WO2014122298A1

WO2014122298A1 - Interleukin-4 inhibitors for the treamtent of neutropenia

Info

Publication number: WO2014122298A1
Application number: PCT/EP2014/052499
Authority: WO
Inventors: Carsten KRIEG; Onur BOYMANN
Original assignee: Universität Zürich
Priority date: 2013-02-08
Filing date: 2014-02-07
Publication date: 2014-08-14

Abstract

The present invention relates to an inhibitor of IL-4 or the IL-4 receptor, wherein said inhibitor is capable of binding to and thereby inhibiting the biological effect of IL-4/IL-4 receptor interaction, for use in a method for preventing or treating neutropenia.. The present invention further relates to inhibitors of gene expression and compounds for use in a method for preventing or treating neutropenia.

Description

lnterleukin-4 inhibitors for the treatment of Neutropenia Description

The present invention relates to the use of inhibitors of the interleukin-4-p38 mitogen- activated protein kinase (MAP kinase) pathway in a method for preventing or treating neutropenia.

The concerted action of innate and adaptive immune cells is essential for anti-microbial immunity and the prevention of immunopathologies, such as allergy and autoimmune disease. In fighting the concerned pathogen most efficiently while limiting damage to non- infected tissue cells, the adaptive immune system employs a set of specialized T cells. Thus, T helper (T_H) 1 CD4⁺ cells, characterized by their production of interferon-γ (IFN-γ), show increased activity against intracellular pathogens. Conversely, T_H2 CD4⁺ cells produce little IFN-γ but ample IL-4 (interleukin-4) and are crucial for defense against helminths. A third lineage of T_H cells, called T_H17, produces IL-17 and IL-22 and controls extracellular bacteria and fungi. The cytokines produced by these T_H cell subsets and the different molecular pathways involved repress one another. Thus, IL-4 is able to restrict the generation of IFN-γ- producing T_H1 cells, while IFN-γ suppresses the formation of IL-4⁺ T_H2 cells. Hence, T_H1 immune responses are more difficult to elicit in a T_H2-skewed immune system, and vice versa.

Even before the activation of adaptive immunity, most infections are controlled by innate immune cells, most notably granulocytes, mast cells, monocytes and macrophages. Similar to T_H cells, innate effector cells exhibit a considerable degree of specialization, although neutrophil granulocytes (also termed neutrophils) usually rapidly migrate to the site of infection and constitute a first line of defense against many pathogens. Neutrophils, even in the absence of infection, are also recruited by inflammatory cues, thereby amplifying this sterile inflammation. However, acute T_H2-type inflammation, as found in allergic disorders such as asthma and atopic dermatitis (AD), is marked by a paucity of neutrophils. Moreover, AD patients display weakened immune responses against pathogens that are typically controlled by neutrophils, thus suggesting a negative impact of T_H2 skewing on neutrophils.

Neutrophils are critically reduced in neutropenia, which is a life-threatening condition as it is accompanied by an increased risk of infections that can rapidly lead to death. The most severe form of neutropenia is called agranulocytosis. Causes of neutropenia are manifold, most commonly including medications (chemotherapy, indomethacin and other drugs), cancer (especially leukemias), radiation, autoimmune diseases, genetic diseases, hemodialysis, and vitamin deficiency (vitamin B12 or folic acid). The objective of the present invention is to provide safe and efficacious means for the prevention or treatment of neutropenia.

In the course of a study investigating the impact of immune polarization on neutrophil responses, wherein T_H1 -prone C57BL/6 (B6) versus T_H2-prone Balb/c mice were infected with different bacteria such as Escherichia coli, Listeria monocytogenes (LM), Salmonella thyphimurium and Staphylococcus aureus which are known to recruit neutrophils, the surprising finding was made that blocking of IL-4 signals by, for example, the use of neutralizing monoclonal antibody (mAb) against IL-4 and the IL-4 receptor, the genetic deletion of IL-4 or the IL-4 receptor-a, or the use of a p38 MAP kinase inhibitor, enhances the neutrophil immune response against several pathogens in, for instance, animals suffering from an inborn or acquired lack of neutrophils (neutropenia).

In general, inhibitors of the IL-4-p38 MAP kinase pathway are provided for use in a method for preventing or treating neutropenia. Such an inhibitor may be an oligopeptide, a polypeptide, a nucleic acid or a small molecule pharmaceutical drug.

According to a first aspect of the invention, an inhibitor of IL-4 (UniProt ID P051 12) or IL-4 receptor (UniProt ID P24394) is provided for use in a method for preventing or treating neutropenia, wherein said inhibitor is capable of binding to and thereby inhibiting the biological effect of IL-4/IL-4 receptor interaction.

In some embodiments said inhibitor is selected from

- the group comprising an antibody, an antibody fragment, an antibody-like

molecule, an oligopeptide of 6 to 30 amino acids, a nucleic acid aptamer molecule of 10 to 75 nucleotides in length,

a soluble polypeptide comprising a contiguous amino acid sequence of at least 30 amino acid residues, which are comprised within the amino acid sequence of IL-4- receptor or IL-4.

UniProt ID numbers in this document refer to entries in the Universal Protein Resource Knowledgebase.

According to a second aspect of the invention, an inhibitor of p38 MAP kinase is provided for use in a method for preventing or treating neutropenia, wherein said inhibitor is capable of binding to and inhibiting the biological effect of p38-a (UniProt ID Q16539), ρ38-β (UniProt ID Q15759), ρ38-γ (UniProt ID P53778) and/or ρ38-δ (UniProt ID 015264), and wherein the inhibitor is selected from the group comprising an antibody, an antibody fragment, an antibody-like molecule, an oligopeptide of 6 to 30 amino acids and a nucleic acid aptamer molecule of 10 to 75 nucleotides in length. The term inhibitor in the context of the present specification refers to a molecule that is capable of binding to IL-4 or the IL-4 receptor or p38 MAP kinase (ρ38-α, ρ38-β, ρ38-γ and/or ρ38-δ) with a dissociation constant of at least 10^"7 M^"1, 10^"8 M^"1 or 10^"9 M^"1 and which inhibits the biological activity of its respective target.

For example, inhibition, as defined in the preceding paragraphs, of the biological activity of IL-4 or of the IL-4 receptor may result in inhibition of signal transduction either by blocking the IL-4 receptor or by means of a significant reduction in the concentration of IL-4 at a particular site within the body which is involved in neutropenia, particularly the blood stream, lymphatic system, lymphatic tissue and bone marrow. Both inhibiting IL-4 and the IL-4 receptor may result in inhibition of the IL-4-triggered signal transduction and, ultimately, in a non-activation or inhibition of p38 MAP kinase signaling.

Alternatively, the biological activity of ρ38-α, ρ38-β, ρ38-γ and/or ρ38-δ may be directly inhibited, thus resulting in an inhibition of the phosphorylation of proteins downstream of p38 in the MAP kinase cascade.

Such an inhibitor according to the above aspects of the invention may be an antibody, an antibody fragment, an antibody-like molecule, an oligopeptide or a nucleic acid aptamer molecule of 10 to 75 nucleotides in length, any of which binds to and thereby inhibits IL-4 or the IL-4 receptor, or ρ38-α, ρ38-β, ρ38-γ and/or ρ38-δ.

An antibody fragment may be a Fab domain or an Fv domain of an antibody, or a single- chain antibody fragment, which is a fusion protein consisting of the variable regions of light and heavy chains of an antibody connected by a peptide linker. The inhibitor may also be a single domain antibody, consisting of an isolated variable domain from a heavy or light chain. Alternatively, an antibody may also be a heavy-chain antibody consisting of only heavy chains such as antibodies found in camelids. An antibody-like molecule may be a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zurich).

Methods for generating antibodies against IL-4 or the IL-4 receptor, or against ρ38-α, ρ38-β, ρ38-γ and/or ρ38-δ, are known in the art. They include, for example, immunization of mice with human IL-4or its receptor, or with human ρ38-α, ρ38-β, ρ38-γ and/or ρ38-δ, or soluble parts thereof.

Suitable inhibitors according to the above aspect of the invention may also be developed by evolutive methods such as phage display, ribosome display or SELEX, wherein polypeptides or oligonucleotides are selected due to their binding affinity to a target of interest. Additionally, the binding affinity of an identified inhibitor may be improved by cycles of evolution of the amino acid sequence or nucleotide sequence and selection of the evolved inhibitors may be effected based on the required affinity. An oligopeptide according to the above aspect of the invention may be a peptide derived from the recognition site of the IL-4 receptor that competes with the receptor for IL-4. Vice versa, an oligopeptide may be derived from the part of the IL-4 molecule that is recognized by its receptor and binding of this oligopeptide results in inhibition of the receptor. Binding of such an oligopeptide must not result in activation of the downstream signal of the receptor.

Alternatively, an inhibitor according to the above aspects of the invention may be a soluble polypeptide comprising a contiguous amino acid sequence of at least 30 amino acid residues taken from the protein sequence of IL-4 or of the IL-4 receptor.

Such a soluble polypeptide is capable of interacting with IL-4 or the IL-4 receptor.

"Interacting" in the context of the present specification means the specific binding of a molecule to another molecule. For example, the sequence of at least 30 amino acids may bind to the IL-4 receptor (if the sequence is a part of the interleukin-4 polypeptide) or to the interleukin polypeptide (if the sequence is part of the IL-4 receptor polypeptide), either binding taking place without eliciting the biological effect of the native interleukin-interleukin- receptor interaction. Optionally the sequence of at least 30 amino acids is linked to an Fc antibody domain. The rationale is to provide a soluble decoy for either of the interleukin- interleukin receptor pair, wherein the decoy outcompetes native interleukin signalling. Thus, such a soluble polypeptide can be used to inhibit IL-4 or the IL-4 receptor by binding.

In one embodiment, the soluble polypeptide according to the above aspect of the invention is the extracellular domain of the IL-4-receptor fused to a constant fragment Fc of an antibody, for example an immunoglobulin G.

In one embodiment, an inhibitor of IL-4 or the IL-4 receptor according to the first aspect or embodiments of the invention is selected from the group comprised of

a polypeptide comprising the amino acid sequence SEQ ID 1 (mAb 6-2 light chain), SEQ ID 2 (mAb 6-2 heavy chain), SEQ ID 3 (mAb 12B5 light chain), SEQ

ID 4 (mAb 12B5 heavy chain), SEQ ID 5 (mAb 27A1 light chain), SEQ ID 6 (mAb 27A1 heavy chain), SEQ ID 7 (mAb 5A1 light chain), SEQ ID 8 (mAb 5A1 ), SEQ ID 9 (mAb 63 light chain), SEQ ID 10 (mAb 63 heavy chain), SEQ ID 1 1 (mAb 1 B7 light chain), SEQ ID 12 (mAb 1 B7 heavy chain) or SEQ ID 13 (soluble interleukin-4 receptor), wherein sequences SEQ ID 1 to SEQ ID 12 refer to monoclonal antibodies directed against the IL-4 receptor;

pascolizumab (a humanized murine antibody, CAS-Nr. 331243-22-2),

AMG 317 (a fully human monoclonal antibody to the IL-4 receptor),

a modified IL-4 (UniProt ID P051 12) comprising the amino acid substitutions R121 D and Y124D, a modified IL-4 (UniProt ID P051 12), comprising the amino acid substitutions R121 D, Y124D and S125D,

Pitrakinra, a recombinant human IL-4 variant that is an inhibitor of the IL-4 receptor, and

Nuvance, a genetically engineered soluble human IL-4 receptor.

In some embodiments, the antibodies shown in table 1 are employed as inhibitors according to the above aspect of the invention.

Table 1 :

# specificity clone name order# distributor

1 anti-human IL-4 4D9 ab13779 Abeam

2 anti-human IL-4 34019 MAB204 R&D

3 anti-human IL-4 3007 MAB304 R&D

4 anti-human IL-4 8D4-8 14-7049 eBioscience

5 anti-human IL-4 MP4-25D2 16-7048 eBioscience

6 anti-human IL-4 8D4-8 554515 BD

7 anti-human IL-4 7A3-3 130-095-753 Miltenyi

8 anti-human IL-4 MP4-25D2 101 17-01 Southern Biotech

9 anti-human IL-4 BVD4-1 D1 1 10203-01 Southern Biotech

10 anti-human IL-4 None given 500-M04 PeproTech

1 1 anti-human IL-4 None given SBH-AIL4 SBH Sciences

12 anti-human IL-4 860A 4B3 AHC0642 LifeTechnologies

13 anti-human IL-4 860F10H 12 AHC0749 LifeTechnologies

14 anti-human IL-4 IL4-I 3410-3-1000 MabTech

15 anti-human IL-4 3007.1 1 01406 StemCellTech

16 anti-human IL-4 1 1 -60 LS-C7862 LSBiosciences

17 anti-human IL-4 EPR1 1 18Y LS-C501 18 LSBiosciences

18 anti-human IL-4 8F12 LS-C148103 LSBiosciences

19 anti-human IL-4 M61 10641 LS-C84601 LSBiosciences 20 anti-human IL-4 M313012 LS-C84602 LSBiosciences

21 anti-human IL-4 860F10H 12 LS-C7880 LSBiosciences

22 anti-human IL-4 None given LS-C7850 LSBiosciences

23 anti-human IL-4 None given LS-C130195 LSBiosciences

24 anti-human IL-4 None given LS-C125798 LSBiosciences

25 anti-human IL-4 None given LS-C7871 LSBiosciences

26 anti-human IL-4 None given LS-C7869 LSBiosciences

27 anti-human IL-4 None given LS-C7865 LSBiosciences

28 anti-human IL-4 None given LS-C7854 LSBiosciences

29 anti-human IL-4 None given LS-C7852 LSBiosciences

30 anti-human IL-4 None given LS-C7855 LSBiosciences

31 anti-human IL-4 None given LS-C7866 LSBiosciences

32 anti-human IL-4 None given LS-C41985 LSBiosciences

33 anti-human IL-4 None given LS-C41734 LSBiosciences

34 anti-human IL-4 None given LS-C26982 LSBiosciences

35 anti-human IL-4 None given LS-C104458 LSBiosciences

36 anti-human IL-4 None given LS-C43680 LSBiosciences

37 anti-human IL-4 None given LS-C123888 LSBiosciences

38 anti-human IL-4 None given LS-C123886 LSBiosciences

39 anti-human IL-4 5B5 ab25033 Abeam

40 anti-human IL-4 3010 MAB604 R&D

41 anti-human IL-4 3010.21 1 340451 BD

42 anti-human IL-4Ra 25463 MAB230 R&D

A modified interleukin-4 in the context of the present specification refers to an interleukin-4 characterized by the native amino acid sequence (described in the UniProt entry P051 12) with the exception of the indicated substitutions.

According to another aspect of the invention, an inhibitor of the gene expression of IL-4 or of the IL-4 receptor is provided for use in a method for preventing or treating neutropenia. According to another aspect of the invention, an inhibitor of the gene expression of p38-a, ρ38-β, ρ38-γ and/or ρ38-δ is provided for use in a method for preventing or treating neutropenia.

An inhibitor of gene expression according to the above aspect of the invention may be a single-stranded or double-stranded interfering ribonucleic acid oligomer or a precursor thereof, comprising a sequence tract complementary to an mRNA molecule, which encodes IL-4 or the IL-4 receptor, or ρ38-α, ρ38-β, ρ38-γ or ρ38-δ.

The art of silencing or "knocking down" genes, by degradation of mRNA or other effects, is well known. Examples of technologies developed for this purpose are siRNA, miRNA, shRNA, shmiRNA, or dsRNA. A comprehensive overview of this field can be found in Perrimon et al, Cold Spring Harbour Perspectives in Biology, 2010, 2, a003640.

Alternatively, an inhibitor of gene expression according to the above aspects of the invention may be a single-stranded or double-stranded antisense ribonucleic or deoxyribonucleic acid, comprising sequences complementary to a sequence comprised in an operon, which expresses a gene encoding IL-4 or the IL-4 receptor, or ρ38-α, ρ38-β, ρ38-γ or ρ38-δ. Such an operon sequence may include, without being restricted to, an intron, an exon, an operator, a ribosome binding site or an enhancer sequence. Such antisense molecules may for example be 12-50 nucleotides in length.

According to a third alternative of the above aspect of the invention, the inhibitor may be an expression vector, comprising a sequence encoding an interfering ribonucleic acid oligomer or precursor thereof, as is described in the preceding paragraph. Optionally, the sequence is under the control of an RNA-polymerase promoter sequence operable in a mammalian cell. Such an expression vector allows for the production of an interfering RNA within the cell. Methods for making and using such expression vectors are known in the art.

In certain embodiments, the inhibitor is a small molecule pharmaceutical. In certain embodiments, the inhibitor is a small molecule inhibitor obeying the "Lipinski" rule, i.e. the inhibitor has a molecular mass between 160 u and 500u, comprises up to five hydrogen bond donators (e.g., oxygen and or nitrogen atoms with one H attached), up to ten hydrogen bond acceptors (e.g., oxygen or nitrogen atoms) and an octanol-water partition coefficient logP of below 5,6.

In one embodiment, the inhibitor according to the above aspect of the invention is AIR645, a 2'-0-methoxyethyl antisense drug which targets the interleukin-4 receptor-a mRNA. According to one aspect of the invention, a compound for use in a method for preventing or treating neutropenia is provided, wherein the compound is selected from the group comprised of: - 6-(2,4-difluorophenoxy)-2-[3-hydroxy-1-(2-hydroxyethyl)propylamino]-8-methyl- pyrido[2,3-d]pyrimidin-7-one (Pamapimod, p38 inhibitor):

- 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1 H-imidazol-5-yl]pyridine (SB203580, p38 inhibitor):

- 4-[4-(4-fluorophenyl)-5-(4-pyridyl)-1 H-imidazol-2-yl]phenol (SB202130, p38 inhibitor):

- 4-[2-(4-chlorophenyl)-4-(4-fluorophenyl)-1 H-imidazol-5-yl]pyridine

- 4-[4-(4-fluorophenyl)-2-(4-nitrophenyl)-1 H-imidazol-5-yl]pyridine (PD169316):

1-[5-tert-butyl-2-(p-tolyl)pyrazol-3-yl]-3-[4-(2-morpholinoethoxy)-1 -naphthyl]urea (doramapimod, p38 inhibitor):

- 6-(N-carbamoyl-2,6-difluoro-anilino)-2-(2,4-difluorophenyl)pyridine-3-carboxamide (VX-702, p38 inhibitor):

- 5-[2-tert-butyl-4-(4-fluorophenyl)-1 H-imidazol-5-yl]-3-(2,2-dimethylpropyl)imidazo[4,5- b]pyridin-2-amine (LY2228820, CAS No 862505-00-8, p38 inhibitor):

- 3H-lmidazo[4,5-0]pyridin-2-amine, 5-[2-(1 , 1-dimethylethyl)-4-(4-fluorophenyl)-1 H- imidazol-5-yl]-3-(2,2-dimethylpropyl)-, methanesulfonate (CAS No 862507-23-1 );

- 3H-lmidazo[4,5-0]pyridin-2-amine, 5-[2-(1 , 1-dimethylethyl)-4-(4-fluorophenyl)-1 H- imidazol-5-yl]-3-(2,2-dimethylpropyl)-, hydrochloride (CAS No 862507-29-7);

- 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenyl)sulfanyl-pyrimido[1 ,6-b]pyridazin-6-one (VX-745, p38 inhibitor):

- Vinorelbine (Navelbine®, CAS No 71486-22-1 , p38 inhibitor):

3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2-oxo-1-pyridyl]-N,4-dimethyl- benzamide (PH-797804, p38 inhibitor):

- 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1 ,2- f][1 ,2,4]triazine-6-carboxamide:

- 8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-2-{[2-hydroxy-1-

(hydroxymethyl)ethyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one (GSK681323, CAS No 444606-18-2, p38a inhibitor):

- 6-[5-(cyclopropylcarbamoyl)-3-fluoro-2-methylphenyl]-N-(2,2-dimethylpropyl)pyridine- 3- carboxamide (Losmapimod, CAS No 585543-15-3, p38 a inhibitor):

- ARRY371797 (p38 inhibitor);

- ARRY-614 (p38 inhibitor);

- 2-[6-chloro-5-[[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazin-1-yl]carbonyl]-1- methyl-1 H-indol-3-yl]-N,N-dimethyl-2-oxoacetamide (talmapimod, CAS No 309913- 83-5, p38 inhibitor):

- UR-13870 (p38 inhibitor);

3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2-oxo-1-pyridyl]-N,4-dimethyl- benzamide (PH797804, CAS No 586379-66-0, p38 inhibitor):

- 1 ,3, 10-trihydroxy-5-[(2E,6E)-3,7, 1 1 -trimethyldodeca-2,6, 10-trienyl]-1 1 H- benzo[b][1 ,4]benzodiazepin-6-one (TLN-4601 , inhibits the RAS-mitogen-activated phosphokinase (MAPK) pathway and also selectively binds to the Peripheral Benzodiazepine Receptor (PBR)):

2'-Amino-3'-methoxyflavone (PD98059):

trans-4-[4-(4-Fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-7 - -imidazol-1- yl]cyclohexanol (SB239063, CAS No 219138-24-6):

2'-Fluoro-N-(4-hydroxyphenyl)-[1 , 1 '-biphenyl]-4-butanamide (CM PD1 ):

H

(2-Methylphenyl)-[4-[(2-amino-4-bromophenyl)amino]-2-chlorophenyl]methanone (EO 1428):

6-(4-Fluorophenyl)-2,3-dihydro-5-(4-pyridinyl)imidazo[2,1 -b]thiazole dihydrochloride

5-(2,6-Dichlorophenyl)-2-[2,4-difluorophenyl)thio]-6H^

- -[4-[2-Ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK 715):

- 6-Chloro-5-[[(2R,5S)-4-[(4-fluorophenyl)methyl]-2,5-dimethyl-1 -piperazinyl]carbonyl]- N,N, 1-trimethyl-a-oxo-7 - -lndole-3-acetamide hydrochloride (SCIO 469):

- 4-[4-(4-Fluorophenyl)-1 -(3-phenylpropyl)-5-(4-pyridinyl)-1 H-imidazol-2-yl]-3-butyn-1 -ol

- 6-Chloro-5-[[4-[(4-fluorophenyl)met hyl]-1 -piperidinyl]carbonyl- V, \/,1 -trimethyl-a-oxo- -indole-3-acetamide (SX 01 1 ):

1-[2-Methoxy-4-(methylthio)benzoyl] -4-(phenylmethyl)piperidine (JX 401 ):

- 6-(4-Fluorophenyl)-5-(4-pyridyl)-2,3-dihydroimidazo[2, 1-b]-thiazole (SFK-86002):

5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophe^

trihydrochloride (SB220025):

any of the compounds schematically shown in Fig. 1 1 .

According to one aspect of the invention, a pharmaceutical composition for use in a method for preventing or treating neutropenia is provided, comprising an inhibitor or compound according to the above aspects or embodiments of the invention.

Such pharmaceutical composition may be for enteral administration, such as nasal, buccal, rectal or oral administration, or for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular administration. The pharmaceutical compositions comprise from approximately 1 % to approximately 95% active ingredient, preferably from approximately 20% to approximately 90% active ingredient.

An inhibitor or a compound according to the above aspects or embodiments of the invention can be administered alone or in combination with one or more other therapeutic agents. Possible combination therapies can take the form of fixed combinations of the inhibitor, or compound with one or more other therapeutic agents known in the prevention or treatment of neutropenia. The administration can be staggered or the combined agents can be given independently of one another or in the form of a fixed combination.

In one embodiment, a pharmaceutical composition comprises an inhibitor or compound according to the above aspects or embodiments of the invention, in combination with a therapeutically active amount of the granulocyte colony-stimulating factor (G-CSF, UniProt ID P09919). According to one aspect of the invention, a dosage form for use in a method for preventing or treating neutropenia is provided, comprising an inhibitor or compound according to the above aspects of the invention.

Such dosage forms according to the above aspects of the invention may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation formulation or suppository. Alternatively, the dosage form may be for parenteral administration, such as intravenous, intrahepatic, subcutaneous or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.

For parenteral administration, preference is given to the use of solutions of an inhibitor or a compound according the above aspects of the invention. Also considered are suspensions or dispersions. Especially preferred are isotonic aqueous solutions, dispersions or suspensions which, for example, can be made up shortly before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, viscosity-increasing agents, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.

For oral pharmaceutical preparations suitable carriers are especially fillers such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, and also binders such as starches, cellulose derivatives and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, flow conditioners and lubricants, for example stearic acid or salts thereof and/or polyethylene glycol. Tablet cores can be provided with suitable, optionally enteric, coatings. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient. Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The capsules may contain the active ingredient in the form of granules or dissolved or suspended in suitable liquid excipients, such as in oils.

Transdermal/intraperitoneal and intravenous applications are also considered, for example using a transdermal patch, which allows administration over an extended period of time, e.g. from one to twenty days.

Intravenous or subcutaneous applications are particularly preferred.

According to another aspect of the invention, a method for the manufacture of a medicament for use in a method for preventing or treating neutropenia is provided, comprising the use of an inhibitor or compound according to the above aspects or embodiments of the invention. Medicaments according to the invention are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing. According to yet another aspect of the invention, a method for preventing or treating neutropenia is provided, comprising the administration of an inhibitor or compound according the above aspects or embodiments of the invention.

Such treatment according to the invention may be for prophylactic or therapeutic purposes. For the administration, the inhibitor or compound is preferably in the form of a pharmaceutical preparation comprising the inhibitor or compound in chemically pure form and, optionally, a pharmaceutically acceptable carrier or adjuvants. The dosage of the inhibitor or compound depends upon the species, its age, weight, individual condition, the individual pharmacokinetic data, the mode of administration, and whether the administration is for prophylactic or therapeutic purposes. The daily dose administered may range from approximately 0.1 mg/kg to approximately 1000 mg/kg, preferably from approximately 0.5 mg/kg to approximately 100 mg/kg, of an inhibitor or compound according to the above aspects or embodiments of the invention.

According to another aspect of the invention, an inhibitor or a compound according to the above aspects or embodiments of the invention is provided for use in a method for preventing or treating atopic dermatitis.

Atopic dermatitis can be worsened and maintained by infection with Staphylococcus aureus.

Wherever alternatives for single separable features are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely with alternatives of other single separable features to form discrete embodiments of the invention disclosed herein.

The invention is further characterized, without limitations, by the following examples, from which further features, advantages or embodiments can be derived. The examples do not limit but illustrate the invention.

Description of the figures

Fig. 1 shows the percent overall survival of Balb/c mice, B6 mice, Rag1^~'^~ mice, p47^phox"'^" mice, and CCR2^"'^" mice after infection with different bacteria, including Salmonella thyphimurium (A), Staphylococcus aureus (B), Listeria monocytogenes (C-F, H, J and K) and Escherichia coli (G and I), starting from the day of infection and following different treatment regimens.

Fig. 2 shows the percent overall survival of Rag1^~'^~ mice (A) or B6 mice (B) following infection with Listeria monocytogenes (LM).

Fig. 3 shows the effect of IL-4 administration on the bacterial load in spleen (A) and liver (B) of B6 mice, on the production of reactive oxygen species (ROS) in neutrophils as assessed by flow cytometry (C) and the ex vivo killing rate of Listeria monocytogenes by purified splenic neutrophils following in vivo treatment (D).

Fig. 4 A and B show the effect of IL-4 on percentages of neutrophils (A) and the total neutrophil count (B) in bone marrow, blood and spleen of uninfected mice.

Fig. 4 C and D show the effect of IL-4 on percentages of neutrophils (C) and the total neutrophil count (D) in bone marrow, blood and spleen of mice infected with Listeria monocytogenes.

Fig. 4 E shows a scheme of the reconstitution of bone marrow chimeric mice and data on the effect of IL-4 on the count of blood neutrophils derived from either wild-type (WT) or IL-4 receptor-a-deficient (H4ra^~/~) bone marrow in chimeric animals.

Fig. 5 shows the effect of IL-4 on neutrophil migration in the presence or absence of an anti- IL-4 receptor-a antibody (A) as well as the effect of other cytokines on neutrophil migration.

Fig. 6 shows the effect of IL-4 in the presence or absence of a p38 MAP kinase inhibitor on the neutrophil migration in vitro (C) and the neutrophil percentages in the blood and bone marrow (D) in vivo.

Fig. 7 shows the effect of IL-4 in the presence or absence of a p38 MAP kinase inhibitor on the neutrophil frequency in the blood of B6 mice (E) and on the migration of human neutrophils (F).

Fig. 8 shows the effect of a p38 MAP kinase inhibitor and a PI3K inhibitor on neutrophil migration.

Fig. 9 shows quantification of phosphorylated (active) p38 MAP kinase in mouse neutrophils following different stimuli.

Fig. 10 shows bacterial load of Listeria monocytogenes in spleen following indicated cytokine treatments in presence or absence of the p38 MAP kinase inhibitor.

Fig. 1 1 shows compounds of the invention suitable for use in a method for treating neutropenia.

Fig. 12 shows the half-life in the serum of mice of free IL-4 and G-CSF, as well as of IL-4 complexed to a specific anti-IL-4 monoclonal antibody (1 1 B1 1 ) or G-CSF complexed to a specific anti-G-CSF monoclonal antibody.

Fig. 13 shows neutralisation of IL-4 by the anti-IL-4 monoclonal antibody and of G-CSF by an anti-G-CSF monoclonal antibody in vitro, and IL-4 production by splenocytes in vivo following Listeria monocytogenes (LM), IL-4 complexes and G-CSF complexes. Fig. 14 shows overall survival of neutrophil elastase (Elane)-deficient mice and CXCR2 knockout mice following Listeria monocytogenes (LM; A and C) or Salmonella typhimurium (B) infection.

Fig. 15 shows expression of different IL-4 receptor subunits on the mRNA (A) and protein levels (B) of murine neutrophils, monocytes and T cells, as well as expression of phosphorylated (active) STAT6 in Gr-1 ^{high (hi)} myeloid cells following different treatment regimens.

Fig. 16 shows higher expression of different effector molecules in bone marrow (BM) and spleen (SPL) cells of IL-4 receptor-a-deficient (//4ra^_/~) mice as compared to wild-type mice following Listeria monocytogenes (LM) infection.

Fig. 17 shows inhibition by IL-4 of migration of human neutrophils towards IL-8, in the presence or not of a p38 MAP kinase inhibitor in vitro.

Fig. 18 shows inhibition of IL-4-induced p38 MAP kinase phosphorylation by the specific inhibitor SB203580 in vivo.

Fig. 19 shows inhibition of imiquimod (Aldara^®) cream-induced skin inflammation by IL-4, following different treatment regimens.

Fig. 20 IL-4 reduces neutrophil recruitment into a subcutaneous air pouch (so-called air pouch model) following stimulation with monosodium urate crystals (MSU) or I L- β .

Fig. 21 IL-4 inhibits human neutrophil-mediated skin inflammation in a modified model of imiquimod (Aldara^®) cream-induced skin inflammation where imiquimod is applied to mice following depletion of endogenous murine neutrophils and adoptive transfer of purified human neutrophils.

Examples

Materials and Methods

Mice

C57BL/6 (B6), CD45.1 (Ly5.1 )-congenic, recombinase activating gene 1 -deficient (Rag1^~'^~) p47^phox"'^", CCR2^"'^" (all on a B6 background); Balb/c, and H4ra^~'^~ mice on a Balb/c background were used. Mice were maintained under specific pathogen-free conditions and used at 3-4 months of age. Experiments were performed in accordance with the Swiss Federal Veterinarian Office and Cantonal Veterinary Office guidelines.

Infections

Age- and sex-matched mice with a minimum body weight of 19 g were infected intravenously, as indicated, with either 1x10⁶ colony-forming units (cfu) or 1 x10⁷ or 1x10⁹ Escherichia coli (E. coli) or 10⁷ cfu or 10⁸ cfu Staphylococcus aureus or 3x10³ cfu, 1x10⁴ cfu or 1 x10⁵ cfu Listeria monocytogenes (LM) or were per os infected with 10⁴ cfu, 10⁵ cfu or 10⁶ cfu Salmonella typhimurium. Bacterial titers were determined prior to injection using agar plates containing Luria Bertani media (LB), nutrient broth media (NB) or brain heart infusion media (BHI).

In vivo treatments

Where mentioned, animals were pretreated by one intraperitoneal injection per day during four days of either phosphate-buffered saline (PBS) or cytokine/monoclonal antibody (mAb) complexes prior to being infected with E. coli or LM on the last day of treatment. Also, where indicated, mice received intraperitoneal injections of 100 g neutralizing mAb (clone names in brackets) against mouse IL-4 (1 1 B1 1 ) or 100 g depleting mAbs against Gr-1 (RB6-8C5), Ly6G (1A8) or NK1.1 (PK136), starting the day prior to infection and continuing for five daily injections, as previously published. Furthermore, in order to block activity of p38 mitogen- activated protein (MAP) kinase in vivo, mice were given three daily (1/day during 3 days) injections of 300 g of the specific p38 MAP kinase inhibitor SB203580.

To prepare cytokine/mAb complexes using granulocyte-colony stimulating factor (G-CSF), interleukin-2 (IL-2), IL-4 and IL-7, 1 -1 .5 g recombinant cytokine, were premixed with its specific anti-cytokine mAb at a 2:1 molecular ratio before intraperitoneal injection. For IL-15 complexes, 1 -1.5 μg recombinant mouse (rm) IL-15 was premixed with soluble rmlL-15 receptor a-Fc. Cytokine/mAb complexes were used in this study only in vivo, whereas the recombinant cytokines were used in vitro. For readability, cytokine/mAb complexes are referred to in the manuscript only by their cytokine.

Flow cytometry

Cell suspensions of organs were prepared according to standard protocols and stained for analysis by flow cytometry using PBS containing 4% fetal calf serum and 2.5 mM EDTA with the following fluorochrome-labeled mAbs: mouse CD3 (145-2C1 1 ), mouse CD1 1 b (M1/70), mouse CD45.1 (A20), mouse Ly6C (AL-21 ), mouse Ly6G (1A8), human CD1 1 b (ICRF44) and human CD15 (HI98). At least 1x10⁵ viable cells were acquired on a BD FACSCanto™ II flow cytometer and analyzed using FlowJo software.

Neutrophil isolation

Murine neutrophils, defined as CD3^" CD1 1 b⁺ Ly6G⁺ cells, were obtained by positive selection using Ly6G microbeads or by discontinuous Percoll density gradient separation. Human neutrophils were isolated from fresh blood of healthy donors by Dextran/Ficoll Paque Plus density gradient separation. Remaining erythrocytes were lysed using red blood cell (rbc) lysis buffer. Purity and cell viability were routinely over 97% and over 95%, respectively, as assessed by flow cytometry and trypan blue staining, respectively.

Production of reactive oxygen species (ROS) by neutrophils

ROS production in CD3^" CD1 1 b⁺ Ly6G⁺ blood or spleen neutrophils was assessed one day after infection using dihydrorhodamine 123 (DHR) following stimulation for 20 minutes with phorbol 12-myristate 13-acetate (20 ng/ml).

Neutrophil kill assay

For assessing killing of ingested bacteria by neutrophils, 1 x10⁵ purified murine neutrophils from the spleen or bone marrow (BM) were incubated at a ratio of 1 neutrophil to 100 cfu LM for 30 minutes at 37°C under shaking conditions, followed by extensive washing. One aliquot of neutrophils was lysed in 1 % triton-X, providing the initial count of ingested live LM. The other aliquots of neutrophils were cultured in serial dilutions on BHI plates for 2 or 4 hours at 37°C, before lysing, plating and counting cfu of LM. Killing of LM by PBS-treated neutrophils was set at 100% and kill in the other groups was calculated relative to PBS. Neutrophils alone (without LM) and LM alone served as controls.

BM chimeras

For the generation of BM chimeras, immune lineage-negative (Lin^") BM cells of wild-type CD45.1-congenic and H4ra^~'^~ CD45.2-congenic mice were purified by negative selection using magnetic beads and biotinylated mAbs against B220, CD3, CD4, CD8, CD1 1 b, CD1 1 c, Gr-1 , NK1.1 and Ter1 19. Subsequently, Lin^" BM cells of wild-type and H4ra^~'^~ mice were mixed at a 1 :1 ratio and injected intravenously into lethally irradiated (950 rad) wild-type CD45.2-congenic host mice. BM chimeric mice were left for two weeks in order to allow for reconstitution of neutrophils before use in experiments.

p38 MAP kinase phosphorylation.

Phosphorylated p38 MAP kinase was assessed using flow cytometry. Cells were cultured with the respective cytokines for 15 minutes, followed by fixation with paraformaldehyde, permeabilisation using methanol and the cytofix/cytoperm protocol and incubation with a phosphorylated p38 MAP kinase-specific monoclonal antibody. For in vivo studies, spleens were processed 15 minutes after intravenous injection of cytokines.

Neutrophil migration assay

1x10⁵ purified murine or human neutrophils were pretreated for 20 minutes with PBS or the indicated recombinant cytokines (30 ng/ml) before seeding into the upper chamber of a 5 μηη transwell. Subsequently, migration of neutrophils towards chemokines in the lower chamber, including CXCL1 (Chemokine (C-X-C motif) ligand 1 , 30 ng/ml), CXCL2 (30 ng/ml) or CXCL8 (100 ng/ml), was determined over 2 hours. Where indicated, anti-murine IL-4Ra mAb (10 μg/ml, clone mll_4R-M2), the p38 MAP kinase inhibitor SB203580 (30 μΐηοΙ) or the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly294002 (30 μηηοΙ) was included during the 20-minute pretreatment period. Cytokine and chemokine concentrations were obtained after careful titration.

Air pouch model

Air pouches were formed on the back of mice by subcutaneous injection of 3 ml sterile air on days 0 and 3. Before the injection of air, mice were briefly anesthetized with isoflurane. On day 6, 2 mg monosodium urate crystals (MSU) or 10 ng IL-1 β in 1 ml PBS were injected into air pouches, and leukocytes were harvested from air pouches 9-16 hours later.

Imiquimod (IMQ)-induced psoriasiform skin inflammation and xenotransfer model

For induction of skin inflammation, mouse ears were treated for 6 consecutive days using 60-80 mg Aldara^® cream containing 5% (3-4 mg) IMQ or Vaseline. Mice were evaluated daily by measuring ear thickness using a digital micrometer. For the xenotransfer model mice were depleted of neutrophils the day before IMQ-treatment by using one injection of 500 g of the monoclonal antibody 1A8. Further neutrophil depletion was kept upright by daily 1A8 injections (200 μg) throughout the experiment. Mouse ears were treated with IMQ for 6 days. Human neutrophils were isolated using dextran/Ficoll density gradient centrifugation. A volume of 10 μΙ containing a minimum of 1 x10⁶ human neutrophils was injected intradermally with an insulin syringe (31 .5 GA needle, BD Biosciences) into the ventral side of the mouse ear pinnae on days 4-6 of IMQ-treatment.

ELISPOT

Frequencies of IL-4 spot-forming cells (SFC) in the spleen were determined by the enzyme- linked immunospot (ELISPOT) assay. Anti-IL-4 mAb BVD-ID1 1 (PharMingen) was used for coating, and biotinylated anti-IL-4 mAb (eBioscience) was used as second-step mAb.

Horseradish peroxidase and amino-ethyl-carbazole (AEC) substrate with H₂0₂ as catalyst were used for detection.

Cytokine bioreactivity

CTLL-2 cells or NFS-60 cells were seeded at 5-10 x 10⁴ cells/well in a 96-well plate, and 2 μΙ/100 μΙ of mouse serum or control was added to each well. Cells were cultured under standard conditions (37°C, 5% C0₂) for 24-48 hours. [3H]-thymidine was added for the last 16 hours and cell proliferation was measured using [3H]-thymidine incorporation on a liquid scintillation counter (Beckman).

qPCR

Cells were immersed in mRNAIater solution (Qiagen) and kept at -80°C. Total mRNA was purified using the RNA Micro kit (QIAGEN). After reverse transcription into cDNA using a Reverse Transcription kit (Applied Biosystems), Quantitative RT-PCR was performed using the Fast SYBR green detection system from Invitrogen and verified specific primers for the genes IL-1 a, IL-1 b, CCL-3, Nox, G-CSF and TNF-a (QuantiTec, QIAGEN). Gene expression was normalized using the ribosomal protein L32 (RPL32) housekeeping gene and data are represented as fold differences by the 2-AACt-method, as previously published.

Statistical analysis

Differences between groups were examined for statistical significance by using a one-way analysis of variance (ANOVA) with Bonferroni's post-test correction. Example 1 :

IL-4 inhibits innate bacterial immunity in vivo. B6 (black lines) or Balb/c mice (grey lines) were infected with the indicated infectious doses (in brackets) of Salmonella typhimurium per os (Fig. 1A), Staphylococcus aureus intravenously (Fig. 1 B), or Listeria monocytogenes (LM) intravenously (Fig. 1 C). Wild-type (WT) Balb/c (black line) or H4ra^~'^~ Balb/c mice (grey line) were infected with 10⁴ cfu LM (Fig. 1 D). B6 or Rag^"'^" mice (dotted lines) mice were treated with PBS (dark line) or anti-IL-4 mAb (grey line), or anti-IL-4 mAb plus 1 A8 (dotted grey line) followed by infection with 10⁴ cfu LM (Fig. 1 E). Prior to infection with 10⁵ cfu LM (Fig. 1 F) or 10⁷ cfu Escherichia coli (Fig. 1 G), B6 mice received PBS (dotted black line), G-CSF (solid black line), G-CSF plus IL-4 (solid grey line), or G-CSF plus anti-Gr-1 mAb (dashed black line). Following administration of PBS (dotted black line), G-CSF (solid black line), or G-CSF plus either IL-4 (solid red line), IL-2 (solid light grey line), IL-7 (dashed light grey line) or IL-15 (dotted light grey line), mice were infected with 10⁵ cfu LM (Fig. 1 H). B6 (black lines) or Balb/c mice (grey lines) were infected with the indicated infectious doses (in brackets) of Escherichia coli intravenously (Fig. 1 1). p47^phox"'^" mice (Fig. 1 J) or CCR2^"'^" mice (Fig. 1 K) received PBS or anti-IL-4 mAb prior to infection with 10⁴ cfu LM.

Shown is percent overall survival starting the day of infection. Data are representative of three to four independent experiments, with a total of 9-12 animals per condition.

As shown in Fig. 2A, T cells, B cells and natural killer cells are dispensable for granulocyte- colony stimulating factor (G-CSF)-mediated immunity against Listeria monocytogenes (LM) (Fig. 2A). T and B cell-deficient Rag1^~'^~ mice received either PBS (dashed line), anti-NK1.1 mAb (dotted line) to deplete natural killer cells or anti-NK1 .1 mAb along with G-CSF (solid line) for four days, followed by infection with 1x10⁵ cfu LM. Shown is percent overall survival starting the day of infection. Neutrophils are essential for G-CSF-mediated immunity against LM (Fig. 2B). Prior to infection with 1x10⁵ cfu LM, B6 mice received PBS (solid black line), G- CSF (solid grey line) or G-CSF plus anti-Ly6G mAb clone 1A8 (dotted black line), the latter mAb targeting neutrophils. Shown is percent overall survival starting from the day of infection.

Upon per os infection with S. thyphimurium or intravenous infection with either S. aureus or LM, Balb/c mice were consistently more susceptible to these pathogens over a wide range of infectious doses, as compared to B6 mice (Fig. 1 , A-C). Thus, infection with 10⁴ colony- forming units (cfu) S. thyphimurium led to death of 50% of Balb/c mice by day 9 after infection, while B6 mice all survived this challenge (Fig. 1A). Similarly, unlike B6 mice that all survived an infection with 3x10³ cfu LM, 50% of Balb/c mice died within a week after infection with this dose of LM (Fig. 1 C). To investigate the hypothesis that the IL-4-driven T_H2 polarization in Balb/c mice might render these animals more susceptible towards infection, wild-type (WT) Balb/c versus Balb/c mice deficient in IL-4 receptor a (IL-4Ra), which together with common γ chain (y_c) forms the IL-4R, were infected. Strikingly, H4ra^~'^~ animals all survived a challenge with 1x10⁴ cfu LM, whereas their WT counterparts all died within a week upon infection (Fig. 1 D). Even in B6 mice, endogenous IL-4 levels negatively influenced immunity against LM, as evidenced by markedly improved survival of animals receiving a neutralizing monoclonal antibody (mAb) against IL-4 compared to controls (Fig. 1 E).

Granulocyte-colony stimulating factor (G-CSF) is abundantly produced during bacterial infection and is crucial for proliferation and mobilization of innate immune cells, notably neutrophils. In order to investigate whether IL-4-mediated inhibition of anti-infectious immunity could be overcome by boosting innate immune cells, mice were treated with long- acting G-CSF, consisting of G-CSF/anti-G-CSF mAb complexes (termed G-CSF from here onwards). In contrast to control mice succumbing to 1x10⁵ cfu LM by 4 days following infection, all animals receiving G-CSF survived this challenge (Fig. 1 F). The anti-listerial action of G-CSF was mediated by enhancing innate immune cells - and not T, B or natural killer cells - as demonstrated in recombinase-activating gene-deficient animals receiving a natural killer cell-depleting mAb (Fig. 2A). However, co-administration of long-acting IL-4, made of IL-4/anti-IL-4 mAb complexes (termed IL-4 from here onwards), antagonized the beneficial survival effect of G-CSF and led to death of animals within a few days upon LM infection (Fig. 1 F). The effect of IL-4 was comparable to depletion of neutrophils or neutrophils and monocytes using mAbs to Ly6G or Gr-1 respectively (Fig. 2B and Fig. 1 F). Similar to LM, B6 mice treated with G-CSF all survived a challenge with a lethal dose of E. coli, S. thyphimurium or S. aureus, whereas animals receiving concomitant IL-4 injections all died within a few days of infection (Fig. 1 G and data not shown). Unlike IL-4, other cytokines signaling via y_c, such as IL-2, IL-7 and IL-15, did not impact survival of G-CSF-treated B6 mice challenged with 1x10⁵ cfu LM (Fig. 1 H). Thus, endogenous and exogenous IL-4 signals dominantly inhibit innate host defense to both gram-positive and gram-negative bacteria. Example 2:

IL-4 reduces neutrophil effector functions as shown in Fig 3. B6 mice received PBS, IL-4, G- CSF or G-CSF plus IL-4, followed by infection with 1 x10⁵ cfu LM (Fig. 3A, B). Shown is bacterial load in spleen (Fig. 3A) and liver (Fig. 3B) on day 3 after infection. Mice were either treated as in Fig. 3A or left uninfected and ROS production assessed through oxidation of 1 ,2,3-dihydrorhodamine (DHR) to rhodamine by flow cytometry (Fig. 3C). Shown is ROS production in CD3^" CD1 1 b⁺ Ly6G⁺ neutrophils one day after infection with numbers referring to rhodamine-positive cells. Upon treatment as in Fig. 3A, in vitro killing of LM by purified CD3^" CD1 1 b⁺ Ly6G⁺ splenic neutrophils was determined after 2 hours of culture (Fig. 3D). Shown is percent kill relative to PBS. Data are representative of three independent experiments with, in Fig. 3A and B, a total of 9 mice per condition. P-values were determined using one-way ANOVA; ns, not significant, ***P<0.001.

Paralleling the survival experiments mentioned above, mice treated with G-CSF cleared LM from spleen and liver within three days following infection, whereas animals receiving G-CSF plus IL-4 showed similar bacterial titers in these organs as control mice (Fig. 3, A and B). Interestingly, animals injected with IL-4 alone had even higher titers of LM in the spleen compared to controls (Fig. 3A). Moreover, production of reactive oxygen species (ROS), a major effector mechanism of neutrophils, was markedly decreased in purified CD1 1 b⁺ Ly6G⁺ neutrophils from mice given G-CSF plus IL-4 or IL-4 alone in comparison to PBS- or G-CSF- treated animals, both with and without LM infection (Fig. 3C). Consequently, purified CD1 1 b⁺ Ly6G⁺ neutrophils of animals receiving IL-4 or G-CSF plus IL-4 were inefficient in killing LM upon phagocytosis (Fig. 3D). Collectively, IL-4 signals suppress neutrophil effector functions.

Example 3:

Fig. 4, 5 and 6 show that IL-4 impedes neutrophil egress from BM in vivo. Mice received PBS, IL-4, G-CSF or G-CSF plus IL-4, followed by harvesting of organs 16 hours after the final injection (Fig. 4 A, B). Dot plots display frequencies (Fig. 4A) and bars represent total counts (Fig. 4B) of CD3^" CD1 1 b⁺ Ly6G⁺ neutrophils in BM, blood and spleen. Animals were treated with PBS or IL-4 and subsequently left uninfected or challenged with 1 x10⁵ cfu LM (Fig. 4 C, D). Shown are frequencies (Fig. 4 C) and total counts (Fig. 4 D) of CD3^" CD1 1 b⁺ Ly6G⁺ neutrophils in BM, blood and spleen 16 hours following LM infection. Immune cell- lineage (Lin)-depleted BM cells of WT (CD45.1 ; Fig. 6E, black) and //4ra^_/" (CD45.2; Fig. 4 E, dark grey) mice were mixed at a 1 :1 ratio and adoptively transferred to lethally irradiated CD45.2⁺ WT hosts (top panel). Following reconstitution, BM chimeric mice were injected with PBS, G-CSF or G-CSF plus IL-4, followed by assessment of CD3^" CD1 1 b⁺ Ly6G⁺ blood neutrophils by flow cytometry 16 hours after the final injection (bottom panel). Data are representative of at least five (for Fig. 4 A-D) or two (for Fig. 4E) independent experiments. P-values were calculated using one-way ANOVA; ns, not significant, *P<0.05; **P<0.01 ; ****P<0.0001 .

As G-CSF is known to mobilize innate immune cells from the bone marrow (BM) to the circulation and subsequently to the spleen, these compartments were analyzed. In comparison to PBS, treatment with G-CSF led to an expansion of CD1 1 b⁺ Ly6G⁺ neutrophils in BM, followed by a prominent increase of these cells in the blood and spleen of these mice (Fig. 4, A and B). Strikingly, co-administration of G-CSF plus IL-4 caused a comparable increase of neutrophils in the BM, however this effect was barely evident in blood and spleen where percentages and cell counts were only slightly higher than in controls (Fig. 4, A and B). Notably, mice receiving IL-4 alone showed a tendency towards even lower percentages and counts of neutrophils in blood and spleen compared to PBS (Fig. 4, A and B).

As expected, infection with 1 x10⁵ cfu LM led to an egress of neutrophils into the blood stream followed by increased neutrophil count in the spleen of these mice (Fig. 4 C and D). However, surprisingly, animals receiving IL-4 prior to infection with LM failed to efficiently recruit neutrophils into the circulation and spleen (Fig. 4 C and D).

To investigate whether IL-4 acted directly or indirectly on BM neutrophils, BM chimeras carrying a 1 : 1 mixture of H4ra^~'^~ and WT immune cells were generated by reconstituting lethally irradiated WT mice with a 1 : 1 mixture of immune lineage-depleted CD45.2 (Ly5.2)- congenic H4ra^~'^~ and CD45.1 (Ly5.1 )-congenic WT BM cells (Fig. 6E, top). Following reconstitution of the immune system in these BM chimeras, frequencies of CD1 1 b⁺ Ly6G⁺ neutrophils were comparable for both CD45.1 ^" H4ra^~'^~ and CD45.1 ⁺ WT cells in control animals (Fig. 6E, bottom left panel). Upon administration of G-CSF, H4ra^~'^~ neutrophils expanded about 50% more than their WT counterparts due to endogenously produced IL-4 impeding the egress of WT neutrophils from the BM (Fig. 4 E, bottom middle panel). This effect was even more pronounced in BM chimeras receiving G-CSF plus IL-4, in which WT neutrophils were greatly outnumbered by their H4ra^~'^~ counterparts, the latter of which accounted for over 90% of the blood neutrophils (Fig. 4E, bottom right panel). These data show that IL-4 directly binds to BM neutrophils to inhibit their recruitment into the circulation.

Example 4:

Fig. 5, 6 and 7 show that IL-4 suppresses, via p38 MAP kinase, neutrophil migration to CXCR(CXC chemokine receptor)2-binding chemokines. Purified CD3^" CD1 1 b⁺ Ly6G⁺ murine neutrophils were treated with PBS, IL-4 or IL-4 plus anti-IL-4Ra mAb, followed by assessment of migration towards CXCL1 (chemokine CXC-motif ligand 1 , Fig. 5A, left) and CXCL2 (Fig. 5A, right). Fig. 5B shows the chemotaxis of purified murine neutrophils as in Fig. 5A following administration of PBS, G-CSF or G-CSF plus either IL-2, IL-4, IL-7 or IL-15. CXCL-1 -induced migration of purified murine neutrophils was analyzed upon treatment with PBS, IL-4 or IL-4 plus p38 inhibitor SB203580 (Fig. 6). Animals received PBS, G-CSF, G- CSF plus IL-4 or G-CSF plus IL-4 and SB203580. Shown are dot plots (Fig. 6) and frequencies (Fig. 7) of CD3^" CD1 1 b⁺ Ly6G⁺ neutrophils from BM and blood 16 hours after the final injection. Purified human neutrophils were treated as in Fig. 8C, followed by assessment of migration towards CXCL8 (Fig. 7). Shown are percent migrating cells relative to PBS. Data are representative of at least three independent experiments. P-values were determined using one-way ANOVA; ns, not significant, *P<0.05; **P<0.01 ; ***P<0.001 .

Migration of murine neutrophils towards CXCR2-binding chemokines requires the PI3K but not the p38 MAP kinase pathway (Fig. 8). Purified CD3^" CD1 1 b⁺ Ly6G⁺ murine neutrophils were treated with PBS, the p38 MAP kinase inhibitor SB203580 or the PI3K inhibitor LY294002, followed by assessment of migration towards CXCL1 . Shown are percent migrating cells relative to PBS.

G-CSF mobilizes BM neutrophils into the blood via induction of CXCR2 -binding chemokines, notably CXCL1 and CXCL2 in mice or their human homolog CXCL8 (also known as IL-8). IL- 4 significantly decreased the migration of purified murine neutrophils towards CXCL1 and CXCL2 in an IL-4Ra-dependent manner in vitro (Fig. 5). However, unlike IL-4, other y_c cytokines or T helper 2 cytokines, like IL-5 or IL-13 did not influence chemotaxis of murine neutrophils towards CXCL1 and CXCL2 (Fig. 5), thus corroborating the survival data observed in mice treated with G-CSF plus these _c cytokines (Fig. 1 F).

Neutrophil chemotaxis towards CXCR2-binding chemokines depends on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Activation of the p38 mitogen-activated protein (MAP) kinase pathway by end target chemoattractants is able to suppress PI3K/Akt- mediated migration. As IL-4 has been shown in human neutrophils to activate p38 (Fig. 9), this pathway was assessed by using the selective p38 blocking agent SB203580. Chemotaxis of murine neutrophils towards CXCR2-binding chemokines was p38- independent, but relied on PI3K/Akt, as demonstrated by using the selective PI3K blocker LY294002 (Fig. 8). In IL-4-treated neutrophils migrating towards CXCL1 , use of SB203580 reconstituted their chemotactic capacity to levels comparable with controls (Fig. 6). Even more strikingly, administration of SB203580 to animals receiving G-CSF plus IL-4 completely reversed the in vivo deficiency induced by IL-4, thus resulting in percentages and cell counts of CD1 1 b⁺ Ly6G⁺ neutrophils in these animals that were comparable to G-CSF alone (Fig. 6, and Fig. 7). Further bacterial clearance from spleens was restored to levels seen in G-CSF pre-treated animals when administering SB203580 to infected animals pre-treated with G- CSF plus IL-4 (Fig. 10). Similar to results seen with murine neutrophils, migration of purified human neutrophils to CXCL8 was also significantly inhibited by the addition of IL-4 in vitro and this IL-4-mediated effect was reversed upon treatment of neutrophils with SB203580 (Fig. 7).

Example 5:

Fig 12 shows in vivo half-life of free IL-4, G-CSF and IL-4 or G-CSF complexed to a specific monoclonal antibody, (top) Wild-type mice were i.p. injected with 1 ^g or 7^g IL-4 or 1 ^g IL-4 complexed to "^g anti-IL-4. At indicated time points serum was harvested from the mice and bioactive IL-4 detected by measuring proliferation on the IL-4 sensitive cell line CTLL-2. (bottom). Analog the half-life of ^ μg G-CSF in free form or complexed to 6 g anti- G-CSF monoclonal antibody was measured on the G-CSF sensitive cell line NFS-60. Data is representative of two independent experiments.

Fig. 13 shows the neutralisation of IL-4 by or G-CSF with IL-4 or G-CSF specific monoclonal antibodies in vitro and in vivo, (top left) The inhibition of IL-4-induced proliferation on CTLL-2 cells by increasing amounts of anti-IL-4 (1 1 B1 1 ) and (bottom left) of the G-CSF-induced proliferation on NFS-60 cells using a specific anti-G-CSF monoclonal antibody was tested in vitro, (top right) Using a standard IL-4 ELISPOT assay we tested IL-4 production in the spleen of untreated (PBS), LM infected (LM), LM-infected mice receiving anti-IL-4 before infection (LM+a-IL-4), IL-4 treated or G-CSF treated wild-type mice.

Fig. 14 shows overall survival of neutrophil elastase (Elane) knockout mice or CXCR2 knockout mice following LM or Salmonella typhimurium infection. Prior to infection with 10⁴ cfu LM or 10⁴ cfu Salmonella, wild-type, Elane knockout or CXCR2 knockout mice were treated with PBS, anti-IL-4 or IL-4. Shown is percent overall survival starting the day of infection. Data is representative of two independent experiments.

Fig. 15 demonstrates that neutrophils express functional IL-4 receptors. (A) Common gamma chain, IL-4 receptor alpha and IL-13 receptor a1 was assessed by qPCR from RNA isolated from purified blood neutrophils, monocytes and T cells. (B) Protein expression of the common gamma chain (CD132), IL-4Ra and IL-13 receptor a1 (CD132a1 ) on mouse neutrophils given as histograms (left) and bars from two experiments showing mean florescence intensities (MFI) of the respective receptor. Grey shaded areas in histograms represent isotype controls. (C) STAT6 phosphorylation in unstimulated (PBS) or IL-4 stimulated Gr-1 high neutrophils shown as histogram with isotype control or as bar graphs displaying MFI following stimulation of neutrophils with low doses (5 ng/ml), medium dose (50 ng/ml) or high doses (500 ng/ml) of IL-4 or IL-13. Unstimulated cells (PBS) served as controls. Data is representative of two independent experiments.

Fig. 16 shows increase RNA expression of different effector molecule in \l-4ra knockout mice as compared to wild-type mice following LM infection. Neutrophils were purified from (A) bone marrow and (B) spleen of wild-type mice and ll-4ra knockout mice 16h after LM infection. RNA was isolated and transcribed into cDNA. Displayed is the relative expression to Rpl32 of the respective gene as quantified by qPCR.

Fig. 17 demonstrates inhibition of migration of human neutrophils by IL-4. (A) The migration of human neutrophils towards a fixed concentration of IL-8 in the presence of increasing amounts of IL-4 was tested. (B) The migration of human neutrophils towards IL-8 alone or in the presence of IL-4 was tested at indicated time points. (C) Migration of human neutrophils using increasing amounts of IL-8 alone or in the presence of IL-4 or IL-4 plus the specific inhibitor SB203580. (D) Migration of human neutrophils towards increasing concentrations of fMLP without or with IL-4.

In Fig. 18 the specific inhibitor SB203580 shows potent inhibition of IL-4 induced p38 phosphorylation in vivo. The specific p38 inhibitor SB203580 was i.v. injected 15 min. before i.v. injection of 7^g IL-4. 5 min after IL-4 injection mice were sacrificed, spleens fixed in paraformaldehyde and stained for p38 phosphorylation. Untreated mice (PBS) and mice without inhibitor (IL-4) served as controls.

Inhibition of Imiquimod-induced skin inflammation by IL-4 (Fig. 19). Wild-type mice were treated for 6 consecutive days with imiquimod (IMQ), IMQ plus IL-4, IMQ plus IL-4 and SB203580, IMQ plus anti-IL-4, IMQ plus anti-IL-4 plus anti-Ly6G or IMQ plus anti-Ly6G on the right ear, whereas Vaseline treated mice served as controls. The difference in ear thickness was determined (A) daily or on (B) day 6. (C) Representative haematoxylin eosin (HE) stain, neutrophil stain (1A8), or myeloperoxidase stain (MPO) of the ears of mice treated with indicated stimuli as in A. (D) Bar graphs displaying the quantification of 1A8- positive or MPO-positive cells in one square millimeter of ears following treatment as in A. Data is representative of two independent experiments.

IL-4 reduces neutrophil recruitment into air pouches following stimulation with monosodium urate crystals (MSU) or IL- β (Fig. 20). (A) Neutrophil infiltration into the air pouch 16 hours after MSU injection (2 mg) of wild-type mice pre-treated for three days with IL-4 complexes. PBS treated mice and mice without MSU injection (control) served as controls. (B) Neutrophil migration 12 hours after I L-1 β injection (10 ng) of mice as pre-treated in A.

The xenotransfer model in Fig. 21 shows IL-4 mediated inhibition of human neutrophils in IMQ-induced psoriasis model. (A) Treatment of wild-type mice with 1A8 was started one day before IMQ treatment for 6 days. Human neutrophils were adoptively transferred daily from day 4 to 6 alone or together with local administration of IL-4 or IL-4 plus SB203580. Ear thickness of mice was monitored daily or (B) on day 7. Data is representative of two independent experiments. Collectively, IL-4 inhibits several neutrophil functions, including ROS production and killing of bacteria as well as recruitment of neutrophils from BM due to its p38-dependent blocking chemotaxis to CXCR2-binding chemokines a. The data show that IL-4 impaired the migration of myeloid and T cells to the pleural cavity in a murine pleuritis model. Interestingly, IL-4 leads to local proliferation of tissue-resident macrophages, rather than recruitment of monocytes from the blood.

In considering the implications of these findings, it is worth noting that AD patients are frequently infected with S. aureus, both on their lesional and non-lesional skin. In fact, 80- 100% of AD patients are colonized with S. aureus on non-lesional skin compared to only 5- 30% of healthy individuals. Usually, cutaneous infection with S. aureus efficiently induces neutrophil recruitment to the skin. However, in the skin of AD patients infected with S. aureus there is a striking absence of neutrophils, which suggests that neutrophil migration to the skin of AD patients is impaired. Moreover, phagocytosis and ROS production of neutrophils from AD patients was found to be deficient. The data would indicate that AD patients suffer from suppressed neutrophil responses, thus explaining their susceptibility towards S. aureus and other bacterial and viral pathogens. It will be of interest to see whether IL-4-targeted approaches, currently tested in clinical trials for allergic asthma, could improve immunity towards these pathogens in patients with concomitant AD.

Claims

1. An inhibitor of IL-4 or the IL-4 receptor for use in preventing or treating neutropenia, wherein said inhibitor is capable of binding to and thereby inhibiting the biological effect of IL-4/IL-4 receptor interaction.

2. The inhibitor according to claim 1 , wherein said inhibitor is selected from

an antibody specific for IL-4 or the IL-4 receptor, an antibody fragment specific for IL-4 or the IL-4 receptor, or an antibody-like molecule specific for IL-4 or the IL-4 receptor, an oligopeptide of 6 to 30 amino acids, or a nucleic acid aptamer molecule of 10 to 75 nucleotides in length, or

a soluble polypeptide comprising a contiguous amino acid sequence of at least 30 amino acid residues comprised within the amino acid sequence of IL-4-receptor or IL-4.

3. The inhibitor according to any one of the previous claims, wherein said inhibitor is selected from the group comprised of:

a polypeptide comprising the amino acid sequence SEQ ID 1 (mAb 6-2 light chain), SEQ ID 2 (mAb 6-2 heavy chain), SEQ ID 3 (mAb 12B5 light chain), SEQ ID 4 (mAb 12B5 heavy chain), SEQ ID 5 (mAb 27A1 light chain), SEQ ID 6 (mAb 27A1 heavy chain), SEQ ID 7 (mAb 5A1 light chain), SEQ ID 8 (mAb 5A1 ), SEQ ID 9 (mAb 63 light chain), SEQ ID 10 (mAb 63 heavy chain), SEQ ID 1 1 (mAb 1 B7 light chain), SEQ ID 12 (mAb 1 B7 heavy chain) or SEQ ID 13 (soluble interleukin-4 receptor),

pascolizumab,

a modified IL-4 comprising the amino acid substitutions R121 D and Y124D, a modified IL-4 comprising the amino acid substitutions R121 D, Y124D and S125D,

a recombinant human IL-4 variant that is an inhibitor of the IL-4 receptor, and a genetically engineered soluble human IL-4 receptor.

4. An inhibitor of the gene expression of IL-4 or the IL-4 receptor for use in preventing or treating neutropenia, comprising

- a single-stranded or double-stranded interfering ribonucleic acid oligomer or precursor thereof, comprising a sequence tract complementary to an mRNA molecule encoding IL-4 or the IL-4 receptor, or - a single-stranded or double-stranded antisense ribonucleic or deoxyribonucleic acid comprising a sequence complementary to a regulatory region of a gene encoding IL-4 or the IL-4 receptor, or

an expression vector, comprising a sequence encoding said interfering ribonucleic acid oligomer or precursor thereof.

5. An inhibitor of p38 MAP kinase, wherein said inhibitor is capable of binding to and inhibiting the biological effect of ρ38-α, ρ38-β, ρ38-γ and/or ρ38-δ, for use in preventing or treating neutropenia, wherein said inhibitor is selected from the group comprising an antibody, an antibody fragment, an antibody-like molecule, an oligopeptide of 6 to 30 amino acids, a nucleic acid aptamer molecule of 10 to 75 nucleotides in length.

6. An inhibitor of gene expression of p38 MAP kinase for use in a method for preventing or treating neutropenia, selected from

- a single-stranded or double-stranded interfering ribonucleic acid oligomer or precursor thereof, comprising a sequence tract complementary to an mRNA molecule encoding ρ38-α, ρ38-β, ρ38-γ and/or ρ38-δ, or

- a single-stranded or double-stranded antisense ribonucleic or deoxyribonucleic acid, comprising sequences complementary to a regulatory region of a gene encoding ρ38-α, ρ38-β, ρ38-γ and/or ρ38-δ, or

7. A compound for use in preventing or treating neutropenia selected from the group comprised of

- 6-(2,4-difluorophenoxy)-2-[[3-hydroxy-1 -(2-hydroxyethyl)propyl]amino]-8-methyl- pyrido[2,3-d]pyrimidin-7-one;

- 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1 H-imidazol-5-yl]pyridine ;

- 4-[4-(4-fluorophenyl)-5-(4-pyridyl)-1 H-imidazol-2-yl]phenol ;

- 4-[2-(4-chlorophenyl)-4-(4-fluorophenyl)-1 H-imidazol-5-yl]pyridine

- 4-[4-(4-fluorophenyl)-2-(4-nitrophenyl)-1 H-imidazol-5-yl]pyridine;;

1-[5-tert-butyl-2-(p-tolyl)pyrazol-3-yl]-3-[4-(2-morpholinoethoxy)-1 -naphthyl]urea;

- 6-(N-carbamoyl-2,6-difluoro-anilino)-2-(2,4-difluorophenyl)pyridine-3-carboxamide; - 5-[2-tert-butyl-4-(4-fluorophenyl)-1 H-imidazol-5-yl]-3-(2,2-dimethylpropyl)imidazo[4,5- b]pyridin-2-amine;

- 3H-imidazo[4,5-b]pyridin-2-amine, 5-[2-(1 ,1 -dimethylethyl)-4-(4-fluorophenyl)-1 H- imidazol-5-yl]-3-(2,2-dimethylpropyl)-, methanesulfonate;

- 3H-imidazo[4,5-b]pyridin-2-amine, 5-[2-(1 ,1 -dimethylethyl)-4-(4-fluorophenyl)-1 H- imidazol-5-yl]-3-(2,2-dimethylpropyl)-, hydrochloride;

- 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenyl)sulfanyl-pyrimido[1 ,6-b]pyridazin-6-one;

- vinorelbine;

- 3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2-oxo-1-pyridyl]-N,4-dimethyl- benzamide;

- 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1 ,2- f][1 ,2,4]triazine-6-carboxamide;

- 8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-2-{[2-hydroxy-1- (hydroxymethyl)ethyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one;

- 6-[5-(cyclopropylcarbamoyl)-3-fluoro-2-methylphenyl]-N-(2,2-dimethylpropyl)pyridine- 3- carboxamide;

- 2-[6-chloro-5-[[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazin-1-yl]carbonyl]-1- methyl-1 H-indol-3-yl]-N,N-dimethyl-2-oxoacetamide;

- 1 ,3, 10-trihydroxy-5-[(2E,6E)-3,7, 1 1 -trimethyldodeca-2,6, 10-trienyl]-1 1 H- benzo[b][1 ,4]benzodiazepin-6-one;

- 4-ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole;

- 2'-amino-3'-methoxyflavone;

- trans-4-[4-(4-fluorophenyl)-5-(2-me thoxy-4-pyrimidinyl)-1 H-imidazol-1 - yl]cyclohexanol;

- 2'-fluoro-N-(4-hydroxyphenyl)-[1 ,1 '-b iphenyl]-4-butanamide;

- (2-methylphenyl)-[4-[(2-amino-4-bro mophenyl)amino]-2-chlorophenyl]methanone;

- 6-(4-fluorophenyl)-2,3-dihydro-5-(4 -pyridinyl)imidazo[2, 1 -b]thiazole dihydrochloride;

- 5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl)thio]-6H-pyrimido[1 ,6-b]pyridazin-6-one; - N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide;

- 6-Chloro-5-[[(2R,5S)-4-[(4-fluorophenyl)methyl]-2,5-dimethyl-1 -piperazinyl]carbonyl]- N,N, 1-trimethyl-a-oxo-1 H-lndole-3-acetamide hydrochloride;

- 4-[4-(4-fluorophenyl)-1 -(3-phenylpropyl)-5-(4-pyridinyl)-1 H-imidazol-2-yl]-3-butyn-1-ol;

1 H-indole-5-carboxamide;

- 6-chloro-5-[[4-[(4-fluorophenyl)met hyl]-1 -piperidinyl]carbonyl-N,N, 1 -trimethyl-a-oxo-1

H-indole-3-acetamide;

1-[2-methoxy-4-(methylthio)benzoyl] -4-(phenylmethyl)piperidine;

- 6-(4-fluorophenyl)-5-(4-pyridyl)-2,3-dihydroimidazo[2, 1-b]-thiazole;

- 5-(2-aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1 -(4-piperidinyl)imidazole

trihydrochloride.

8. A pharmaceutical composition for use in preventing or treating neutropenia,

comprising an inhibitor or compound according to any one of claims 1 to 7.

9. A pharmaceutical composition comprising an inhibitor or compound according to any one of claims 1 to 7 in combination with a therapeutically active amount of the granulocyte colony-stimulating factor.

10. A dosage form for use in preventing of treating neutropenia, comprising an inhibitor or compound according to any one of claims 1 to 7.

1 1 . A method for manufacture a medicament use in a method for preventing or treating neutropenia, comprising the use of an inhibitor or compound according to any one of claims 1 to 7.

12. A method for preventing or treating neutropenia, comprising the administration of an inhibitor or compound according to any one of claims 1 to 7 to a patient in need thereof.

13. An inhibitor or compound according to any one of claims 1 to 7 for use in preventing or treating atopic dermatitis.

14. An inhibitor or compound according to any one of claims 1 to 7 for use in a method for treating atopic dermatitis in a patient infected with Staphylococcus aureus.