Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

• the present invention pertains to the treatment of diseases associated with a dysregulated immune response such as auto immune disorders, inflammatory diseases or pathological immune responses as adverse effects of medical treatments.
• a dysregulated immune response such as auto immune disorders, inflammatory diseases or pathological immune responses as adverse effects of medical treatments.
• the invention pro- vides a combined use of inhibitors of Receptor-interacting serine/threonine-protein kinase 1 (RIPKl) and inhibitors of Inhibitor of KB (IKB) Kinase (IKK) in subjects suffering from such disorders.
• the invention provides such inhibitoiy compounds and their combinations for use in medical applications, as well as pharmaceutical compositions comprising the compounds of the invention.
• NF-kB is a heterodimeric transcription factor that regulates the expression of multiple in- flammatory genes.
• NF-kB has been implicated in many pathophysiologic processes includ- ing angiogenesis (Koch et al, Nature 1995, 376, 517-519), atherosclerosis (Brand et al. Clin Inv. 1996, 97, 1715-1722), endotoxic shock and sepsis (Bohrer et al, J. Clin. Inv. 1997, 200972-985), inflammatoiy bowel disease (Panes et al, Am J Physiol.
• IKKs The IKB kinases
• LPS lipopolysaccharide
• IL-I b IL-I b
• CD3/CD28 antigen presentation
• CD40L CD40L
• FasL viral infection
• oxida- tive stress oxida- tive stress
• the IKK complex composed of the regulatory subunit NEMO/IKKg and the catalytic subu- nits IKKa/IKKi and IKKb/IKK2 is essential for activation of NF-kB.
• the IKK/NF-kB signal- i is a key regulator of inflammatory responses. Therefore, inhibitors of IKK/NF- KB signalling have a great potential for the treatment of inflammatory diseases.
• the present invention seeks to provide novel therapeutic approaches to tackle im- mune related disorders via inhibition of IKK/NF-kB signaling, but overcoming known ad- verse effects of such treatments.
• a Receptor-interacting serine/threonine- protein kinase 1 (RIPKi) inhibitor for use in the treatment or prevention of a disease associ- ated with a pathological or deregulated immune response, such as an increased Interleukin- ib (IL-ib) release or systemic neutrophilia, in a subject.
• RIPKi Receptor-interacting serine/threonine- protein kinase 1
• RIPKi kinase activity Inhibition of RIPKi kinase activity either genetically (using knock-in mice expressing ki- nase-inactive RIPKi D138N) or pharmacologically (using Necrostatin-i, a chemical inhibi- tor of RIPKi) strongly diminished the LPS-induced production of IL-ib by macrophages treated with two different IKK inhibitors.
• the disease associated with an increased IL-ib re- lease or or systemic neutrophilia is a side effect or adverse effect caused by a treatment of vith an Inhibitor of KB (IKB) Kinase (IKK)/Nuclear Factor KB (NFKB) - signal- ing inhibitor.
• IKB Inhibitor of KB
• IKK Inhibitor of KB
• NFKB Nuclear Factor KB
• RIPKi inhibitor or“inhibitor of RIPKi” or“antagonist of RIPKi” or any similar expressions shall in context of the present invention encompass any compound or combina- tion of compounds that have an activity as a modulator of the expression, function and/or stability of RIPKi, or of a variant of RIPKi.
• a modulator is preferably an inhibitor/ antagonist.
• receptor interacting serine/threonine kinase l or“RIPKi” pertains to a human gene encoding for a protein according to the ami- no acid sequence shown in SEQ ID NO:i.
• RIPKi is also known as "receptor (TNFRSF)- interacting serine-threonine kinase l” or “receptor-interacting protein kinase l” (RIP) (HUGO Gene Nomenclature Committee symbol: HGNC: 10019, database version of Novem- ber 2017).
• the human RIPKi gene is located 6p25.2, homologs are known from mouse (MGI:IO8212; NCBI Gene: 19766) and rat (Rat Genome Database (RGD) ID: 1310158).
• RIPKi-protein or“protein of RIPKi” as used in context of the herein disclosed invention shall pertain to a protein (such as a full-length protein, fusion protein or partial protein) comprising a sequence as shown in SEQ ID NO: 1.
• the terms shall also refer to a protein comprising the amino acid sequence according to SEQ ID NO: 1 with any protein modifications.
• Such protein modifications preferably do not alter the amino acid sequence of the polypeptide chain, but constitute a functional group, which is conjugated to the basic amino acid polymer chain.
• Protein modifications in context of the invention may be selected from a conjugation of additional amino acid sequences to the RIPKi amino acid chain, such as ubiquitination, sumolation, neddylation, or similar small protein conjugates.
• pro- tein modifications include, but are not limited to, glycosylation, methylation, lipid- conjugation, or other natural or artificial post-translational modifications known to the skilled person.
• the terms“protein of a variant of RIPKi” and the like, shall have the corre- sponding meaning with respect to a variant of RIPKi.
• RIPKi-mRNA or“mRNA of RIPKi” as used in context of the herein disclosed invention shall pertain to a messenger ribonucleic acid (such as a full-length mRNA, fusion mRNA or partial mRNA, and/or splice-variants thereof) comprising a region encoding for an amino acid sequence as shown in SEQ ID NO: 1.
• the terms shall also refer to an mRNA comprising a region encoding for the amino acid sequence according to SEQ ID NO: 1 with any codon or nucleotide modifications. Such modifications preferably would not alter the sequence of the encoded polypeptide chain.
• a variant of RIPKi is, in some embodiments, a protein comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, preferably at least 80% such as at least 90% sequence identity to SEQ ID NO: 1, and most preferably at least 95% (such as at least 98%) sequence identity to SEQ ID NO: 1 (the human RIPKi amino acid sequence).
• the variant of RIPKi comprises an amino acid sequence with at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1.
• amino acid identity amino acid identity
• the term“subject” or“patient” preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human, for example a human patient.
• the subject of the invention may be at danger of suffering from a dysregulated immune response, preferably induced or caused by the administration of a IKK inhibitor as defined herein elsewhere.
• A“modulator of expression, function and/or stability of RIPKi or IKK, or of a variant of RIPKi or IKK”, or grammatically similar expressions, in context of the invention may be any compound that affects, for example when an inhibitor/ antagonist impairs or interferes with, the expression, function and/or stability of RIPKi or IKK respectively, or of a variant of these targets, in particular the expression, function and/or stability of protein of RIPKi or IKK or their variant, and/or the expression, function and/or stability of mRNA of RIPKi or IKK or their variant.
• the RIPKi inhibitor is selected from a small molecule, a polypeptide, peptide, glycoprotein, a peptide-mimetic, an antigen binding protein (ABP) (for example, an antibody, antibody-like molecule or other antigen binding derivative, or an or antigen binding fragment thereof), a nucleic acid such as a DNA or RNA, for example an antisense or inhibitory DNA or RNA, a ribozyme, an RNA or DNA aptamer, RNAi, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA), a genetic construct for targeted gene editing, such as a CRISPR/Cas9 construct and/or a guide nucleic acid (gRNA or gDNA) and/or tracrRNA.
• ABSP antigen binding protein
• a nucleic acid such as a DNA or RNA, for example an antisense or inhibitory DNA or RNA, a ribozyme, an
• the modulator of expression, function and/or stability of RIPKi, or of a variant of RIPKi is an anti-sense nucleotide molecule such as described in detail herein below, more preferably one that binds to, such as specifically binds to, a nucle- ic acid that encodes or regulates the expression of RIPKi, or of a variant of RIPKi, or alter- natively more preferably one that binds to, such as specifically bind to, a nucleic acid that encodes or (regulates the expression of a gene that controls the expression, function and/or stability of) RIPKi, or of a variant of RIPKi.
• the terms“inhibitor of RIPKi expression” and the like shall relate to any of the herein disclosed modulators (for example, the antigen binding constructs or anti-sense molecules described herein), which has an antagonistic activity toward the expression of an RIPKi protein, such that it impairs, suppresses, reduces and/or lowers the expression of an RIPKi protein such as may be determined by measuring an amount (or change in an amount) of RIPKi protein or RIPKi mRNA.
• the term“expression” means in this context the cellular process of tran- scribing a gene into an mRNA and the following translation of the mRNA into a protein.
• Gene expression therefore may refer only to the generation of mRNA, irrespectively from the fate of the so produced mRNA, or alternatively/ additionally to the translation of the ex- pressed mRNA into a protein.
• protein expression on the other hand shall refer to the complete cellular process of synthesis of proteins.
• an in- hibiting modulator of the invention such as an anti-sense molecule, may bind to the RIPKi gene or mRNA and reduce transcription and/or translation or the RIPKi mRNA.
• the terms “inhibitor of expression of a variant of RIPKi” and the like, shall have the corresponding meaning with respect to a variant of RIPKi.
• inhibitor of RIPKistability shall refer to any of the herein disclosed modulators (for ex- ample, the antigen binding constructs or anti-sense molecules described herein), which has a negative activity towards the stability of an RIPKi protein.
• an“inhibitor of RIPKi function” and the like shall refer to any of the herein disclosed modulators (for ex- ample, the antigen binding constructs or anti-sense molecules described herein) that im- pairs, such as induces a decrease or reduction in the amount or rate of one or more activi- ties of RIPKi protein or mRNA (for example, by impairing the expression and/or stability of RIPKi protein or mRNA), such as one or more of those activities described herein, for ex- ample, the kinase activity of RIPKi.
• Such an inhibiting modulator can act directly, for example, by binding to RIPKi and de- creasing the amount or rate of one or more of the properties of RIPKi such as its expres- sion, function and/or stability.
• An RIPKi antagonist or inhibitor can also decrease the amount or rate of RIPKi function or activity by impairing its expression, stability, for ex- ample, by binding to RIPKi protein or mRNA and modifying it, such as by removal or addi- tion of a moiety, or altering its three-dimensional conformation; and by binding to RIPKi protein or mRNA and reducing its stability or conformational integrity.
• An RIPKi antago- nist or inhibitor can also act indirectly, for example, by binding to a regulatory molecule or gene region to modulate regulatory protein or gene region function and affect a decrease in the amount or rate of RIPKi expression, function and/or stability, in particular by impair- ing one or more activity of RIPKi protein or mRNA (such as by changing the amount or rate of expression and/or stability of RIPKi protein or mRNA).
• an RIPKi inhibitor or an- tagonist can act by any mechanisms that impair, such as result in a decrease in, the amount or rate of RIPKi expression, function and/or stability.
• the RIPKi inhibitor is a corn- pound that only affects and impairs the kinase enzymatic activity or function of RIPKi without interfering with RIPKi protein expression or protein stability.
• kinase activity refers to a phosphoiylation of a substrate including RIPKi by RIPKi protein.
• the present invention in some preferred embodiments pertains to compounds which selectively bind to and impair kinase enzymatic activity or the autophosphorylation of RIPKi or the acquisition of conformational changes induced by au- tophosphorylation, such as small molecular compounds, for example kinase inhibitors.
• Preferred RIPKi inhibitors of the invention are small molecular compounds. Such corn- pounds are preferably selected from GSK2982772, necrostatin-i (5-(iH-indoi-3-ylmethyl)- thioxo-4-imidazolidinone, 5-(indoi-3-ylmethyl)-3-methyl-2-thio-hydantoin) and necrostatin-i stable (5-((7-chloro-i H-indol-3-yl)methyl)-3-methyl-2,4- imidazolidinedione, 5-((7-chloro-iH-indoi-3-yl)methyl)-3-methylimidazolidine-2,4-dione).
• any other known RIPKi inhibitor may be used in context of the invention.
• the subject or patient subjected to the treatments or uses of the invention in preferred embodiments is suffering from a disease associated with a dysregulated immune response such as auto immune disorders, inflammatoiy diseases or pathological immune responses as adverse effects of medical treatments.
• Treatment or pre- vention in some embodiments comprises the administration of a therapeutically effective amount of the RIPKi inhibitor to the subject.
• Such an administration may be a sequential or concomitant administration of a therapeutically effective amount of both the RIPKi inhibi- tor and an IKK/NFKB signaling inhibitor to the subject.
• the term“therapeutically effective amount” means that amount of a corn- pound or combination that will elicit the biological or medical response of a tissue, system, animal or human subject that is being sought, for instance, by a researcher or clinician, in accordance with the herein disclosed invention.
• therapeutically effective amount means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or ame lioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
• the effective amount of the compounds or combinations adminis- tered at least once to a subject in need of treatment with a RIPKi and/or IKK/NFKB signal- ing inhibitor is, typically, between about 0.01 mg/kg and about 500 mg/kg per administra- tion, or about 0.01 mg/kg and about 100 mg/kg per administration such as between about 1 mg/kg and about 10 mg/kg per administration.
• the effective amount administered at least once to said subject of a RIPKi and/or IKK/NFKB signaling inhibitor is between about 0.01 mg/kg and about 0.1 mg/kg per administration, between about 0.1 mg/kg and about 1 mg/kg per administration, between about 1 mg/kg and about 5 mg/kg per administration, between about 5 mg/kg and about 10 mg/kg per administration, be- tween about 10 mg/kg and about 50 mg/kg per administration, or between about 50 mg/kg and about 100 mg/kg per administration.
• a RIPKi and/or IKK/NFKB signaling inhibitor (or a pharmaceutical composition comprised thereof) will he type of disease to be treated, the severity and course of the disease, whether the RIPKi and/or IKK/NFKB signaling inhibitor and/or pharmaceutical composition is ad- ministered for preventive or therapeutic purposes, previous therapy, the patient's clinical history, age, size/weight and response to the RIPKi and/or IKK/NFKB signaling inhibitor and/or pharmaceutical composition, and the discretion of the attending physician.
• the RIPKi and/or IKK/NFKB signaling inhibitor and/or pharmaceutical composition is suitably administered to the patient at one time or over a series of treatments.
• the total number of administrations for a given course of treatment may consist of a total of about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than about 10 treatments.
• a treatment may be given once every day (or 2, 3 or 4 times a day) for a week, a month or even several months.
• the course of treatment may con- tinue indefinitely.
• IKK inhibitor is used in the broadest sense and includes any molecule that par- tially or fully blocks, inhibits or neutralizes a biological activity mediated by IKK, preferably by preventing the activation of IKK.
• IKK inhibitors act directly on one or more subunits of IKK for example by binding to one or more subunits of IKK.
• the IKK inhibitors may prevent IKK from interacting with a sub- strate, such as I-KB and/or may act on molecules in an IKK signaling pathway, preferably downstream from IKK.
• the IKK inhibitors may modulate the level of IKK gene expression or otherwise reduce the levels of IKK in affected cells.
• the abil- ity of a molecule to inhibit IKK activation can be measured using assays that are well known in the art.
• IKK inhibitors can be identified using im- mune complex kinase assays and gene reporter assays.
• IKK complexes are examined for the ability to phosphory- late GST-IkBa in vitro.
• IKK complexes can be immunoprecipitated from cleared striatal extracts from animals or cells treated with the putative IKK inhibitor by in- cubation with a mouse anti-IKKa antibody (Santa Cruz Biotechnology) coupled to protein-A beads and rocked for 3 hr at 4 0 C.
• IKK activity can be evaluated in vitro with 1 pg of purified GST-Ik-Ba (N-terminal 61 amino acids) in the presence of 10 pCi of [32R]g-ATR for 30 min at 30° C. Products are examined by SDS-PAGE followed by auto- radiography.
• IKK inhibitor includes any molecule that mimics a biological activ- ity mediated by an IKK subunit and specifically changes the function or expression of IKK, cy of signaling through IKK, thereby inhibiting an already existing biological activity or triggering a new biological activity.
• a preferred IKK/NFKB signaling inhibitor in context of the invention is an IKK inhibitor, preferably an IKK2/IKKb inhibitor.
• the protein is known as IKK-beta, IKK2, IKKB, NFKBIKB or“inhibitor of nuclear factor kappa B kinase subunit beta” (Human Gene No- menclature Committee symbol HGNC:596o) and is a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (see https://www.genenames.org/).
• Preferred IKK inhibitors of the invention are selected from SPC839 (Signal Pharmaceutical Inc.), Anilino- Pyrimidine Derivative (Signal Pharmaceutical Inc.), MLN120B or PS1145 (Millennium Pharmaceutical Inc.), BMS- 34554 1* (Bristol-Myers Squibb Pharmaceutical Research Institute, IKK inhib-itor III), SC- 5i4 (Smithkilne Beecham Corp.), Amino- imidazolecarboxamide de-rivative(Smithkilne Beecham Corp.), Ureudo- thiophenecarboxamide deriva-tives (AstraZeneca), Diarylpybidine derivative(Bayer ), Pyri- dooxazinone deriva-tive(Bayer), Indolecarboxamide derivative(Aventis Pharma), Benzoim- idazole car-boxamide derivative (Aventis Pharma), Pyrazolo[4,3-c]quinoline deriva- tive(Pharmacia Corporation
• an IKK/NFKB signaling inhibitor for use in the prevention or treatment of an inflammatory disease, wherein the prevention or treatment comprises the concomitant or sequential administration of the IKK/NFKB signaling inhibi- tor and a RIPKi inhibitor as defined herein.
• the present invention provides the combined use of a IKK/NFKB signaling inhibitor, such as an IKK inhibitor, and a RIPKi inhibitor, in the treatment of subjects suffering from inflammatoiy diseases.
• the combination of the invention could overcome the problem of the prior art of severe side effects caused by IKK/NFKB signaling inhibition, such as the development of neutrophilia and/or the release of IL-ib.
• a combination (of compounds) for use in the treatment or preven- tion of an inflammatoiy disease wherein the combination comprises an RIPKi inhibitor and an IKK/NFKB signaling inhibitor as defined herein.
• the treatment or preven- tion comprises the administration of a therapeutically effective amount of an IKK/NFKB signaling inhibitor to a subject suffering from the inflammatoiy disease.
• the treatment or prevention also may preferably comprise the administration of a sufficient amount of an RIPKi inhibitor to the subject to suppress or prevent increased IL-ib release and/or neu- trophilia caused by the administration of the IKK/NFKB signaling inhibitor to the subject.
• Combinations of the invention in particular combinations of RIPKi and IKK inhibitors are preferably provided in a synergistically effective amount.
• the combinations of the invention in preferred embodiments is a synergistic combination.
• the present invention provides a pharmaceutical composition for use in the treatment or prevention of a disease associated with an increased Interleukin-ib (IL-ib) release in a subject, wherein the pharmaceutical composition comprises a RIPKi inhibitor as described before together with a pharmaceutically acceptable carrier and/or excipient.
• the IL-ib or or systemic neutrophilia is a side effect of an IKK inhibitor treat- ment.
• Another aspect pertains to a pharmaceutical composition for use in the treatment or pre- vention of a disease associated with an increased Interleukin- ib (IL-ib) release or systemic neutrophilia in a subject, wherein the pharmaceutical composition comprises a IKK/NFKB signaling inhibitor as described before together with a pharmaceutically acceptable carrier and/or excipient.
• IL-ib Interleukin- ib
• the pharmaceutical composition of the invention is formulated to be compatible with its in- tended route of administration.
• routes of administration include parenteral, e.g., intrathecal, intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical), intracerebroventricular, intraparenchymal, and transmucosal administration.
• a pharmaceutical composition of the invention shall comprise one or more corn- pounds for inhibiting RIPKi and/or IKK/NFKB signaling, and at least one pharmaceutically acceptable carrier and/or excipient.
• the pharmaceutical compositions as described herein are particularly useful for use in the herein described methods for treating immune related diseases.
• intrathecal means introduced into or occurring in the space un- der the arachnoid membrane which covers the brain and spinal cord.
• the term“intracere- ar” refers to administration of a composition into the ventricular system of the brain, e.g., via injection, infusion, or implantation (for example, into a ventricle of the brain).
• the term“intraparenchymal” can refer to an administration directly to brain tissue. In other instances, intraparenchymal administration may be directed to any brain region where delivery of one or more compounds of the invention is effective to miti- gate or prevent one or more of disorders as described herein. Forms of administration di- rectly to brain tissue is on some embodiments preferred.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solu- tion, fixed oils, polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; anti-bacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascor- bic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buff- ers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, dispos- able syringes or multiple dose vials made of glass or plastic.
• compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous prepara- tion of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsip- pany, N.J.) or phosphate buffered saline (PBS).
• the injectable composition should be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mix- tures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phe- nol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for exam- ple, aluminum monostearate and gelatin.
• table solutions can be prepared by incorporating the active compound (e.g., a sulfotransferase inhibitor) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered steriliza- tion.
• dispersions are prepared by incorporating the active compound into a ster- ile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• a ster- ile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Oral compositions generally include an inert diluent or an edible carrier. They can be en- closed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate or Stertes
• a glidant such as colloidal silicon dioxide
• the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• Systemic administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid deriva- tives.
• Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
• the pharmaceutical compositions are for- mulated into ointments, salves, gels, or creams as generally known in the art.
• the pharmaceutical composition is formulated for sustained or controlled release of the active ingredient.
• Biodegradable, biocompatible polymers can be s ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyortho- esters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from e.g. Alza Cor- poration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes tar- geted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
• Dosage unit form as used herein includes physically discrete units suited as unitaiy dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly depend- ent on the unique characteristics of the active compound and the particular therapeutic ef fect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
• Toxicity and therapeutic efficacy of such compounds can be determined by standard phar- maceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
• Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such corn- pounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• the data obtained from the cell culture assays and animal studies can be used in formulat- ing a range of dosage for use in humans.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of ad-ministration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
• IC50 i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms
• Such information can be used to more accurately determine useful doses in humans.
• the pharmaceutical composi- included in a container, pack, or dispenser together with instructions for ad- ministration.
• the herein disclosed compounds, combinations and compositions are in particular useful in a method of preventing or treating a subject suffering from a disease associated with a pathological or deregulated immune response, such as an increased Interleukin- ib (IL-ib) release or systemic neutrophilia, in a cell of the subject, the method comprising the admin- istration of a therapeutically effective amount of a RIPKi inhibitor to the subject.
• a pathological or deregulated immune response such as an increased Interleukin- ib (IL-ib) release or systemic neutrophilia
• the term“dysregulated immune response” shall refer to any pathological condition characterized by an abnormal, preferably harmful, immune re- sponse in a subject.
• an immune response is characterized by an increased release of IL-ib or systemic neutrophilia in cells of the immune system, such as a myeloid cell, such as a bone marrow derived myeloid progenitor cell, monocyte or macro-phage.
• a dysregulated immune response is an adverse effect of a therapy of a sub- ject with an inhibitor of IKK/NFKB signaling.
• the diseases treatable with the compounds, compositions, combinations and methods of the invention are preferably inflammatory disorders.
• the term“inflammatory disease(s)” includes also“autoimmune disease(s).”
• the term“autoimmunity” is generally understood to encompass inflammatoiy immune- mediated processes involving“self’ antigens. In autoimmune diseases, self antigen(s) trig- ger host immune responses.
• autoimmune and/or inflammatory disorders include, but are not limited to, systemic lupus erythematosus (SLE), discoid lupus, lupus nephritis, sarcoidosis, inflamma- tory arthritis, including juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis, and gouty arthritis, rejection of an organ or tissue trans- plant, hyperacute, acute, or chronic rejection and/or graft versus host disease, multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac's disease (gluten-sensitive enteropa- thy), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriasis, scleroderma, mya
• Cells associated with the diseases to be treated according to the invention are preferably a myeloid cell, such as a bone marrow derived myeloid progenitor cell, monocyte or macro- phage.
• a myeloid cell such as a bone marrow derived myeloid progenitor cell, monocyte or macro- phage.
• a method for the treatment of an in- flammatory disease in a subject comprising the concomitant or sequential ad- ministration to the subject of a therapeutically effective amount of (i) a RIPKi inhibitor and (ii) a IKK/NFKB signaling inhibitor.
• a preferred embodiment of the invention pertains to a use of the here- in disclosed compounds, combinations, compositions and methods for the treatment of adverse effects induced by a therapy with a IKK/NFKB signaling inhibitors.
• the embodiment relates to a medical use where a subject suffers from a disorder that is primarily treated using an IKK/NFKB signaling inhibitor.
• therapies often are associated with severe adverse effects.
• the present invention therefore in some embodi- ments provides a preventive/therapeutic treatment of such side effects by administering to the same subject RIPKi inhibitor in accordance with the herein provided disclosure.
• the disease to be treated is a side effect or adverse event asso- ciated with RIPKi activity.
• the primary disorder meaning herein the disorder treated by the use of a IKK/NFKB signaling inhibitors
• the terms“adverse effect”,“adverse event”, or“side effect” shall mean a medical unfavorable condition induced, caused or worsened by treating a patient suffering from a given disease (here primary disease) with a therapy/therapeutic indicated for that given (primary) disease. Therefore, in context of the herein disclosed invention it may be preferred that a side effect or adverse effect occurring in a subject as a secondaiy disease, wherein the subject suffers from a primary disease different from the secondary disease, is treated by the herein disclosed compounds, compositions, combinations and/methods, most preferably the herein disclosed RIPKi inhibitors.
• Figure 1 Genetic (A) or pharmacologic (B) inactivation of the kinase activity of RIPKi abrogates LPS-induced IL-ib secretion in BMDMs treated with different con- centrations of IKK2 inhibitors. Each bar represents the mean+/-SEM values of triplicate samples for each condition.
• Figure 2 Generation of knock-in mice expressing kinase inactive IKK2 from the en- dogenous Ikk2 genomic locus by mutating lysine 44 to alanine (K44A) using CRISPR/Cas9-mediated gene targeting a) Schematic depiction of the murine Ikk2 genomic locus indicating the location of lysine 44 on exon 3 (upper pan- el), as well as the sequence of the wild type exon 3 and the mutated exon 3 indicating the designed mutation changing lysine 44 to alanine (AAG to GCG) (lower panel) b) Tables depicting the number of litters and the total number as well as the number of /kk2 K44 V/K44A mice obtained from the indi- cated breedings.
• FIG. 3 Loss of IKK2 kinase activity in cells expressing mutated IKK2 K44A strongly suppresses NF-kB activation in response to TNF and LPS in bone marrow de- rived macrophages (BMDMs). Bone marrow was isolated from femur and tibia of 2-week-old control Hkk2 wt / K44A ;Ripki wt / Ol3m and Ikk2 wt K44A ;Ripki T>13m T>13m ) and IKK2 kinase inactive
• mice [ kk2 K44L/ K44A ; R ipk 1 1 ’ l48x/ 1 ’ 148x ) mice.
• BMDMs were differentiated in the pres- ence of 20 ng/ml M-CSF (Thermo Fisher Scientific, #14-8983-62) for 7 days and seeded at lxio 6 cells in 6-well cell culture dishes.
• time course- stimulations with TNF (20 ng/ml) and LPS (100 ng/ml) were performed in the presence of M-CSF (20 ng/ml), and cell-lysates were prepared on ice us- ing RIPA buffer supplemented with protease and phosphatase inhibitors.
• Proteins (20 pg) were separated by SDS-PAGE and Western Blots were per- formed using the following antibodies: phospho-IkBa S32/36 (Cell Signaling, #9246L), IKBOC (Santa Cruz Biotechnologies, #10315), phospho -RelA S536 (Cell Signaling, #30368), and Alpha Tubulin (Sigma-Aldrich, #T6q74). Note strongly suppressed phosphorylation and degradation of IkBa as well as phosphorylation of RelA in cells homozygously expressing kinase inactive IKK2, compared to the control cells that cariy one wild type and one kinase inactive IKK2 allele. The RIPK1D138N mutation does not affect NF-kB acti- vation (Polykratis et al, 2014, J Immunol 193: 1539-1543).
• FIG. 4 Loss of IKK2 kinase activity in cells expressing mutated IKK2 K44A strongly suppresses NF-kB activation in response to TNF and LPS in lung fibroblasts.
• Lung fibroblasts were isolated from two-week old control (IKK2 wt / K44A ; Ripki Dl38N / Dl38N ), and IKK2 and Ripki kinase dead (ikk2 K44A / K44A ; Ripki Dl38N / Dl38N ) mice. Lungs were disrupted using scissors and collagenase treatmen, and subsequently cultured for 7 days.
• lung fibroblasts were seeded at lxio 6 cells in 6-well cell culture dishes.
• Time course-stimulations with TNF (20 ng/ml) and LPS (100 ng/ml) were per- formed, and cell-lysates were prepared on ice using RIPA buffer supplement- ed with protease and phosphatase inhibitors.
• Proteins (20 pg) were separated by SDS-PAGE and Western Blots were performed using the following anti- bodies: phospho-IkBa S32/36 (Cell Signaling, #9246L), IkBa (Santa Cruz Biotechnologies, #10315) and Alpha Tubulin (Sigma-Aldrich, #T6q74).
• Figure 5 Inhibition of RIPKi kinase activity prevents the development of neutrophilia in mice with myeloid cell specific inhibition of IKK2 kinase activity. The presence of neutrophils was assessed by flow cytometiy on peripheral blood collected from 2.5 month old control mice ( Ikk2 FL !
• mice with macrophage specific inhibition of IKK2 kinase activity mice with macrophage specific inhibition of IKK2 kinase activity
• mice with macrophage specific inhibition of IKK2 kinase activity that also lack RIPKi kinase activity in all cells mice with macrophage specific inhibition of IKK2 kinase activity that also lack RIPKi kinase activity in all cells.
• Neutrophils were determined as CD 11 5- Ly6G + Ly6C + leukocytes.
• the graph shows the per- centage of CDii5 _ Ly6G + Ly6C + leukocytes in mice with the indicated geno- types. Each dot represents an individual mouse. Note that lack of RIPKi ki- nase activity fully prevents the neutrophilia caused by inhibition of IKK2 ki- nase activity.
• SEQ ID NO: 3 to 7 depict sequences disclosed in figure 2.
• Example 1 Inhibition of RlPKl kinase activity prevents LPS-induced IL-ib production in macrophages treated with pharmacological IKK inhibitors.
• Bone marrow was isolated from the tibiae and femurs of one WT and one Ripki Dl38N / Dl38N mouse (6-7 week old). Bone marrow from one leg of each mouse was immediately frozen. Bone marrow cells from the other leg were spun down at 1200 rpm, for 5 min and seeded in 15 cm 2 non-treated cell culture plates in 20 ml of medium containing 10 ng/ml M-CSF. Cells were allowed to differentiate for six days and subsequently adherent BMDMs were lifted, counted and seeded at a density of 2x1o 5 cells/well in a 48 well plate.
• Neci the frozen bone marrow cells were used. After thawing, mac- rophages were differentiated as described above for fresh bone marrow cells. Necrostatin-i (Nec-i) was added together with the IKK2 inhibitors during the 30 min pretreatment BMDMs were stimulated with LPS for 20-24I1 and supernatants were kept for ELISA meas- urements.
• Example 2 Inhibition of RIPKi kinase activity prevents the development of systemic neutrophilia caused by inhibition of IKK2 kinase activity in macro phages.
• sgRNA targeting the Ikk2 gene together with the repair oligo containing the desired muta- tion as well as Cas9 were microinjected into the pronucleus of zygotes from C57BI/6 mice, resulting in the generation of founder mice that transmitted the K44A mutation to their progeny.
• Heterozygous /kk2 , ⁇ 44 V/wl mice were viable, developed to adulthood, were fertile and did not show any abnormalities.
• mice Although the double kinase inactive /kfc2 K44A/, ⁇ 44A Ripki Ol3m 138N mice were born normally, they showed reduced growth and needed to be sacrificed at the age of 2 weeks, likely due to infection with opportunistic bacteria as shown before in double RelA and TNFRi knockout animals that were also born but died during the first 3 weeks after birth due to bacterial infections (E Alcamo et ah, Journal of Immunology (Baltimore, Md : 1950) 167, no. 3 (August 1, 2001): 1592-1600.).
• BMDMs from ikk 2 ⁇ A/K44A Ripki D138N/D138N mjce showed very strongly suppressed activation of NF- KB, as indicated by impaired phosphorylation and degradation of IkBa as well as phosphor- ylation of RelA/p6s in response to stimulation by TNF or LPS.
• Similar results were ob- tained by measuring activation of NF-kB in lung fibroblasts from /kfc2 K44 V/K44A Rzpki Dl38N / Dl38N and control mice in response to TNF or LPS (Figure 4). These results con- firmed that the K44A mutation abolished the kinase activity of IKK2 and very strongly sup- presses the activation of NF-kB by TNF or LPS.
• mice with inhibition of IKK2 kinase activity in myeloid cells by crossing mice carrying one K44A and one loxP- flanked IKK2 allele (Ikk2 V i K44A ) with Cx3cn-Cre mice expressing Cre recombinase specifi- cally in macrophages (Simon Yona et al.,“Fate Mapping Reveals Origins and Dynamics of Monocytes and Tissue Macrophages Under Homeostasis.,” Immunity 38, no. 1 (Januaiy 24, 2013): 79-91, doi:io.ioi6/j.immuni.2012.12.001).
• Cre recombinase de- letes the loxP-flanked IKK2 allele that expresses wild type IKK2 resulting in expression ex- clusively of the kinase inactive IKK2K44A in macrophages. Consistent with the earlier stud- ies based on myeloid cell specific knockout of IKK2 as well as treatment of mice with IKK2 kinase inhibitors, the inventors found that inhibition of IKK2 kinase activity in macrophag- es resulted in the development of systemic neutrophilia in mice, as shown by the analysis of neutrophils in the blood ( Figure 5).
• RIPKi kinase inhibi- tors will overcome the serious adverse effects of inhibitors of IKK/NF-kB, thereby allowing iication of drugs inhibiting IKK/NF-kB signaling for the treatment of inflamma- tory diseases. Since inhibition of RIPKi kinase activity has also been reported to inhibit in- flammation in some models of acute and chronic inflammatory pathologies, co- administration of RIPKi inhibitors with IKK/NF-kB inhibitors may not only prevent the adverse side effects of the latter but may also have a synergistic effect by combining the therapeutic effects of the two inhibitors.

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