TWI786132B - Activity - Google Patents

Abstract

The present invention relates to inhibitors of C5_A activity and their use in the treatment of cutaneous, neutrophilic, inflammatory diseases in a subject.

Description

Treatment of Inflammatory Diseases Using C5A Activity Inhibitors

The present invention relates to inhibitors of C5a activity and their use in treating skin, neutrophil, and inflammatory diseases in individuals.

Targets C5a in inflammation

C5a is a 74-amino acid cleavage product of its "parent" molecule, C5, and represents an assessment of the complement activation cascade. It can be generated by activation of at least three distinct pathways (alternative, canonical and MBL pathways). All pathways merge at the C3 stage to form C5 or an alternative C5 convertase, resulting in cleavage of C5 into C5a and C5b. The latter binds to C6, C7, C8 and several C9 molecules, eventually allowing the formation of holes in eg bacterial membranes (terminal Membrane Attack Complex = MAC). C5a is produced by activation of the complement system in inflammatory environments and other immune and inflammatory disorders/diseases.

Among complement activation products, C5a is one of the most potent inflammatory peptides with a wide range of functions (Guo and Ward, 2005). C5a exerts its effects through high-affinity C5a receptors (C5aR and C5L2) (Ward, 2009). C5aR belongs to a rhodopsin family of G protein-coupled receptors with seven transmembrane segments; C5L2 has a similar structure but does not appear to be G protein-coupled. It is currently believed that C5a exhibits its biological function mainly through the C5a-C5aR interaction, since few biological responses were found for the C5a-C5L2 interaction. However, recent reports demonstrate that signaling also occurs via C5L2 activation (Rittirsch and others, 2008).

C5aR is widely expressed on myeloid cells including neutrophils, eosinophils, basophils, and monocytes, as well as on non-myeloid cells in many organs, especially in the lung and liver, expressing C5a /C5aR Importance of Communications. Upregulation of C5aR is ubiquitous during sepsis, and blockade of the C5a/C5aR interaction by anti-C5a or anti-C5aR antibodies or C5aR antagonists confers high protection in rodent models of sepsis (Czermak and others, 1999; Huber-Lang and others, 2001; Riedemann and others, 2002).

C5a has various biological functions (Guo and Ward, 2005). C5a is a strong chemoattractant for neutrophils and also has chemoattractant activity for monocytes and macrophages. C5a causes an oxidative burst ( ₀₂ consumption) in neutrophils and enhances phagocytosis and release of granzymes. C5a has also been found to be a vasodilator. C5a has been shown to be involved in the regulation of cytokine expression in different cell types and to increase the expression of attachment molecules expressed on neutrophils. High doses of C5a may lead to non-specific chemotaxis "desensitization" of neutrophils, causing obvious functional abnormalities. Many inflammatory diseases, including sepsis, acute lung injury, inflammatory bowel disease, rheumatoid arthritis, and others, are due to the action of C5a. In the experimental setting of sepsis, exposure of neutrophils to C5a results in abnormal neutrophil function and signaling pathway paralysis, resulting in defective assembly of NADPH oxidase, paralysis of MAPK signaling cascade, oxidative burst, and phagocytosis And chemotaxis was greatly inhibited (Guo and others, 2006; Huber-Lang and others, 2002). Thymocyte apoptosis and delayed neutrophil apoptosis are two important pathogenic events in sepsis, which depend on the presence of C5a. During experimental sepsis, C5a upregulates β2-integrin expression on neutrophils to promote cell migration into organs, a major cause of multiple organ failure (MOF). C5a was also found to contribute to the activation of the coagulation pathway that occurs in experimental sepsis. C5a stimulates the synthesis and release from human leukocytes of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and also macrophage migration inhibitory factor (MIF). If complement activation is an event that occurs during the onset of acute inflammation, C5a may act before most inflammatory "cytokine storms" occur. C5a appears to play a key role in coordinating and amplifying the expression of cytokine networks and the development of systemic inflammatory response syndrome (SIRS).

In the immunoregulatory network following acquired immunity, C5a affects the crosstalk between dendritic cells (DC) and δεT cells, which may result in the production of a large number of inflammatory mediators, such as IL-17 (Xu and others ,2010). A major role for C5a has been established and defined in the generation of pathogenic Th17 responses in systemic lupus erythematosus (SLE) (Pawaria and others, 2014). In addition, it has been reported that C5a is a key regulator of Treg cells, providing a strong inhibitory effect on Treg proliferation and induction (Strainic and others, 2013). If it is true that Treg as well as TH17 are key players in the autoimmune disease setting, inhibition of C5a signaling would be expected to significantly reduce the overactive immune state in autoimmune disease.

IFX-1

IFX-1 is a chimeric monoclonal IgG4 antibody that specifically binds to soluble human complement split product C5a. IFX-1 is composed of 1328 amino acids and has an approximate molecular weight of about 148,472 Daltons. The CDR and FR sequences of IFX-1 are disclosed in WO 2015/140304 A1 (in Table 3 on page 31), the content of which is incorporated by reference in its entirety.

IFX-1 is expressed as a recombinant protein in mammalian CHO cell lines and ultimately formulated in phosphate-buffered saline for intravenous administration. Binding of this antibody to human C5a leads to efficient blocking of C5a-induced biological effects by rendering C5a unable to bind to and interact with its corresponding cell-bound receptors.

Various nonclinical studies are performed to evaluate the pharmacological and toxicological aspects of IFX-1, which can be divided into in vitro/ex vivo tests and in vivo studies (including GLP toxicology studies in cynomolgus monkeys (using IFX-1)). None of the nonclinical tests and studies conducted have shown any toxicological or safety concerns with IFX-1. The human Phase I trial noted that safety laboratory parameters, vital signs, and ECG parameters showed no clinically meaningful time- or dose-related changes.

In vitro assays of IFX-1 demonstrated the ability to potently bind to soluble human C5a and strongly block C5a-induced biological effects such as lysozyme release from human neutrophils or upregulation in neutrophils from human whole blood CD11b) activity. An IFX-1 antibody achieved the ability to neutralize the effects of two C5a molecules with nearly 100% efficiency under experimental in vitro conditions. Clinical trials using IFX-1 have been conducted to test its clinical efficacy in several inflammatory diseases including septic organ dysfunction and complex cardiac surgery.

Neutrophils

Neutrophils are short-lived, terminally differentiated cells in circulation and are the most abundant white blood cells in humans. As a first line of defense against invading microorganisms, neutrophils are characterized by their ability to act as phagocytes upon stimulation, releasing lytic enzymes from their granules and generating reactive oxygen species. In addition to microbial products, other stimuli, such as immune complexes, can also induce a respiratory burst of neutrophils, leading to increased inflammation and recruitment of inflammatory cells (Kaplan, 2013).

After infiltration into inflamed tissue, neutrophils associate with many other cell types (such as macrophages, dendritic cells (DCs), natural killer cells, lymphocytes, and mesenchymal stem cells), modulating innate as well as adaptive immune responses . For example, neutrophils can regulate DC maturation and T cell proliferation and polarization, and they can also directly prime antigen-specific type 1 helper T cells and type 17 helper T cells (Abi Abdallah and others, 2011). Different stimuli induce neutrophil degranulation, including C5a, formyl-methionyl-leucyl-phenylalanine (FMLP), lipopolysaccharide, platelet-activating factor, and tumor necrosis factor (TNF) ( Kaplan, 2013). Contents and oxides released by degranulation and cytokines and chemokines due to neutrophil activation are major inflammatory mediators that cause tissue damage, and this mechanism is believed to drive many types of Inflammatory tissue damage.

Hidradenitis suppurativa (HS)

HS is a chronic destructive skin disease affecting areas rich in the apocrine gland, and it is considered one of the neutrophil-associated inflammatory diseases of the skin. Nodules appear in the affected area, and they gradually become enlarged and rupture and release pus. This process occurs repeatedly, leading to sinus tract formation and scarring (Jemec, 2004). This disease progression creates a daunting situation for the patient as well as the physician. The reported point prevalence ranges from 1% to 4% (Jemec and others, 1996).

The exact pathophysiology of HS remains poorly defined. Smoking, dietary habits, and genetic predisposition are all associated with HS (Kurzen and others, 2008; Slade and others, 2003). The increased percentage of NK cells and decreased percentage of CD4-lymphocytes compared to healthy controls may imply an autoimmune tendency in this condition. IL-1β and IL-17 have been found to be upregulated in HS lesions and are associated with inflammasome activation (Lima and others, 2016). Hidradenitis suppurativa (HS) presents with massive neutrophil infiltration in inflamed skin, especially in advanced disease stages (Lima and others, 2016; Marzano, 2016). Activated neutrophils may be an important effector cell type, causing tissue damage through direct deleterious effects or indirect modulation of other effector cells such as activated T cells and TH17 in disease settings.

The past few years have created a hypothesis that some autoimmune or autoinflammatory mechanism is involved in the pathogenesis of HS. This hypothesis is strengthened by the positive results of the administration of TNF antagonists in the prospective placebo-controlled study, which led to the approval of adalimumab (an antibody against tumor necrosis factor alpha) for the treatment of moderate to severe disease. in patients with severe HS. An important but still unanswered question is how neutrophils are recruited to diseased skin lesions and to what extent activated neutrophils contribute to disease progression.

The possible wide range of pathological mechanisms proposed by different studies may imply that HS is related to host mechanisms rather than exogenous factors. Given the paradox that anti-infective (antibiotics) versus pro-infectious (anti-TNF, corticosteroids, immunosuppressive drugs) may be beneficial, the role of pro-inflammatory cytokines such as interleukin (IL)-1β HS appears to be an autoinflammatory disease based on a defect in the innate immunity of the hair follicle, supported by the fact that tumor necrosis factor-alpha (TNF-α) is markedly increased in both focal and perilesional skin (Wollina et al., 2013) diseases (Revuz, 2009).

Neutrophilic dermatoses

Neutrophilic dermatoses (ND) are a group of disorders characterized by histological examination of skin lesions that reveal an extremely inflamed infiltrate composed primarily of neutrophils with no evidence of overt infection. ND mainly includes Sweet syndrome (SS), pyoderma gangrenosum (PG), subcorneal pustular dermatosis (SPD), other defined entities and their atypical or transitional sexual forms (Prat and others, 2014). Hidradenitis suppurativa has recently been classified in the ND family because of the massive neutrophil infiltration observed in inflamed skin (Lima et al., 2016; Marzano, 2016).

Pyoderma gangrenosum (PG) and hidradenitis suppurativa (HS) are standard neutrophilic dermatoses thought to arise from autoinflammatory disorders characterized by accumulation of neutrophils in the skin (Braun- Falco and others, 2012; Marzano and others, 2014). Autoinflammatory syndromes represent an emerging group of inflammatory conditions distinct from autoimmunity, allergies, and infectious diseases. From a pathophysiological point of view, all autoinflammatory syndromes such as PAPA (septic arthritis, PG, and acne), PASH (PG, acne, and hidradenitis suppurativa) or PAPASH (septic arthritis, acne, and PG and hidradenitis suppurativa) share a mechanism consisting of an overactivated innate immune system and inflammation of "ineffective" neutrophil-rich skin (Cugno and others, 2017).

BADAS (Bowel-Associated Dermatosis-Arthritis Syndrome) is characterized by fever, flu-like symptoms, arthritis and inflammatory skin involvement. The latter is characterized by memory distinct neutrophilic dermatoses such as papules and plaques (Sweet's syndrome), pustules and ulcers (pyoderma pyoderma) or nodules, abscesses or fistulas (hidradenitis suppurativa). In addition, acne and neutrophilic panniculitis may also be associated. Patients typically present with symmetric, nonerosive polyarthritis, predominantly involving the facet joints (Cugno et al, 2018).

Synovitis, acne, impetigo, hyperosteogeny, and osteitis (SAPHO) syndrome was originally described in 1987. SAPHO syndrome is a rare condition that can be misdiagnosed. Although its pathogenesis remains elusive, SAPHO is increasingly recognized to share similarities with other autoinflammatory diseases (Cugno et al., 2018).

Neutrophils and autoimmune diseases

Autoimmune diseases are defined by defective discrimination between self and non-self molecules, resulting in inappropriate recognition of self molecules as well as tissues as foreign structures and simultaneous immune attack of host organs. The pathogenesis of autoimmune diseases can usually be divided into two phases, the immune phase and the effect phase. The immune phase is characterized by the appearance of autoreactive T lymphocytes. Those T cells in turn trigger subsequent stages that lead to tissue damage by activating various other cell types (B cells, cytotoxic T cells, NK cells, neutrophils, macrophages, osteoclasts, fibroblasts, etc.). Hairy reaction. Activation of those effector cells by autoreactive T cells can be viewed as the effector phase, which is mediated by multiple stages including autoantibody production, cytokine networks, or direct cell-cell contact (Nemeth and Mocsai, 2012 ).

The role of neutrophils in the development of the pathophysiology of autoimmune diseases has been poorly defined but gradually recognized. Neutrophils may be involved in multiple steps in the autoimmune disease process, including antigen presentation, regulation of the activity of other immune cell types, and direct tissue injury. Autoantigens can be exposed/released when neutrophils are activated or die due to apoptosis, or when neutrophil extracellular traps (neutrophil extracellular trap, NET) are formed. They may also contribute to the accumulation of autoantibodies in tissues, or as effector cell types that themselves can induce tissue damage. Cumulative studies have demonstrated the role of neutrophils in autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), bullous pemphigus, epidermolysis bullosa, ANCA -associated vasculitis, familial Mediterranean fever, cryopyrin-associated periodic disorder (CAPS) and gout, etc.) (Nemeth and Mocsai, 2012; Nemeth and others, 2016). Because the skin is a vulnerable target of the immune response, skin inflammation is one of the most common symptoms presented by these autoimmune diseases. However, rheumatoid neutrophilic dermatosis is a rare cutaneous manifestation in patients with severe rheumatoid arthritis. It mainly affects patients with severe seropositive rheumatoid arthritis, mainly women (ratio 2:1), but has also been observed in seronegative rheumatoid arthritis (Cugno et al., 2018).

Technical problem of the present invention

As explained above, there is a need in the prior art for effective therapies to treat neutrophil skin diseases such as hidradenitis suppurativa (HS) as well as neutrophil autoimmune diseases of the skin.

The inventors have now surprisingly discovered that molecules that inhibit C5a signaling, such as anti-C5a antibodies, are particularly useful in the treatment of hidradenitis suppurativa. The inventors have additionally investigated the physiological mechanisms leading to neutrophil activation and discovered that C5a is a key driver of neutrophil activation.

Therefore, the inventors anticipate that inhibition of C5a activity will be a suitable therapeutic approach for the treatment of various neutrophil disorders, particularly skin, neutrophil and inflammatory diseases.

Summary of the invention

In a first aspect, the present invention relates to a compound for use in the treatment of a skin, neutrophil, inflammatory disease in an individual, wherein the compound is an inhibitor of C5a activity, and wherein the skin, neutrophil Balloon, inflammatory disease is selected from the group consisting of: hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG, and acne); PASH (PG, acne, and acne hidradenitis); PAPASH (suppurative arthritis, acne, PG and hidradenitis suppurativa); Sweet syndrome (SS); subcorneal pustular dermatosis (SPD); acquired vesicular dermatosis Epidermolysis; Erythema Elevations Persistent (EED); Neutrophilic Stenomatitis; Bowel-Associated Dermatosis-Arthritis Syndrome (BADAS); SAPHO (synovitis, acne, impetigo, osteoarthritis) hyperplasia and osteitis) syndromes; rheumatoid neutrophilic dermatosis; familial Mediterranean fever, cold inflammatory factor-related disorders, gout, and Schnitzler syndrome.

In a second aspect, the invention relates to a method for treating a skin, neutrophil, inflammatory disease in an individual comprising the steps of: administering to an individual in need thereof a therapeutic amount of a compound, wherein The compound is an inhibitor of C5a activity, and wherein the skin, neutrophil, inflammatory disease is selected from the group consisting of: hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (purulent arthritis, PG, and acne); PASH (PG, acne, and hidradenitis suppurativa); PAPASH (arthritis suppurativa, acne, PG, and hidradenitis suppurativa); Schwaite's syndrome (SS); Epidermolysis bullosa (SPD); Erythematosus elongata (EED); Neutrophils; Bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovial rheumatoid neutrophilic dermatosis; familial Mediterranean fever, cold inflammatory factor-related disorders, gout, and Schnitzler syndrome.

In the third aspect, the present invention relates to the use of a compound for the preparation of a pharmaceutical composition for treating skin, neutrophil, and inflammatory diseases, wherein the compound is an inhibitor of C5a activity, and wherein the skin , neutrophils, inflammatory diseases are selected from the group consisting of: hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, Acne and hidradenitis suppurativa); PAPASH (suppurative arthritis, acne, PG, and hidradenitis suppurativa); Schwitters syndrome (SS); Subcorneal bullous dermatosis (SPD); Epidermolysis bullosa acquired persistent erythema elevated (EED); neutrophilic steatophyllitis; bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hyperosteogeny and osteoarthritis rheumatoid neutrophilic dermatosis; familial Mediterranean fever, cold inflammatory factor-related disorders, gout, and Schnitzler syndrome.

This summary of the invention does not necessarily describe all features of the invention. Other specific examples will become apparent upon review of the detailed description that follows.

Figure 1. Blocking activity of IFX-1 on recombinant human C5a (rhC5a)-induced upregulation of CD11b on blood neutrophils. IFX-1-004 and IFX-1-012 represent two different product batches. Human whole blood was incubated with buffer, antibody alone, rhC5a alone, or a combination of antibody and rhC5a at different concentrations. After incubation, cells were stained with anti-mouse CD11b:FITC and analyzed for CD11b MFI by flow cytometry. Results are expressed as mean ± SD. The percentage of IFX-1 blocking activity of C5a-induced CD11b expression is indicated (arrow). Statistical differences were calculated by one-way ANOVA, p values of p<0.05 were statistically significant.

Figure 2. Blocking activity of IFX-1 on endogenous human C5a (eC5a) driven upregulation of CD11b on neutrophils. Zymosan-activated human plasma (ZAP) was used as a source of eC5a. Whole blood was incubated with buffer, IFX-1 alone, ZAP alone or a combination of IFX-1 and ZAP. After incubation, cells were stained with anti-mouse CD11b:FITC and analyzed by flow cytometry. Results are expressed as mean ± SD. The percentage of IFX-1 blocking activity of C5a-induced CD11b expression is indicated (arrow). Statistical differences were calculated by one-way ANOVA, p values of p<0.05 were statistically significant.

Figure 3. Blood neutrophils due to zymosan activation and IFX-1 blocking activity. Whole blood was incubated with HBSS, rhCa5, and zymosan alone, or combinations of different concentrations of IFX-1 and rhC5a or zymosan A. After incubation, cells were stained with anti-mouse CD11b:FITC and analyzed for CD11b MFI by flow cytometry. Results are expressed as mean ± SD. The percentage of IFX-1 blocking activity of C5a-induced CD11b expression is indicated (arrow). Statistical differences were calculated by one-way ANOVA, p values of p<0.05 were statistically significant.

Figure 4. IFX-1 inhibits zymosan-induced IL-8 production in human whole blood. IL-8 concentrations were obtained by ELISA after incubation of human whole blood in the presence (open circles) or absence (closed circles) of IFX-1 with different concentrations of zymosan A as indicated on the x-axis. Results are expressed as mean ± SD.

Figure 5. Concentrations of C3a (A), C5a (B) and C5b-9 (C) in the plasma of 14 healthy controls and 54 hidradenitis suppurativa (HS) patients. Circles indicate outliers and asterisks indicate extreme values. The P value signifies a significant difference between patients and controls.

Figure 6. Effect of HS plasma on blood neutrophil activation and possible role of C5a. HS plasma samples were incubated with human whole blood in the presence and absence of IFX-1, and CD11b expression on blood neutrophils was determined by flow cytometric analysis. C5a levels in control and HS samples are marked in the embedded table.

50%，以及膿腫或排膿瘻管相較於基線並無增加。 Figure 7. HiSCR response after IFX-1 treatment in HS patients. HiSCR responders were defined as a reduction in the total number of inflammatory lesions (abscesses + inflamed nodules)

50%, and no increase from baseline in abscesses or draining fistulas.

Figure 8. Blocking of C5a-induced CD11b upregulation by different anti-C5aR antibodies. Whole blood as a source of neutrophils was incubated with (spiked) plasma samples in the absence or presence of the respective anti-C5aR antibodies. The blocking activity of each inhibitor is expressed as a percentage of the corresponding sample.

Figure 9. Blockade of C5a-induced CD11b upregulation by the C5aR antagonist PMX-53. Whole blood as a source of neutrophils was incubated with (spiked) plasma samples in the absence or presence of low (A) or overly concentrated (B) concentrations of the C5aR antagonist PMX-53. The blocking activity of PMX-53 is expressed as a percentage of the corresponding samples.

Figure 10. Blocking of C5a-induced CD11b upregulation by the C5a blocking antibody IFX-1. Whole blood as a source of neutrophils was incubated with (spiked) plasma samples in the absence or presence of IFX-1. The blocking activity of IFX-1 is expressed as a percentage of the corresponding samples.

Figure 11. Blockade of C5a-induced CD11b upregulation by the C5aR inhibitor Avacopan. Whole blood as a source of neutrophils was incubated with (spiked) plasma samples in the absence or presence of Avacopan. The blocking activity of Avacopan is expressed as a percentage of the corresponding samples.

definition

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as such may vary. It should also be understood that the terminology used herein is for the purpose of describing specific examples only, and is not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferably, the terms used herein are such as "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kölbl, H.eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Unless the context requires otherwise, throughout this specification and in subsequent claims, the word "comprise" and variations such as "comprises and comprising" should be understood to indicate that the stated integers or steps or Integers or groups of steps, but do not exclude any other integers or steps or groups of integers or steps.

Several documents are cited throughout the text of this specification (eg, patents, patent applications, scientific publications, manufacturer's instructions, operating instructions, GenBank accession number serial submissions, etc.). Nothing herein is considered an admission that the present invention should not be patentable by reason of earlier disclosure of prior invention. Some documents cited herein are characterized as "incorporated by reference." To the extent a definition or teaching of an incorporated reference conflicts with a definition or teaching recited in this specification, the text of this specification takes precedence.

Sequences: All sequences mentioned herein are disclosed in the accompanying Sequence Listing, and the entire content and disclosure of the sequences are part of this specification.

In the context of the present invention, C5a refers in particular to human C5a. Human C5a is a 74 amino acid peptide with the following amino acid sequence: TLQKKIEEIA AKYKHSVVKK CCYDGACVNN DETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGR (SEQ ID NO: 1)

The amino acid sequence of human C5 can be found under accession number UniProtKB P01031 (CO5_HUMAN).

As used herein, the term "inhibitor of C5a activity" means any compound that reduces the activity of C5a in any way. This reduction in activity can be achieved by directly or indirectly reducing the concentration of C5a, or by reducing the activity of C5a, or by preventing C5a from exhibiting its effects on one or more of its receptors (eg, on C5cR or C5L2), or by reducing one or more C5a Receptor concentration or activity is achieved.

In the context of the present invention, the term "C5a receptor" means any possible C5a-binding ligand on the cell surface, in particular any receptor to which C5a can bind and elicit a response (e.g. activating or inhibiting the receptor) at the receptor protein. The term "C5a receptor" specifically includes two receptors, C5aR and C5L2. The aliases of C5aR are C5aR1 and CD88. The alias of C5L2 is C5aR2.

Certain embodiments of the invention relate to inhibitors of C5a that interfere with the C5a receptor (eg, by binding to the C5a receptor, or by blocking expression of the C5a receptor). In these cases, the term "C5a receptor" may refer to (i) C5aR or (ii) C5L2 or (iii) both C5aR and C5L2. This means that some C5a inhibitors interfere with only one of the C5a receptors (ie C5aR or C5L2), while other inhibitors of C5a interfere with the C5a receptor (ie both C5aR and C5L2).

100個胺基酸)以及多肽(>100個胺基酸)。用語「蛋白配體」也包含環狀肽，不論其大小為何。用語「蛋白配體」尤其含括抗體、抗體的抗原結合片段、抗體樣蛋白以及擬肽物。 In the context of the present invention, the term "protein ligand" means any molecule consisting of amino acids linked by peptide bonds and capable of specifically binding to another molecule, regardless of the overall size of the molecule. Thus, the term "protein ligand" includes oligopeptides (

100 amino acids) and peptides (>100 amino acids). The term "protein ligand" also encompasses cyclic peptides, regardless of their size. The term "protein ligand" includes antibodies, antigen-binding fragments of antibodies, antibody-like proteins and peptidomimetics, among others.

As used herein, a first compound is considered to "bind" to a second compound if the first compound (e.g., a protein ligand or nucleic acid aptamer) has the following dissociation constant _Kd for the second compound (e.g., a protein of interest): 1 mM or Less, preferably 100 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less , more preferably 1 μM or less, better 900 nM or less, better 800 nM or less, better 700 nM or less, better 600 nM or less, better 500 nM or less, more Preferably 400nM or less, more preferably 300nM or less, more preferably 200nM or less, yet more preferably 100nM or less, yet more preferably 90nM or less, yet more preferably 80nM or less, Still more preferably 70nM or less, yet more preferably 60nM or less, yet more preferably 50nM or less, yet more preferably 40nM or less, yet more preferably 30nM or less, yet more preferably 20nM or less, and more preferably 10 nM or less.

The term "binding" according to the invention preferably relates to a specific binding. "Specific binding" means that a compound (eg, protein ligand or nucleic acid aptamer) binds more strongly to one target (such as an epitope specific for it) than to another target. If a compound binds to a first target with a dissociation constant (K _d ) that is lower than the dissociation constant for a second target, then the compound binds more strongly to the first target than to the second target. strong. Preferably, the compound specifically binds to the target with a dissociation constant (K _{d )} that is more than 10 times, preferably more than 20 times, more preferably more than 50 times, and more preferably more than 100 times, 200 times, 500 times or 1000 times.

As used herein, the term " _Kd " (usually measured in "mol/L", sometimes abbreviated "M") is intended to mean the dissociation equilibrium constant for a particular interaction between a compound (eg, a protein ligand) and a molecule of interest.

Methods for determining the binding affinity of a compound (for example for determining the dissociation constant K _d ) are known to those skilled in the art and can be selected from, for example, the following methods known in the art: based on surface plasmon resonance (SPR) Biolayer Interferometry (BLI), Enzyme-Linked Immunosorbent Assay (ELISA), Flow Cytometry, Isothermal Titration Calorimetry (ITC), Analytical Ultracentrifugation, Radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL). Usually the dissociation constant K _d is determined at 20°C, 25°C, 30°C or 37°C. If not otherwise specified, the _Kd values listed herein are determined by ELISA at 20°C.

An "epitope", also known as an antigenic determinant, is a part of a macromolecule that is recognized by the immune system, especially antibodies, B cells or T cells. As used herein, an "epitope" is a portion of a macromolecule capable of binding to a compound as described herein (eg, an antibody or antigen-binding fragment thereof). In the context, the term "binding" preferably relates to specific binding. Epitopes usually consist of chemically active surface clustered molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational epitopes are distinguished from non-conformational epitopes in that the former lose binding in the presence of denaturing solvents, but the latter do not.

A "paratope" is the part of an antibody that binds to an epitope. In the context of the present invention, a "paratope" is a part of a compound (eg, a protein ligand) that binds to an epitope as described herein.

The term "antibody" generally means a glycoprotein or an antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" also includes all recombinant forms of antibodies, especially recombinant forms of the antibodies described herein, e.g. antibodies expressed in prokaryotes, aglycosylated antibodies, antibodies expressed in eukaryotes (e.g. CHO cells), Glycosylated antibodies and any antigen-binding antibody fragments and derivatives as described below. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH or _VH ) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as VL or _VL ) and a light chain constant region. The VH and VL regions can be further divided into regions of hypervariability, named complementarity determining regions (CDRs), interspersed with more conserved regions, named framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first complement (Clq) of the classical complement system.

The term "antigen-binding fragment" of an antibody (or simply "binding portion") as used herein means one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH domains; (ii) F(ab ' ) ₂ fragments, A bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hub; (iii) Fd fragment, consisting of VH and CH domains; (iv) Fv fragment, consisting of VL and VH of a single arm of the antibody domain composition; (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of VH domains; (vi) isolated complementarity determining regions (CDRs); and (vii) Consists of two or more isolated CDRs, optionally joined by a synthetic linker. In addition, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, they can be joined together through a synthetic linker using recombinant technology, allowing them to be produced as a single protein chain. True, where the VL and VH regions are paired to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Yet another example is a binding domain immunoglobulin fusion protein comprising: (i) a binding domain polypeptide fused to an immunoglobulin hub polypeptide; (ii) an immunoglobulin heavy chain CH2 constant region fused to a hub; and ( iii) An immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments can be obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as whole antibodies. Further examples of "antigen-binding fragments" are so-called microantibodies, which are derived from a single CDR. For example, Heap et al., 2005 describe a minibody with 17 amino acid residues derived from the heavy chain CDR3 of an antibody directed against the gp120 outer membrane glycoprotein of HIV-1 (Heap CJ et al. (2005) Analysis of a 17-amino acid residue, virus-neutralizing microantibody. J. Gen. Virol. 86: 1791-1800). Other examples include small antibody mimetics comprising two or more CDR regions fused to each other (preferably through homologous framework regions). Such a small antibody mimetic comprising V _H CDR1 and V _L CDR3 linked by a cognate V _H FR2 was described by Qiu et al., 2007 (Qiu X.-Q. et al. (2007) Small antibody mimetics comprising two complementary-determining regions and a framework region for tumor targeting. Nature biotechnology 25(8):921-929).

Accordingly, the term "antibody or antigen-binding fragment thereof" as used herein refers to immunoglobulin molecules as well as immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds an antigen. Also encompassed are immunoglobulin-like proteins that are screened for specific binding to a target molecule or target epitope by techniques including, for example, phage display. The immunoglobulin molecules of the invention may be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. IgG1, IgG2, preferably IgG2a and IgG2b, IgG3, IgG4, IgA1 and IgA2) or subclass. class of immunoglobulin molecules.

Antibodies and antigen-binding fragments thereof useful in the present invention may be from any animal source, including birds as well as mammals. Preferably, the antibody or fragment is from a human, chimpanzee, rodent (mouse, rat, guinea pig, or rabbit), chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog. source. It is especially preferred that the antibodies are of human or murine origin. Antibodies of the invention also include chimeric molecules in which an antibody constant region derived from one species (preferably human) is combined with an antigen binding site derived from another species (eg mouse). Furthermore, antibodies of the invention include humanized molecules in which the antibody combining site derived from an antibody of a non-human species (eg, from a mouse) is combined with constant and framework regions of human origin.

As exemplified herein, antibodies of the invention can be obtained directly from antibody-expressing fusionomas, or can be propagated and expressed recombinantly in host cells such as CHO cells or lymphocytes. Further examples of host cells are microorganisms such as E. coli and fungi such as yeast. Alternatively, they are produced recombinantly in transgenic non-human animals or plants.

The term "chimeric antibody" means that a portion of the amino acid sequences of each of the heavy and light chains is homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular class, while maintaining segments of the chains with the other The corresponding sequences of a species or genus are homologous. Typically the variable regions of the light and heavy chains mimic those of antibodies derived from one mammalian species, while the constant portions are homologous to antibody sequences derived from the other. A definite advantage of such chimeric forms is that the variable regions can be conveniently used from presently known sources in combination with constant regions derived from, for example, human cell preparations, using B cells or hybridomas from non-human host organisms. Although the variable region has the advantages of being easy to prepare and that the specificity is not affected by the source, the human constant region is less capable of eliciting an immune response in a human individual than a non-human source constant region when injecting antibodies. However, this definition does not limit this particular example.

The term "humanized antibody" means a molecule having an antigen-binding site that is substantially derived from an immunoglobulin of a non-human species, wherein the remainder of the molecule's immunoglobulin structure is that of a human immunoglobulin and/or sequence based. Antigen binding sites may comprise entire variable domains fused to constant domains, or simply the complementarity determining regions (CDRs) grafted onto appropriate framework regions within the variable domains. The antigen binding site can be wild-type or modified by one or more amino acid substitutions, eg, to closely resemble human immunoglobulin. Some forms of humanized antibodies retain all CDR sequences (eg, a humanized mouse antibody that contains all six CDRs of a mouse antibody). Other forms have one or more CDRs that are altered from the original antibody.

Different methods for humanizing antibodies are known to those skilled in the art, as reviewed by Almagro & Fransson, 2008, Frontiers in Bioscience, 13: 1619-1633, the contents of which are hereby incorporated by reference in their entirety. Almagro & Fransson's review article is briefly summarized in US 2012/0231008 A1, which is the national phase entry of international patent application WO 2011/063980 A1. The contents of US 2012/0231008 A1 and WO 2011/063980 A1 are incorporated herein by reference in their entirety.

As used herein, "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or mutations introduced by somatic mutation in vivo). Human antibodies of the present invention include antibodies isolated from the human immunoglobulin repertoire or from animals that have been transferred with one or more human immunoglobulin genes and do not express endogenous immunoglobulins, as described, for example, by Kucherlapati & Jakobovits in U.S. Patent No. 5,939,598.

The term "monoclonal antibody" as used herein means a preparation of antibody molecules having a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. In one embodiment, the monoclonal antibody is produced by a fusion tumor comprising B cells obtained from a non-human animal (eg, mouse) fused with an immortal cell.

The term "recombinant antibody" as used herein includes all antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) from an animal (e.g., a mouse) that has been transgenic or transchromosomal with respect to an immunoglobulin gene or from Antibodies isolated from fusionomas produced therefrom; (b) antibodies isolated from host cells transformed to express antibodies (e.g., from transfectomas); (c) antibodies isolated from recombinant, combinatorial antibody libraries; and (d) Antibodies prepared, expressed, created or isolated by any other method involving the splicing of immunoglobulin gene sequences to other DNA sequences.

The term "transfectoma" as used herein includes recombinant eukaryotic host cells expressing antibodies, such as CHO cells, NS/0 cells, HEK293 cells, HEK293T cells, plant cells, or fungi (including yeast cells).

As used herein, "heterologous antibody" is defined relative to the GMO producing such antibody. The term means an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism other than the GMO, and usually derived from a species other than the GMO.

As used herein, "heterohybrid antibody" means an antibody having light and heavy chains from different organisms. For example, an antibody having human heavy chains associated with murine light chains is a heterohybrid antibody.

Therefore, "antibodies and antigen-binding fragments thereof" applicable to the present invention include, but are not limited to polyclonal, monoclonal, monovalent, bispecific, heterotypic binding, multispecific, recombinant, heterologous, heterologous hybrid, chimeric Synthetic, humanized (especially CDR grafted), deimmunized or human antibodies, Fab fragments, Fab' fragments, F(ab') ₂ fragments, fragments made from Fab expression libraries, Fd, Fv, disulfide linkage Fvs (dsFv), single chain antibody (such as scFv), bifunctional antibody or tetrafunctional antibody (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA90 (14), 6444-6448), Natl Antibodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies described herein), and epitope-binding fragments of any of the foregoing.

The antibodies described herein are preferably isolated. "Isolated antibody" as used herein is intended to mean an antibody that is substantially free of other antibodies having a different antigen specificity (e.g. an isolated antibody that specifically binds to C5a is substantially free of antibodies that specifically bind to an antigen other than C5a ). However, isolated antibodies that bind to epitopes, isoforms or variants of human C5a may have cross-reactivity to other related antigens (eg from other species, eg C5a species homologues such as rat C5a). Furthermore, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies relates to antibodies with different specificities combined in a defined composition.

The term "natural" as used herein, when applied to an object, means that the object can be found in nature. For example, a polypeptide or polynucleotide sequence is native to an organism, including a virus, that can be isolated from a source in nature and has not been artificially modified in the laboratory.

As used herein, the term "aptamer" means a nucleic acid molecule that has been engineered to bind to a target molecule through repeated cycles of in vitro screening or SELEX (systematic evolution of ligands by exponential enrichment) (for a review see: Brody EN and Gold L. (2000), Aptamers as therapeutic and diagnostic agents. J. Biotechnol. 74(1):5-13). Aptamers can be DNA or RNA molecules. Aptamers may contain modifications, such as modified nucleotides (such as 2'-fluoro substituted pyrimidines), and/or may comprise one or more standard D with L-ribose units (or L-deoxyribose) substituted - Nucleotides of ribose units (or D-deoxyribose units).

As used herein, the term "antibody-like protein" means a protein that has been engineered (eg, by mutagenesis of a loop) to specifically bind to a target molecule. Usually such an antibody-like protein contains at least one variable peptide loop attached to both ends of the protein framework. This dual structure substantially increases the binding affinity of antibody-like proteins to a degree comparable to that of antibodies. The length of the variable peptide loop usually consists of 10 to 20 amino acids. The architectural protein can be any protein with good solubility. Preferably, the scaffold protein is a small globulin. Antibody-like proteins include, but are not limited to, affibodies, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin (designed ankyrin repeat protein), fynomer, Kunitz domain peptide (Kunitz domain peptide) and monobody (see for review: Binz HK et al. (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23(10): 1257-1268). Antibody-like proteins can be derived from large mutant libraries, for example panning from large phage display libraries, and can be isolated in a manner similar to conventional antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed residues of globulins. Antibody-like proteins are sometimes called "peptide aptamers" or "antibody mimics".

As used herein, a "peptidomimetic" is a small protein-like chain designed to mimic a peptide. Peptidomimetics are generally derived from the modification of existing peptides to alter molecular properties. For example, they may result from modifications that alter the stability or biological activity of the molecule. This could come into play when developing drug-like compounds from existing peptides. These modifications involve changes to the peptide that do not occur naturally (such as changing the backbone and incorporating unnatural amino acids).

In the context of the present invention, the term "small molecule" means a molecule having a molecular weight of 2 kDa or less, preferably a molecule of 1 kDa or less. The term "small molecule" especially means a molecule which is neither an oligopeptide nor an oligonucleotide.

In the context of the present invention, the general phrase "wherein A competes with B for binding to C" (eg in the phrase "wherein the antibody or antigen-binding fragment thereof competes with one of the antibodies referred to in (a) for binding to C5a") is used Define the binding properties of the compounds listed in position A. The compound A binds to C and the compound also binds to C, but compound A and compound B cannot bind to C at the same time; that is, A and B bind to the same epitope on C (or at least bind to overlapping epitopes). Such binding competition can be determined by competition ELISA or by surface plasmon resonance (SPR) based techniques or by any of the other techniques listed above under Determining binding affinity. If not stated otherwise, the competitive binding properties of compounds were determined by ELISA at 20°C using equimolar concentrations of two competing compounds.

As used herein, "skin, neutrophil, inflammatory disease" means any disease that is associated with skin inflammation and infiltration of neutrophils into the skin (eg, into the epidermis) of a subject afflicted with the disease. The term "skin, neutrophil, inflammatory disease" means especially hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, Acne and hidradenitis suppurativa); PAPASH (suppurative arthritis, acne, PG, and hidradenitis suppurativa); Schwitters syndrome (SS); Subcorneal bullous dermatosis (SPD); Epidermolysis bullosa acquired Erythema elevated (EED); Neutrophil panniculitis; Neutrophil panniculitis; Bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne , impetigo, hyperosteogeny, and osteitis) syndromes; rheumatoid neutrophilic dermatosis; familial Mediterranean fever, cold inflammatory factor-related disorders, gout, and Schnitzler syndrome.

As used herein, the term "HS-associated disease" includes, but is not limited to, pyoderma gangrenosum (PG); PAPA (septic arthritis, PG, and acne); PASH (PG, acne, and hidradenitis suppurativa); PAPASH ( Suppurative Arthritis, Acne, PG, and Hidradenitis Suppurativa); Schwaite's Syndrome (SS); and Subcorneal Pustular Dermatosis (SPD).

IFX-1 (alias CaCP29; InflaRx GmbH, Germany) is an antibody that specifically binds to C5a. The CDR sequence and FR sequence of IFX-1 are disclosed in WO 2015/140304 A1 (in Table 3 on page 31), the content of which is incorporated by reference in its entirety.

INab708 (InflaRx GmbH, Germany) is another antibody that specifically binds to C5a. The CDR sequence and FR sequence of INab708 are disclosed in WO 2015/140304 A1 (Table 3), the content of which is incorporated by reference in its entirety.

MEDI-7814 (MedImmune) is a recombinant humanized anti-C5a antibody. The crystal structure of human C5a in complex with MEDI7814 can be found in the RCSB Protein Data Bank according to 4UU9 (DOI: 10.2210/pdb4uu9/pdb).

ALXN-1007 (Alexion) is a humanized anti-C5a antibody.

NOX-D21 (Noxxon) is a PEGylated mixed L-RNA/DNA-aptamer (Spiegelmer ^TM ) with a sequence of 40kDaPEG-aminohexyl-GCG AUG(dU)GG UGG UGA AGG GUU GUU GGG(dU) GU CGA CGC A(dC)GC (SEQ ID NO: 2). NOX-D21 targets C5a (Hyzewicz J, Tanihata J, Kuraoka M, Nitahara-Kasahara Y, Beylier T, Ruegg UT, Vater A, and Takeda S. 2017. Low-Intensity Training and the C5a Complement Antagonist NOX-D21 Rescue the mdx Phenotype through Modulation of Inflammation. Am. J. Pathol., 187(5): 1147-1161; electronically published ahead of print: March 18, 2017).

Eculizumab (alias: Soliris ^TM , 5G1-1; h5G1.1; Alexion Pharmaceuticals) is a recombinant humanized monoclonal IgG2/4κ antibody produced by mouse myeloma cell culture and passed through standard Bioprocess technology to be purified. Eculizumab specifically binds to human C5. Eculizumab contains human constant regions derived from human IgG2 sequences and human IgG4 sequences, and murine complementarity determining regions grafted onto the human framework light and heavy chain variable regions. Eculizumab is composed of two heavy chains of 448 amino acids and two light chains of 214 amino acids, and has a molecular weight of approximately 148 kDa. The heavy chain and light chain of eculizumab are disclosed in, for example, WO 2016/061066 A1 as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Nucleic acids encoding the heavy and light chains of eculizumab are disclosed, eg, in US Patent No. 6,355,245.

ALXN1210 (alias: BNJ441; Alexion Pharmaceuticals) is an anti-C5 antibody. The heavy chain and light chain of ALXN1210 are disclosed in WO 2016/209956 A1 as SEQ ID NO: 14 and 11, respectively.

ALXN5500 (Alexion) is a humanized anti-C5 antibody. It is a next-generation eculizumab candidate.

LFG316 (alias: Tesidolumab, NOV-4; Morphosys, Novartis) is an anti-C5 antibody.

Coversin ^TM (alias: EV 576; PAS-coversin; rEV 576; tissue-targeted Coversin ^TM -Akari; Akari Therapeutics, Evolutec ) is a recombinant protein molecule (16.7 kDa) derived from the Salivary molecules that assist the parasite to feed without provoking a host immune response. The amino acid sequence of the EV576 protein (ie Coversin) and its encoding nucleotide sequence are shown in Figure 2 of WO 2008/029167. Coversin ^™ binds to C5.

R-A101495 (Ra Pharma) is a macrocyclic synthetic peptide inhibitor of C5 (Ricardo A, Arata M, DeMarco S, Dhamnaskar K, Hammer R, Fridkis-Hareli M, Rajagopal V, Seyb K, Tang GQ, Tobe S and Treco D. 2015. Preclinical Evaluation of RA101495, a Potent Cyclic Peptide Inhibitor of C5 for the Treatment of Paroxysmal Nocturnal Hemoglobinuria. Blood 126:939).

Zimura® (alias: anti-C5 aptamer; ARC-187; ARC-1905; Avacincaptad pegol sodium; OphthoTech Corporation, Archemix Corporation) is a pegylated RNA aptamer that inhibits complement factor C5. The nucleotide sequence of ARC1905 (ie Zimura) is shown eg in WO 2005/079363 A2 as SEQ ID NO: 67 and its structure is shown in Figure 22 of WO 2005/079363 A2.

AMY-201 (Amyndas Pharmaceuticals) is an engineered form of Factor H that directly links regulatory and surface recognition domains; thus, it is a class of mini-FH molecules.

Mirococept (alias: APT070 and APT 070C; Originator: Adprotech; Developer: Inflazyme Pharmaceuticals) consists of the first three contiguous short domains of human complement receptor 1, manufactured in recombinant bacteria and based on native membrane-linked myristica Acyl-electrostatic switching peptides modified by membrane-targeting amphiphilic peptides (Souza DG, Esser D, Bradford R, Vieira AT, and Teixeira MM. 2005. APT070 (Mirococept), a membrane-localissd complement inhibitor, inhibits inflammatory responses that follow Intestinal ischaemia and reperfusion injury. Br J Pharmacol 145(8):1027-1034).

BikacioMab (Novelmed) is an F(ab) ₂ fragment of an anti-factor Bb antibody (named NM001). Antibody NM001 was produced by the fusionoma cell line 1D3 deposited under ATCC accession number PTA-8543.

Lampalizumab (alias: anti-factor D Fab; FCFD4514S; RG7417; TNX-234; originator: Tanox, developer: Genentech) is a humanized anti-factor D Fab fragment, which binds to To the exosite of Factor D to inhibit Factor D and the alternative complement pathway.

ALN-CC5 (Alnylam) is an RNAi therapeutic targeting C5 in humans, primates, and rodents. Exemplary iRNA compositions targeting the C5 gene are described in WO 2016/044419.

Avacopan的IUPAC/化學名為(2R,3S)-2-[4-(環戊基胺基)苯基]-1-(2-氟-6-甲基苯甲醯基)-N-[4-甲基-3-(三氟甲基)苯基]哌啶-3-甲醯胺。Avacopan是C5aR的一個選擇性抑制劑。在本發明內文中，術語「avacopan」意指如式I的化合物還有其生理學上可耐受之鹽。 Avacopan (known aka CCX168; Chemocentryx) is a small molecule (MW=581.66 g/mol) which has the structure of formula I:

The IUPAC/chemical name of Avacopan is (2R,3S)-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methylbenzoyl)-N-[4 -methyl-3-(trifluoromethyl)phenyl]piperidine-3-carboxamide. Avacopan is a selective inhibitor of C5aR. In the context of the present invention, the term "avacopan" means a compound according to formula I also its physiologically tolerable salts.

及其醫藥上可接受鹽、水和物及構象異構物(rotomers)；其中C¹選自由芳基及雜芳基組成之群組，其中雜芳基具有1-3個選自N、O及S之雜原子作為環成員；且該芳基及雜芳基視情況經1至3個R¹取代基取代；C²選自由芳基及雜芳基組成之群組，其中雜芳基具有1-3個選自N、O及S之雜原子作為環成員；且該芳基及雜芳基視情況經1至3個R²取代基取代；C³選自由下列組成之群組：C_1-8烷基或雜烷基、C_3-8環烷基、C_3-8環烷基-C_1-4烷基、芳基、芳基-C_1-4烷基、雜芳基、雜芳基-C_1-4烷基、雜環烷基或雜環烷基-C_1-4烷基，其中該雜環烷基或部分具有1-3個選自N、O及S之雜原子，其中該雜芳基具有1-3個選自N、O及S之雜原子作為環成員，且各C³視情況經1-3個R³取代基取代；各R¹獨立選自由下列組成之群組：鹵素、-CN、-R^c、-CO₂R^a、-CONR^aR^b、-C(O)R^a、-OC(O)NR^aR^b、-NR^bC(O)R^a、-NR^bC(O)₂R^c、-NR^a-C(O)NR^aR^b、-NR^aC(O)NR^aR^b、-NR^aR^b、-OR^a及-S(O)₂NR^aR^b；其中各R^a及R^b獨立選自由下列組成之群組：氫、C_1-8烷基及C_1-8鹵烷基，或當連接至相同氮原子時，可與氮原子合併形成5或6-員環，其另外具有0至2個選自N、O或S之雜原子作為環成員，且視情況經一或二個側氧基取代；各R^c獨立選自由下列組成之群組：C_1-8烷基或雜烷基、C_1-8鹵烷基、C_3-6環烷基、雜環烷基、芳基及雜芳基，及其中R^a、R^b及R^c之脂族及環狀部分視情況又經一至三個鹵素、羥基、甲基、胺基、烷胺基及二烷胺基取代；及當二個R¹取代基在相鄰原子上時，視情況合併形成稠合5或6-員碳環或雜環；各R²獨立選自由下列組成之群組：鹵素、-CN、-NO₂、-R^f、-CO₂R^d,-CONR^dR^e、-C(O)R^d、-OC(O)NR^dR^e、-NR^eC(O)R^d、-NR^eC(O)₂R^f、-NR^dC(O)NR^dR^e、-NR^dC(O)NR^dR^e、-NR^dR^e、-OR^d及-S(O)₂NR^dR^e；其中各R^d及R^e獨立選自由下列組成之群組：氫、C_1-8烷基及C_1-8鹵烷基，或當連接至相同氮原子時，可與該氮原子合併形成5或6-員環，其具有另外0至2個選自N、O或S之雜原子作為環成員，及視情況經一或二個側氧基取代；各R獨立選自由下列組成之群組：C_1-8烷基或雜烷基、C_1-8鹵烷基、C_3-6環烷基、雜環烷基、芳基及雜芳基，及當R^d、R^e及R^f之脂族及環狀部分視情況又經一至三個鹵素、羥基、甲基、胺基、烷胺基及二烷胺基取代，及視情況當二個R²基位於相鄰原子上時，彼等可合併形成5-或6-員環；各R³獨立選自由下列組成之群組：鹵素、-CN、-Rⁱ、-CO₂R^g、-CONR^gR^h、-C(O)R^g、-C(O)Rⁱ、-OC(O)NR^gR^h、-NRhC(O)R^g、-NR^hCO₂Rⁱ、-NR^gC(O)NR^gR^h、-NR^gR^h、-OR^g、-OR^j、-S(O)₂NR^gR^h、-X⁴-R^j、-NH-X⁴-R^j、-O-X⁴-R^j、-X⁴-NR^gR^h、-X⁴-NHR^j、-X⁴-CONR^gR^h、-X⁴-NR^hC(O)R^g、-X⁴-CO₂R^g、-O-X⁴-CO₂R^g、NH-X⁴-CO₂R^g、-X⁴-NR^hCO₂Rⁱ、-O-X⁴-NR^hCO₂Rⁱ、-NHR^j及-NHCH₂R^j，其中X⁴為C_1-4伸烷基；各R^g及R^h獨立選自氫、C_1-8烷基或雜烷基、C_3-6環烷基及C_1-8鹵烷基，或當連接至相同氮原子時，可與該氮原 子合併形成4-、5-或6-員環，其另外具有0至2個選自N、O或S之雜原子作為環成員及視情況經一或二個側氧基取代；各Rⁱ獨立選自由下列組成之群組：C_1-8烷基或雜烷基、C_1-8鹵烷基、C_3-6環烷基、雜環烷基、芳基及雜芳基；及各R^j選自由下列組成之群組：C_3-6環烷基、咪唑基、嘧啶基、吡咯啉基、哌啶基、嗎福啉基、四氫呋喃基、四氫哌喃基及S,S-二側氧基-四氫硫代哌喃基，及其中R^g、R^h、Rⁱ及R^j之脂族及環狀部分視情況又經一至三個鹵素、甲基、CF₃、羥基、C_1-4烷氧基、C_1-4烷氧基-C_1-4烷基、-C(O)O-C_1-8烷基、胺基、烷胺基及二烷胺基取代，及視情況當二個R³基未於相鄰原子上時，彼等可合併形成5-或6-員環；及X為氫或CH₃。 Compounds similar to Avacopan which are also suitable for realizing the present invention are disclosed in international patent applications WO 2010/075257 A1 and WO 2011/163640 A1, the contents of which are hereby incorporated by reference in their entirety. Therefore, in some embodiments, the inhibitor of C5a activity is a compound of formula II

And its pharmaceutically acceptable salt, water and product and conformational isomers (rotomers); wherein C ¹ is selected from the group consisting of aryl and heteroaryl, wherein heteroaryl has 1-3 selected from N, O and a heteroatom of S as a ring member; and the aryl and heteroaryl are optionally substituted by ¹ to ³ R substituents; C is selected from the group consisting of aryl and heteroaryl, wherein the heteroaryl has 1-3 heteroatoms selected from N, O and S as ring members; and the aryl and heteroaryl are optionally substituted by 1 to ^{3 R} substituents; C is selected from the group consisting of: C _1-8 alkyl or heteroalkyl, C _3-8 cycloalkyl, C _3-8 cycloalkyl-C _1-4 alkyl, aryl, aryl-C _1-4 alkyl, heteroaryl, Heteroaryl-C _1-4 alkyl, heterocycloalkyl or heterocycloalkyl-C _1-4 alkyl, wherein the heterocycloalkyl or part has 1-3 heterocycloalkyl selected from N, O and S atom, wherein the heteroaryl has 1-3 heteroatoms selected from N, O, and S as ring members, and each C3 is optionally substituted by ^{1-3 R3} substituents ^; each R1 is independently selected from the following Composition group: halogen, -CN, -R ^c , -CO ₂ R ^a , -CONR ^a R ^b , -C(O)R ^a , -OC(O)NR ^a R ^b , -NR ^b C(O )R ^a , -NR ^b C(O) ₂ R ^c , -NR ^a -C(O)NR ^a R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a R ^b , -OR ^a and -S(O) ₂ NR ^a R ^b ; wherein each R ^a and R ^b is independently selected from the group consisting of hydrogen, C _1-8 alkyl and C _1-8 haloalkyl, or when attached to the same nitrogen Atoms may be combined with a nitrogen atom to form a 5- or 6-membered ring, which additionally has 0 to 2 heteroatoms selected from N, O or S as ring members, optionally substituted by one or two pendant oxy groups; Each R is independently selected from the group consisting of ^C _1-8 alkyl or heteroalkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl , and wherein the aliphatic and cyclic moieties of R ^a , R ^b and R ^c are optionally substituted by one to three halogen, hydroxyl, methyl, amine, alkylamino and dialkylamino groups; and when two R ¹ When the substituents are on adjacent atoms, they are optionally combined to form a condensed 5- or 6-membered carbocyclic or heterocyclic ring; each R ² is independently selected from the group consisting of: halogen, -CN, -NO ₂ , -R ^f 、-CO ₂ R ^d ,-CONR ^d R ^e ,-C(O)R ^d ,-OC(O)NR ^d R ^e ,-NR ^e C(O)R ^d ,-NR ^e C(O) ₂ R ^f , -NR ^d C(O)NR ^d R ^e , -NR ^d C(O)NR ^d R ^e , -NR ^d R ^e , -OR ^d , and -S(O) ₂ NR ^d R ^e ; ^Wherein each R and R ^are independently selected from the group consisting of hydrogen, C _1-8 alkyl and C _1-8 haloalkyl, or when connected to the same nitrogen atom, may combine with the nitrogen atom to form 5 or a 6-membered ring having an additional 0 to 2 heteroatoms selected from N, O or S as ring members, and optionally substituted with one or two pendant oxy groups; each R is independently selected from the group consisting of : C _1-8 alkyl or heteroalkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and when R ^d , R ^e and R ^f The aliphatic and cyclic moieties are optionally substituted by one to three halogen, hydroxyl, methyl, amino, alkylamino and dialkylamino groups, and optionally when ^two R groups are located on adjacent atoms, They may combine to form a 5- or 6-membered ring; each R ³ is independently selected from the group consisting of: halogen, -CN, -R ⁱ , -CO ₂ R ^g , -CONR ^g R ^h , -C(O )R ^g , -C(O)R ⁱ , -OC(O)NR ^g R ^h , -NRhC(O)R ^g , -NR ^h CO ₂ R ⁱ , -NR ^g C(O)NR ^g R ^h , -NR ^g R ^h , -OR ^g , -OR ^j , -S(O) ₂ NR ^g R ^h , -X ⁴ -R ^j , -NH-X ⁴ -R ^j , -OX ⁴ -R ^j , -X ⁴ -NR ^g R ^h , -X ⁴ -NHR ^j , -X ⁴ -CONR ^g R ^h , -X ⁴ -NR ^h C(O)R ^g , -X ⁴ -CO ₂ R ^g , -OX ⁴ -CO ₂ R ^g , NH-X ⁴ -CO ₂ R ^g , -X ⁴ -NR ^h CO ₂ R ⁱ , -OX ⁴ -NR ^h CO ₂ R ⁱ , -NHR ^j and -NHCH ₂ R ^j , wherein X ⁴ is C _1-4 alkylene; each R ^g and R ^h are independently selected from hydrogen, C _1-8 alkyl or heteroalkyl, C _3-6 cycloalkyl and C _1-8 haloalkyl, or when connected to When the nitrogen atom is the same, it can be combined with the nitrogen atom to form a 4-, 5- or 6-membered ring, which additionally has 0 to 2 heteroatoms selected from N, O or S as ring members and optionally one or two Each R ^is independently selected from the group consisting of C _1-8 alkyl or heteroalkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, heterocycloalkyl, ^Aryl and heteroaryl; and each R is selected from the group consisting of C _cycloalkyl , imidazolyl, pyrimidinyl, pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydrofuranyl, Hydropyranyl and S,S-dioxo-tetrahydrothiopyranyl, and wherein the aliphatic and cyclic moieties of R ^g , R ^h , R ⁱ and R ^j are optionally modified by one to three halogens , methyl, CF ₃ , hydroxyl, C _1-4 alkoxy, C _1-4 alkoxy-C _1-4 Alkyl, -C(O)OC _1-8 alkyl, amino, alkylamino and dialkylamino are substituted, and as the case may be, when two ^R groups are not on adjacent atoms, they may combine to form 5- or 6-membered ring; and X is hydrogen or _CH3 .

Compounds similar to Avacopan but with an improved solubility profile are disclosed in WO 2017/176620 A2, the contents of which are hereby incorporated by reference in their entirety. Thus, in some other embodiments, inhibitors of C5a activity are compounds of formula III:

or a pharmaceutically acceptable salt thereof, wherein: R ¹ is selected from the group consisting of H, -O-CH ₂ -OP(O)OR ^a OR ^b , -OC(O)-C _1-6 alkylene -L ² -X ¹ , OP(O)OR ^a OR ^b and -OC(O)-A ¹ -(C _1-3 alkylene)nC _4-7 heteroalkyl, wherein the C _4-7 heteroalkane The group is optionally substituted by ¹ to 6 R groups; A is selected from the group consisting of ^C _6-10 aryl, C _3-10 cycloalkyl, C _5-10 heteroaryl and C _5-10 Heteroalkyl, each of which ^is optionally substituted with 1 to 5 Rx, which may be the same or different; n=0 or ¹ ; L2 is independently selected from the group consisting of: a bond, -OC(O)-C _1-6 alkylene-, and -NR ^d -C(O)-C _1-6 alkylene-; X ¹ is independently selected from the group consisting of: -NR ^e R ^f , -P(O)OR ^a OR ^b , -OP(O)OR ^a OR ^b and -CO ₂ H; R ² is selected from the group consisting of H, -L ³ -C _1-6 alkylene -L ⁴ -X ² , - L ³ -(C _1-6 alkylene)mA ² -X ² , -P(O)OR ^a OC(O)-C _1-6 alkyl, -P(O)OR ^a NR ^g R ^h and - P(O)OR ^a OR ^b ; L ³ is independently selected from the group consisting of -C(O)-O- and -C(O)-; L ⁴ is independently selected from the group consisting of: key, -OC( O)-C _2-6 alkenyl-, -OC(O)-C _1-6 alkylene- and -NR ^d -C(O)-C _1-6 alkylene-, where -NR ^d - C(O)-C _1-6 alkylene-and -OC(O)-C _1-6 alkylene- in the C _1-6 alkylene group is optionally substituted by NR ^e R ^f ; X ² is independently selected A group consisting of -NR ^k R ^l , -P(O)OR ^a OR ^b , -OP(O)OR ^a OR ^b and -CO ₂ H; m=0 or 1; A ² is selected from the following groups The group of: C _6-10 aryl, C _3-10 cycloalkyl, C _5-10 heteroaryl and C _5-10 heteroalkyl, each of which is optionally substituted by 1 to 5 R ^x , which can be Same or different; R ³ is H or -L5-P(O)OR ^a OR ^b , wherein L ⁵ is independently selected from the group consisting of a bond and -CH ₂ -O-; each R ^{x is} independently selected from the group consisting of Group: halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 heteroalkyl, CN, NR ^y R ^z , SR ^y and OR ^y ; each R ^c is independently selected from the group consisting of Groups: halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 heteroalkyl, CN, NR ^y R ^z , SR ^y and OR ^y each R ^a , R ^b , R ^d , R ^e , R ^f , R ^g , R ^k , R ^l , R ^y and R ^z are independently selected from the group consisting of H and C _1-6 alkyl; each R ^h independently selected from the group consisting of H and C _1-6 alkyl; wherein the C _1-6 alkyl is optionally substituted by 1 to 5 substituents, the substituents are independently selected from CO ₂ H, NR ⁱ R ^j , C _6-10 aryl, C _3-10 cycloalkyl, C _5-10 heteroaryl and C _5-10 heteroalkyl, wherein each R ⁱ and R ^j are independently H or C _1-6 alkyl; wherein Two of R ¹ , R ² and R ³ are H, and one of R ¹ , R ² and R ³ is not H.

PMX-53 is a potent antagonist of C5aR(CD88). It is a cyclic peptide composed of six amino acids with the following sequence: Ac-Phe-ring (Orn-Pro-D-Cha-Trp-Arg), with a lactam bridge between Orn-2 and Arg-6 . Since PMX-53 contains at least one D-amino acid (ie, D-Cha), it is not included in the appended sequence listing of this application. PMX-53 is a commercial item of bio-techne GmbH, Wiesbaden-Nordenstadt, Germany, catalog number 5473.

Compounds like PMX-53 also suitable for carrying out the present invention are disclosed in International Applications WO 99/00406 A1 , WO 03/033528 A1 and WO 2008/009062 A1 , which are hereby incorporated by reference in their entirety. Therefore, in some embodiments, the inhibitor of C5a activity is a cyclic peptide or peptidomimetic compound of formula IV

Where A is H, alkyl, aryl, NH ₂ , NH-alkyl, N(alkyl) ₂ , NH-aryl, NH-acyl, NH-benzyl, NHSO ₃ , NHSO ₂ -alkyl , NHSO ₂ -aryl, OH, O-alkyl or O-aryl; B is alkyl, aryl, phenyl, benzyl, naphthyl or indolyl, or D- or L-amino acid side chain, but not glycine, D-phenylalanine, L-homophenylalanine, L-tryptophan, L-homotryptophan, L-tyrosine, or L-homotyrosine side chain; C is a D-, L- or homo-amino acid side chain, but not isoleucine, phenylalanine or cyclohexylalanine side chain; D is a neutral D-amino acid side chain, But not glycine or D-alanine side chains, huge planar side chains or huge charged side chains; E is a huge substituent, but not D-tryptophan, LN-methyltryptophan, L-homo Phenylalanine, L-2-naphthyl L-tetrahydroisoquinoline, L-cyclohexylalanine, D-leucine, L-fenylalanine or L-histidine side chain; F is L -Arginine, L-homoarginine, L-citrulline or L-canavanine side chain, or its biological analog; and X ¹ is -(CH ₂ )nNH- or (CH ₂ ) nS-, where n is an integer from 1 to 4; -(CH ₂ ) ₂ O-; -(CH ₂ ) ₃ O; -(CH ₂ ) ₃ -; -(CH ₂ ) ₄ -, -CH ₂ -COCHRNH -; or _-CH2 -CHCOCHRNH-, wherein R is any common or uncommon amino acid side chain.

Herein, the term "common amino acid" refers to the twenty protein-producing amino acids, which are defined by the standard genetic code. The term "uncommon amino acid" includes, but is not limited to, D-amino acid, homo-amino acid, N-alkyl amino acid, dehydroamino acid, aromatic amino acid other than phenylalanine , tyrosine and tryptophan, o-, m- or p-aminobenzoic acid, ornithine, citrulline, canaline, norleucine, delta-glutamic acid, aminobutyric acid , L-fenylalanine, L-3-benzothienyl (benzothienyl)alanine and α,α-disubstituted amino acids.

Specific C5aR(CD88) antagonists suitable for the practice of the present invention include PMX95, PMX218, PMX200, PMX273, PMX205 and PMX201, as disclosed in WO 2008/009062 A1.

Colony S5/1 is a monoclonal antibody that recognizes the C5a (CD88) human receptor. Colony S5/1 bulged against a synthetic peptide (Met1-Asn31 ) comprising the N-terminal region of C5aR. This antibody has been shown to inhibit the binding of C5a to its receptor. It is commercially available via Hycult Biotech (Uden, The Netherlands), catalog number HM2094.

Colony 7H110 is a monoclonal antibody that recognizes the C5a (CD88) human receptor. It is commercially available through Biomol GmbH (Hamburg, Germany), catalog number C2439-60N.

As used herein, "patient" means any mammal or bird that would benefit from treatment with a compound described herein (ie, with an inhibitor of C5a activity described herein). Preferably, the "patient" is selected from experimental animals (such as mice or rats), domestic animals (including, for example, guinea pigs, rabbits, chickens, native chickens, pigs, sheep, goats, camels, cows, horses, donkeys, cats or dogs) or primates, including chimpanzees, as well as humans. Most preferably, the patient is human.

As used herein, "treating, treating, or treating" a disease or condition means achieving one or more of the following: (a) reducing the severity and/or duration of the condition; (b) limiting or preventing the effects of the condition being treated. (c) inhibiting worsening of symptoms characteristic of the disorder being treated; (d) limiting or preventing recurrence of symptoms in patients previously suffering from the disorder; and (e) limiting or preventing recurrence of symptoms in patients previously suffering from symptoms of the disorder.

As used herein, "prevent, preventing, prevention or prophylaxis" of a disease or condition means preventing the condition from occurring in an individual.

An "effective amount" is an amount of therapeutic agent sufficient to achieve the desired purpose. The effective amount of a given therapeutic agent will vary with such factors as the nature of the agent, the route of administration, the size and species of the animal receiving the therapeutic agent, and the purpose of administration. The effective amount in each individual case is to be determined experimentally by the skilled artisan according to established methods in the art.

"Pharmaceutically acceptable" means approved by a competent agency of the federal or state government, or listed in the United States Pharmacopoeia or other pharmacopoeia approved for use in animals, especially humans.

Specific examples of the present invention

The present invention will now be further described. Different aspects of the invention are defined in more detail in the following sections. Each aspect defined below may be combined with any other aspect(s) unless explicitly stated to be in opposition to each other. In particular, any feature which is stated to be preferred or advantageous may be combined with any other other feature (etc.) which is stated to be preferred or advantageous.

In a first aspect the present invention relates to a compound for use in the treatment of a skin, neutrophil, inflammatory disease in an individual, wherein the compound is an inhibitor of C5a activity, and wherein the skin, neutrophil Balloon, inflammatory disease is selected from the group consisting of: hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG, and acne); PASH (PG, acne, and acne hidradenitis); PAPASH (suppurative arthritis, acne, PG, and hidradenitis suppurativa); Schwaite's syndrome (SS); subcorneal blister dermatosis (SPD); epidermolysis bullosa acquired; persistent Erythema embosses (EED); Neutrophilic steatophyllitis; Bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hyperosteogeny, and osteitis) syndrome ; rheumatoid neutrophilic dermatosis; familial Mediterranean fever, cold inflammatory factor-related disorders, gout, and Schnitzler syndrome.

In a second aspect, the invention relates to a method for treating a skin, neutrophil, inflammatory disease in an individual comprising the steps of: administering to an individual in need thereof a therapeutic amount of a compound, wherein The compound is an inhibitor of C5a activity, and wherein the skin, neutrophil, inflammatory disease is selected from the group consisting of: hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (purulent arthritis, PG, and acne); PASH (PG, acne, and hidradenitis suppurativa); PAPASH (arthritis suppurativa, acne, PG, and hidradenitis suppurativa); Schwaite's syndrome (SS); Epidermolysis bullosa (SPD); Erythematosus elongata (EED); Neutrophils; Bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovial rheumatoid neutrophilic dermatosis; familial Mediterranean fever, cold inflammatory factor-related disorders, gout, and Schnitzler syndrome.

In the third aspect, the present invention relates to the use of a compound for the preparation of a pharmaceutical composition for treating skin, neutrophil, and inflammatory diseases, wherein the compound is an inhibitor of C5a activity, and wherein the skin , neutrophils, inflammatory diseases are selected from the group consisting of: hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, Acne and hidradenitis suppurativa); PAPASH (suppurative arthritis, acne, PG, and hidradenitis suppurativa); Schwitters syndrome (SS); Subcorneal bullous dermatosis (SPD); Epidermolysis bullosa acquired persistent erythema elevated (EED); neutrophilic steatophyllitis; bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hyperosteogeny and osteoarthritis rheumatoid neutrophilic dermatosis; familial Mediterranean fever, cold inflammatory factor-related disorders, gout, and Schnitzler syndrome.

In some embodiments of any aspect of the present invention, the C5a activity inhibitor: - reduces the concentration of C5 (for example by inhibiting the formation and/or activity of C3 convertase; by inhibiting the formation and/or activity of C5 convertase; by inhibiting the transcription of the C5 gene; by blocking the translation of C5 mRNA; by increasing the degradation of C5 mRNA; by increasing the degradation of C5 protein; or by preventing the secretion of C5 from the liver); - inhibiting the cleavage of C5 to C5a and C5b (e.g. by inhibiting C5 convertase or by binding to the cleavage site on C5 thereby blocking cleavage); - reducing the concentration of C5a (e.g. by increasing the degradation of C5a protein); - inhibiting the interaction between C5a and 5Ca receptor binding between (for example by binding to C5a or by binding to the C5a receptor); - reducing the concentration of the C5a receptor (for example by inhibiting the transcription of the C5a receptor gene; by blocking the translation of the C5a receptor mRNA; by increasing the degradation of C5a receptor mRNA; by increasing the degradation of C5a receptor protein); and/or - inhibiting the activity of C5a receptor.

In some embodiments of any aspect of the invention, the inhibitor of C5a activity is selected from the group consisting of protein ligands (as defined above); oligonucleotides; and small molecules (as defined above). An oligonucleotide acting as an inhibitor of C5a activity can achieve its inhibition, for example, by binding to a nucleic acid molecule (thus inhibiting transcription and/or translation) or by binding to a protein (e.g. when the oligonucleotide is an aptamer). effect.

In some embodiments of any aspect of the present invention, the C5a activity inhibitor is a protein ligand that specifically binds to a C5 protein or binds to a C5a protein or binds to a C5a receptor protein. In more embodiments, the protein ligand is selected from the group consisting of (i) an antibody (such as an anti-C5 antibody, an anti-C5a antibody, an anti-C5aR antibody, or an anti-C5L2 antibody), (ii) an antibody of Antigen-binding fragments, (iii) antibody-like proteins, (iv) inhibitory variants of C5a, (v) inhibitory variants of C5a receptors (e.g. decoy receptors), (vi) proteins acting on the complement pathway (e.g. Coversin); and (vii) peptides (eg RA101495 (Ra Pharma, Cambridge, MA)); PMX-53 (bio-techne GmbH (Wiesbaden-Nordenstadt, Germany)).

In some embodiments of any aspect of the present invention, the inhibitor of C5a activity is a protein ligand or oligonucleotide, preferably a protein ligand, which specifically binds to human C5a amino acid sequence NDETCEQRA (SEQ ID NO: 2 ) and SHKDMQL (SEQ ID NO: 3) formed a conformational epitope. Binding to the conformation formed according to the amino acid sequences of SEQ ID NO: 2 and 3 means that the protein ligand or oligonucleotide binds to at least one amino acid within the amino acid sequence according to SEQ ID NO: 2 and binds according to At least one amino acid within the amino acid sequence of SEQ ID NO:3. SEQ ID NO: 2 corresponds to amino acids 30-38 of human C5a. SEQ ID NO: 3 corresponds to amino acids 66-72 of human C5a.

In some embodiments of any aspect of the invention, the protein ligand or oligonucleotide, preferably the protein ligand, binds to at least one amino acid within the amino acid sequence according to DETCEQR (SEQ ID NO: 4). SEQ ID NO: 4 corresponds to amino acids 31-37 of human C5a.

In some embodiments of any aspect of the present invention, the protein ligand or oligonucleotide, preferably the protein ligand, binds to at least one amino acid in the amino acid sequence according to HKDMQ (SEQ ID NO: 5), more preferably Combined with at least one amino acid in the amino acid sequence KDM. SEQ ID NO: 5 corresponds to amino acids 67-71 of human C5a; sequence KDM corresponds to amino acids 68-70 of human C5a.

In some embodiments of any aspect of the present invention, the protein ligand or oligonucleotide, preferably the protein ligand, is combined with at least one amino acid and at least one Amino acid binding. Amino acid sequence HKDMQ (SEQ ID NO: 5).

In some embodiments of any aspect of the present invention, the protein ligand or oligonucleotide, preferably the protein ligand, is associated with at least one amino acid within the amino acid sequence DETCEQR (SEQ ID NO: 4) and the amino acid sequence At least one amino acid is bound within the KDM.

In some embodiments of any aspect of the invention, two sequences form a conformational epitope of C5a (for example, a sequence pair according to SEQ ID NO: 2 and 3; SEQ ID NO: 4 and 5; or SEQ ID NO: 4 and The sequence KDM) is separated by 1-50 consecutive amino acids which are not involved in the binding to the binding moiety of the invention. Hereinafter, amino acids that do not participate in binding to the binding moiety of the present invention will be referred to as "non-binding amino acids". The two sequences forming a conformational epitope are preferably separated by 6-45 consecutive non-binding amino acids, more preferably 12-40 consecutive non-binding amino acids, more preferably 18-35 consecutive non-binding amino acids , more preferably 24-30 consecutive non-binding amino acids, more preferably 25-29 consecutive non-binding amino acids, even more preferably 26-28 consecutive non-binding amino acids, and most preferably 27 consecutive unbound amino acids.

In some embodiments of any aspect of the present invention, the protein ligand or oligonucleotide that specifically binds to the conformational epitope of C5a, preferably the protein ligand has a binding constant to human C5a with a Kd value of 10 nM or less, Preferably 9nM or less, more preferably 8nM or less, more preferably 7nM or less, more preferably 6nM or less, more preferably 5nM or less, more preferably 4nM or less, more preferably 3nM or less, More preferably 2nM or less, even more preferably 1nM or less. In some embodiments of any aspect of the present invention, the dissociation constant Kd between the binding moiety and human C5a is between 1 pM (picomole) and 5 nM (nanomole), more preferably between 2 pM and 4 nM, more preferably between 5 pM Between 10pM and 2nM, between 50pM and 1nM, between 100pM and 900pM, between 200pM and 800pM, between 300pM and 700pM, and even better , between 400pM and 600pM.

In some embodiments of any aspect of the invention, the protein ligand or oligonucleotide specifically binding to C5a, preferably the protein ligand, exhibits at least 75% of the biological effect induced by a molecule of C5a, especially human C5a Blocking activity, preferably at least 80% blocking activity, more preferably at least 85% blocking activity, more preferably at least 90% blocking activity, more preferably at least 95% blocking activity. These specific blocking activities refer to those embodiments wherein the binding moiety comprises a single paratope that binds C5a, preferably human C5a. In embodiments wherein the binding moiety comprises two or more C5a-specific paratopes, when one binding moiety molecule is contacted with a number of C5a molecules equal to the number of C5a-specific paratopes present in the binding moiety, the desired Said blocking activity is at least 75%, preferably at least 80%, more preferably at least 85%. In other words, when the paratope of the binding moiety described herein and C5a are present in equimolar concentrations, the binding moiety exhibits at least 75% blocking activity, preferably at least 80% blocking activity, of the biological effects induced by C5a Activity, more preferably at least 85% blocking activity, more preferably at least 90% blocking activity, more preferably at least 95% blocking activity. The preferred biological effect to be blocked is the C5a induced release of lysozyme from human whole blood cells. Assays for determining this C5a-induced release of lysozyme and its blockade are described eg in WO2011/063980A1 and the corresponding US National Phase Application US2012/0231008A1.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises (i) a heavy chain CDR3 sequence as shown in SEQ ID NO: 6; or (ii) The heavy chain CDR3 sequence as shown in SEQ ID NO: 7; wherein the heavy chain CDR3 sequence includes 1, 2 or 3 amino acid exchanges as appropriate, preferably conservative amino acid exchange, 1, 2 or 3 Amino acid deletions, and/or additions of 1, 2 or 3 amino acids.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises (iii) a light chain CDR3 sequence as shown in SEQ ID NO: 8; or (iv) a light chain CDR3 sequence as shown in SEQ ID NO: 9; wherein the light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 Amino acid deletions, and/or additions of 1, 2 or 3 amino acids.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof comprises (i) a heavy chain CDR3 sequence as shown in SEQ ID NO: 6 and as shown in The light chain CDR3 sequence shown in SEQ ID NO: 8; or (ii) the heavy chain CDR3 sequence shown in SEQ ID NO: 7 and the light chain CDR3 sequence shown in SEQ ID NO: 9; wherein the heavy chain CDR3 sequence optionally comprising 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amino acid additions; and wherein The light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amino acid exchanges Add to.

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises at least one of the following sequences: (v) heavy according to SEQ ID NO: 10 Chain CDR2 sequence; (vi) heavy chain CDR2 sequence according to SEQ ID NO: 11; (vii) light chain CDR2 sequence according to SEQ ID NO: 12; (viii) light chain CDR2 sequence according to SEQ ID NO: 13; ( ix) the heavy chain CDR1 sequence according to SEQ ID NO: 14; (x) the heavy chain CDR1 sequence according to SEQ ID NO: 15; (xi) the light chain CDR1 sequence according to SEQ ID NO: 16; or (xii) the light chain CDR1 sequence according to SEQ ID NO: 16; ID NO: 17 light chain CDR1 sequence; wherein the heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and /or 1, 2 or 3 amino acid additions; wherein the light chain CDR2 sequence contains 1, 2 or 3 amino acid exchanges as appropriate, preferably conservative amino acid exchange, 1, 2 or 3 amino acids Deletion, and/or addition of 1, 2 or 3 amino acids; wherein the heavy chain CDR1 sequence contains 1, 2 or 3 amino acid exchanges as appropriate, preferably conservative amino acid exchange, 1, 2 or 3 amino acid deletion, and/or 1, 2 or 3 amino acid additions; and wherein the light chain CDR1 sequence optionally includes 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1 , 2 or 3 amino acid deletions, and/or 1 , 2 or 3 amino acid additions.

In certain embodiments, according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 The total number of these optional changes described in each of the above amino acid sequences, that is, exchanges, deletions and additions The total is 1 or 2 in each sequence.

In certain embodiments, the total number of exchanges, deletions and additions of all CDRs present in the antibody or antigen-binding fragment thereof is between 1 and 5 (eg 1, 2, 3, 4 or 5).

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof comprising one of Groups A to H of heavy chain CDR3, heavy chain CDR2, and heavy chain CDR1 sequences listed in Table 1 below, wherein Each heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amino acid exchanges Acid addition; wherein each heavy chain CDR2 sequence optionally comprises 1 , 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1 , 2 or 3 amino acid deletions, and/or 1 , 2 or 3 amino acid additions; and wherein each heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or or 1, 2 or 3 amino acid additions:

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or an antigen-binding fragment thereof, comprising one of the light chain CDR3, light chain CDR2 and light chain CDR1 sequences in groups I to IV listed in Table 2 below, wherein Each light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amino acid exchanges Acid addition; wherein each light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amino acid additions; and wherein each light chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or Or 1, 2 or 3 amino acid additions.

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof comprising one of the heavy chain CDR sets listed in Table 1 above and one of the light chain CDR sets I-IV listed above one. In Table 2, that is one of the following combinations: AI, A-II, A-III, A-IV, BI, B-II, B-III, B-IV, CI, C-II, C-III, C -IV, DI, D-II, D-III, D-IV, EI, E-II, E-III, E-IV, FI, F-II, F-III, F-IV, GI, G-II , G-III, G-IV, HI, H-II, H-III or H-IV (wherein the combination of AI and H-IV is particularly preferred), wherein each heavy chain CDR3 sequence optionally contains 1, 2 or 3 Amino acid exchange, preferably conservative amino acid exchange, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amino acid additions; wherein each heavy chain CDR2 sequence optionally includes 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amino acid additions; wherein each heavy chain CDR1 sequence depends on Cases involving 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions and/or 1, 2 or 3 amino acid additions; wherein each light chain The CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, in particular conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amino acid additions; wherein each light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions, and/or 1, 2 or 3 amines and wherein each light chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions and/or 1, 2 or 3 amino acid additions.

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof comprising a VH domain comprising, consisting essentially of (i) the VH domain of IFX-1 or (ii) the VH domain of INab708 domain composition.

The FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4 sequences delimiting the VH domains of IFX-1 and INab708 are shown in Table 3 below.

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof comprising a VL domain comprising, consisting essentially of (i) the VL domain of IFX-1 or (ii) the VL of INab708 domain composition.

The FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4 sequences delimiting the VL domains of IFX-1 and INab708 are shown in Table 3 below.

In some embodiments of any aspect of the invention, the inhibitor of C5a activity is an oligonucleotide that specifically binds C5, or C5a or a C5a receptor. In a further embodiment, the oligonucleotide is an aptamer. The nucleic acid aptamer can be selected from the group consisting of DNA-aptamers, D-RNA aptamers and L-RNA aptamers (eg, Spiegelmers™).

In some embodiments of any of the aspects of the invention, the inhibitor of C5a activity reduces the expression of C5 protein or C5a receptor protein. In a further embodiment, the C5a activity inhibitor that reduces the expression of C5 protein or C5a receptor protein is an oligonucleotide selected from the group consisting of antisense DNA, antisense RNA, siRNA and miRNA.

In some embodiments of any aspect of the invention, the C5a receptor is C5aR and/or C5L2. In a preferred embodiment of any aspect of the invention, the C5a receptor is C5aR (also known as CD88 or C5aR1).

In some embodiments of any aspect of the present invention, the C5a activity inhibitor is selected from the group consisting of (a) IFX-1, INab708, MEDI-7814, ALXN-1007 or NOX-D21, or an antigen thereof A binding fragment; (b) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes for binding to C5a with one of the antibodies referred to in (a); (c) eculizumab, ALXN1210, ALXN5500 or LFG316 , or an antigen-binding fragment thereof; (d) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes for binding to C5 with one of the antibodies referred to in (c); (e) Coversin or RA101495; (f) An antibody or antigen-binding fragment thereof or protein or macrocyclic peptide, wherein the antibody or antigen-binding fragment thereof or macrocyclic peptide competes for binding to C5 with one of the proteins or peptides referred to in (e); (g) Zimura; (h) An antibody or an antigen-binding fragment thereof or an aptamer, wherein the antibody or an antigen-binding fragment thereof or an aptamer competes with Zimura for binding to C5; (i) AMY-201 or Mirococept; (j) an antibody or an antigen-binding fragment or protein thereof, wherein The antibody or antigen-binding fragment thereof or protein competes for binding to C3b with one of the proteins indicated in (i); (k) Bikaciomab; (l) the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with Bikaciomab Binds to Factor B; (m) Lampazumab; (n) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with Lampalizumab for binding to Factor D; (o) ALN-CC5; and (p) Avacopan or a compound according to formula II or III or PMX-53 or a compound according to formula IV; (q) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is combined with Avacopan or PMX-53 competes for binding to C5aR; (r) colony S5/1 or colony 7H110, or an antigen-binding fragment thereof; and (s) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is associated with ( One of the antibodies indicated in r) competes for binding to C5aR.

In some embodiments of any aspect of the invention, the skin, neutrophil, inflammatory disease is: - autoimmune disease (more precisely: skin, neutrophil, autoimmune disease) ; or - autoimmune disease with skin inflammation (more precisely: autoimmune disease with skin, neutrophil inflammation).

In some embodiments of any aspect of the invention, the skin, neutrophil, inflammatory disease is an autoinflammatory disease selected from the group consisting of: hidradenitis suppurativa (HS); Dermatosis (PG); PAPA (septic arthritis, PG, and acne); PASH (PG, acne, and hidradenitis suppurativa); PAPASH (suppurative arthritis, acne, PG, and hidradenitis suppurativa); Schwitters syndrome (SS); Subcorneal blister dermatosis (SPD); Epidermolysis bullosa acquired; Erythema elevated persistent (EED); syndrome (BADAS); and SAPHO (synovitis, acne, impetigo, hyperosteogeny and osteitis) syndrome.

In some embodiments of any aspect of the present invention, the skin, neutrophil, and inflammatory diseases are HS or HS-related diseases selected from the group consisting of: pyoderma gangrenosum (PG); PAPA ( Septic arthritis, PG, and acne); PASH (PG, acne, and hidradenitis suppurativa); PAPASH (suppurative arthritis, acne, PG, and hidradenitis suppurativa); Schwaite's syndrome (SS); Herpes dermatosis (SPD).

In some embodiments of any aspect of the present invention, the skin, neutrophil, inflammatory disease is an autoimmune disease with skin inflammation selected from the group consisting of: rheumatoid neutrophils Glomeral dermatosis, familial Mediterranean fever, cold-inflammation-related disorders, gout, and Schnitzler syndrome.

In some embodiments of the first or third aspect of the invention, the compound is administered at a dose of 800 mg once a week or at a dose of 800 mg twice a week. In more specific examples of the first aspect or the third aspect, - the C5a activity inhibitor is a compound specifically binding to C5a (preferably selected from IFX-1, INab708, MEDI-7814, ALXN-1007, The group consisting of NOX-D21 and antigen-binding fragments thereof; more preferably, the C5a activity inhibitor is selected from the group consisting of IFX-1, INab708, MEDI-7814, ALXN-1007 and antigen-binding fragments thereof; and more preferably , the inhibitor of C5a activity is selected from the group consisting of IFX-1 and antigen-binding fragments thereof; optimally, the inhibitor of C5a activity is IFX-1); and - the skin, neutrophil, inflammatory disease is purulent hidradenitis (HS); and - the compound is administered at a dose of 800 mg once weekly or at a dose of 800 mg twice weekly.

In further embodiments of the first aspect or the third aspect, the C5a activity inhibitor is administered intravenously. In more specific examples of the first aspect or the third aspect, the C5a activity inhibitor is administered twice a week at a dose of 800 mg in the first week of treatment, and administered at a dose of 800 mg in the second and subsequent weeks of treatment. The dose is administered once a week. In further embodiments of the first aspect or the third aspect, the total duration of treatment is between 5 and 12 weeks (e.g., 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks).

In some embodiments of the second aspect of the invention, the compound is administered at a once-weekly dose of 800 mg or twice-weekly at a dose of 800 mg. In more specific examples of the second aspect, - the C5a activity inhibitor is a compound specifically binding to C5a (preferably selected from IFX-1, INab708, MEDI-7814, ALXN-1007, NOX-D21 and The group consisting of antigen-binding fragments; more preferably, the C5a activity inhibitor is selected from the group consisting of IFX-1, INab708, MEDI-7814, ALXN-1007 and antigen-binding fragments thereof; and more preferably, the C5a activity inhibitor selected from the group consisting of IFX-1 and antigen-binding fragments thereof; optimally, the inhibitor of C5a activity is IFX-1); and - the skin, neutrophil, inflammatory disease is hidradenitis suppurativa (HS); and - the compound is administered at a once-weekly dose of 800 mg or twice-weekly at a dose of 800 mg.

In further embodiments of the second aspect, the inhibitor of C5a activity is administered intravenously. In a further embodiment of the second aspect, the compound is administered at a dose of 800 mg twice weekly during the first week of treatment and once weekly at a dose of 800 mg during the second and subsequent weeks of treatment . In further embodiments of the second aspect, the total duration of treatment is between 5 and 12 weeks (eg, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks).

Pharmaceutical composition and mode of administration

When implementing any aspect of the present invention, the compound (such as the C5a activity inhibitor described herein) or the pharmaceutical composition comprising the compound can be administered to the patient through any route established in the art, which provides sufficient content of compounds. It can be administered systemically or locally. Such administration can be parenteral, transmucosal (eg, oral, nasal, rectal, intravaginal, sublingual, submucosal, transdermal or by inhalation). Preferably, administration is parenteral, eg, via intravenous or intraperitoneal injection, and also includes, but is not limited to, intraarterial, intramuscular, intradermal and sublingual administration. If a compound described herein (eg, an inhibitor of C5a activity described herein) or a pharmaceutical composition comprising the compound is administered locally, it may be injected directly into the organ or tissue to be treated.

Pharmaceutical compositions suitable for oral administration may be provided as capsules or lozenges; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); as edible foams or foams ; or as an emulsion. Tablets or hard gelatine capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or its salts. Soft gelatin capsules may contain vegetable oils, waxes, lipids, semi-solid or liquid polyols, and the like. Solutions and syrups may contain water, polyols and sugars.

Active agents intended for oral administration may be coated with or mixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract (for example, glyceryl monostearate or glyceryl distearate may be used) . Thus, sustained release of the active agent can be achieved over many hours and the active agent can be protected from degradation in the stomach if desired. Pharmaceutical compositions for oral administration can be formulated to induce release of the active agent at a specific gastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions suitable for transdermal administration may be presented as release patches, which are intended to remain in intimate contact with the epidermis of the recipient for an extended period of time. Pharmaceutical compositions adapted for topical administration may be presented as ointments, creams, suspensions, emulsions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye or other external tissues, topical ointments or creams are preferred. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water base or a water-in-oil base. Pharmaceutical compositions suitable for topical administration to the eye include eye drops. In these compositions, the active ingredient may be dissolved or suspended in a suitable carrier, for example an aqueous solvent. Pharmaceutical compositions suitable for topical administration in the mouth include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions suitable for nasal administration may comprise a solid carrier such as a powder (preferably having a particle size in the range of 20 to 500 microns). Powders may be administered by inhaling the nasal powder through the nose (eg, by rapid inhalation) from a powder container close to the nose. Alternatively, compositions suitable for nasal administration may comprise a liquid carrier such as nasal spray or nasal drops. These compositions may contain aqueous or oily solutions of the active ingredients. Compositions for administration by inhalation may be employed in atopic devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which may be configured to provide predetermined doses of the active ingredient. Pharmaceutical compositions can also be administered nasally to the lungs.

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions which may contain antioxidants, buffers, bacteriostatic agents and agents which render the composition substantially isotonic with the blood of the intended recipient. of solute. Other ingredients that may be present in these compositions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. Compositions suitable for parenteral administration may be presented in unit-dose containers or in multi-dose containers, for example, sealed ampoules and vials, and presented in sterile liquid for injection (requiring only addition, for example, sterile saline solution shortly before use). agent) and stored in freeze-dried (lyophilized) conditions. Solutions for immediate injection and suspensions can be prepared from sterile powders, granules and lozenges.

In a preferred embodiment, the compounds described herein (eg, C5a activity inhibitors described herein) can be formulated according to conventional procedures as applicable to pharmaceutical compositions for intravenous administration to humans. Typically compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include solubilizers and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are presented, alone or in admixture, in unit dosage form, such as a lyophilized powder or dry concentrate in a hermetically sealed container, such as an ampoule or sachet, with the indicated quantity of active agent. If the composition is to be administered by infusion, it can be suspended using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule with sterile saline can be provided to allow the ingredients to be mixed prior to administration.

In another embodiment, for example, a compound (eg, an inhibitor of C5a activity described herein) or a pharmaceutical composition containing the compound can be administered in a controlled release system. For example, the compounds can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one particular example, a pump can be used (see Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14 : 201; Buchwald et al. (1980) Surgery 88 : 507; Saudek et al. (1989) N. Eng . .J.Med. 321 :574). In another embodiment, the compound can be delivered in vesicles, especially liposomes (see Langer (1990) Science 249 : 1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, NY, 353-365; WO 91/04014; US 4,704,355). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release (1974) Langer and Wise (eds.), CRC Press: Boca Raton, Fla.; Controlled Drug Bioavailability, Drug Product Design and Performance, (1984 ) Smolen and Ball (eds.), Wiley: NY; Ranger and Peppas (1953) J.Macromol. Sci. Rev. Macromol. Chem. 23 : 61; see also Levy et al. (1985) Science 228 : 190; During et al. (1989) Ann. Neurol . 25 : 351; Howard et al. (1989) J. Neurosurg. 71 : 105).

In yet another embodiment, a controlled release system can be placed in the vicinity of the therapeutic agent, i.e., the target cell, tissue or organ, thus requiring only a small systemic dose (see, e.g., Goodson (1984) 115-138 in Medical Applications of Controlled Release, vol. 2). Other controlled release systems are disclosed in a review by Langer (1990, Science 249 : 1527-1533).

In a specific embodiment, it is desirable to administer a compound described herein (eg, an inhibitor of C5a activity described herein), or a pharmaceutical composition containing the same, locally to an area in need of treatment. This can be through, for example, but not limited to, local infusion during surgery, local application (eg, in conjunction with a wound dressing after surgery), via catheter, via suppository, or via graft (which may be porous, non-porous or colloidal materials, including membranes or fibers such as silicone membranes).

Selection of a preferred effective dose will be determined by the skilled artisan upon consideration of several factors known to those of ordinary skill in the art. These factors include the specific form of the pharmaceutical composition (e.g., polypeptide or carrier), and its pharmacokinetic parameters (such as bioavailability, metabolism, half-life, etc.), which will be used in obtaining regulatory approval for the pharmaceutical compound. Established during customary development procedures. Additional dosage considerations include the condition or disease to be prevented or treated, or the benefit to be achieved in normal individuals, the body mass of the patient, the route of administration, whether the administration is acute or chronic, concomitant medications, and effects known to affect the efficacy of the administered agent other factors. Accordingly, the exact dosage will be determined according to standard clinical techniques based on the judgment of the physician and each patient's circumstances, eg depending on the individual patient's condition and immune status.

example

The following examples are provided to further illustrate the invention. However, the present invention will not be limited to the following examples, and the following examples merely show the feasibility of the present invention based on the above description.

1. Method 1.1 Preparation of zymosan A stock solution and zymosan A activated plasma (ZAP)

Zymosan A was dissolved at 2 mg/ml in 50 ml sterile saline and boiled at 100°C for 1 hour. After centrifugation, the supernatant was discarded and the pellet was resuspended in 50 ml sterile saline. After the second centrifugation step, the pellet was resuspended in 5 ml sterile saline to obtain a 20 mg/ml stock solution. The stock solution was aliquoted and stored at -20°C until use. To activate plasma, zymosan A stock solution was mixed with 100 μl of plasma and incubated at 37° C. for 30 minutes. After incubation, the tubes were centrifuged and the supernatant was aliquoted and stored at -20°C until use.

1.2 CD11b analysis using rhC5a or ZAP as stimulator

Human whole blood was stimulated with rhC5a or ZAP. To test the blocking activity of IFX-1 and an irrelevant control IgG4 against rhC5a, antibodies were diluted to final Ab/Ag ratios of 1:1 and 0.5:1. To test the blocking activity of IFX-1 on eC5a, IFX-1 was diluted to achieve a final Ab/Ag molar ratio of about 4:1/3:1/2:1/1:1/0.5:1. Buffered only blood was used as a non-stimulated control group to assess baseline CD11b expression. Antibody-containing blood alone was used to determine the effect of antibody on CD11b expression under unstimulated conditions. The complete mixture (Ab/Ag/blood) was incubated at 37°C for 20 minutes to assess C5a-induced CD11b upregulation. After addition of anti-mouse CD11b:FITC, samples were incubated on ice for 30 minutes to minimize background staining. Circle the pellet and check the mean fluorescence intensity (MFI) of the FITC-labeled pellet (expressing CD11b) by flow cytometry.

1.3 CD11b analysis using rhC5a or zymosan A in whole blood

Whole blood was stimulated with rhC5a or zymosan A, and the complete mixture (Ab/Ag/blood) was incubated at 37°C for 20 min to stimulate CD11b-induced C5a-upregulation. After incubation, 2 μl of anti-mouse CD11b:FITC or isotype:FITC controls were added and samples were incubated on ice for 30 minutes to minimize background staining. After lysis, cells were analyzed using flow cytometry. According to the FSC/SSC dot-plot, the pellet was circled and the mean fluorescence intensity (MFI) of the FITC-labeled pellet (expressing CD11b) was examined against the whole cell panel.

1.4 Cytokines IL-8 ELISA

IL-8 ELISA (eBioscience Inc., San Diego, CA) was performed as suggested in the manual under "Analytical Procedures". Briefly, coating was performed overnight at 4°C using 100 μl of 1x capture antibody. Plates were blocked for 1 hour at room temperature using 200 [mu]l 1x Assay Diluent. Dilute the standard stock solution to the desired concentration with 1x Assay Diluent, then perform six 1:2 serial dilutions. Dilute sample supernatants as needed in 1x Assay Diluent. According to the "Analytical Procedure", 100 μl standard dilution and sample dilution were added to the coated plate and incubated at RT for 1 hour, then mixed with 100 μl 1x detection antibody (RT, 1h) and 100 μl 1x avidin- HRP (RT, 30 min) incubation. Color development was performed for 10 min at RT in the dark using 100 μl of TMB substrate solution and stopped with 100 μl of stop solution. Absorbance was read at 450 nm using a plate reader within 30 minutes. All standards and samples were subtracted from the zero standard value (blank). The cytokine concentrations of the samples were calculated using the log(x)/log(y) standard curve of the included standard samples.

1.5 C5a ELISA

Purified anti-human C5a monoclonal antibody (InflaRx GmbH, Jena, Germany) was coated on ELISA plates overnight at a final concentration of 0.5 μg/mL. After blocking with assay diluent (1x PBS, 0.05% Tween20 and 2% heat-inactivated FBS), calibration samples (recombinant human C5a, Sigma, Taufkirchen, Germany) diluted in assay diluent and samples were placed in the laboratory. Incubation at room temperature lasted 90 minutes. Mouse anti-human C5/C5a antibody strain 561 (Hycult Biotech, Uden, The Netherlands) diluted to 2 μg/mL in assay diluent was applied as a primary detection antibody for 60 minutes at room temperature and incubated with Secondary horseradish peroxidase-labeled antibody (goat anti-mouse IgG2a polyclonal antibody, SouthernBiotech, Birmingham, USA) diluted to 0.05 μg/mL in assay diluent was incubated for 30 minutes. Color development was performed using tetramethylbiphenyl substrate solution (TMB, Biozol, Eching, Germany) and stopped with 3.7N sulfuric acid. OD was read at absorbance at 450 nm by a Tecan Infinite® 200 reader with Tecan Magellan ^™ (Tecan Group, Maennedorf, Switzerland). The self-developed C5a ELISA was evaluated according to the EMA guidelines for the evaluation of bioanalytical methods.

The intra- and inter-analytical precision tested using five different concentrations showed coefficients of variation (CV) ranging from 0.65% to 4.96% and 1.50% to 4.88% for six and 18 replicates, respectively. The recovery assay spiked with recombinant human C5a in buffer had a recovery of 86.98±1.20% (mean±SD) at the lower limit of quantification and 91.50±3.29% at the upper limit of quantification. No cross-reactivity was detected for C3, C3a and C4, whereas <0.01% cross-reactivity was detected for C5b-6. Human IgG4 antibodies did not interfere with the assay. The mean C5a content of citrated plasma in 20 human volunteers was 17.08 ng/ml ± 6.96 ng/ml, ranging from 7.52 ng/mL to 30.17 ng/mL.

1.6 Measurement of complement activation products

The concentrations of complement activation products C3a, C5a and membrane attack complex C5b-9C were measured by ELISA method. C3a ELISA was performed according to the manufacturer's instructions (BD OptEIA ^™ Human C3a ELISA Kit, BD Bioscience, Germany). C5b-9 concentrations were determined using the InflaRx validated C5b-9 ELISA based on the BD OptEIA ^™ Human C5b-9 ELISA Set (BD Bioscience). C5a concentrations were determined using the InflaRx validated C5a ELISA above.

1.7 Statistical analysis

All results are presented as mean ± standard deviation. After baseline adjustment, statistical differences between the two groups were calculated by one-way ANOVA (including Tukey's multiple comparison test or by Student's t-test). A p-value of 0.05 was calculated to determine whether there were any significant differences between any two groups. Graphing and statistical analysis were performed using GraphPad PRISM ^® V6.05 (CA, USA).

2. Preclinical data 2.1 C5a activation of neutrophils and blocking effect of IFX-1

Because CD11b upregulation is a hallmark of neutrophil activation, CD11b levels on neutrophils were used to assess neutrophil activation. The human whole blood model was used in this study to evaluate the blocking activity of IFX-1 on recombinant human C5a (rhC5a). Human whole blood was incubated with buffer, antibody alone, rhC5a alone, or a combination of antibody and rhC5a at different concentrations. After incubation, cells were stained with anti-mouse CD11b:FITC and CD11b MFI was analyzed by flow cytometry to examine the degree of activation of blood neutrophils. As shown in Figure 1, recombinant human C5a strongly stimulated the upregulation of CD11b on human neutrophils. This effect was completely blocked in the presence of anti-human C5a antibody IFX-1. This inhibition was highly specific and an irrelevant human IgG4 antibody did not show any blocking activity.

As a source of endogenous C5a (eC5a), zymosan-activated plasma (ZAP) was used to stimulate blood neutrophils. The amount of eC5a in ZAP was measured using a commercial C5a-ELISA kit. The data presented here (Figure 2) indicate that eC5a-induced upregulation of CD11b in ZAP is equivalent to rhC5a. The presence of IFX-1 significantly reduced CD11b expression on human neutrophils even at an Ab:Ag molar ratio of 0.5:1. The overall blocking activity of IFX-1 on ZAP-induced CD11b upregulation ranged from 100% to 82%, depending on the Ab:Ag ratio. Despite the presence of high levels of eC3a and other complement activation products in ZAP, IFX-1 was able to specifically block CD11b upregulation by 100%. It can therefore be concluded that eC5a is the only driver for neutrophil activation after ZAP stimulation, which can be completely blocked by IFX-1.

2.2 C5a blockade attenuates zymosan-induced inflammatory response in human whole blood

Zymosan A (as an active fungal cell wall component) induces a strong inflammatory response in human whole blood, eg by activating neutrophils, characterized by increased levels of cytokines and chemokines. In the present study, human whole blood was spiked with Zymosan A in the presence or absence of IFX-1, and CD11b expression on blood neutrophils was measured by flow cytometry. As shown in Figure 3, CD11b on blood neutrophils was strongly upregulated when zymosan was present in human whole blood. CD11b upregulation due to zymosan stimulation was suppressed by 79%-93%, depending on the added IFX-1 concentration. As a positive control, upregulation of CD11b by rhC5a stimulation was 100% blocked by IFX-1. Therefore, it is certain that the upregulation of CD11b on blood neutrophils after zymosan A stimulation is mainly due to eC5a. In addition, it was concluded that eC5a, after being produced in whole blood from zymosan A, first binds to IFX-1, thereby blocking its access to its natural receptor.

In the same experimental setup, IL-8 levels were measured and used to assess the inflammatory response. After stimulation with different doses of zymosan A, IL-8 concentrations ranged from 458 pg/ml to 3218 pg/ml in the absence of IFX-1. As shown in Figure 4, the presence of IFX-1 significantly reduced IL-8 production after stimulation with various concentrations of zymosan A, and a reduction rate of up to 54% was observed. Thus, in the context of inflamed whole blood, most of the zymosan-induced inflammatory response was associated with the presence of C5a.

3. Clinically relevant data 3.1 Data obtained from clinical samples 3.1.1 Complement activation in HS patients

A total of 54 HS patients and 14 healthy volunteers were recruited in the study. Patients were followed up at the Immuno-infection Outpatient Unit of the University of Athikon (ATTIKON) Medicine, Greece. The study was approved by the hospital's ethics committee. All patients provided informed consent. HS is diagnosed on the basis of: a) early onset after puberty; b) appearance of subcutaneous nodules in areas of the skin rich in apocrine glands; and c) a compatible history of recurrent pus from the affected area.

Circulating concentrations of the complement factors C3a and C5a and the membrane attack complex sC5b-9 were determined in the plasma of 54 patients and 14 healthy controls, and in the pus of seven patients. As shown in Figure 5, circulating C5a was significantly greater in patient plasma than in control plasma (P<0.01), and C3a and C5b-9 differed equally significantly between patients and controls. Therefore, it can be concluded that systemic complement activation occurs in HS. Assuming that complement activation plays a major role in innate as well as acquired immunity, the inventors hypothesized that targeting complement activation might be a new therapeutic strategy for the treatment of HS.

However, in light of the above results, it is uncertain which of C3a, C5a, or C5-9b, or other complement activation products, would be a most promising target for this new therapeutic strategy and whether targeting only one of these factors is sufficient, or two or more factors involved in complement activation can be targeted.

3.1.2. Blocking CD11b upregulation induced by HS plasma on blood neutrophils

To determine the role of C5a on neutrophil activation in HS plasma samples, HS plasma samples with high levels of C5a were selected and evaluated by using a human whole blood model. As shown in Figure 6, HS plasma samples with high levels of C5a (Pat.088 and Pat.092) strongly up-regulate blood neutrophil CD11b expression. Recombinant human C5a was used as a positive control, while plasma from healthy volunteers was selected as a negative control. The up-regulation of CD11b induced by HS plasma can be inhibited by 100% by IFX-1, pointing out that C5a is the most important activator for the initiation of neutrophil activation in HS plasma. The inventors conclude from these novel results that blockade of C5a in HS patients is sufficient to achieve a strong suppression of neutrophil activation.

3.2 Data obtained from clinical trials 3.2.1 Experimental design

An open-label phase II trial was conducted in 11 patients with moderate-to-severe hidradenitis suppurativa at the Department of Internal Medicine, Adiken University Hospital, Greece.

The primary objective of the trial was to investigate the safety and tolerability of IFX-1 administered over 8 weeks. Secondary objectives of the trial were to assess the pharmacokinetics and pharmacodynamics of IFX-1 and to generate preliminary efficacy data on IFX-1 on clinically assessed measures (e.g., HiSCR, DLQI, VAS for disease status, VAS, HS-PGA, modified Sartorius score) to generate more hypotheses. Enrolled patients were treated with 800 mg IFX-1 twice in the first week and once a week thereafter for a total of 8 weeks; that is, on days 1, 4, 8, 15, 22, 29, 36, 43, and 50 Nine intravenous doses of 800 mg of IFX-1 were administered. All patients were followed for an additional 12 weeks.

The inclusion criteria for screening are:

18歲的男性或女性患者 1.

18-year-old male or female patient

2. Informed Consent

3. Diagnosis of HS for at least 1 year

4. HS lesions in at least 2 different anatomical regions, one of which is Hurley stage II or stage II

3 5. Total AN (abscess and nodule) number

3

6. The patient has primary or secondary failure of biological therapy, or is not suitable for treatment with other biological agents

Note: Primary failure is defined as no response to treatment with biologic compound for at least 12 weeks, while secondary failure is achieved after at least 12 weeks of treatment with biologic compound followed by relapse

7. Failure of previous antimicrobial therapy

The exclusion criteria for filtering are:

1. Over 150kg or under 60kg

2. The number of draining fistulas greater than 30 at baseline

3. Have scheduled surgery within the next 24 weeks

4. An episode of HS resulting in the use of intravenous antimicrobial therapy within the past 14 days

5. Any other disease or condition that may interfere with the conduct of the assessment of the investigational product, assessment of outcomes, or goodness of the study

a) active infection

b) Severe congestive heart failure (ie NYHA type IV)

c) depression

d) History of systemic lupus erythematosus or rheumatoid arthritis

e) Any immunodeficiency disease

f) Active hematological or solid malignancies

g) The patient has not previously had any other active skin disease or condition (such as bacterial, fungal or viral infection) that would interfere with the assessment of HS

6. One of the following abnormal laboratory results

a) White blood cell count <2,500/mm ³

b) Neutrophil count <1000/mm ³

c) Plasma creatinine >3 x upper limit of normal (UNL)

d) Total bilirubin >2 x UNL

e) Alanine-aminotransferase (ALAT) >2x UNL

f) Positive screening test for hepatitis B, hepatitis C or HIV1/2

7. Have previously administered any biological compound within the last 3 months

8. Taking corticosteroids, defined as taking more than 1 mg/kg of prednisone or equivalent daily for the last three weeks

9. Taking immunosuppressive drugs (such as cyclosporine, tacrolimus) within the last 30 days

10. General exclusions

a) Pregnant (urine pregnancy testing is mandatory in women of childbearing potential) or breastfeeding women

b) Pregnant potential (defined as having last menstrual period within two years) but unwilling to use appropriate contraception (eg, inplanon, injection, oral contraceptive, intrauterine device, partner vas deferens) while participating in the trial resection, abstinence) women

c) Participate in any interventional clinical trial within the last three months

d) Abuse of known intravenous drugs

e) a staff member of the Research Office, or the spouse/partner or relative of any research staff member (such as a researcher, associate researcher or research nurse), or has a relationship with the sponsor

3.2.2 Results of clinical trials

IFX-1 is well tolerated in HS patients. No drug-related serious adverse events were reported throughout the treatment period.

A commonly used efficacy parameter is hidradenitis suppurativa clinical response (HiSCR). HiSCR is defined by three types of lesion status (defining conditions): abscess (abscess, with or without drainage, tender or painful), inflamed nodule (tender, erythematous, pyogenic granulomatous focus), and draining fistula (sinus, communication with skin surface, outflow of pus). The proposed definition of responders to treatment (HiSCR successors) was: (i) at least 50% reduction in AN, (ii) no increase in the number of abscesses, and (iii) no increase in the number of draining fistulas relative to baseline. HiSCR has recently been demonstrated as a reactive and clinically meaningful measure of inflammatory conditions in HS (Kimball and others, 2014).

¹ Humira EMA評估報告：http：//www.ema.europa.eu/docs/en_GB//document_library/EPAR_-_Assessment_Report_-_Variation/human/000481/WC500195564.pdf ² Anakinra Study(Tzanetakou and others,2016).³ 新聞稿XBiotech(http：//investors.xbiotech.com/phoenix.zhtml？c=253990&p=irol-newsArticle&ID=2246777) HiSCR responses were investigated during the 8-week treatment period in this study, and 8 of 11 treated patients responded up to day 56, representing a response rate of 72.7% with a 95% confidence interval of 43% to 91%. In order to compare these results with past data, a literature study was performed to examine placebo-controlled clinical studies using HiSCR as an efficacy parameter. Table 4 below summarizes the five most recently completed studies:

¹ Humira EMA Assessment Report: http://www.ema.europa.eu/docs/en_GB//document_library/EPAR_-_Assessment_Report_-_Variation/human/000481/WC500195564.pdf ² Anakinra Study (Tzanetakou and others, 2016). ³ Press Release XBiotech (http://investors.xbiotech.com/phoenix.zhtml?c=253990&p=irol-newsArticle&ID=2246777)

Overall, 179 patients have been treated in the placebo arm of these studies, with a response rate of 19.0%, with a 95%-confidence interval ranging from 14% to 25%. Since the confidence intervals (eg, past placebo patients and patients treated with IFX-1) did not overlap, it was concluded that IFX-1 had a significant therapeutic effect.

Photographic documentation of the affected area corroborates these findings with a dramatic reduction in inflammation on the skin, as can be seen visually after treatment with less inflammation, swelling and redness.

Therefore, anti-C5a represents a potent anti-inflammatory agent in the setting of HS disease. The results of this clinical study demonstrate that blockade of C5a is highly effective in reducing neutrophil activation, thereby effectively alleviating cutaneous neutrophil inflammatory conditions.

4. Blockade of CD11B upregulation induced by activation of complement factors in hidradenitis patients via C5A-C5AR axis inhibition

4.1 Purpose

The aim of the following study was to demonstrate the blocking of hidradenitis on the surface of neutrophils by anti-human C5a monoclonal antibody IFX-1, anti-human C5a receptor C5aR (CD88) antibody and C5aR antagonists and C5aR inhibitors (HS) Plasma-induced upregulation of CD11b in patients.

4.2 Principles of analysis

Accumulation of neutrophils at sites of inflammation depends on the expression of adhesion molecules, including CD11b (also known as integrin alpha M) (Larson and Springer, 1990; Carlos and Harlan, 1990). Upregulation and movement of CD11b/CD18 from intracellular pools to the neutrophil surface is essential for human neutrophil rolling and migration (Smith et al., 1989). Thus, enhanced expression of CD11b/CD18 reflects inflammatory triggering events. Human CD11b assay was performed using flow cytometry to detect FITC-conjugated anti-CD11b antibodies on the surface of neutrophils. Activated complement products, especially elevated endogenous C5a (eC5a) in plasma samples from HS patients can strongly upregulate CD11b expression through the binding of C5a to its receptor C5aR (CD88) on neutrophils. Therefore, blocking the C5a-C5aR axis is expected to abolish or attenuate CD11b upregulation on the surface of neutrophils.

As the first anti-human C5a monoclonal antibody to be introduced into clinical development, IFX-1 has been shown to control inflammatory responses that lead to tissue and organ damage. The antibody is currently being evaluated in a Phase IIb study in patients with moderate or severe hidradenitis. It specifically and directly neutralizes the terminal complement anaphylatoxin C5a and blocks its deleterious role as a key inflammatory mediator in acute and chronic inflammatory diseases (Klos et al., 2009; Guo and Ward, 2005; Riedemann et al., 2017) .

C5a exerts its effects by interacting with high affinity C5a receptors (C5aR and C5L2) (Guo and Ward, 2005). C5aR belongs to the rhodopsin family of G protein-coupled receptors with seven transmembrane segments, whereas C5L2 is not G protein-coupled. It is generally accepted that C5a-C5aR signaling is important in the pathogenesis of proinflammatory outcomes (Ward, 2009). Therefore, targeting C5aR is another strategy to suppress complement-dependent inflammatory diseases. A series of small molecules derived from the C-terminus of C5a were developed as C5aR antagonists. Among them, the lead compound cyclic hexapeptide PMX-53 (AcF-[OP(D-Cha)WR]) (Finch et al., 1999) was shown to reduce damage in a number of animal models of inflammation after intravenous, subcutaneous, intraperitoneal and oral administration (Proctor et al., 2006). Due to their structural similarity to C5a, these antagonists compete with C5a for the C5a receptor on neutrophils (March et al., 2004). In addition, anti-C5aR antibodies can block the binding of C5a to C5aR, thereby reducing the accumulation and activation of myeloid-derived suppressor cells and neutrophils (Markiewski et al., 2008). Two commercially available monoclonal anti-C5aR antibodies, clonal S5/1 and 7H110, were tested in this study. They were raised in mice against a synthetic peptide containing the N-terminal extracellular domain of C5aR (Met1-Asn31) and recombinant human C5aR (Met1-Val350), respectively, and both antibodies were described as neutralizing antibodies. Avacopan (CCX168), an orally administered small molecule drug candidate that selectively inhibits the complement C5a receptor (C5aR), is being developed for inflammatory and autoimmune diseases (Bekker et al., 3008; Jayne et al., 2017)

Inhibition of these blockers targeting the C5a-C5aR axis to block upregulation of CD11b on neutrophils was monitored by flow cytometry.

4.3 Experimental Details

4.3.1 Samples

According to the consensus definition and diagnostic criteria of the Hidradenitis Suppurativa Foundation in 2009, plasma samples from two HS patients (Pat.088 and Pat.092) and two healthy controls (Ctrl 009 and Ctrl 010) were included in this study. The complement system is activated in the pathogenesis of HS, manifested by elevated levels of C3a, C5a and C5b-9 (see Table 5 below). The C5a levels of Pat.088 and 092 (93.77 ng/mL and 70.01 ng/mL, respectively) were significantly higher than those of healthy controls (21.02 ng/mL and 11.77 ng/mL).

4.3.2 Reagents

‧AnalaR water, VWR (Darmstadt, Germany), catalog number 102923C, NORMAPUR for analysis, sterile filtered

‧ACD, Sigma Aldrich (Taufkirchen, Germany), catalog number C3821-50ML

‧Reagents for flow cytometry

○ FACS Flow Sheat Fluid, BD Bioscience (NJ, USA), catalog number 342003

○ FACS Closure Solution, BD Bioscience (NJ, USA), catalog number 334224

○ FACS cleaning solution, BD Bioscience (NJ, USA), catalog number 340345

○ Rat anti-mouse CD11b: FITC, BD Bioscience (NJ, USA), catalog number 553310, 0.5 mg/mL

○ 10× FACS Lysis Solution, BD Bioscience (NJ, USA), catalog number 349202 → Working Solution: 1× FACS Lysis Solution (diluted 1:10 in AnalaR water)

○Staining buffer: 1% heat-inactivated FBS+0.1% sodium azide in 1×PBS solution

‧FBS, Thermo Fisher Scientific (Darmstadt, Germany), catalog number 10099133, heat inactivation: 56°C, 30 minutes

‧PBS powder, Sigma Aldrich (Taufkirchen, Germany), catalog number P3813-10PAK

‧Sodium Azide, VWR (Darmstadt, Germany), catalog number 1.06688.0250

‧Recombinant human C5a (rhC5a), Hycult Biotech (Uden, Netherlands), catalog number HC2101, expressed in Escherichia coli, dissolved in sterile AnalaR water

‧0.9% sterile sodium chloride (saline), B. Braun (Melsungen, Germany), catalog number 3200950

‧IFX-1, anti-human C5a antibody used as control group, InflaRx (Jena, Germany), 10mg/mL, PBS+0.05% Tween 80

‧PMX-53, bio-techne (Wiesbaden-Nordenstadt, Germany), catalog number 5473

‧Anti-C5aR (CD88) antibody colony S5/1, Hycult Biotech (Uden, Netherlands), catalog number HM2094

‧Anti-C5aR(CD88) antibody colony 7H110, biomol (Hamburg, Germany), catalog number C2439-60N

‧Avacopan, MedKoo Biosciences Inc. (Morrisville, USA), catalog number 319575

‧Human blood from a healthy donor containing 12% ACD (use immediately)

‧Human plasma bank (citrated plasma) at the University Hospital of Jena.

4.3.3 Equipment

‧Flow Cytometry (FACS Canto II with DIVA Software V6.1.2)

4.3.4 Procedure

a) Human CD11b Potency Assay (Flow Cytometry Assay)

In the absence or presence of C5a-C5aR axis blockers (anti-C5a antibody IFX-1, anti-C5aR antibody colony S5/1 and colony 7H110, C5aR antagonist PMX-53 and C5aR inhibitor Avacopan ; 10 μL ), two patient plasma samples Pat.088 and Pat.092 (5 μL) were incubated with fresh human blood (60 μL, as source of neutrophils) in a total volume of 100 μL . Two control plasma samples (Ctrl 009 and Ctrl 010 , 5 μL) prepared according to the procedure applied to patient samples were used as controls for non-specific activation. Blood containing saline only (40 μL) or normal human plasma pool ( huPP5 μL +saline 35 μL ) was used as non-stimulated control group to define the baseline expression of CD11b. Blood samples with pools of normal human plasma spiked with recombinant human C5a (rhC5a) simulated the stimulated state. All samples were incubated at 37°C for 20 minutes to activate CD11b upregulation. After cooling on ice, 2 μL of FITC-conjugated anti-mouse CD11b antibody was added to the samples. Labeled samples were kept on ice for an additional 30 min in the dark to minimize background signal. Red blood cells were then lysed with 1x FACS lysis solution for 10 min at room temperature. Wash the remaining cells twice with 2 mL of staining buffer. After centrifugation at 2500 rpm for 3 minutes, cells were resuspended in 0.5 mL of staining buffer and ready for FACS analysis. A gate was set on the FSC vs. SSC plot so that only cells with the size of neutrophils were analyzed.

The percent blocking activity (BA) of the C5a-C5aR axis blocking test material was calculated using the following formula. where MFI is the mean fluorescence intensity emitted by CD11b-bound FITC on neutrophils.

BA(%)=(MFI patient plasma-MFI test material spiked patient plasma)÷(MFI patient plasma-MFI huPP)×100

b) Statistical analysis

Graphs were created using GraphPad Prism 7 (CA, USA).

4.4 Results and discussion

Expression of Integrin CD11b on Neutrophils

3500)。相反，藉由HS患者樣本(Pat.088，Pat.092)或20nM重組人類C5a(rhC5a)誘導CD11b表現升高2.3至3.9倍(圖8、9、10和11；表6、7和8)。這些數據提示，顯著上調的CD11b表現是由HS患者中的炎性因子介導的。推測補體活化產物，尤其是C5a，在CD11b上調中扮演主要作用(表5)。 CD11b expression on neutrophils was assessed with plasma samples from two healthy blood donors (Ctrl 009 and Ctrl 010) and two diagnosed HS patients (Pat.088 and Pat.092). The mean fluorescence intensity (MFI) of Ctrl- samples was 2156.9±114.3, which fell within the range of baseline expression of unstimulated CD11b (MFI

3500). In contrast, a 2.3 to 3.9-fold increase in CD11b expression was induced by HS patient samples (Pat.088, Pat.092) or 20 nM recombinant human C5a (rhC5a) (Figures 8, 9, 10 and 11; Tables 6, 7 and 8) . These data suggest that the significantly upregulated CD11b expression is mediated by inflammatory factors in HS patients. It is speculated that complement activation products, especially C5a, play a major role in the upregulation of CD11b (Table 5).

Inhibition of CD11b Upregulation by Anti-Human C5aR Monoclonal Antibodies, C5aR Antagonists, and C5aR Inhibitors

As shown in Figure 8, rhC5a (20 nM) with high levels of eC5a and two HS patient plasma samples (Pat.088 and Pat.092) strongly upregulated CD11b expression on blood neutrophils, while the final concentration of targeted C5aR was The presence of blocking antibody at 50 nM significantly attenuated CD11b expression driven by plasma samples from rhC5a or HS patients. Colony 7H110 and colony S5/1 achieved a high blocking activity of 71% to 79% by using anti-C5aR antibody (Figure 8, Tables 9 and 6). In contrast, the small hexapeptide C5aR antagonist PMX-53 was not as effective as the C5aR-specific monoclonal antibody. The blocking activity produced by the same concentration of PMX-53 (5OnM) was within 50% to 57% (Figure 9A, Tables 9 and 6). In order to examine whether the elimination of CD11b upregulation can be achieved by C5aR inhibition, the blocking activity of high concentration of PMX 53 (20 μM ) was further evaluated under the same experimental setup. As a result, in the presence of high levels of PMX-53, HS patient plasma samples as well as rhC5a-induced upregulation of CD11b were completely abolished (Fig. 9B, Tables 9 and 7). Similar data were obtained in the presence of Avacopan, a C5aR inhibitor (Figure 11, Tables 8 and 9). The blocking activity in the presence of 100 μM Avacopan was between 77% and 80%, while the plasma-induced upregulation of CD11b in HS patients was completely blocked in the presence of 500 μM Avacopan (Figure 11, Tables 8 and 9).

These results suggest that the C5a/C5aR axis is primarily responsible for the upregulation of CD11b in blood neutrophils, and that C5aR may serve as a potential target to block C5a activity.

IFX-1, an anti-human C5a monoclonal antibody, completely blocks HS-induced CD11b upregulation

By employing the same experimental setup as above, as expected, rhC5a and HS patient plasma samples (Pat.088 and Pat.092) strongly activated CD11b expression on blood neutrophils, and addition of IFX- 1 could completely abolish the upregulation of CD11b (Fig. 10). These results show that IFX-1 is more effective in suppressing C5a/C5aR-driven inflammatory responses.

4.5 Conclusion

Taken together, our results show that a C5a-targeting approach, application of the anti-C5a monoclonal antibody IFX-1, effectively abolished C5a-mediated upregulation of CD11b. In addition, anti-C5aR antibodies, C5aR antagonists and C5aR inhibitors also showed strong blocking activity under the same experimental conditions. Therefore, targeting C5aR with blocking antibodies or antagonists represents an alternative strategy to block the C5a/C5aR axis under inflammatory conditions such as HS.

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<110> Deshang Infleas Co., Ltd.

<120> Treatment of Inflammatory Diseases Using C5A Activity Inhibitors

<130> 680-17 PCT

<150> EP 17177657.8

<151> 2017-06-23

<150> EP 17189938.8

<151> 2017-09-07

<160> 34

<170> PatentIn version 3.5

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<221> Various features

<222> (1)..(1)

<223> substituted with 40kDaPEG-aminohexyl

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<221> Various features

<222> (7)..(7)

<223> Deoxyuridine

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<223> Deoxyuridine

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Claims (1)

A compound for the preparation of medicines for treating skin, neutrophils, and inflammatory diseases in individuals, wherein the compound is a C5a activity inhibitor, and the C5a activity inhibitor directly binds to a C5a receptor and inhibits the C5a receptor activity, wherein the C5a activity inhibitor is selected from the group consisting of Avacopan, PMX-53, colony S5/1, colony 7H110 and antigen-binding fragments thereof, and wherein the skin, neutrophils, inflammation The disease is selected from the group consisting of: hidradenitis suppurativa (HS); PASH (PG, acne, and hidradenitis suppurativa); PAPASH (arthritis suppurativa, acne, PG, and hidradenitis suppurativa).

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