection, such as pancreatitis, burns or trauma. Other causes include toxic shock syndrome (TSS), anaphylaxis (sudden severe allergic reaction), adrenal insufficiency (acute worsening of chronic adrenal insufficiency, destruction or removal of the adrenal glands, suppression of adrenal function by exogenous steroids, hypopituitarism and metabolic failure of hormone production), reaction to drugs or toxins, heavy metal poisoning, hepatic (liver) failure and damage to the central nervous system. For review, see Koya and Paul 2018.Shock.StatPearls[Internet].Treasure Island (FL):StatPearls Publishing;2019-2018 Oct 27.

[0268] Refractory shock is defined as the need for >0.5 μg / kg / min noradrenaline infusion despite adequate volume resuscitation. Mortality in these patients can be as high as 94%, and evaluation and management of these patients requires a much more aggressive approach for survival. The term "refractory shock" is used when tissue perfusion cannot be restored with the initial corrective measures used (e.g., vasopressors), and thus may be termed "highly vasopressor-dependent" or "vasopressor-resistant" shock (Udupa and Shetty 2018. Indian J Respir Care 7:67-72). Patients with refractory shock may have features of inadequate perfusion such as hypotension (mean arterial pressure <65 mmHg), tachycardia, cold peripheries, prolonged capillary refill times, and tachypnea resulting from hypoxia and acidosis. Fever may be seen in septic shock. Other signs of hypoperfusion such as altered sensorium, hyperlactemia, and oliguria may also be seen. These familiar signs of shock do not help distinguish whether the problem is with the pump (heart) or the circuit (vessels and tissues). Different types of shock may coexist, and all forms of shock can become refractory, as evidenced by unresponsiveness to high doses of vasopressors (Udupa and Shetty 2018. Indian J Respir Care 7:67-72).

[0269] The subject of the present invention is a pharmaceutical formulation for use in the treatment or prophylaxis of a patient having shock, the patient being characterized by having a level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample below a threshold value before drug administration, the anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binding to the N-terminal part (amino acids 1 to 21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1).

[0270] A subject of the present invention is a pharmaceutical formulation for use in the treatment or prevention of DPP3 in a body fluid sample of the patient, the threshold value of which is 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, most preferably the threshold value is 50 ng / mL.

[0271] A subject of the present invention is a pharmaceutical formulation for use in therapy or prevention, in which the level of DPP3 is determined by contacting the body fluid sample with a capture binding agent that specifically binds to DPP3.

[0272] One embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the shock is selected from the group comprising hypovolemic shock, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.

[0273] Another embodiment of the present application is an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, comprising: In the case of cardiogenic shock, the patient may be suffering from an acute coronary syndrome (e.g., acute myocardial infarction) or the patient may have heart failure (e.g., acute decompensated heart failure), myocarditis, arrhythmia, myocardial injury, valvular disease, aortic dissection with acute aortic stenosis, traumatic chordae tendineae rupture or massive pulmonary embolism; or In the case of hypovolemic shock, the patient may have a bleeding disorder, including spontaneous bleeding in the setting of gastrointestinal bleeding, trauma, vascular etiology (e.g., ruptured abdominal aortic aneurysm, tumor encroaching on a large blood vessel) and anticoagulant use, or a non-hemorrhagic disorder, including vomiting, diarrhea, kidney loss, skin loss / anesthesia loss (e.g., burns, heat stroke) or third space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma, or In the case of obstructive shock, the patient may have cardiac tamponade, tension pneumothorax, pulmonary embolism, or aortic stenosis, or In the case of distributive shock, the patient may have septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis, and relates to anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds.

[0274] A preferred embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the threshold level of DPP3 in a body fluid sample of the patient is 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, more preferably 40 to 90 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably the threshold level is 50 ng / mL.

[0275] A particular embodiment of the present application relates to an anti-ADM antibody or antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the level of DPP3 is determined by contacting a sample of the body fluid with a capture binding agent that specifically binds DPP3.

[0276] Another embodiment of the present application relates to an anti-ADM antibody or antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein either the level of DPP3 protein and / or the level of active DPP3 is determined and compared with a predefined threshold.

[0277] One embodiment of the present application relates to an anti-ADM antibody or antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the patient is further characterized by having a level of ADM-NH2 above a threshold value.

[0278] To identify patients in shock, levels of ADM-NH2 are measured.

[0279] A preferred embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the threshold value of ADM-NH2 in a body fluid sample of the patient is 40 to 100 pg / mL, more preferably 50 to 90 pg / mL, even more preferably 60 to 80 pg / mL, and most preferably the threshold value is 70 pg / mL.

[0280] Another embodiment of the present application relates to an anti-ADM antibody or antibody fragment, or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein the level of ADM-NH2 is determined by contacting a sample of the body fluid with a capture binding agent that specifically binds ADM-NH2.

[0281] Another preferred embodiment of the present application relates to an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold for use in the treatment or prevention of shock in a patient, wherein a body fluid sample of the patient is selected from the group of blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.

[0282] Another embodiment of the present application is a method of treating or preventing shock in a patient, the method comprising administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold, the method comprising: determining the level of DPP3 in a body fluid sample of the subject; comparing the determined level of DPP3 with a predefined threshold, the patient is treated if the determined level of DPP3 is below the predetermined threshold; The anti-ADM antibody or anti-ADM fragment or anti-ADM non-Ig scaffold binds to the N-terminal portion (aa 1-21) of ADM: YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1).

[0283] Certain embodiments of the present application are directed to a method of treating or preventing shock in a patient, the method comprising administering to the patient an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold, the method further comprising: determining the level of ADM-NH2 in a body fluid sample of the subject; comparing said level of ADM-NH2 with a predetermined threshold, The patient is treated if the determined level of ADM-NH2 is below the predetermined threshold level.

[0284] In one embodiment of the invention, either the level of DPP3 protein and / or the level of active DPP3 is determined and compared to a threshold level.

[0285] In a specific embodiment of the present invention, the threshold value of DPP3 in a body fluid sample from the patient is 20 to 120 ng / mL, more preferably 40 to 90 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably the threshold value is 50 ng / mL.

[0286] In certain embodiments of the invention, the threshold for the level of DPP3 is 5 times the median concentration of the normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0287] The level of DPP3 as the amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject can be determined by different methods, such as immunoassays, activity assays, mass spectrometry, etc.

[0288] According to the invention, any type of binding assay can be used (immunoassays and similar assays that use other types of antigen-specific binding agents instead of antibodies), as well as DPP3 enzyme activity assays that are specific for DPP3 by specifically capturing DPP3 from the sample using a specific binding agent (anti-DPP3 antibody or other type of binding agent) prior to determination of enzyme activity.

[0289] DPP3 activity can be measured by detecting the cleavage products of DPP3-specific substrates.Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, barulfin, β-casomorphin, dynorphin, proctolin, ACTH (adrenocorticotropic hormone) and MSH (melanocyte-stimulating hormone; Abramic et al. 2000, Barsun et al. 2007, Dhanda et al. 2008).The cleavage of the mentioned peptide hormones as well as other untagged oligopeptides (e.g., Ala-Ala-Ala-Ala, Dhanda et al. 2008) can be monitored by detecting the respective cleavage products. Detection methods include, but are not limited to, HPLC analysis (e.g., Lee & Snyder 1982), mass spectrometry (e.g., Abramic et al. 2000), H1-NMR analysis (e.g., Vandenberg et al. 1985), capillary zone electrophoresis (CE; e.g., Barsun et al. 2007), thin layer chromatography (e.g., Dhanda et al. 2008) or reverse phase chromatography (e.g., Mazocco et al. 2006).

[0290] Detection of fluorescence due to hydrolysis of fluorogenic substrates by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are specific di- or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) conjugated to fluorophores. Fluorophores include, but are not limited to, β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide) and 7-amido-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). Cleavage of these fluorogenic substrates results in the release of fluorescent β-naphthylamine or 7-amino-4-methylcoumarin, respectively. In a liquid phase assay, ECA substrate and DPP3 are incubated, for example, in a 96-well plate format, and fluorescence is measured using a fluorescence detector (Ellis & Nuenke 1967). Furthermore, DPP3-loaded samples can be fixed and resolved on a gel by electrophoresis, the gel stained with a fluorogenic substrate (e.g., Arg-Arg-βNA) and Fast Garnet GBC, and the fluorescent protein bands detected by a fluorescence reader (Ohkubo et al. 1999). The same peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala) can be used to detect the fluorescence of DPP3. DPP3 activity can be monitored using detection of a color change due to hydrolysis of the chromogenic substrate.

[0291] Another option for detecting DPP3 activity is the Protease-Glo™ Assay (commercially available from Promega). In this embodiment of the method, DPP3-specific di- or tripeptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Luciferin is a cytochrome P450 luciferase that binds a cytochrome P450 complex (Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) to an aminoluciferin that, upon cleavage by DPP3, is released and serves as a substrate for the bound luciferase reaction, emitting detectable luminescence.

[0292] In a preferred embodiment, DPP3 activity is measured by addition of the fluorogenic substrate Arg-Arg-βNA and monitoring fluorescence in real time.

[0293] In certain embodiments of the method for determining active DPP3 in a body fluid sample of a subject, the capture binding agent reactive with DPP3 is immobilized on a solid phase.

[0294] The test sample is passed over the immobilized binding agent, and if DPP3 is present, DPP3 binds to the binding agent and is itself immobilized for detection.Substrate can then be added and reaction products can be detected to indicate the presence or amount of DPP3 in the test sample.For the purposes of this description, the term "solid phase" can be used to include any material or container in which an assay can be performed, including, but not limited to, porous materials, non-porous materials, test tubes, wells, slides, agarose resins (e.g., Sepharose from GE Healthcare Life Sciences), magnetic particles (e.g., Dynabeads™ or Pierce™ magnetic beads from Thermo Fisher Scientific).

[0295] In another embodiment of the invention, the level of DPP3 is determined by contacting the body fluid sample with a capture binding agent that specifically binds DPP3.

[0296] In a preferred embodiment of the invention, the capture binding agent for determining the level of DPP3 may be selected from the group of antibodies, antibody fragments or non-IgG scaffolds.

[0297] In certain embodiments of the invention, the capture binding agent is an antibody.

[0298] The amount of DPP3 protein and / or DPP3 activity in a body fluid sample of the subject can be determined, for example, by one of the following methods: 1. Luminescence immunoassay (LIA) for quantification of DPP3 protein concentration (Rehfeld et al., 2019 JALM 3(6):943-953).

[0299] The LIA is a one-step chemiluminescence sandwich immunoassay that uses white high-binding polystyrene microtiter plates as the solid phase. The plates are coated with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer anti-DPP3 antibody AK2553 is labeled with MA70-acridinium-NHS-ester and used at a concentration of 20 ng / well. 20 microliters of sample (e.g. serum, heparin-plasma, citrate-plasma or EDTA-plasma from the patient's blood) and calibrators are pipetted into the coated white microtiter plate. After adding the tracer antibody AK2553, the microtiter plate is incubated for 3 hours at room temperature and 600 rpm. Unbound tracer is then removed by 4 washing steps (350 μL / well). The remaining chemiluminescence is measured at 1 second / well by using a microtiter plate luminometer. The concentration of DPP3 is determined using a 6-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0300] 2. Enzyme capture activity assay (ECA) for quantification of DPP3 activity (Rehfeld et al., 2019 JALM 3(6):943-953).

[0301] ECA is a DPP3 specific activity assay that uses black high-binding polystyrene microtiter plates as the solid phase. The plates are coated with monoclonal anti-DPP3 antibody AK2555 (capture antibody). 20 microliters of sample (e.g., serum, heparin-plasma, Citrate-plasma, EDTA-plasma, cerebrospinal fluid and urine) and calibrators are pipetted into the coated black microtiter plate. After addition of assay buffer (200 μL), the microtiter plate is incubated for 2 h at 22° C. and 600 rpm. DPP3 present in the sample is immobilized by binding to the capture antibody. Unbound sample components are removed by four washing steps (350 μL / well). The specific activity of the immobilized DPP3 is measured by addition of the fluorogenic substrate, Arg-Arg-β-naphthylamide (Arg2-βNA), in the reaction buffer, followed by incubation for 1 h at 37° C. DPP3 specifically cleaves Arg2-βNA into Arg-Arg dipeptide and fluorescent β-naphthylamine. Fluorescence is measured in a fluorometer using an excitation wavelength of 340 nm and emission is detected at 410 nm. The activity of DPP3 is determined using a six-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0302] 3. Liquid Phase Assay (LAA) for quantification of DPP3 activity (modified from Jones et al., Analytical Biochemistry, 1982).

[0303] LAA is a liquid-phase assay that uses a black non-binding polystyrene microtiter plate to measure DPP3 activity. Twenty microliters of sample (e.g., serum, heparin-plasma, citrate-plasma) and calibrators are pipetted into the black non-binding microtiter plate. After addition of the fluorogenic substrate Arg2-βNA in assay buffer (200 μL), the initial βNA fluorescence (T=0) is measured in a fluorometer using an excitation wavelength of 340 nm and emission is detected at 410 nm. The plate is then incubated for 1 hour at 37° C. The final fluorescence (T=60) is measured. The difference between the final and initial fluorescence is calculated. The activity of DPP3 is determined using a six-point calibration curve. Calibrators and samples are preferably run in duplicate.

[0304] In certain embodiments, an assay is used to determine the level of DPP3, the assay sensitivity of which is capable of quantifying DPP3 in healthy subjects is <20 ng / mL, preferably <30 ng / mL, more preferably <40 ng / mL.

[0305] 4. Another immunoassay method for measuring DPP3 from plasma of whole blood samples is available: IB10 sphingotest® DPP3 (https: / / www.nexus-dx.com / wp-content / uploads / 2020 / 11 / DPP3-022-00072-IFU-REV-B_8x11.pdf).

[0306] IB10 sphingotest® DPP3 is a rapid point-of-care (POC) immunoassay for the in vitro quantitative measurement of dipeptidyl peptidase 3 (DPP3) in human EDTA whole blood and plasma. The Nexus IB10 immunochemistry system combines chemicals with microfluidic devices and centrifugal flows to rapidly prepare cell-free plasma from whole blood, which can then be moved through channels to rehydrate, solubilize and mix lyophilized immune complexes.

[0307] In certain embodiments, the binding agent has an affinity of at least 10 for DPP3. 7 M -1 , preferably 10 8 M -1 and more preferably has a binding affinity of 10 9 M -1 Greater than 10, most preferably 10 M -1 Those skilled in the art know that it may be considered to compensate for lower affinity by applying a higher dose of the compound, and this measure does not fall outside the scope of the present invention.

[0308] In another embodiment of the invention, the body fluid sample is selected from the group of whole blood, plasma, and serum.

[0309] Mature ADM, bio-ADM, and ADM-NH2 are used interchangeably throughout this application and are the molecule set forth in SEQ ID NO:13.

[0310] In one particular embodiment, the body fluid according to the invention is a blood sample. The blood sample may be selected from the group comprising whole blood, serum and plasma. In a particular embodiment of the method, the sample is selected from the group comprising human citrated plasma, heparinized plasma and EDTA plasma.

[0311] In certain embodiments, an assay is used to determine levels of ADM-NH2, the assay sensitivity of which is capable of quantifying mature ADM-NH2 in healthy subjects is <70 pg / mL, preferably <40 pg / mL, more preferably <10 pg / mL.

[0312] In a particular embodiment of the present invention, the threshold value for ADM-NH2 is 40-100 pg / mL, more preferably 50-90 pg / mL, even more preferably 60-80, with a most preferred threshold value of 70 pg / mL being applied.

[0313] In certain embodiments of the invention, the threshold value for plasma ADM-NH2 is 5 times the median concentration of the normal healthy population, preferably 4 times the median concentration, more preferably 3 times the median concentration, and most preferably 2 times the median concentration.

[0314] In certain embodiments, the binding agent has a binding affinity of at least 10 to ADM-NH2. 7 M -1 , preferably 10 8 M -1 The binding affinity is preferably 10 9 M -1 Greater than 10, most preferably 10 M -1 Those skilled in the art know that it may be considered to compensate for lower affinity by applying a higher dose of the compound, and this measure does not fall outside the scope of the present invention.

[0315] To determine the affinity of the antibody for adrenomedullin, the binding kinetics of adrenomedullin to immobilized antibody was determined by label-free surface plasmon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany). Reversible immobilization of the antibody was performed using anti-mouse Fc antibody covalently coupled at high density to the CM5 sensor surface according to the manufacturer's instructions (Mouse Antibody Capture Kit; GE Healthcare), (Lorenz et al. 2011. Antimicrob Agents Chemother. 55(1):165-173).

[0316] As used herein, the term "binding affinity" refers to the affinity of the proteins according to the present invention to their binding targets, and is expressed numerically using "Kd" values. As used herein, the term "Kd" is intended to refer to the dissociation constant of antibody-antigen interaction. When two or more proteins are shown to have comparable binding affinities to their binding targets, the Kd values ​​for the binding of each protein to those binding targets are within ±2-fold of each other. When two or more proteins are shown to have comparable binding affinities to a single binding target, the Kd values ​​for the binding of each protein to that single binding target are within ±2-fold of each other. When a protein is shown to bind to two or more targets with comparable binding affinities, the Kd values ​​for the binding of the protein to two or more targets are within ±2-fold of each other. Generally, a higher Kd value corresponds to a weaker binding. In some embodiments, "Kd" is measured by a radiolabeled antigen binding assay (MA) or a surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ).

[0317] In certain embodiments, the binding agent is selected from the group comprising an antibody or antibody fragment or a non-Ig scaffold that binds to ADM-NH2.

[0318] In certain embodiments, an assay is used to determine the level of ADM-NH2, such an assay being a sandwich assay, preferably a fully automated assay.

[0319] In one embodiment, such an assay for determining the level of biomarkers (DPP3 and / or ADM-NH2) is a sandwich immunoassay using any type of detection technology, including but not limited to enzyme labeling, chemiluminescence labeling, electrochemiluminescence labeling, and is preferably a fully automated assay. In one embodiment of the diagnostic method, such an assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, BiomerieuxVidas®, Alere Triage®.

[0320] A variety of immunoassays are known and can be used in the assays and methods of the present invention, including radioimmunoassays ("RIA"), homogeneous enzyme amplified immunoassays ("EMIT"), enzyme-linked immunosorbent assays ("ELISA"), apoenzyme reactivation immunoassays ("ARIS"), dipstick immunoassays, and immunochromatographic assays.

[0321] In one embodiment of the present invention, such an assay is a sandwich immunoassay using any type of detection technology, including but not limited to enzyme labeling, chemiluminescent labeling, electrochemiluminescent labeling, and is preferably a fully automated assay. In one embodiment of the present invention, such an assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.

[0322] In one embodiment of the present invention, this may be a so-called POC test (point-of-care), a testing technology that allows testing to be performed close to the patient within less than an hour, without the need for a fully automated assay system. An example of this technology is the immunochromatographic testing technology.

[0323] In a preferred embodiment, the label is selected from the group comprising a chemiluminescent label, an enzyme label, a fluorescent label, a radioactive iodine label.

[0324] The assays may be homogeneous or heterogeneous assays, competitive and non-competitive assays. In one embodiment, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, in which the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, e.g., a bead, a surface of a well or other container, a chip or a strip, and the second antibody is an antibody labeled, e.g., with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general compositions and procedures involved in "sandwich assays" are well established and known to those skilled in the art (The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb; 10(1): 4-10. PMID: 16376134).

[0325] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as dispersion in a liquid reaction mixture, a first label component attached to the first capture molecule, said first label component being part of a labeling system based on fluorescence or chemiluminescence quenching or amplification, and a second label component of said marking system attached to the second capture molecule, such that, upon binding of both capture molecules to the analyte, a measurable signal is generated that allows detection of the sandwich complex formed in the solution containing the sample.

[0326] In another embodiment, the labeling system comprises a rare earth cryptate or rare earth chelate in combination with a fluorescent or chemiluminescent dye, particularly a cyanine type dye.

[0327] In the context of the present invention, fluorescence-based assays include, for example, FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), TET, 6-carbo The use of dyes may be selected from the group including: 4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine glow, rhodamine red, rhodamine 110, BODIPY dyes, e.g. BODIPY TMR, Oregon Green, coumarins such as umbelliferone, benzimides such as Hoechst 33258; phenanthridines, e.g. Texas Red, Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, and the like.

[0328] In the context of the present invention, chemiluminescence-based assays include the use of dyes based on the physical principles described for chemiluminescent materials (Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, JI Kroschwitz; editor, M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, p. 518-562, incorporated herein by reference, including citations on pages 551-562). Preferred chemiluminescent dyes are acridinium esters.

[0329] As referred to herein, an "assay" or "diagnostic assay" can be of any type applied in the field of diagnostics. Such an assay can be based on the binding of the analyte to be detected to one or more capture probes with a specific affinity. For the interaction between the capture molecule and the target molecule or molecule of interest, the affinity constant is preferably greater than 10 8 M -1 Greater than.

[0330] In certain embodiments, at least one of the two binding agents is labeled for detection.

[0331] The ADM-NH2 levels of the present invention are determined by the described ADM-NH2 assay (Weber et al. 2017. JALM 2(2):1-4). The DPP3 levels of the present invention were determined using the described DPP3 assay as outlined in the Examples (Rehfeld et al. 2019. JALM 3(6):943-953). The above thresholds may be different for other assays if they are calibrated differently from the assay system used in the present invention. The above cut-off values ​​should therefore be applied to such differently calibrated assays, taking into account the calibration difference. One possibility to quantify the calibration difference is a method comparison analysis (correlation) between the assay in question and the respective biomarker assay used in the present invention, by measuring the respective biomarkers (e.g. bio-ADM, DPP3) in the sample using both methods. Another possibility is to use the assay in question to determine the median biomarker level of a representative normal population, assuming that the test has sufficient analytical sensitivity, compare the results with the mean biomarker levels as reported in the literature, and recalculate the calibration based on the difference obtained by this comparison. Using the calibration used in the present invention, samples from normal (healthy) subjects were measured: plasma median bio-ADM (mature ADM-NH2) was 24.7 pg / mL, with nadirs of 11 pg / mL and 99 pg / mL. th The percentile was 43 pg / mL (Marino et al. 2014. Critical Care 18:R34). Using the calibration used in the present invention, samples from 5,400 normal (healthy) subjects (Swedish single-center prospective population-based study (MPP-RES)) were measured: median (interquartile range) plasma DPP3 was 14.5 ng / mL (11.3 ng / mL to 19 ng / mL).

[0332] Therefore, in the present invention, the levels of ADM-NH2 are measured to identify patients at high risk of going into shock.

[0333] As used herein, the term "prevention" or any grammatical variations thereof (e.g., prevent, preventing, prevention, etc.) includes, but is not limited to, delaying the onset of symptoms, preventing recurrence of disease, increasing the latency between symptomatic episodes, or combinations thereof. Prevention, as used herein, does not require the complete absence of symptoms.

[0334] The efficacy of non-neutralizing antibodies targeted against the N-terminus of ADM was investigated in survival studies in CLP-induced sepsis in mice. Pretreatment with non-neutralizing antibodies resulted in reduced catecholamine infusion rates, renal dysfunction, and ultimately improved survival (Struck et al. 2013. Intensive Care Med Exp 1(1):22; Wagner et al. 2013. Intensive Care Med Exp 1(1):21).

[0335] Due to these positive results, a humanized version of the N-terminal anti-ADM antibody, called adrecizumab, is being developed for further clinical development. The beneficial effects of adrecizumab on vascular barrier function and survival were recently demonstrated in a preclinical model of systemic inflammation and sepsis (Geven et al. 2018. Shock 50(6):648-654). In this study, pretreatment with adrecizumab attenuated renal vascular leakage in endotoxemic rats as well as mice with CLP-induced sepsis, which was accompanied by increased renal expression of the protective peptide Ang-1 and decreased expression of the deleterious peptide vascular endothelial growth factor. Pretreatment with adrecizumab also improved 7-day survival in CLP-induced sepsis in mice from 10 to 50% for single dose administration and from 0 to 40% for repeated dose administration. Moreover, excellent safety and tolerability were demonstrated in a Phase I study (see Example 6): no serious adverse events were observed, no signals of more frequent adverse events were detected in adrecizumab-treated subjects, and no relevant changes in other safety parameters were found (Geven et al. 2017. Intensive Care Med Exp 5(Suppl 2):0427). Of particular interest is the proposed mechanism of action of adrecizumab. Both animal and human data reveal a strong dose-dependent increase in circulating ADM after administration of this antibody. Based on the pharmacokinetic data and the lack of an increase in MR-proADM (an inactive peptide fragment derived from the same prohormone as ADM), the higher circulating ADM levels cannot be explained by increased production.

[0336] A mechanistic explanation for this increase could be that since ADM is small enough to cross the endothelial barrier, but antibodies are not, excess antibodies in the blood circulation could dump ADM from the interstitium into the blood circulation (Geven et al. 2018.Shock.50(2):132-140 and Voors et al (J.Eur J Heart Fail.2019 Feb;21(2):163-171)). Furthermore, binding of antibodies to ADM results in an extension of the half-life of ADM. Even when NT-ADM antibodies partially inhibit ADM-mediated signaling, a significant increase in blood circulating ADM would result in an overall "net" increase in ADM activity in the blood compartment, exerting beneficial effects on ECs (mainly barrier stabilization), whereas the deleterious effects of ADM on VSMCs in the interstitium (vasodilation) are reduced.

[0337] The present invention is not limited to the use of adrecizumab in particular. What is true for adrecizumab undoubtedly also applies to antibodies that share the main essential characteristics (particularly affinity and epitope specificity). Antibodies targeting the same region would be expected to have the same technical effect, provided they have the same affinity and the same or very comparable structural characteristics (size, shape, ...).

[0338] The term "antibody" generally includes monoclonal and polyclonal antibodies and their binding fragments, in particular Fc fragments, as well as so-called "single-chain antibodies" (Bird et al. 1988), chimeric antibodies, humanized antibodies, in particular CDR-grafted antibodies, and diabodies or tetrabodies (Holliger et al. 1993). Also included are immunoglobulin-like proteins selected through techniques including, for example, phage display, which specifically bind to a molecule of interest contained in a sample. In this context, the term "specific binding" refers to an antibody raised against a molecule of interest or a fragment thereof. An antibody is considered specific if its affinity for the molecule of interest or said fragment thereof is at least preferably 50 times higher, more preferably 100 times higher, and most preferably at least 1000 times higher than its affinity for other molecules contained in the sample containing the molecule of interest. Methods for generating antibodies and selecting antibodies with a given specificity are well known in the art.

[0339] In one embodiment of the invention, the anti-Adrenomedullin (ADM) antibody or anti-Adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is monospecific.

[0340] A monospecific anti-adrenomedullin (ADM) antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold means that the antibody or antibody fragment or non-Ig scaffold binds to one specific region encompassing at least five amino acids in the target ADM. A monospecific anti-adrenomedullin (ADM) antibody or monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM non-Ig scaffold is an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold that all have affinity for the same antibody. Although monoclonal antibodies are monospecific, monospecific antibodies can also be produced by measures other than producing them from a common germ cell.

[0341] The anti-ADM antibody or antibody fragment that binds to ADM, or a non-Ig scaffold that binds to ADM, can be a non-neutralizing anti-ADM antibody or antibody fragment that binds to ADM, or a non-Ig scaffold that binds to ADM.

[0342] In certain embodiments, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is a non-neutralizing antibody, fragment, or non-Ig scaffold. A neutralizing anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold blocks the biological activity of ADM by nearly 100%, by at least greater than 90%, and preferably by at least greater than 95%.

[0343] In contrast, a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold blocks the biological activity of ADM by less than 100%, preferably less than 95%, preferably less than 90%, more preferably less than 80%, and even more preferably less than 50%. This means that the biological activity of ADM is reduced to less than 100%, 95% or less, 90% or less, 80% or less, 50% or less. This means that the residual biological activity of ADM bound to a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold is more than 0%, preferably more than 5%, preferably more than 10%, more preferably more than 20%, and more preferably more than 50%.

[0344] In this regard, molecules that are antibodies or antibody fragments or non-Ig scaffolds with "non-neutralizing anti-ADM activity" are collectively referred to herein for simplicity as "non-neutralizing" anti-ADM antibodies, antibody fragments or non-Ig scaffolds, e.g., molecules that block the biological activity of ADM by less than 80%, as defined below. One or more molecules that bind to ADM, which, when added to a culture of a eukaryotic cell line expressing a functional human recombinant ADM receptor composed of CRLR (calcitonin receptor-like receptor) and RAMP3 (receptor activity modifying protein 3), reduce the amount of cAMP produced by the cell line through the action of a human synthetic ADM peptide added in parallel, said human synthetic ADM being added in an amount that results in half-maximal stimulation of cAMP synthesis in the absence of the non-neutralizing antibody being analyzed, and where the reduction in cAMP by said molecule that binds to ADM occurs to the extent of 80% or less even when the non-neutralizing molecule that binds to the ADM being analyzed is added in an amount that is 10-fold greater than the amount required to obtain the maximum reduction in cAMP synthesis obtained with the non-neutralizing antibody being analyzed.

[0345] The same definition applies to other ranges; 95%, 90%, 50%, etc.

[0346] An antibody or fragment according to the invention is a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes that specifically bind to an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length.

[0347] A full-length immunoglobulin heavy chain is generally about 50 Kd or 446 amino acids in length. Light chains are encoded by a variable region gene (about 110 amino acids in length) at the NH2-terminus and a kappa or lambda constant region gene at the COOH-terminus. Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0348] The basic structural unit of an antibody is generally a tetramer of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain, in which the light and heavy chain variable regions bind the antigen and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (see, e.g., Lanzavecchia et al. 1987. Eur. J. Immunol. 17:105; Huston et al. 1988. Proc. Natl. Acad. Sci. USA, 85:5879-5883; Bird et al. 1988. Science 242:423-426; Hood et al. 1984, Immunology, Benjamin, NY, 2nd ed.; Hunkapiller and Hood 1986. Nature 323:15-16). An immunoglobulin light or heavy chain variable region contains a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (Sequences of Proteins of Immunological Interest, E. Kabat et al., 1987, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 19 ... (See, e.g., U.S. Department of Health and Human Services, 1983). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. The immune complex is an antibody, such as a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody, or a functional antibody fragment, that specifically binds to an antigen.

[0349] Chimeric antibodies are antibodies in which the light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments of genes from a mouse monoclonal antibody can be linked to human constant segments, e.g., kappa and gamma 1 or gamma 3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein composed of a variable domain or antigen binding domain from a mouse antibody and a constant domain or effector domain from a human antibody, although other mammalian species can be used or the variable regions can be generated by molecular techniques. Methods for generating chimeric antibodies are well known in the art, see, for example, U.S. Pat. No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin that comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, they will be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. A "humanized antibody" is an antibody that comprises a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are substitutions such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr.Humanized immunoglobulins can be constructed by genetic engineering measures (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies whose light and heavy chain genes are of human origin. Human antibodies can be made using methods known in the art. Human antibodies can be produced by immortalizing human B cells secreting the antibody of interest. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trioma cells. Human antibodies can also be produced by phage display methods (see, for example, WO 91 / 17271; WO 92 / 001047; WO 92 / 20791) or selected from human combinatorial monoclonal antibody libraries (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying human immunoglobulin genes (see, eg, WO 93 / 12227; WO 91 / 10741).

[0350] Thus, the anti-ADM antibody can have any format known in the art, for example, a human antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody. In a preferred embodiment, the antibody according to the invention is a recombinantly produced antibody, e.g. an IgG, a typical full-length immunoglobulin, or an antibody fragment containing at least the F variable domain of the heavy and / or light chain, e.g. as a chemically conjugated antibody (fragment antigen binding), including, but not limited to, a Fab minibody, a single chain Fab antibody, a monovalent Fab antibody with an epitope tag, e.g. a Fab fragment comprising Fab-V5Sx2; a bivalent Fab (minibody) dimerized with a CH3 domain; a bivalent Fab or multivalent Fab formed, e.g. via multimerization with a heterologous domain, e.g. via dimerization of dHLX domains, e.g. Fab-dHLX-FSx2; F(ab')2 fragments, scFv fragments, multimerized multivalent or / and multispecific scFv fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T cell engager), trifunctional antibodies, multivalent antibodies, e.g. from classes different from G; single domain antibodies, e.g. nanobodies derived from camelid or fish immunoglobulins and many others.

[0351] In addition to anti-ADM antibodies, other biopolymer scaffolds are known in the art to complex target molecules and have been used to generate highly target-specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins. For illustrations of antibody formats, see Figures 1a, 1b and 1c.

[0352] In a preferred embodiment, the anti-ADM antibody format is selected from the group comprising Fv fragments, scFv fragments, Fab fragments, scFv fragments, F(ab)2 fragments and scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from the group comprising scFab fragments, Fab fragments, scFv fragments and bioavailability optimized conjugates thereof, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0353] The non-Ig scaffold may be a protein scaffold and may be used as an antibody mimic since it is capable of binding a ligand or antigen. Non-Ig scaffolds include non-Ig scaffolds based on tetranectin (e.g., as described in U.S. Patent Application Publication No. 2010 / 0028995), fibronectin scaffolds (e.g., as described in EP 1266025); lipocalin-based scaffolds (e.g., as described in WO 2011 / 154420); ubiquitin scaffolds (e.g., as described in WO 2011 / 073214), transferrin scaffolds (e.g., as described in U.S. Patent Application Publication No. 2004 / 0023334), protein A scaffolds (e.g., as described in EP 2231860), ankyrin repeat-based scaffolds (e.g., as described in WO 2010 / 060748), microprotein, preferably a microprotein forming a cysteine ​​knot) scaffold (e.g., as described in EP 2314308), Fyn The scaffolds may be selected from the group including SH3 domain based scaffolds (e.g. as described in WO 2011 / 023685), EGFR-A domain based scaffolds (e.g. as described in WO 2005 / 040229) and Kunitz domain based scaffolds (e.g. as described in EP 1941867).

[0354] In one embodiment of the invention, anti-ADM antibodies according to the invention can be produced by synthesizing a fragment of ADM as an antigen, as outlined in Example 1. Binders to the fragment are then identified using the methods described below or other methods known in the art.

[0355] Humanization of a mouse antibody can be carried out according to the following procedure. For humanization of antibodies of mouse origin, antibody sequences are analyzed for structural interactions between framework regions (FRs) and complementarity determining regions (CDRs) and antigens. Based on structural modeling, suitable FRs of human origin are selected, and mouse CDR sequences are grafted to human FRs. Mutations in the amino acid sequences of CDRs or FRs can be introduced to restore structural interactions that were disrupted by species switching for FR sequences. This restoration of structural interactions can be achieved by a random approach using phage display libraries, or through a directed approach guided by molecular modeling (Almagro and Fransson 2008. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33).

[0356] In a preferred embodiment, the ADM antibody format is selected from the group comprising Fv fragments, scFv fragments, Fab fragments, scFv fragments, F(ab)2 fragments and scFv-Fc fusion proteins. In another preferred embodiment, the antibody format is selected from the group comprising scFab fragments, Fab fragments, scFv fragments and bioavailability optimized conjugates, such as PEGylated fragments. One of the most preferred formats is the scFab format.

[0357] In another preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold is a full-length antibody, antibody fragment, or non-Ig scaffold.

[0358] In a preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and capable of binding to an epitope of at least 5 amino acids in length contained in ADM.

[0359] In a more preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold is directed to and capable of binding to an epitope of at least four amino acids in length contained in ADM.

[0360] In one particular embodiment of the invention, there is provided an anti-ADM antibody or anti-ADM antibody fragment that binds to adrenomedullin, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, wherein the antibody or fragment or scaffold is not ADM binding protein 1 (complement factor H), for use in treating or preventing shock in a patient.

[0361] In one particular embodiment of the invention, there is provided an anti-adrenomedullin (ADM) antibody or anti-ADM antibody fragment that binds to adrenomedullin, or an anti-ADM non-Ig scaffold that binds to adrenomedullin, for use in treating or preventing shock in a patient, wherein the antibody or fragment or scaffold binds to a region of preferably at least four, or at least five amino acids within the sequence of amino acids 1 to 21 of mature human ADM:YRQSMNNFQGLRSFGCRFGTC, SEQ ID NO:1.

[0362] In a preferred embodiment of the invention, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold binds to a region or epitope of ADM located in the N-terminal portion (amino acids 1-21) of adrenomedullin.

[0363] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment, or anti-ADM non-Ig scaffold binds to and recognizes a region or epitope within amino acids 1-14 of adrenomedullin: YRQSMNNFQGLRSF (SEQ ID NO: 15), which refers to the N-terminal portion (amino acids 1-14) of adrenomedullin.

[0364] In another preferred embodiment, the anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1 to 10 of adrenomedullin: YRQSMNNFQG (sequence number 16), which means the N-terminal portion (amino acids 1 to 10) of adrenomedullin.

[0365] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to a region or epitope within amino acids 1-6:YRQSMN (SEQ ID NO: 17) of adrenomedullin, meaning the N-terminal portion (amino acids 1-6) of adrenomedullin. As noted above, the region or epitope preferably comprises at least 4 or at least 5 amino acids in length.

[0366] In another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-terminus (amino acid 1) of adrenomedullin. The N-terminus refers to amino acid 1, which is "Y" in SEQ ID NO: 13 or 18, respectively (SEQ ID NO: 18 (1-42 of human ADM): YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA), and is essential for binding. The antibody or fragment or scaffold does not bind to N-terminally extended or N-terminally modified adrenomedullin, nor to N-terminally degraded adrenomedullin. This means that in another preferred embodiment, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold only binds to a region within the sequence of mature ADM when the N-terminus of ADM is free. In such embodiments, the anti-ADM antibody or anti-ADM antibody fragment or non-Ig scaffold does not bind to a region within the sequence of mature ADM, such as when that sequence is contained within pro-ADM.

[0367] For clarity, numbers in parentheses surrounding a particular region of ADM, such as "N-terminal portion (amino acids 1-21)," will be understood by those of skill in the art to mean that the N-terminal portion of ADM consists of amino acids 1-21 of the mature ADM sequence.

[0368] In another specific embodiment according to the invention, the anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold provided herein does not bind to the C-terminal portion of ADM, i.e., amino acids 43-52 of ADM: PRSKISPQGY-NH2 (SEQ ID NO: 19).

[0369] An epitope, also known as an antigenic determinant, is the part of an antigen that is recognized by the immune system, especially by an antibody. For example, an epitope is the specific piece of an antigen that an antibody binds to. The part of an antibody that binds to an epitope is called a paratope. Epitopes of protein antigens are divided into two categories based on their structure and interaction with the paratope: conformational epitopes and linear epitopes.

[0370] Conformational and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the epitope residues involved and the shape or tertiary structure of other segments of the antigen. Conformational epitopes are formed by the 3-D conformation adopted by the interaction of non-contiguous amino acid residues. Linear or continuous epitopes are epitopes that are recognized by antibodies by their linear sequence or primary structure of amino acids and are formed by the 3-D conformation adopted by the interaction of adjacent amino acid residues.

[0371] In one particular embodiment, it is preferred to use an anti-ADM antibody or anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to the invention, which results in an increase in ADM levels or ADM immunoreactivity in serum, blood or plasma of at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

[0372] In one particular embodiment, it is preferable to use an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADM non-Ig scaffold according to the present invention, and the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold has a half-life (t 1 / 2 ; half-maximum retention time) by at least 10%, preferably at least 50%, more preferably >50%, and most preferably >100%.

[0373] The half-life (half-maximum retention time) of ADM can be determined in human serum, blood or plasma in the absence and presence of an ADM-stabilized antibody or antibody fragment or an ADM-stabilized non-Ig scaffold, respectively, using an immunoassay for quantification of ADM.

[0374] The following steps may be performed: ADM can be diluted in human citrated plasma and incubated at 24° C. in the absence and presence of ADM stabilizing antibodies or adrenomedullin stabilizing antibody fragments or adrenomedullin stabilizing non-Ig scaffolds, respectively. At selected time points (eg, within 24 hours), aliquots can be removed and the degradation of ADM in the aliquots arrested by freezing at -20°C. The amount of ADM can be determined directly by hADM immunoassay if the assay chosen is not affected by the stabilizing antibody. Alternatively, aliquots can be treated with a denaturing agent (such as HCl) and after clarification of the sample (e.g., by centrifugation), the pH can be neutralized and ADM can be quantified by ADM immunoassay. Alternatively, non-immunoassay techniques (e.g., RP-HPLC) can be used for ADM quantification. Half-lives of ADM are calculated for ADM incubated in the absence and presence of ADM stabilizing antibody or adrenomedullin stabilizing antibody fragment or adrenomedullin stabilizing non-Ig scaffold, respectively. The half-life extension is calculated for the stabilized ADM compared to ADM incubated in the absence of the ADM-stabilized antibody or adrenomedullin-stabilized antibody fragment or the adrenomedullin-stabilized non-Ig scaffold.

[0375] A two-fold increase in the half-life of ADM is a 100% increase in the half-life.

[0376] Half-life (half-maximum retention time) is defined as the period of time it takes for the concentration of a particular chemical or drug to fall to half of its baseline concentration in a particular body fluid or blood.

[0377] An assay that can be used to determine the half-life (half-maximal retention time) of adrenomedullin in serum, blood, or plasma is described in Example 3.

[0378] In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold is a non-neutralizing antibody, fragment or scaffold. A neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM to almost 100%, at least to more than 90%, preferably at least to more than 95%. In other words, this means that the non-neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 100%, preferably to less than 95%, preferably to less than 90%. In one embodiment, the non-neutralizing anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM to less than 95%, and anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold that blocks the biological activity of ADM to more than 95% is outside the scope of the embodiment. This means that in one embodiment the biological activity is reduced by 95% or less, preferably 90% or less, more preferably 80% or less, more preferably 50% or less.

[0379] In one embodiment of the present invention, a non-neutralizing antibody is an antibody that binds to a region of at least 5 amino acids within the sequence of amino acids 1 to 21 of mature human ADM (SEQ ID NO: 1).

[0380] In another preferred embodiment of the invention, the non-neutralizing antibody is an antibody that binds to a region of at least four amino acids within the sequence of amino acids 1 to 21 of mature human ADM (SEQ ID NO: 1).

[0381] In a particular embodiment according to the invention, a non-neutralizing anti-ADM antibody or anti-ADM antibody fragment or an ADM non-Ig scaffold is used, which blocks the biological activity of ADM to less than 80% (of baseline value), preferably less than 50%. It should be understood that this limited blocking (meaning reduced biological activity) of the biological activity of ADM occurs even with an excess concentration of the antibody, fragment or scaffold, i.e. an excess of the antibody, fragment or scaffold relative to ADM. This limited blocking is an inherent property of the ADM binding agent itself in this particular embodiment. This means that the antibody, fragment or scaffold has a maximum inhibition of 80% or 50%, respectively. In a preferred embodiment, the anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks / reduces the biological activity of anti-ADM to at least 5%. The above statement means that there remains a residual ADM biological activity of about 20% or 50% or even 95%, respectively.

[0382] Thus, in accordance with the present invention, the anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADM non-Ig scaffolds provided do not neutralize the respective ADM biological activity.

[0383] Biological activity is defined as the effect that a substance exhibits on a living organism or tissue or organ or functional unit after its interaction in vivo or in vitro (e.g., in an assay). In the case of ADM biological activity, this may be the effect of ADM in a human recombinant ADM receptor cAMP functional assay. Thus, according to the present invention, biological activity is defined by an ADM receptor cAMP functional assay. The following steps may be carried out to determine the biological activity of ADM in such an assay. A dose response curve is set up with ADM in the human recombinant ADM receptor cAMP functional assay. The ADM concentration for half-maximal cAMP stimulation can be calculated. At a constant half-maximal cAMP stimulating ADM concentration, dose response curves (up to 100 μg / mL final concentration) are set up with ADM stabilized antibody or antibody fragment or ADM stabilized non-Ig scaffold, respectively.

[0384] A 50% maximal inhibition in the ADM bioassay means that the anti-ADM antibody or the anti-ADM antibody fragment or the anti-ADM non-Ig scaffold, respectively, blocks ADM biological activity to 50% of baseline values. A 80% maximal inhibition in the ADM bioassay means that the anti-ADM antibody or the anti-Adrenomedullin antibody fragment or the anti-Adrenomedullin non-Ig scaffold, respectively, blocks ADM biological activity to 80%. This means that ADM biological activity is blocked to 80% or less. This means that about 20% residual ADM biological activity remains.

[0385] However, in the present specification and in the above context, the expression "blocking the biological activity of ADM" in relation to the anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADM non-Ig scaffolds disclosed herein should be understood as merely reducing the biological activity of ADM from 100% to a maximum of 20% remaining ADM biological activity, preferably reducing the biological activity of ADM from 100% to 50% remaining ADM biological activity, but in each case there is remaining ADM biological activity, which can be determined as detailed above.

[0386] The biological activity of ADM can be determined in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay) according to Example 2.

[0387] In a preferred embodiment, the regulatory anti-ADM antibody or regulatory anti-ADM antibody fragment or regulatory anti-ADM non-Ig scaffold is used in the treatment or prevention of shock in a patient.

[0388] The "modulating" anti-ADM antibody or modulating anti-ADM antibody fragment or modulating anti-ADM non-Ig scaffold reduces the half-life (t 1 / 2 An antibody or antibody fragment or a non-Ig scaffold that extends the half-life (half-maximum retention time) by at least 10%, preferably at least 50%, more preferably >50%, most preferably >100% and blocks the biological activity of ADM by less than 80%, preferably less than 50%, and said anti-ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold blocks the biological activity of ADM by at least 5%. These values ​​relating to half-life and blocking of biological activity should be understood in conjunction with the assays described above to determine these values. This means blocking of ADM biological activity by less than 80% or less than 50%, respectively.

[0389] Such a regulatory anti-ADM antibody or antibody fragment or a regulatory anti-ADM non-Ig scaffold offers the advantage of easier administration of the drug. The combination of partial blocking or partial reduction of ADM biological activity and the extension of in vivo half-life (increased ADM biological activity) results in a beneficial simplification of anti-ADM antibody or antibody fragment or anti-ADM non-Ig scaffold dosing. In situations of excess endogenous ADM (maximal stimulation, late septic phase, shock, hemodynamic depression phase), the activity-reducing effect is the main influence of the antibody or fragment or scaffold, limiting the (negative) effect of ADM. In the case of low or normal endogenous ADM concentration, the biological effect of the anti-ADM antibody or antibody fragment or anti-ADM non-Ig scaffold is a combination of reducing (by partial blocking) and increasing by the extension of ADM half-life. Thus, non-neutralizing and modulating anti-ADM antibodies or anti-ADM antibody fragments or anti-ADM non-Ig scaffolds act like an ADM bioactivity buffer to maintain the bioactivity of ADM within a particular physiological range.

[0390] The term "pharmaceutical formulation," as used herein, refers to a preparation that is in a form that allows the biological activity of the active ingredients it contains to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0391] The present invention also relates to pharmaceutical formulations comprising a therapeutically effective dose of the active ingredient in combination with at least one pharma- ceutically acceptable excipient.

[0392] A "pharmaceutically acceptable excipient" refers to an excipient that does not produce adverse, allergic, or other untoward reactions when administered to a subject. It includes, in addition to the therapeutic protein, carriers, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. The characteristics of the carrier will vary depending on the route of administration.

[0393] In some embodiments, the formulation of the present invention can be administered directly into the bloodstream, muscle, tissue, fat or internal organs of a patient. Suitable procedures for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraosseous, intradermal and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle, microprojection, dissolvable needle and other micropore forming technology) injectors, needle-free injectors and infusion techniques. In some embodiments, the formulation of the present invention is administered subcutaneously to a subject.

[0394] In further embodiments, the aqueous pharmaceutical composition can be administered to a subject in need of treatment according to known administration methods, which may further include shock dose injection or infusion over a specific period of time, intracerebrospinal, transdermal, oral, topical application, or inhalation.

[0395] In some embodiments, the dosing pattern of the aqueous formulation according to the invention comprises administering a dose of the formulation once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 7 weeks, once per 8 weeks, once per 9 weeks, once per 10 weeks, once per 15 weeks, once per 20 weeks, or once per 24 weeks. In some embodiments, the aqueous formulation according to the invention is administered once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, or once per 6 months.

[0396] In some embodiments, the volume of the dose in the formulation is about 20 mL, about 15 mL, about 10 mL, about 5 mL, about 2.5 mL, about 1.5 mL, about 1.0 mL, about 0.75 mL, about 0.5 mL, about 0.25 mL, or about 0.01 mL or less.

[0397] In some embodiments, the volume of the dose in the formulation is about 20 mL, about 19 mL, about 18 mL, about 17 mL, about 16 mL, about 15 mL, about 14 mL, about 13 mL, about 12 mL, about 11 mL, about 10 mL, about 9 mL, about 8 mL, about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL, about 2 mL, or about 1 mL. Alternatively, it is about 20.5 mL, about 19.5 mL, about 18.5 mL, about 17.5 mL, about 16.5 mL, about 15.5 mL, about 14.5 mL, about 13.5 mL, about 12.5 mL, about 11.5 mL, about 10.5 mL, about 9.5 mL, about 8.5 mL, about 7.5 mL, about 6.5 mL, about 5.5 mL, about 4.5 mL, about 3.5 mL, about 2.5 mL, about 1.5 mL, or about 0.5 mL. Alternatively, about 900 microliters, about 800 microliters, about 700 microliters, about 600 microliters, about 500 microliters, about 400 microliters, about 300 microliters, about 200 microliters, or about 100 microliters, or about 950 microliters, about 850 microliters, about 750 microliters, about 650 microliters, about 550 microliters, about 450 microliters, about 350 microliters, about 250 microliters, about 150 microliters, or about 50 microliters. In some embodiments, the volume of the dose in the formulation is about 1.0 mL.

[0398] Dosing regimens may vary depending on the pattern of pharmacokinetic decay the physician wishes to achieve. For example, in some embodiments, dosing 1-4 times per week is contemplated. Less frequent dosing may be used. In some embodiments, doses are administered once every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 15 weeks, every 20 weeks, every 25 weeks, or more. In some embodiments, doses are administered once every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or more. The progress of this therapy is easily monitored by conventional techniques and assays. Dosing regimens may vary over time. For example, in some embodiments, doses of the formulations of the invention are administered (e.g., by intravenous injection) at 1-10 mg / kg body weight every other week. In some embodiments, a dose of the formulation of the invention is administered (e.g., by intravenous injection) at 1-10 mg / kg body weight every other week. In some embodiments, a dose of the formulation of the invention is administered at an initial dose of 1-10 mg / kg body weight intravenous injections four times on one day (day 1), or 1-10 mg / kg body weight injections twice per day on two consecutive days (days 1 and 2), followed by a second dose of 1-10 mg / kg body weight two weeks later (day 15), followed by a maintenance dose of 1-10 mg / kg body weight every other week. In some embodiments, a dose of the formulation of the invention is administered (e.g., by intravenous injection) at an initial dose of 1-10 mg / kg body weight, followed by 1-10 mg / kg body weight every other week starting one week after the initial dose. For the purposes of the present invention, the appropriate dosage of the medicament varies depending on the antibody used, the type and severity of the disorder being treated, whether the medicament is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the medicament, and the discretion of the attending physician.Typically, the clinician will administer the medicament until a dosage is reached that achieves the desired result.Dosage can be empirically determined.For example, an individual can be administered increasing doses and blood glucose levels can be tracked to evaluate the effectiveness of the medicament.

[0399] Dosage and / or frequency may vary over the course of treatment. Empirical considerations, such as antibody half-life, generally contribute to the determination of dosage. Dosage frequency may be determined and adjusted over the course of treatment, generally, but not necessarily, based on the treatment and / or suppression and / or amelioration and / or delay of one or more symptoms of autoimmune disease. In some individuals, more than one dose may be required. Dosage frequency may be determined and adjusted over the course of treatment. For example, but not limited to, for repeated administration over several days or more, treatment is continued until the desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved, depending on the disease and its severity.

[0400] Administration of the formulations of the invention may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the formulations of the invention may be essentially continuous over a preselected period of time, or may be in a series of spaced doses.

[0401] Preferably, administration of the dose is parenteral, preferably selected from intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraosseous, intradermal, and subcutaneous. Preferably, the formulation is in a unit dose sterile form for parenteral administration (e.g., subcutaneous administration).

[0402] The following embodiments are also subject of the present invention. 1. A medicinal aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, wherein the antibody or fragment binds to the N-terminal portion (aa 1-21) of ADM:YRQSMNNFQGLRSFGCRFGTC (SEQ ID NO: 1), and the antibody or antibody fragment is present at a concentration of 1 mg / mL to 100 mg / mL, preferably 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL, and most preferably 20 mg / mL, and the medicinal aqueous formulation is Arginine in the range of 1g / L to 100g / L, and Trehalose in the range of 1g / L to 100g / L, and a surfactant selected from polysorbates and poloxamers in the range of 0.01 g / L to 5 g / L; and ●Further containing histidine in the range of 0.1g / L to 6.4g / L, The pharmaceutical aqueous formulation has a pH in the range of 4.0 to 8.0.

[0403] 2. The antibody or fragment is a human monoclonal antibody or fragment that binds to the N-terminal region (aa 1-21) of ADM (SEQ ID NO:1), or an antibody fragment thereof, and the heavy chain has the sequence: CDR1: SEQ ID NO:2 GYTFSRYW CDR2: SEQ ID NO:3 ILPGSGST CDR3: SEQ ID NO:4 Includes TEGYEYDGFDY, The light chain has the sequence: CDR1: SEQ ID NO:5 QSIVYSNGNTY CDR2: SEQ ID NO:6 RVS CDR3: SEQ ID NO:7 An aqueous pharmaceutical formulation comprising the human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment of embodiment 1, comprising FQGSHIPYT.

[0404] 3. The following sequence as the heavy chain: SEQ ID NO:8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or a sequence that is >95% identical thereto, The light chain has the following sequence: SEQ ID NO:9 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC or a sequence that is >95% identical thereto, comprising an anti-adrenomedullin antibody against the N-terminus of adrenomedullin.

[0405] 4. An aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 3, wherein the surfactant is a poloxamer.

[0406] 5. An aqueous pharmaceutical formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 4, wherein the formulation is essentially free of NaCl and / or glycine.

[0407] 6. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 5, wherein arginine is present in a range of 1 g / L to 100 g / L, preferably 15 g / L to 60 g / L, preferably 13.1 g / L to 52.2 g / L, preferably 11.2 g / L to 44.4 g / L, more preferably 26.1 g / L.

[0408] 7. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 6, wherein trehalose is present in a range of 1 g / L to 100 g / L, preferably 32.6 g / L to 97.8 g / L, preferably 28.4 g / L to 85.1 g / L, preferably 24.1 g / L to 72.4 g / L, more preferably 56.7 g / L.

[0409] 8. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 7, wherein the poloxamer is present in a range of 0.01 g / L to 5 g / L, preferably 0.115 g / L to 1.15 g / L, preferably 0.1 g / L to 1.0 g / L, preferably 0.085 g / L to 0.85 g / L, more preferably 0.5 g / L.

[0410] 9. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 8, wherein histidine is present in the range of 0.1 g / L to 6.4 g / L, preferably 0.22 g / L to 6.88 g / L, preferably 0.8 g / L to 3.2 g / L, preferably 0.68 g / L to 3.68 g / L, more preferably 1.6 g / L.

[0411] 10. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 9, wherein the pH of the formulation is in the range of 4.0 to 8.0, preferably 5.0 to 7.0, and more preferably 6.0.

[0412] 11. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment described in any one of embodiments 1 to 10, wherein the formulation comprises an anti-adrenomedullin antibody against the N-terminus of adrenomedullin having the following sequence: SEQ ID NO: 1, arginine at a concentration of 26.1 g / L, trehalose at a concentration of 56.7 g / L, poloxamer at a concentration of 0.5 g / L, histidine at a concentration of 1.6 g / L, and the formulation has a pH of 6.0.

[0413] 12. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 11, wherein the formulation is stable after storage for at least 4 weeks at 2 to 8°C, and wherein less than 5% wt / wt, preferably less than 4% wt / wt, more preferably less than 3% wt / wt, more preferably less than 2% wt / wt, more preferably less than 1% wt / wt, more preferably less than 0.05% molar, or most preferably less than 0.01% wt / wt of the total antibody is aggregated as measured by size-exclusion high performance liquid chromatography (SEC-HPLC), and the antibody is present at a concentration of 1 mg / mL to 100 mg / mL, preferably 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL, and most preferably 20 mg / mL.

[0414] 13. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 12, wherein the formulation is stable after storage at 2 to 8° C. for at least 4 weeks, where stable means that the formulation remains free of visible particles.

[0415] 14. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 13, wherein the formulation is stable after storage at 2 to 8° C. for at least 4 weeks, where stable means that the formulation remains free of particles subvisible to the naked eye.

[0416] 15. The activity of the antibody or antibody fragment in the formulation is stable after a stress, which is achieved by storing the formulation at 45°C, preferably 42°C, more preferably 38°C, more preferably 36°C, most preferably 40°C and 85% rH, preferably 80rH, preferably 70% rH, most preferably 75% rH for at least 4 weeks, and / or by storing the formulation at 29°C, preferably 27°C, more preferably 22°C, more preferably 20°C, most preferably 25°C and / or 70% rH, preferably 65% ​​rH, preferably 55% rH, more preferably 50% rH, most preferably 60% rH for at least 3 months. and / or by storing the formulation at 9° C., preferably 7° C., more preferably 2° C., more preferably 0° C., and most preferably 5° C. for at least 6 months, and / or by performing at least one freeze / thaw cycle on the formulation, and / or by subjecting the formulation to mechanical stress including orbital shaking and overhead rotation, wherein stability is determined by evaluating the visual appearance including color, clarity and visible particles of the formulation after stress.

[0417] 16. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to any one of embodiments 1 to 15, wherein the antibody is present at a concentration of 1 mg / mL to 100 mg / mL, preferably 2 mg / mL to 50 mg / mL, more preferably 10 mg / mL to 30 mg / mL, more preferably 15 mg / mL to 25 mg / mL, and most preferably 20 mg / mL.

[0418] 17. A pharmaceutical lyophilized formulation obtained from the pharmaceutical aqueous formulation according to any one of embodiments 1 to 16.

[0419] 18. A method for producing a ready-to-use solution, comprising: a. Providing a medicinal aqueous formulation according to any one of embodiments 1 to 16; b. preparing the aqueous pharmaceutical formulation from step a) in a physiologically acceptable solution, optionally by diluting a volume of the pharmaceutical formulation from step a) with a physiologically acceptable solution, and optionally by aliquoting the pharmaceutical formulation from step a), wherein the diluted or prepared aqueous pharmaceutical formulation is suitable for administration in a patient.

[0420] 19. A method for producing a ready-to-use solution, comprising: a. Providing a lyophilized formulation obtained from the aqueous formulation according to any one of embodiments 1 to 16 by lyophilization, optionally without the addition of any other bulking reagents; b. Reconstitution of the lyophilisate from step a) in water for injection and / or a physiologically acceptable solution for injection; and c. adjusting the reconstituted formulation from step b) in a physiologically acceptable solution, optionally by diluting a volume of the reconstituted formulation from step b) with a physiologically acceptable solution, and optionally by aliquoting the reconstituted formulation from step b), wherein the diluted or adjusted aqueous pharmaceutical formulation is suitable for administration in a patient.

[0421] 20. A ready-to-use liquid pharmaceutical formulation obtained by the method according to embodiment 18 or 19, comprising the human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment at a dose of 1 to 10 mg / kg body weight.

[0422] 21. A ready-to-use aqueous pharmaceutical formulation obtained by the method according to embodiment 18 or 19, comprising the human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment at a dose of 2 to 8 mg / kg body weight, preferably 2 or 4 or 8 mg / kg body weight.

[0423] 22. The pharmaceutical formulation according to any one of embodiments 1 to 16, 20 and 21 for use in the treatment or prevention of acute diseases or conditions selected from the group comprising SIRS, severe infections, sepsis, hypovolemic shock, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock, septic shock, Covid-19 shock, burn shock and traumatic shock, acute vascular diseases, such as heart failure, congestion, in particular diuretic resistant congestion, inflammatory conditions, autoimmune diseases, metabolic diseases, brain diseases, cardiovascular diseases and drug induced diseases, symptoms of a disease or diseases characterised by such symptoms, wherein the symptoms of the disease are selected from the group consisting of nausea, headache, muscle pain, back pain, tremors and / or vomiting and migraine.

[0424] 23. A pharmaceutical formulation according to any one of embodiments 1 to 16, 20 and 21 for use in the treatment or prophylaxis of an acute disease or condition in a patient for the prevention or reduction of organ dysfunction or the prevention of organ failure in said patient, wherein said acute disease or condition is selected from the group comprising, for example, severe infections, diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, shock and organ dysfunction, renal dysfunction, hepatic dysfunction, burns, surgery, trauma, poisoning and chemotherapy induced damage, and wherein said disease is not SIRS, sepsis or septic shock.

[0425] 24. A pharmaceutical formulation according to any one of embodiments 1 to 16, 20 and 21 for use in the treatment or prevention of an acute disease or condition in a patient to stabilize the patient's systemic circulation, wherein the patient is suffering from a disease selected from the group comprising SIRS, sepsis, diabetes, cancer, acute vascular disease such as heart failure, shock such as septic shock, organ dysfunction such as renal dysfunction.

[0426] twenty five. a) a pharmaceutical preparation for use in the treatment of an acute disease or condition in a patient for stabilizing the systemic circulation, the patient being in need of stabilizing the systemic circulation and exhibiting a heart rate of >100 beats / min and / or a mean arterial pressure of <65 mmHg, where stabilizing the systemic circulation means increasing the mean arterial pressure to above 65 mmHg, or b) A pharmaceutical formulation according to any one of embodiments 1 to 16, 20 and 21, which is a pharmaceutical formulation for prophylactic use in the treatment of an acute disease or condition in a patient, for preventing an increase in heart rate to >100 beats / min and / or a decrease in mean arterial pressure to <65 mmHg.

[0427] 26. A pharmaceutical formulation for use in the treatment or prevention of an acute disease or condition in a patient for regulating fluid balance, the patient being in need of regulation of fluid balance and suffering from a disease selected from the group comprising systemic inflammatory response syndrome (SIRS), sepsis, diabetes, cancer, heart failure, shock and renal dysfunction, according to any one of embodiments 1 to 16, 20 and 21.

[0428] 27. A pharmaceutical formulation according to any one of embodiments 1 to 16, 20 and 21 for use in the treatment or prevention of congestion in a patient, the patient having a disease or condition selected from the group comprising congestive hypertension, swelling or water retention (edema), heart failure, in particular acute heart failure, renal or hepatic disease.

[0429] 28. determining the level of a fragment of pre-pro-Adrenomedullin selected from the group including mid-region pro-Adrenomedullin (MR-proADM), C-terminal pro-Adrenomedullin (CT-proADM), Bio-ADM, ADM-gly, or pro-Adrenomedullin N-terminal 20 peptide (PAMP) or fragments thereof in a body fluid obtained from the subject prior to drug administration; - comparing said level of a fragment of pre-pro-Adrenomedullin selected from the group comprising MR-proADM, CT-proADM, PAMP, Bio-ADM or ADM-gly with a predefined threshold or a pre-determined said level of the fragment, A pharmaceutical formulation for use in treatment or prevention according to any one of embodiments 1 to 16 and 20 to 27, wherein for correlation, an elevated level of said fragment of pre-pro-Adrenomedullin selected from the group comprising MR-proADM, CT-proADM, PAMP, Bio-ADM or ADM-gly or a fragment thereof, above a certain threshold or above a predetermined level, is used for stratification of patients for therapeutic use according to the present invention.

[0430] 29. A pharmaceutical preparation for use in the treatment or prophylaxis of a patient with shock, in particular septic shock, comprising: - suffers from shock, particularly septic shock, within 8.4 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment; and / or - Admission to an ICU within 8.4 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment; and / or A pharmaceutical formulation for use in treatment or prevention according to any one of embodiments 1 to 16, 20 to 28, in which the patient is not receiving any organ support or is not receiving organ support within 8.4 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment.

[0431] 30. A pharmaceutical preparation for use in treatment or prevention according to any one of embodiments 1 to 17, 20 to 29, wherein the patient is characterized by a level of dipeptidyl peptidase 3 (DPP3) in a body fluid sample of the patient prior to administration of the drug that is below a threshold value.

[0432] 31. The pharmaceutical formulation for use in treatment or prevention according to embodiment 30, wherein the threshold value of DPP3 in a body fluid sample of the patient is 20 to 120 ng / mL, more preferably 30 to 80 ng / mL, even more preferably 40 to 60 ng / mL, and most preferably the threshold value is 50 ng / mL.

[0433] 32. The pharmaceutical preparation for use in treatment or prevention according to embodiment 30 or 31, wherein the level of DPP3 is determined by contacting the body fluid sample with a capture binding agent that specifically binds DPP3.

[0434] 33. A pharmaceutical preparation for use in treatment or prevention according to embodiments 30 to 32, wherein either the level of DPP3 protein and / or the level of active DPP3 is determined and compared with a predefined threshold value. EXAMPLES

[0435] Example 1 - Basic characterization of anti-ADM antibody (HAM8101): Methods 1.Material All excipients used for the formulation of HAM8101 were supplied in United States Pharmacopoeia (USP) and / or European Pharmacopoeia (Ph.Eur.) compliant quality. All chemicals used in analytical methods were supplied in the quality appropriate to the individual method (e.g., pa). Drug Substance (DS) samples and the current formulation of HAM8101 in basic buffer were provided by the manufacturer. All samples were stored at -80°C with internal monitoring.

[0436] Water used throughout the project was sterile water of WFI quality (AquaB. Braun Water for Injection, Braun, product number 0082479E), except for HPLC analysis.

[0437] 2.pH adjustment The adjustment of the final pH of all formulations and buffer solutions was carried out at temperatures between 23°C and 25°C (in accordance with Ph.Eur. and USP) using a calibrated pH electrode (VWR) connected to a SevenExcellence pH meter (Mettler Toledo). The pH value was always adjusted to the desired target value ± 0.03. For pH measurement of the formulated DS, samples were measured at temperatures between 23°C and 25°C (in accordance with Ph.Eur. and USP) using a calibrated pH electrode (Mettler Toledo) connected to a SevenExcellence pH meter (Mettler Toledo).

[0438] 3. Determining Osmolality Osmolality determinations were performed by freezing point depression in an osmometer (Osmomat 030-D RS, Gonotec). Each measurement was performed in a 15 μl aliquot after calibration with sodium chloride standard (600 mOsmol / kg) and purified water.

[0439] 4. Dialysis for Buffer Exchange Buffer exchange of DS for reformulation was performed by dialysis in Slide-A-Lyzer Cassettes (Thermo) with a nominal cut-off of 20 kDa. Samples were dialyzed three times for at least 2 h each, with gentle agitation in a freezer at 2-8 °C, from which one step was performed overnight (>12 h). After dialysis, the HAM8101 concentration was adjusted to a target value of 20 g / L ± 2 g / L with the corresponding buffer, if necessary.

[0440] 5.Concentration measurement The concentration of the drug substance was measured by absorbance at 280 nm using a Spark Plate Reader (Tecan, Switzerland). To do so, placebo and DS samples were measured in one 96-well plate under the same conditions. The adsorption of the placebo was then subtracted from the corresponding DS samples.

[0441] 6. Vial filling Prior to vial filling, samples were sterile filtered through a 0.1 μm PES filter. A 2 mL volume of each formulation with a DS of 20 mg / mL±2 mg / mL was inserted into a sterile, particle-free 2R vial (provided by Rentschler) under laminar air flow for liquid storage in steps 3 and 4. The vials were closed with sterile stoppers (provided by Rentschler) and crimped with suitable aluminum caps.

[0442] 7. Sample storage Samples stored at 5±3° C. were kept in a refrigerator with external temperature monitoring. DS in the original formulation was stored at −80° C. in a freezer with internal temperature monitoring.

[0443] The samples subjected to accelerated aging were stored in an ICH cabinet (Memmert) with controlled humidity at 25±2°C / 60±5%rH, 30±2°C / 65±5%rH and 40±2°C / 75±5%rH, respectively. The temperature was further monitored independently during the complete storage period. Freeze / thaw stresses were performed in cooling cells with alternating storage at -80°C and +25°C for at least 2 hours each. The results of freezing and thawing were visually controlled and adapted accordingly. Shear stress was applied by overhead rotation (Over-Head shaker, Hei-Mix Reax 2, Heidolph / VWR) at 30 rpm for 24 hours or orbital shaking (Dual Plate Shaker PSU-2T, Kisker) at 400 rpm for the indicated times at uncontrolled room temperature. For all storage time points up to 1 month, samples were removed within + / - 1 day of the target removal date, and for the 2-, 3-, and 6-month stability periods, samples were removed within + / - 3 days of the target removal date.

[0444] 8. Size Exclusion Chromatography (SE-HLPC) The SE-HPLC method was based on the SOP provided by the manufacturer for HAM8101. For the size exclusion chromatography method, the stored samples (20±2 mg / mL HAM8101) were separated on a TSKgel G3000SWXL column (5 μm, 7.8×300 mm, Tosoh Bioscience, No: 0008541) using a Thermo Ultimate 3000 HPLC system (50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0) at 20±5° C. with an isocratic flow of 0.5 mL / min. Data were recorded at 214 nm and 280 nm by a UV detector. For each injection, 20 μg of DS diluted in 20 μl of mobile phase was loaded. Data analysis was performed using Chromeleon 7.2.6.10049 software. For column performance checks, molecular weight markers (1511901; Bio-Rad) were used before and after each set of 10 samples and reference standards including thyroglobulin, gamma globulin, ovalbumin, myoglobin, and vitamin B12. To prevent high pressure, all turbid samples (e.g., FT samples, F01 and F06) up to Example 3 (WP4a) and all samples from Example 4 (WP4b) onwards as well as all placebos were filtered with a 0.1 PES filter (Sartorius, 16553).

[0445] 9. Cation exchange chromatography (CEX-HLPC) The CEX-HPLC method was based on the SOP provided by Rentschler for HAM8101. For the cation exchange chromatography method, the stored sample (20±2 mg / mL HAM8101) was separated on a MAbPac SCX-10 column (5 μm, 4 mm×150 mm; Thermo Scientific, No.: 085198) on a Thermo Ultimate 3000 HPLC system. A gradient over 40 min with Diluent A: 100% 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) and Diluent B: 100% 20 mM MES at pH 6.0 containing 200 mM sodium chloride (see Table 1) was used to separate the antibody charge variants at a constant flow rate of 0.5 mL / min at 25±5° C. For each injection, 60 μg DS diluted in eluent A in 30 μl was loaded. Data were recorded by a UV detector at 280 nm and 214 nm. Data analysis was performed using Chromeleon 7.2.6.10049 software.

[0446] To prevent high pressure, all turbid samples (e.g., FT and 2 week 40°C samples, F01 and F06) up to Example 3 (WP4a) and all samples from Example 4 (WP4b) onwards as well as all placebos were filtered through a 0.1 PES filter (Sartorius, 16553).

[0447] 10.CE-SDS / reducing condition / non-reducing condition) Rentschler provided the CE-SDS method SOP together with the analytical accessories for HAM8101 for the quantitative determination of low molecular weight species (LMWS) by non-reducing conditions CE-SDS analysis. However, there was agreement to perform CE-SDS on all samples, reduced and non-reduced, during the course of this project. Therefore, standard instrument settings according to the recommendations of the instrument supplier were used, as well as standard sample buffer for CE-SDS (pH 6.8). Samples were diluted to a concentration of 3 mg / mL in water, but were not subsequently concentrated by zentricons as described in the manufacturer's SOP. The eluent was prepared according to the SOP, the samples were diluted, and the sequencing run was also performed corresponding to the SOP, but the results with sample buffer pH 6.8 were not comparable to those provided by the manufacturer. Therefore, the samples were diluted in sample buffer pH 9.0 and the sequencing run was repeated. Using a sample buffer pH 9.0, the results were comparable and the instrument settings and sample preparation were used for further WPs of this project.

[0448] 11. Dynamic Light Scattering (DLS) for Aggregation Testing Dynamic light scattering was performed on a DynaPro Plate Reader III (Wyatt) in 384-well plates (Aurora) at 25°C. For the implementation of the method, samples were measured undiluted at 20 mg / mL and diluted to different concentrations (0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL and 1 mg / mL), and a placebo was measured together with the sample. All subsequent measurements were performed with 35 μL of undiluted sample and 35 μL of all placebos and measured under the same conditions. After sample loading, the plate was sealed with sealing tape and briefly centrifuged before being placed in the DLS plate reader at 25°C. Analysis was performed using Dynamics, version 7.9.14. For each repetition, 10 individual acquisitions of 5 seconds each were recorded, as shown in Figure 1.

[0449] Results are reported as the average of two biological replicates with three technical replicates each. All measurements were reported as intensity-weighted results. In DLS, particles with nanometer-scale hydrodynamic radii are monitored. The degradation products can be either fragments with radii smaller than that of the monomeric DS, or aggregates with larger radii, each appearing as a further population. Results can be visualized, for example, as a histogram of particle size populations. A category-type plot of DLS peaks including D50 values ​​and peak radii in combination with intensity weighting based on histograms was applied. For stability studies, DLS data were presented as D50 values ​​versus radius scatter plots of all peaks measured with intensity-weighted symbol size to facilitate comparison of different formulations during storage time. The radius of each population (x-axis) and the D50 value of all measured particles for each measurement (y-axis) are plotted in a log-log scatter plot.

[0450] The depicted symbol size of each peak is proportional to the relative intensity (% intensity weighted) of the respective peak. In addition, the overlay of replicate measurements using transparent colors allows the evaluation of the reproducibility of each signal by color intensity, which is useful in determining the main peak. Unless otherwise stated, each measurement consists of at least 2 x 100 single measurements. Parameters are derived from the volume distribution and the intensity distribution, which itself is obtained from the correlation function. Overall, the D50 values ​​on the peak radius plot with the intensity weighted symbol size are used for the qualitative comparison of formulations at application time points within different test arms of the stability study.

[0451] 12. Turbidity The turbidity of the samples was analyzed by nephelometry or by measuring the absorbance at 350 nm, 510 nm and 550 nm. The turbidity was measured by absorbance at 350 nm, 510 nm and 550 nm using a Spark Plate Reader (Tecan, Switzerland). To do so, placebo and DS samples were measured in one 96-well plate under the same conditions. When sufficient sample volume (minimum 1.5 mL) was available, the turbidity of the formulated drug substance was measured in a cuvette with an inner diameter of 11 mm (HACH-Lange) on a calibrated turbidimeter LAB 2100AN (HACH-Lange) by comparing the measured turbidity with standards with known turbidity (<0.1, 3, 6, 18, 20 and 30 NTU, according to the European Pharmacopoeia 6.0 as described in the StablCal®-Kit user manual; HACH-Lange).

[0452] 13. Subvisible Particles (SVP) Micron-sized protein aggregates and particles (subvisible particles, SVPs) are an important quality attribute of therapeutic protein drugs due to the risk of enhancing immunogenic responses. Therefore, quantification of subvisible particles larger than 10 μm and 25 μm is required by pharmacopoeias and is generally performed using light obscuration (LO) techniques. Currently, the acceptance criteria for SVPs are 6000 NMT ≥ 10 μm / container and 600 NMT ≥ 25 μm / container. SVPs < 10 μm need to be monitored and no acceptance criteria have been specified. However, quantification and characterization of particles with sizes below 10 μm is of growing interest and on the other hand there is a regulatory expectation. In this study, measurements were performed by microfluidic imaging using FlowCam (Fluid Imaging Technologies) according to the following software settings: The software Visual Spreadsheet (version 4.12.3, Fluid Imaging Technologies) was used for the analysis. Data were size classified according to Ph.Eur. / USP into <5 μm, 5-10 μm, ≥10 μm, and ≥25 μm subvisible particles (SVPs). Sample and placebo measurements were performed undiluted. After each technical replicate, at least one cleaning and washing step with 4% Hellmanex III (Sigma-Aldrich) and water was performed. Sample measurements were performed only if the SST measurements with water did not show any particle contamination.

[0453] It should be emphasized that according to the literature, MFI is thought to result in higher particle counts when compared to LO due to its higher sensitivity for particles with a refractive index similar to that of the solvent (Huang et al., 2009). This is especially true for proteinaceous particles.

[0454] 14. Visual color inspection The appearance test of the formulated DS was carried out for color according to Ph.Eur. method 2.2.2. Visible particles, (Ph.Eur. method 2.9.20), and the degree of transparency and opalescence (Ph.Eur. method 2.2.) were analyzed according to Ph.Eur. methods 2.9.20 and 2.2.1, respectively. The above evaluations were carried out using an inspection light box equipped with a non-blinking fluorescent lamp and black and white background plates. For color evaluation, the samples were compared with Ph.Eur. reference solution and freshly prepared water. The samples were evaluated for 5 seconds without magnification to evaluate the number and shape of visible particles as well as transparency in front of black and white backgrounds.

[0455] 15. Analytical Ultracentrifugation Selected samples analyzed via analytical ultracentrifugation (AUC) were sent for external analysis. At Zentriforce Pharma Research GmbH, the sedimentation profile of the samples is detected by an optical detection system. Thus, the shape, conformational changes and size distribution of the molecules can be determined.

[0456] Example 2: Basic characterization of anti-ADM antibody (HAM8101) The response of anti-ADM antibody (HAM8101) in current buffer (20 mM His / HCL, 300 mM glycine, pH 6.0) to different stress modes was investigated in forced degradation studies including temperature, mechanical and freeze / thaw stresses as summarized below in Table 2. Samples were tested by SE-HPLC, CEX-HPLC / CiEF, CE-SDS (reducing / non-reducing conditions), appearance, turbidimetry, DLS and SvP after 3, 7, 14 and 21 days of storage.

[0457] 1. Size Exclusion Chromatography (SE-HPLC) The SEC-HPLC results were analyzed as relative peak area [%], the average of two replicates, each measured in duplicate, was calculated and shown in Figure 1. In addition to storage at elevated temperature, samples were stressed by overhead rotation, orbital shaking at room temperature, and five freeze / thaw (F / T) cycles. The SEC-HPLC results of mechanical and freeze / thaw stress are shown in Figure 2. The key findings of the SEC analysis are: 1. A temperature-dependent increase in LMWS during thermal storage (strongest increase at 40°C), and an increase in HMWS in SEC after 2.5 F&T cycles.

[0458] 2. Cation exchange chromatography (CEX-HPLC) CEX-HPLC results were expressed as the average relative peak area from two replicates measured in duplicate. The results are shown in Figure 3. In addition to storage at elevated temperature, samples were stressed by overhead rotation, orbital shaking at room temperature, and five freeze / thaw (F / T) cycles. These results are shown in Figure 4. CEX analysis leads to the following conclusion: CEX-HPLC revealed a significant increase in acidic species during storage, especially in samples stored at 40°C. A possible explanation is deamidation.

[0459] 3. Dynamic Light Scattering (DLS) The results of the DLS analysis of the original formulation are shown in Figure 5. A shift of the blue dots to the right (larger particle size of a particular population) and up (larger average particle size) indicates aggregation. A shift to the left indicates fragmentation. A high number of dots indicates that the sample contains various populations of different particle sizes, i.e. hydrodynamic radii, and the plot shows the average of two technical replicates, with 3 x 100 acquisitions per second evaluated for each replicate. Overall, no obvious stress effects were observed. In all samples, larger particle sizes were detectable, which was expected.

[0460] 4.Appearance The results of visual appearance after stress and subsequent freezing at -80°C (Table 3), followed by another cycle of aliquoting and freezing at -80°C (Table 4) are shown below. Notably, the visible particle load after overnight incubation at 5°C appeared to be less after a few hours of thawing than the day before. Thus, the freeze and thaw stress appears to be significant.

[0461] 5.CE-SDS (reducing / non-reducing conditions) CE-SDS results were expressed as the average relative peak area from two replicates measured in duplicate (see Figures 6 and 7). Key findings of the CE-SDS analysis are: 1. Elevated LMWS at 40°C after 7 days and beyond; 2. No effect of mechanical stress was detected; 3. CE-SDS data is consistent with SEC data.

[0462] Example 3: Design, testing and optimization of formulations to stabilize anti-ADM antibody (HAM8101) and reduce particle formation in liquid formulations during two rounds of accelerated aging; Screening DoE design Here, the performance and respective results of the first accelerated aging round are presented. The aim of this screening round was to investigate the influence of different parameters of the screening DoE, as shown in Table 5. The reference anti-ADM antibody (HAM8101) was formulated in the current buffer (20 mM His / HCL, 300 mM glycine, pH 6.0).

[0463] Based on the results shown in Example 2, the following stress conditions were selected: ● Store at 40℃±2℃ / 75±5%rH for 2 weeks • Five freeze / thaw cycles (-80°C / +25°C) (1°C / min; plateau 2 h). • Mechanical stress: orbital shaking and overhead rotation as above.

[0464] The analytical methods used are shown in Table 2 and the composition of the different formulations is given in Table 6.

[0465] 1. Osmolality and Concentration First, the osmolality and concentration of the samples are described. The measured data are shown in Table 7.

[0466] 2. Dynamic Light Scattering (DLS) The results of DLS analysis of the WP4a formulation are shown in Figures 8 to 11. The results showed very high sensitivity for large radii (≦×6) to see the initial changes, and the D50 value (approximately 5 nm) confirmed that most particles were within the size range of the API.

[0467] 3. Size Exclusion HPLC The SEC-HPLC results, expressed as the average relative peak area [%], derived from two replicates measured in duplicate, are shown in Figures 12-14.

[0468] 4. Subvisible particles (SVP) Here, the amount of sub-visible particles in the samples was determined and the results are shown in Figures 15 and 16. In detail, the particle content below 5 μm, between 5 and 10 μm, ≥ 10 μm and ≥ 25 μm after 2 weeks of storage at 40°C or after mechanical stress was evaluated.

[0469] 5. Visual Appearance Also evaluated were mechanical stress, freezing and thawing, and visual appearance after 2 weeks storage at 40° C. The results are depicted in Table 8.

[0470] 6. Summary The main focus of this formulation development was the prevention of visible particles. In screening, DoE stressed formulations 4, 10, 11, 16, 18, 19 and 20 were free of visible particles. Most of these formulations also showed low, sub-visible particle counts. No clear effect was observed in SE-HPLC and DLS analysis. However, formulation 6 and reference formulation 21 show clearly elevated HMWS content after 5 freeze-thaw cycles. Both formulations have high glycine content along with the absence of arginine and poloxamer. Based on the observed results, the following potential effects were further explored in Example 4:

[0471] Example 4: Design, testing and optimization of formulations to stabilize anti-ADM antibody (HAM8101) and reduce particle formation in liquid formulations during two rounds of accelerated aging - Screening DoE design, optimization rounds Based on the results of Example 3, 10 formulations in addition to the current formulation (F11) were selected to be subjected to a second round of forced degradation. The stress conditions in Example 4 were the same as those used in Example 3. The compositions of the different formulations are shown in Table 9.

[0472] 1. Osmolality and Concentration First, the osmolality and concentration of the sample in Example 4 will be described. The measured data are shown in Table 10.

[0473] 2. Size Exclusion-HPLC The SEC-HPLC results, expressed as the average relative peak area [%], are from two replicates measured in duplicate and are shown in Figures 17 and 18. Overall, significant fragmentation is detected after 4 weeks at 40°C, and the effect of increased HMW levels in formulation 11 after five freeze and thaw cycles.

[0474] 3. AUC of preselected samples (1, 2, 3, 4, 10, 11) AUC results for selected samples after 3.5 weeks at 40° C. confirmed the SE-HPLC results. Overall, there was no obvious aggregation detectable, and formulation WP4b-2 showed the lowest amount (%) of LMWS and HMWS. The results are depicted in Table 11. Relative monomer content after external analysis by analytical ultracentrifugation is shown in FIG. 19.

[0475] 4.CE-SDS (reducing / non-reducing conditions) The results of CE-SDS non-reducing (Table 12) and reducing (Table 13) conditions are presented as the average relative peak area from two replicates measured in duplicate and are shown in Tables 12 and 13, respectively. The results of CE-SDS (non-reducing) showed that no obvious aggregation effect was detectable, but significant fragmentation was observed after 3.5 weeks at 40°C. Furthermore, the lowest fragmentation levels could be observed for F01 and F02, while the highest fragmentation levels were observed for F08 and F11. Notably, the results of CE-SDS (reducing) showed that no obvious aggregation and fragmentation effects could be detected. In particular, significant fragmentation was detectable after 3.5 weeks at 40°C, and the lowest fragmentation levels were observed for F02, F03 and F09.

[0476] 5. Dynamic Light Scattering (DLS) The DLS results after storage at 40° C. and after mechanical stress are shown in FIG.

[0477] 6. FlowCam analyzes particles invisible to the naked eye Here, the particles were analyzed after thermal and mechanical stress, and the results are shown in Figures 21 and 22.

[0478] 7. Visual Appearance Also evaluated were mechanical stress, freezing and thawing, and visual appearance after 2 weeks storage at 40° C. The results are shown in Table 14 (clear: water-like opalescence, NVP: no visible particles, hazy: too many particles to count).

[0479] 8. Cation Exchange HPLC The relative areas [%] of main (top), acidic (middle) and basic (bottom) species in CEX-HPLC of formulated HAM8101 stored at 40°C ± 2°C / 75%rH ± 5%rH for up to 3.5 weeks or after mechanical stress and 5 freeze-thaw cycles were evaluated. The results are shown in Figures 23 and 24.

[0480] [Table 2]

[0481]

Table 3

[0482]

Table 4

[0483]

Table 5

[0484]

Table 6

[0485]

Table 7

[0486]

Table 8

[0487]

Table 9

[0488]

Table 10

[0489]

Table 11

[0490]

Table 12

[0491]

Table 13

[0492]

Table 14

[0493]

Table 15

Claims

1. A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment, wherein the antibody or fragment is bound to the N-terminal portion (aa 1-21) of ADM:YRQSMNNNFQGLRSFGCRFGTC (SEQ ID NO: 1), the antibody or antibody fragment is present at a concentration of 1 mg / mL to 100 mg / mL, and the pharmaceutical aqueous formulation is ●Arginine in the range of 1 g / L to 100 g / L, ● Trehalose in the range of 1 g / L to 100 g / L, ●A surfactant selected from polysorbates and poloxamers in the range of 0.01 g / L to 5 g / L, and ●Further contains histidine in the range of 0.1 g / L to 6.4 g / L, The aforementioned formulation is a pharmaceutical aqueous formulation having a pH in the range of 4.0 to 8.

0.

2. The antibody or fragment is a human monoclonal antibody or fragment, or an antibody fragment thereof, that binds to the N-terminal region (aa 1-21) of ADM (SEQ ID NO: 1), and the heavy chain is sequence: CDR1: Sequence ID 2 GYTFSRYW CDR2: Sequence ID 3 ILPGSGST CDR3: Sequence ID 4 Contains TEGYEYDGFDY, The light chain is arranged as follows: CDR1: Sequence ID 5 QSIVYSNGNTY CDR2: Sequence ID 6 RVS CDR3: Sequence ID 7 A pharmaceutical aqueous formulation comprising FQGSHIPYT, a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment as described in claim 1.

3. The heavy chain consists of the following sequence: Sequence ID 8 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGSTNYNQKFQGRVTITADTSSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Or it includes a sequence that is >95% identical to it, The light chain has the following sequence: Sequence ID 9 DVVLTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWYLQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGGGTKL EIKRTVAAPSVFIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC A pharmaceutical aqueous formulation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to claim 1 or 2, comprising an anti-adrenomedullin antibody against the N-terminus of adrenomedullin, or comprising a sequence that is >95% identical thereto.

4. The aqueous pharmaceutical preparation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment according to claim 1, wherein the surfactant is poloxamer.

5. A pharmaceutical aqueous preparation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment as described in claim 1, wherein arginine is present in an amount of 1 g / L to 100 g / L.

6. A pharmaceutical aqueous preparation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment as described in claim 1, wherein trehalose is present in an amount of 1 g / L to 100 g / L.

7. A pharmaceutical aqueous preparation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment as described in claim 1, wherein poloxamer is present in a concentration of 0.01 g / L to 5 g / L.

8. A pharmaceutical aqueous preparation comprising a human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment as described in claim 1, wherein histidine is present in a concentration of 0.1 g / L to 6.4 g / L.

9. A pharmaceutical aqueous preparation comprising a human or humanized anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment according to claim 1, wherein the pH of the preparation is 4.0 to 8.

0.

10. The preparation comprises an anti-adrenomedullin antibody against the N-terminus of adrenomedullin having the following sequence: SEQ ID NO: 1, arginine at a concentration of 26.1 g / L, trehalose at a concentration of 56.7 g / L, poloxamer at a concentration of 0.5 g / L, and histidine at a concentration of 1.6 g / L, and the preparation exhibits a pH of 6.0, and is a pharmaceutical aqueous preparation comprising the human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment as described in claim 1.

11. A pharmaceutical freeze-dried preparation obtained from the pharmaceutical aqueous preparation described in claim 1.

12. A method for producing a ready-to-use solution, a. A step of providing the aqueous pharmaceutical preparation described in claim 1, b. A method comprising the step of preparing the aqueous pharmaceutical preparation from step a) in a physiologically acceptable solution by optionally diluting the volume of the pharmaceutical preparation from step a) with a physiologically acceptable solution, and optionally dividing the pharmaceutical preparation from step a) into equal parts, wherein the diluted or prepared aqueous pharmaceutical preparation is suitable for administration to a patient.

13. A method for producing a ready-to-use solution, a. A step of providing a lyophilized formulation obtained from the aqueous formulation described in claim 1 by freeze-drying without adding any other bulk reagents as needed, b. Reconstitution of the lyophilized product from step a) in water for injection and / or a physiologically acceptable solution for injection, c. A method comprising the step of preparing a reconstituted formulation from step b) in a physiologically acceptable solution by optionally diluting the volume of the reconstituted formulation from step b) with a physiologically acceptable solution, and optionally dividing the reconstituted formulation from step b) into equal parts, wherein the diluted or prepared aqueous pharmaceutical formulation is suitable for administration to a patient.

14. A ready-to-use aqueous pharmaceutical preparation obtained by the method of claim 12 or 13, comprising the human or humanized anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment in a dose of 1 to 10 mg / kg body weight.

15. A pharmaceutical formulation according to claim 1 for use in the treatment or prevention of an acute disease or acute condition selected from the group including SIRS, severe infection, sepsis, shock due to hypovolemia, cardiogenic shock, obstructive shock and distributed shock, acute vascular disease, congestion, inflammatory conditions, autoimmune diseases, metabolic diseases, cerebrovascular diseases, cardiovascular diseases and drug-induced diseases, symptoms of a disease or diseases characterized by such symptoms, wherein the symptoms of the disease are selected from the group including nausea, headache, muscle pain, back pain, tremors, and / or vomiting and migraine.

16. A pharmaceutical formulation according to claim 1 for use in the treatment or prevention of an acute disease or acute condition in a patient for the prevention or reduction of organ failure, wherein the acute disease or acute condition is selected from the group including severe infection, diabetes, cancer, acute and chronic vascular disease, myocardial infarction, stroke, atherosclerosis, shock and organ failure, renal dysfunction, hepatic dysfunction, burns, surgery, trauma, poisoning and chemotherapy-induced injury, wherein the disease is not SIRS, sepsis or septic shock.

17. A pharmaceutical formulation according to claim 1 for use in the treatment or prevention of an acute disease or acute condition in a patient for the regulation of fluid balance, wherein the patient is a patient who requires regulation of fluid balance and suffers from a disease selected from the group including systemic inflammatory response syndrome (SIRS), sepsis, diabetes mellitus, cancer, heart failure, shock, and renal dysfunction.

18. A pharmaceutical preparation according to claim 1 for use in the treatment or prevention of congestion in a patient, wherein the patient has a disease or condition selected from the group including congestive hypertension, swelling or fluid retention (edema), heart failure, renal or hepatic disease.

19. A pharmaceutical preparation for use in the treatment or prevention described in claim 1, ● Determining the level of pre-pro-adrenomedullin fragments selected from the group including intermediate region pro-adrenomedullin (MR-proADM), C-terminal pro-adrenomedullin (CT-proADM), Bio-ADM, ADM-gly, or pro-adrenomedullin N-terminal 20 peptide (PAMP) or fragments thereof in the body fluids obtained from the subject before drug administration, ● Comparing the level of a pre-pro-adrenomedullin fragment selected from the group including MR-proADM, CT-proADM, PAMP, Bio-ADM, or ADM-gly with a predetermined threshold or a pre-determined level of the fragment, For correlation purposes, elevated levels of the pre-pro-adrenomedullin fragment, selected from the group comprising MR-proADM, CT-proADM, PAMP, Bio-ADM, or ADM-gly, or fragments thereof, that exceed a specific threshold or a predetermined level are used for patient stratification for therapeutic use according to the present invention, in a pharmaceutical formulation.

20. A pharmaceutical preparation for therapeutic or prophylactic use according to claim 1, for use in the treatment of a patient having shock, wherein the patient is ● Within 8.4 hours of initiating treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, the patient is suffering from shock, particularly septic shock, and / or ● The patient is admitted to the ICU within 8.4 hours of the start of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment, and / or ●A pharmaceutical product that has not received any organ support, or has not received organ support within 8.4 hours of the initiation of treatment with the anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment.

21. The pharmaceutical formulation for therapeutic or preventive use according to claim 1, characterized in that the patient has a level of dipeptidyl peptidase 3 (DPP3) below a threshold in a sample of the patient's body fluids prior to drug administration.

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