WO2012150325A1 - Method for finding active pharmaceutical ingredients for the treatment of diseases associated with increased or decreased cytokine pathway activity - Google Patents

Method for finding active pharmaceutical ingredients for the treatment of diseases associated with increased or decreased cytokine pathway activity Download PDF

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Publication number
WO2012150325A1
WO2012150325A1 PCT/EP2012/058217 EP2012058217W WO2012150325A1 WO 2012150325 A1 WO2012150325 A1 WO 2012150325A1 EP 2012058217 W EP2012058217 W EP 2012058217W WO 2012150325 A1 WO2012150325 A1 WO 2012150325A1
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lipid profile
cells
treatment
culture medium
lipid
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PCT/EP2012/058217
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French (fr)
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Jos Paquaij
Ronald Bronsaer
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Cellmade Sas
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the invention relates to a method for finding active pharmaceutical ingredients for the treatment of diseases associated with increased or decreased cytokine pathway activity, to active pharmaceutical ingredients thus found and to active pharmaceutical ingredients for use in the treatment of diseases associated with increased or decreased cytokine pathway activity.
  • TNF tumor necrosis factor
  • DIF differentiation-inducing factor
  • Examples of 'bad' TNFa- responses include apoptic cell death, necrotic cell death, and its involvement in diseases, for example inflammation diseases, for example acute inflammation, chronic inflammation, psoriasis, periodontitis, rheumatoid arthritis, cardiovascular diseases, osteoarthritis; atherosclerosis, asthma, septic shock, irritable bowel syndrome (IBS), heamorrhagic fever, cachexia, diabetes mellitus type 2, Chronic Obstructive Pulmonary Disease(COPD), asthma, Crohn's disease, collitus, different types of cancer,
  • inflammation diseases for example acute inflammation, chronic inflammation, psoriasis, periodontitis, rheumatoid arthritis, cardiovascular diseases, osteoarthritis
  • atherosclerosis asthma, septic shock, irritable bowel syndrome (IBS), heamorrhagic fever, cachexia, diabetes mellitus type 2, Chronic Obstructive Pulmonary Disease(COPD), asthma, Crohn's disease, collitus
  • TNFa is a ligand for cytokine receptors and as such the diseases associated with TNFa are diseases that are also associated with increased or decreased cytokine pathway activity.
  • the present invention provides an in vitro method for finding active pharmaceutical ingredients for the prevention, treatment or co-treatment of diseases associated with increased or decreased cytokine pathway activity.
  • the invention provides a method for finding an active pharmaceutical ingredient potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity, comprising the steps of:
  • step A2) providing a second lipid profile by performing step A1 ) except for sub-step c) to obtain the second lipid profile
  • the pro-inflammatory cytokine may be added to the cell culture medium in an amount of from 1 .10 " 2 mol/l to 1 mol/l , for example in an amount of at least 1 .10 "9 mol/l and/or at most 1 .10 3 mol/l, for example at most 1 .10 6 mol/l.
  • US2005/0026296A1 discloses a method for finding substances able to induce a pathological state based on lipid measurement parameter modulation or effector quotient profiles.
  • This method differs from the method of the present invention in that the method of US2005/0026296 A1 uses the sum of the concentration of lipids or groups of lipids to identify a substance, whereas the method of the present invention identifies pharmaceutically active ingredients by using lipid profiles, which are collections of the individual concentrations of individual lipids.
  • lipid profiles which are collections of the individual concentrations of individual lipids.
  • a difference between two lipid profiles as mentioned herein is understood to be a difference in at least one concentration of an individual lipid.
  • the method may further comprise the steps of:
  • step A3 providing a third lipid profile by performing step A1 ) except for sub-step b) to obtain the third lipid profile
  • step A4) providing a fourth lipid profile by performing step A1 ) except for sub-steps b) and c) to obtain the fourth lipid profile
  • the in vitro method of the invention is capable of a reliable prediction of the therapeutical effect of the active pharmaceutical ingredient thus identified in vivo (than any of the other methods known so far).
  • the method of the invention capable of identifying active pharmaceutical ingredients suitable for treatment of acute diseases associated with increased or decreased cytokine pathway activity, but may also be capable of identifying active pharmaceutical ingredients (APIs) that are especially suitable for treatment chronic diseases.
  • APIs active pharmaceutical ingredients
  • chronic diseases include chronic inflammation, diabetes mellitus type 2, cancer, immune response associated diseases, (osteo)arthritis etc.
  • active pharmaceutical ingredient means any compound or mixture of compounds that has a prophylactic or therapeutic effect in vivo, preferably in a human.
  • diseases that are associated with increased or decreased cytokine receptor activity include but are not limited to those cited above, for example inflammation diseases, acute inflammation, chronic inflammation, psoriasis, periodontitis, rheumatoid arthritis, cardiovascular diseases, osteoarthritis;
  • Atherosclerosis asthma, septic shock, irritable bowel syndrome (IBS), heamorrhagic fever, cachexia, diabetes mellitus type 2, COPD, asthma, Crohn's disease, collitus, different types of cancer and Alzheimer's disease.
  • IBS irritable bowel syndrome
  • the pro-inflammatory cytokine added to the cell culture medium comprising the cells having cytokine receptors include but are not limited to IL-1 b, IL-18, IL-4, IL-10, prostaglandins, for example PGE2, TNFa, preferably TNFa.
  • Animal cells are suitable, preferably mammalian cells. Particularly preferred are human cells.
  • the cells may be genetically modified or altered cells.
  • Preferred cells include human cells, such as human primary cells. Examples of (commercially) available cells include animal cells, for example NSO cells, CHO-cells and human cells, for example HeLa cells or PER.C6® cells.
  • Preferred examples of the types of cells that can be used in the method of the present invention include human primary cells, such as adipocytes / preadipocytes, bladder cells, brain cells, cardiac cells, endothelial cells, epidermal cells, epithelial cells, eye cells, fibroblasts, granulocytes, hepatic cells, human mesothelial cells, intestine and colon cells, lung and pulmonary cells, lymph cells, lymphocytes, mononuclear cells, neural nerve cells, phagocytes, renal cells, reproductive cells, skeletal cells, smooth muscle cells and umbilical cells.
  • human primary cells such as adipocytes / preadipocytes, bladder cells, brain cells, cardiac cells, endothelial cells, epidermal cells, epithelial cells, eye cells, fibroblasts, granulocytes, hepatic cells, human mesothelial cells, intestine and colon cells, lung and pulmonary cells, lymph cells, lymphocytes, mononuclear cells, neural nerve cells
  • human stem cells such as mesenchymal stem cells, pericytes, CD34+ progenitor cells, CD133+ progenitor Cells, mononuclear cells and CD14+ Monocytes.
  • the cells are human aorta endothelial cells (HAOEC) .
  • HAOEC may be commercially obtained from Cell Applications Inc. productnumber 304-05a.
  • the density of the cells is in principle not critical and may for example be from 100,000 to 50,000,000 cells/ml or from 500 to 10,000,000 per cm 2 .
  • the cell density as used herein may be determined using methods known to the person skilled in the art, for example by using a flow cytometer to count the number of cells in a given volume or on a given surface.
  • the cells may be present in the cell culture medium in any suitable way.
  • the cells may be present as a suspension in the cell culture medium or may be attached to a surface present in the cell culture medium.
  • the method of the invention can in principle be performed in any type of cell culture medium suitable for the culturing of (animal) cells. Suitable cell culture media support the growth and differentiation of the cells used in the method of the invention.
  • a cell culture medium for (mammalian) cells comprises salts, amino acids, vitamins, lipids, detergents, buffers, growth factors, hormones, cytokines, trace elements and carbohydrates.
  • salts include magnesium salts, for example MgCI 2 .6H 2 0, MgS0 4 and MgS0 4 .7H 2 0 iron salts, for example FeS0 4 .7H 2 0, potassium salts, for example KH 2 P0 4 , KCI; sodium salts, for example NaH 2 P0 4 , Na 2 HP0 4 and calcium salts, for example CaCI 2 .2H 2 0.
  • Examples of amino acids are all 20 known proteinogenic amino acids, for example hystidine, glutamine, threonine, serine, methionine.
  • vitamins include: ascorbate, biotin, choline. CI, myo-inositol, D-panthothenate, riboflavin.
  • Examples of lipids include: fatty acids, for example linoleic acid and oleic acid; soy peptone and ethanol amine.
  • Examples of detergents include Tween 80 and Pluronic F68.
  • An example of a buffer is HEPES.
  • Examples of growth factors/hormones/cytokines include IGF, hydrocortisone and (recombinant) insulin.
  • Examples of trace elements are known to the person skilled in the art and include Zn, Mg and Se.
  • Examples of carbohydrates include glucose, fructose, galactose and pyruvate.
  • the culture medium may be supplemented with growth factors, metabolites, etc.
  • RPMI commercially available from Sigma
  • FCS Foetal Calf Serum
  • the optimal conditions under which the cells are cultured can easily be determined by the skilled person.
  • the pH, temperature, dissolved oxygen concentration and osmolarity of the cell culture medium are in principle not critical and depend on the type of cell chosen.
  • the pH, temperature, dissolved oxygen concentration and osmolarity are chosen such that these conditions optimal for the growth and productivity of the cells.
  • the person skilled in the art knows how to find the optimal pH, temperature, dissolved oxygen concentration and osmolarity.
  • the optimal pH is between 6.6 and 7.6, the optimal temperature between 30 and 39 °C, for example a temperature from 36 to 38 °C, preferably a temperature of about 37 ⁇ C; the optimal osmolarity between 260 and 400mOsm/kg.
  • the component that is potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity may be any component, for example a component from a library of chemical compounds. It may also be a mixture of compounds.
  • the component may for example be a chemically synthesized compound, such as a steroid, but may also be a biological, such as a peptide,
  • the amount of component that is potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity may be anywhere in the range from 1 .10 " 2 mol/ml to 1 mol/ml, for example in an amount of at least 1 .10 9 mol/ml and/or at most 1 .10 3 mol/ml, for example at most 1 .10 6 mol/ml.
  • the amount of said component may be increased step-wise using routine experimentation.
  • the lipid profiles may be determined using any suitable analysis method, which are known to the person skilled in the art.
  • analysis methods like flow cytometry, enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LCMS), gas chromatography-mass spectrometry (GCMS) and immunocytochemistry may be used.
  • GCMS or LCMS are used.
  • the determination of the lipid profile may be done on the cells. If the analysis is done on the cells, the method of the invention may further comprise the steps of harvesting the cells by separation of the cells and the cell culture medium and optional disruption of the cell wall (prokaryotes) or cell membrane (eukaryotes) using methods known in the art. Alternatively, or also, the determination of the lipid profile may be done on the cell culture medium. In the cases where the cells are attached to a surface, before determining the lipid profile of the cells, the cells may be removed by use of e.g.
  • the cells may be directly lysed without removal from the surface using a lysis buffer (e.g. RIPA buffer as for example commercially available from Cellsignal).
  • a lysis buffer e.g. RIPA buffer as for example commercially available from Cellsignal.
  • the method of the invention may be performed more than once at the same time and that the determining of the lipid profiles may be done at different points in time for the different simultaneously performed methods in order to ensure that the determination of the lipid profile by e.g. taking samples of the cell culture medium or of the cells does not change the result of measurements later in time.
  • lipid profile is meant the collection of the amounts of lipids found in one sample.
  • the lipid profile may include the amounts of lipids chosen from individuals comprised in the groups of lysophospholipids, monoacylglycerols, phospholipids, sphingomyelines, diacylglycerols, cholesterol esters, ceramides, for example a ceramide that is a sphingosine with fatty acid chains of from 16 to 24 C-atoms, for example a ceramide having 18, 20, 22 or 24 C-atoms, for example a ceramide having 22 C-atoms;
  • the second lipid profile may be provided by a method comprising the step of a) providing a cell culture medium comprising cells having cytokine receptors, b) adding a pro-inflammatory cytokine to the cell culture medium and
  • the first and the second lipid profiles are lipid profiles of the cells and/or the cell culture medium obtained by method including step b), i.e. it is a lipid profile of the cells and/or the cell culture medium in which pro-inflammatory cytokine is added.
  • the second lipid profile is a lipid profile of the cells and/or the cell culture medium obtained by a method not including step c), i.e. it is a lipid profile of the cells and/or the cell culture medium to which a component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity is not added.
  • comparison between the first lipid profile and the second lipid profile allows determining the effect of the component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity when a pro- inflammatory cytokine is added.
  • the third lipid profile may be provided by a method comprising the steps of
  • the fourth lipid profile may be provided by a method comprising the steps of a) providing a cell culture medium comprising cells having cytokine receptors and d) determining the lipid profiles of the cells and/or of the cell culture medium to obtain the first lipid profile, wherein the method does not include the steps of
  • the third and the fourth lipid profiles are a lipid profile of the cells and/or the cell culture medium obtained by method not including step b), i.e. it is a lipid profile of the cells and/or the cell culture medium to which no pro-inflammatory cytokine is added.
  • the difference between the third and the fourth lipid profiles is that a component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity is added for obtaining the third profile.
  • comparison between the third lipid profile and the fourth lipid profile allows determining the effect of the component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity when no pro-inflammatory cytokine is added.
  • the component which induces a change between the first lipid profile and the second lipid profile and/or a change between the third lipid profile and the fourth lipid profile is identified to be an active pharmaceutical ingredient.
  • the steps for providing the lipid profiles is not critical may be performed in any order.
  • the first lipid profile may be provided after the second lipid profile, or the fourth lipid profile may be provided before any other lipid profiles are provided.
  • the lipids of which the lipid profiles are compared are chosen from the group of ceramides, for example a ceramide that is a sphingosine with fatty acid chains of from 16 to 24 C-atoms, sphingosine and sphingosine-1 -phosphate .
  • the differences in lipid profiles should be assessed in methods that make use of the same cells, conditions etc. Also, the differences should be assessed at the same point in time. For example, if a difference can only be seen in step d) after 6 hours after adding the component that is potentially suitable for the prevention, treatment or co- treatment of a disease associated with increased or decreased cytokine pathway activity, then this difference should be noted and the component identified as active pharmaceutical ingredient.
  • the invention relates to a composition
  • a composition comprising the active pharmaceutical ingredient obtained with the method of the invention, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical composition according to the present invention may further comprise conventional pharmaceutical additives and adjuvants, excipients or diluents, including, but not limited to, water, gelatin of any origin, vegetable gums,
  • the invention relates to an active pharmaceutical ingredient obtained with the method of the invention or to the pharmaceutical composition of the invention for use as a medicament.
  • the invention relates to an active pharmaceutical ingredient obtained with the method of the invention or to the pharmaceutical composition of the invention for use in the prevention, treatment, or co-treatment of a disease associated with increased or decreased cytokine pathway activity.
  • the invention relates to use of an active pharmaceutical ingredient obtained with the method of the invention or to the pharmaceutical composition of the invention for the manufacture of a medicament for the prevention, co-treatment or treatment of a diseases associated with increased or decreased cytokine pathway activity.
  • the invention in another aspect, relates to a method for treatment, co-treatment or prevention of a disease associated with increased or decreased cytokine pathway receptor activity in animals, including humans, said method comprising the step of administering an effective amount of the active pharmaceutical ingredient obtained with the method of the invention or administering an effective amount of the pharmaceutical composition of the invention to animals including humans which are in need thereof.
  • 'effective amount' of the active pharmaceutical ingredient obtained with the method of the invention or of the pharmaceutical composition of the invention is meant the amount needed to treat, co-treat or prevent the disease associated with increased or decreased cytokine pathway receptor activity in animals, including humans.
  • animals In the framework of the invention, with animals is meant all animals, including mammals, examples of which include humans. Preferred examples of mammals beside humans include dogs, dromedaris, camels, elephants, and horses.
  • HAoEC Primary Human Aorta Endothelial Cells
  • Example 1 was repeated, but additionally 20 ⁇ g/ml anti-TNFa antibody was added to the cells. The results for the cells are shown in Figure 2. Lipid profiles were determined by means of the following LCMS method:
  • ESI(+), MRM mode fragmentor optimized for each compound (120 V); EMV +400 V, 300 'C gas temperature, flow 5 L N 2 /min, 45 psi, sheath gas temperature 250 'C, sheath gas flow 10 L N 2 /min, 3.5 kV cap, 0.5 kV nozzle, dwell time 5 ms. Two time segments are used: 0-2.5 minutes for sphingosine and sphingosine-1 -phosphate and 2.5-12.5 minutes for the ceramides. MS MRM conditions, tandem MS in the MRM mode:
  • Fig. 1 describes the lipid profile of the HAoEC cells in time, wherein TNFa was added; it specifically describes the concentrations of several ceramides: sphingosine with fatty acid side chains of 16 (C1 6), 1 8 (C18), 20 (C20), 22 (C22) or 24 (C24) C-atoms in time.
  • sphingosine with fatty acid side chains of 24 C-atoms a sphingosine with an unsaturated fatty acid side chain (C24:1 ) and a sphingosine with a saturated fatty acid side chain (C24) were measured.
  • Fig. 2 describes the lipid profile of the HAoEC cells cells in time, wherein TNFa and anti-, TNFa antibody were added; it specifically describes the concentrations of several ceramides: sphingosine with fatty acid side chains of 16 (C16), 18 (C18), 20 (C20), 22 (C22) or 24 (C24) C-atoms in time.
  • sphingosine with fatty acid side chains of 24 C-atoms a sphingosine with an unsaturated fatty acid side chain (C24:1 ) and a sphingosine with a saturated fatty acid side chain were measured.
  • Fig. 3 describes the lipid profile of the cell culture medium of the HAoEC cells in time, wherein TNFa was added; it specifically describes the concentrations of several ceramides: sphingosine with fatty acid side chains of 16 (C16), 18 (C18), 20 (C20), 22 (C22) or 24 (C24) C-atoms in time.
  • sphingosine with fatty acid side chains of 24 C-atoms a sphingosine with an unsaturated fatty acid side chain (C24:1 ) and a sphingosine with a saturated fatty acid side chain were measured.
  • C24:1 unsaturated fatty acid side chain
  • a sphingosine with a saturated fatty acid side chain were measured.
  • HFLS Primary Human Fibroblast-Like Synoviocytes
  • HFLS Primary Human Fibroblast-Like Synoviocytes
  • HFLS were cultured in 6 well culture plates using Synoviocytes Growth Medium (Cell Applications Incorporated) in an incubator at a temperature of 37 ⁇ C and having an atmosphere of 5% 0 2 and 95% C0 2 .
  • HFLS were about 90% confluent, medium was refreshed.
  • HFLS were either untreated (control), treated with TNFoc (20ng/ml) for one hour (TNFa 1 H), or treated with TNFa for one hour with subsequent addition of the antiinflammatory compound aspirin to the wells containing already TNFa (TNFa + Asp 4H).
  • Aspirin was used in a concentration in the cell culture medium of 50 ⁇ g/ml.
  • a sample was taken after 4 hours exposure to the anti-inflammatory compound aspirin.
  • Cells were taken and frozen at -80 °C until measurements of lipid profiles were executed. The lipid profiles were measured
  • sphing stands for sphingosine. Similar to examples 1 and 2 above, C16 stands for a ceramide: sphingosine with fatty acid side chains of 16 C-atoms, C18 stands for a ceramide: sphingosine with fatty acid side chains of having 18 C-atoms, etc.
  • stimulation of HFLS with TNFa causes an increase in the concentrations of the individual lipids as compared to the concentrations before stimulation (control).
  • concentration of the individual lipids is decreased to the concentration before stimulation (control) or even below that concentration.
  • the decrease of the concentration of the individual lipids shows that aspirin is potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity, such as (acute) inflammation. This is in line with the well-known fact that aspirin is a good drug for the treatment of acute inflammation
  • the method of the invention provides an easy way to determine whether or not a component may be suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity. It does not require extensive calculations; rather, by looking at the behaviour of the individual lipids (the lipid profile), it can be seen instantaneously whether or not the component has an effect. If the component has an effect on one individual lipid, it is already a potential candidate, whereas in a method that only looks at the sum of the lipids, such candidate could easily be missed.
  • the method of the invention therefore provides a very sensitive method for identifying active pharmaceutical ingredient potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity.

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Abstract

The invention relates to a method for finding an active pharmaceutical ingredient potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity, comprising the steps of A1) providing a first lipid profile by a) providing a cell culture medium comprising cells having cytokine receptors, b) adding a pro-inflammatory cytokine to the cell culture medium c) adding a component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity to the cell culture medium and d) determining the lipid profiles of the cells and/or of the cell culture medium to obtain the first lipid profile, A2) providing a second lipid profile by performing step A1) except for sub-step c) to obtain the second lipid profile, B) comparing the first lipid profile to the second lipid profile and C) identifying the component that was added in the method that shows a difference between the first lipid profile and the second lipid profile as the active pharmaceutical ingredient.

Description

METHOD FOR FINDING ACTIVE PHARMACEUTICAL INGREDIENTS FOR THE TREATMENT OF DISEASES ASSOCIATED WITH INCREASED OR DECREASED
CYTOKINE PATHWAY ACTIVITY The invention relates to a method for finding active pharmaceutical ingredients for the treatment of diseases associated with increased or decreased cytokine pathway activity, to active pharmaceutical ingredients thus found and to active pharmaceutical ingredients for use in the treatment of diseases associated with increased or decreased cytokine pathway activity.
Before its discovery in 1984, the tumor necrosis factor (TNF) was the unknown factor that was named after its ability to regress tumor masses in patients whose tumor had become infected with a bacterial infection. TNF is also known as TNFa, cachectin or differentiation-inducing factor (DIF). TNFa is associated with both 'good' and 'bad' responses. Examples of 'good' TNFa-responses include cell growth and differentiation, inflammatory effects, mediation of immune responses. Examples of 'bad' TNFa- responses include apoptic cell death, necrotic cell death, and its involvement in diseases, for example inflammation diseases, for example acute inflammation, chronic inflammation, psoriasis, periodontitis, rheumatoid arthritis, cardiovascular diseases, osteoarthritis; atherosclerosis, asthma, septic shock, irritable bowel syndrome (IBS), heamorrhagic fever, cachexia, diabetes mellitus type 2, Chronic Obstructive Pulmonary Disease(COPD), asthma, Crohn's disease, collitus, different types of cancer,
Alzheimer's disease Since its discovery in 1984, a lot of research has been dedicated to pro-inflammatory cytokines, in particular to the tumor necrosis factor. For example, an overview of the TNFa and its activity is given in a review article by David J. MacEwan in TNF receptor subtype signalling: Differences and cellular consequences in Cell Signalling, 14 (2002), pages 477-492.
The interest in pro-inflammatory cytokines, in particular in TNFa is understandable, since many of the diseases associated with TNFa are abundant and ever-spreading and the understanding of the mechanism behind the action of TNFa may help to find effective treatments of the diseases mentioned above.
TNFa is a ligand for cytokine receptors and as such the diseases associated with TNFa are diseases that are also associated with increased or decreased cytokine pathway activity.
The present invention provides an in vitro method for finding active pharmaceutical ingredients for the prevention, treatment or co-treatment of diseases associated with increased or decreased cytokine pathway activity.
In particular, the invention provides a method for finding an active pharmaceutical ingredient potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity, comprising the steps of:
A1 ) providing a first lipid profile by
a) providing a cell culture medium comprising cells having cytokine receptors, b) adding a pro-inflammatory cytokine to the cell culture medium,
c) adding a component that is potentially suitable for the prevention, treatment or co- treatment of a disease associated with increased or decreased cytokine pathway activity to the cell culture medium and
d) determining the lipid profiles of the cells and/or of the cell culture medium to obtain the first lipid profile,
A2) providing a second lipid profile by performing step A1 ) except for sub-step c) to obtain the second lipid profile,
B) comparing the first lipid profile to the second lipid profile and
C) identifying the component that was added in the method that shows a difference between the first lipid profile and the second lipid profile as the active pharmaceutical ingredient.
The pro-inflammatory cytokine may be added to the cell culture medium in an amount of from 1 .10" 2mol/l to 1 mol/l , for example in an amount of at least 1 .10"9mol/l and/or at most 1 .10 3 mol/l, for example at most 1 .10 6 mol/l. US2005/0026296A1 discloses a method for finding substances able to induce a pathological state based on lipid measurement parameter modulation or effector quotient profiles.
This method differs from the method of the present invention in that the method of US2005/0026296 A1 uses the sum of the concentration of lipids or groups of lipids to identify a substance, whereas the method of the present invention identifies pharmaceutically active ingredients by using lipid profiles, which are collections of the individual concentrations of individual lipids. A difference between two lipid profiles as mentioned herein is understood to be a difference in at least one concentration of an individual lipid.
The method may further comprise the steps of:
A3) providing a third lipid profile by performing step A1 ) except for sub-step b) to obtain the third lipid profile,
A4) providing a fourth lipid profile by performing step A1 ) except for sub-steps b) and c) to obtain the fourth lipid profile,
D) comparing the third lipid profile to the fourth lipid profile and
E) identifying the component that was added in the method that shows a difference between the third lipid profile and the fourth lipid profile as the active pharmaceutical ingredient.
It has been found that the in vitro method of the invention is capable of a reliable prediction of the therapeutical effect of the active pharmaceutical ingredient thus identified in vivo (than any of the other methods known so far). Not only is the method of the invention capable of identifying active pharmaceutical ingredients suitable for treatment of acute diseases associated with increased or decreased cytokine pathway activity, but may also be capable of identifying active pharmaceutical ingredients (APIs) that are especially suitable for treatment chronic diseases.
Examples of such chronic diseases include chronic inflammation, diabetes mellitus type 2, cancer, immune response associated diseases, (osteo)arthritis etc.
The term active pharmaceutical ingredient means any compound or mixture of compounds that has a prophylactic or therapeutic effect in vivo, preferably in a human.
Examples of diseases that are associated with increased or decreased cytokine receptor activity include but are not limited to those cited above, for example inflammation diseases, acute inflammation, chronic inflammation, psoriasis, periodontitis, rheumatoid arthritis, cardiovascular diseases, osteoarthritis;
atherosclerosis, asthma, septic shock, irritable bowel syndrome (IBS), heamorrhagic fever, cachexia, diabetes mellitus type 2, COPD, asthma, Crohn's disease, collitus, different types of cancer and Alzheimer's disease.
The pro-inflammatory cytokine added to the cell culture medium comprising the cells having cytokine receptors include but are not limited to IL-1 b, IL-18, IL-4, IL-10, prostaglandins, for example PGE2, TNFa, preferably TNFa.
Any type of cells having cytokine receptors may be used in the method of the present invention. Animal cells are suitable, preferably mammalian cells. Particularly preferred are human cells. The cells may be genetically modified or altered cells. Preferred cells include human cells, such as human primary cells. Examples of (commercially) available cells include animal cells, for example NSO cells, CHO-cells and human cells, for example HeLa cells or PER.C6® cells. Preferred examples of the types of cells that can be used in the method of the present invention include human primary cells, such as adipocytes / preadipocytes, bladder cells, brain cells, cardiac cells, endothelial cells, epidermal cells, epithelial cells, eye cells, fibroblasts, granulocytes, hepatic cells, human mesothelial cells, intestine and colon cells, lung and pulmonary cells, lymph cells, lymphocytes, mononuclear cells, neural nerve cells, phagocytes, renal cells, reproductive cells, skeletal cells, smooth muscle cells and umbilical cells. Further preferred examples of the types of cells that can be used in the method of the present invention include human stem cells such as mesenchymal stem cells, pericytes, CD34+ progenitor cells, CD133+ progenitor Cells, mononuclear cells and CD14+ Monocytes.
Preferably, the cells are human aorta endothelial cells (HAOEC) . HAOEC may be commercially obtained from Cell Applications Inc. productnumber 304-05a.
The density of the cells is in principle not critical and may for example be from 100,000 to 50,000,000 cells/ml or from 500 to 10,000,000 per cm2.
The cell density as used herein may be determined using methods known to the person skilled in the art, for example by using a flow cytometer to count the number of cells in a given volume or on a given surface. The cells may be present in the cell culture medium in any suitable way. The cells may be present as a suspension in the cell culture medium or may be attached to a surface present in the cell culture medium. The method of the invention can in principle be performed in any type of cell culture medium suitable for the culturing of (animal) cells. Suitable cell culture media support the growth and differentiation of the cells used in the method of the invention.
Guidelines for choosing a cell culture medium and cell culture conditions are well known and are for instance provided in Chapter 8 and 9 of Freshney, R. I. Culture of animal cells (a manual of basic techniques), 4th edition 2000, Wiley-Liss and in Doyle, A. , Griffiths, J. B., Newell, D. G. Cell & Tissue culture: Laboratory Procedures 1993, John Wiley & Sons..
Generally, a cell culture medium for (mammalian) cells comprises salts, amino acids, vitamins, lipids, detergents, buffers, growth factors, hormones, cytokines, trace elements and carbohydrates. Examples of salts include magnesium salts, for example MgCI2.6H20, MgS04 and MgS04.7H20 iron salts, for example FeS04.7H20, potassium salts, for example KH2P04, KCI; sodium salts, for example NaH2P04, Na2HP04 and calcium salts, for example CaCI2.2H20. Examples of amino acids are all 20 known proteinogenic amino acids, for example hystidine, glutamine, threonine, serine, methionine. Examples of vitamins include: ascorbate, biotin, choline. CI, myo-inositol, D-panthothenate, riboflavin. Examples of lipids include: fatty acids, for example linoleic acid and oleic acid; soy peptone and ethanol amine. Examples of detergents include Tween 80 and Pluronic F68. An example of a buffer is HEPES. Examples of growth factors/hormones/cytokines include IGF, hydrocortisone and (recombinant) insulin. Examples of trace elements are known to the person skilled in the art and include Zn, Mg and Se. Examples of carbohydrates include glucose, fructose, galactose and pyruvate.
The culture medium may be supplemented with growth factors, metabolites, etc.
An example of a cell culture medium that may suitably be used is RPMI (commercially available from Sigma) supplemented with 10% Foetal Calf Serum(FCS).
The optimal conditions under which the cells are cultured can easily be determined by the skilled person. For example, the pH, temperature, dissolved oxygen concentration and osmolarity of the cell culture medium are in principle not critical and depend on the type of cell chosen. Preferably, the pH, temperature, dissolved oxygen concentration and osmolarity are chosen such that these conditions optimal for the growth and productivity of the cells. The person skilled in the art knows how to find the optimal pH, temperature, dissolved oxygen concentration and osmolarity. Usually, the optimal pH is between 6.6 and 7.6, the optimal temperature between 30 and 39 °C, for example a temperature from 36 to 38 °C, preferably a temperature of about 37<C; the optimal osmolarity between 260 and 400mOsm/kg.
The component that is potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity may be any component, for example a component from a library of chemical compounds. It may also be a mixture of compounds. The component may for example be a chemically synthesized compound, such as a steroid, but may also be a biological, such as a peptide,
(monoclonal) antibody, siRNA, protein etc. or a compound isolated from a natural, for example from a plant.
The amount of component that is potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity may be anywhere in the range from 1 .10" 2mol/ml to 1 mol/ml, for example in an amount of at least 1 .10 9 mol/ml and/or at most 1 .10 3 mol/ml, for example at most 1 .10 6 mol/ml. To enhance the response, the amount of said component may be increased step-wise using routine experimentation.
The lipid profiles may be determined using any suitable analysis method, which are known to the person skilled in the art. For example, analysis methods like flow cytometry, enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LCMS), gas chromatography-mass spectrometry (GCMS) and immunocytochemistry may be used.
Preferably for the determination of the lipid profiles GCMS or LCMS are used.
The determination of the lipid profile may be done on the cells. If the analysis is done on the cells, the method of the invention may further comprise the steps of harvesting the cells by separation of the cells and the cell culture medium and optional disruption of the cell wall (prokaryotes) or cell membrane (eukaryotes) using methods known in the art. Alternatively, or also, the determination of the lipid profile may be done on the cell culture medium. In the cases where the cells are attached to a surface, before determining the lipid profile of the cells, the cells may be removed by use of e.g.
trypsin. Alternatively, the cells may be directly lysed without removal from the surface using a lysis buffer (e.g. RIPA buffer as for example commercially available from Cellsignal).
The lipid profiles are preferably determined at different points in time (i.e. monitored), for example the lipid profiles may be determined at the start of the method of the invention (t = 0), after 1 ; 3; 6; 9; 12; 24; 48 and after 72 hours to get a good overview on the change of lipid profile.
It is noted that the method of the invention may be performed more than once at the same time and that the determining of the lipid profiles may be done at different points in time for the different simultaneously performed methods in order to ensure that the determination of the lipid profile by e.g. taking samples of the cell culture medium or of the cells does not change the result of measurements later in time.
With lipid profile is meant the collection of the amounts of lipids found in one sample. The lipid profile may include the amounts of lipids chosen from individuals comprised in the groups of lysophospholipids, monoacylglycerols, phospholipids, sphingomyelines, diacylglycerols, cholesterol esters, ceramides, for example a ceramide that is a sphingosine with fatty acid chains of from 16 to 24 C-atoms, for example a ceramide having 18, 20, 22 or 24 C-atoms, for example a ceramide having 22 C-atoms;
triacylglycerols, sphingosine and sphingosine-1 -phosphate. The second lipid profile may be provided by a method comprising the step of a) providing a cell culture medium comprising cells having cytokine receptors, b) adding a pro-inflammatory cytokine to the cell culture medium and
d) determining the lipid profiles of the cells and/or of the cell culture medium to obtain the second lipid profile, wherein the method does not include the step of
c) adding a component that is potentially suitable for the prevention, treatment or co- treatment of a disease associated with increased or decreased cytokine pathway activity to the cell culture medium.
The first and the second lipid profiles are lipid profiles of the cells and/or the cell culture medium obtained by method including step b), i.e. it is a lipid profile of the cells and/or the cell culture medium in which pro-inflammatory cytokine is added. The second lipid profile is a lipid profile of the cells and/or the cell culture medium obtained by a method not including step c), i.e. it is a lipid profile of the cells and/or the cell culture medium to which a component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity is not added. Thus, comparison between the first lipid profile and the second lipid profile allows determining the effect of the component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity when a pro- inflammatory cytokine is added.
The third lipid profile may be provided by a method comprising the steps of
a) providing a cell culture medium comprising cells having cytokine receptors, c) adding a component that is potentially suitable for the prevention, treatment or co- treatment of a disease associated with increased or decreased cytokine pathway activity to the cell culture medium and
d) determining the lipid profiles of the cells and/or of the cell culture medium to obtain the third lipid profile, wherein the method does not include the step of
b) adding a pro-inflammatory cytokine to the cell culture medium.
The fourth lipid profile may be provided by a method comprising the steps of a) providing a cell culture medium comprising cells having cytokine receptors and d) determining the lipid profiles of the cells and/or of the cell culture medium to obtain the first lipid profile, wherein the method does not include the steps of
b) adding a pro-inflammatory cytokine to the cell culture medium and
c) adding a component that is potentially suitable for the prevention, treatment or co- treatment of a disease associated with increased or decreased cytokine pathway activity to the cell culture medium. The third and the fourth lipid profiles are a lipid profile of the cells and/or the cell culture medium obtained by method not including step b), i.e. it is a lipid profile of the cells and/or the cell culture medium to which no pro-inflammatory cytokine is added. The difference between the third and the fourth lipid profiles is that a component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity is added for obtaining the third profile. Thus, comparison between the third lipid profile and the fourth lipid profile allows determining the effect of the component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity when no pro-inflammatory cytokine is added.
Thus, the component which induces a change between the first lipid profile and the second lipid profile and/or a change between the third lipid profile and the fourth lipid profile is identified to be an active pharmaceutical ingredient. It will be appreciated that the steps for providing the lipid profiles is not critical may be performed in any order. For example, the first lipid profile may be provided after the second lipid profile, or the fourth lipid profile may be provided before any other lipid profiles are provided. Preferably, the lipids of which the lipid profiles are compared are chosen from the group of ceramides, for example a ceramide that is a sphingosine with fatty acid chains of from 16 to 24 C-atoms, sphingosine and sphingosine-1 -phosphate .
The differences in lipid profiles should be assessed in methods that make use of the same cells, conditions etc. Also, the differences should be assessed at the same point in time. For example, if a difference can only be seen in step d) after 6 hours after adding the component that is potentially suitable for the prevention, treatment or co- treatment of a disease associated with increased or decreased cytokine pathway activity, then this difference should be noted and the component identified as active pharmaceutical ingredient.
In another aspect, the invention relates to a composition comprising the active pharmaceutical ingredient obtained with the method of the invention, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
A person skilled in the art knows which carriers may be used as pharmaceutically acceptable carriers. Examples of such pharmaceutically acceptable carriers are both inorganic and organic carrier materials, suitable for oral/parenteral/injectable administration and include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, and the like. Besides the active pharmaceutical ingredient and a pharmaceutically acceptable carrier, the pharmaceutical composition according to the present invention, may further comprise conventional pharmaceutical additives and adjuvants, excipients or diluents, including, but not limited to, water, gelatin of any origin, vegetable gums,
ligninsulfonate, talc, sugars, starch, gum Arabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like. In another aspect, the invention relates to an active pharmaceutical ingredient obtained with the method of the invention or to the pharmaceutical composition of the invention for use as a medicament.
In yet another aspect, the invention relates to an active pharmaceutical ingredient obtained with the method of the invention or to the pharmaceutical composition of the invention for use in the prevention, treatment, or co-treatment of a disease associated with increased or decreased cytokine pathway activity.
Examples of diseases associated with increased or decreased cytokine pathway activity are given herein.
In yet another aspect, the invention relates to use of an active pharmaceutical ingredient obtained with the method of the invention or to the pharmaceutical composition of the invention for the manufacture of a medicament for the prevention, co-treatment or treatment of a diseases associated with increased or decreased cytokine pathway activity.
In another aspect, the invention relates to a method for treatment, co-treatment or prevention of a disease associated with increased or decreased cytokine pathway receptor activity in animals, including humans, said method comprising the step of administering an effective amount of the active pharmaceutical ingredient obtained with the method of the invention or administering an effective amount of the pharmaceutical composition of the invention to animals including humans which are in need thereof. With 'effective amount' of the active pharmaceutical ingredient obtained with the method of the invention or of the pharmaceutical composition of the invention is meant the amount needed to treat, co-treat or prevent the disease associated with increased or decreased cytokine pathway receptor activity in animals, including humans.
In the framework of the invention, with animals is meant all animals, including mammals, examples of which include humans. Preferred examples of mammals beside humans include dogs, dromedaris, camels, elephants, and horses.
Although the invention has been described in detail for purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the claims.
It is further noted that the invention relates to all possible combinations of features described herein, especially those combinations of features that are present in the claims.
The invention will now be illustrated with the following examples without however being limited thereto. Examples
Example 1
Primary Human Aorta Endothelial Cells (HAoEC) were cultured in T25 culture flasks using Endothelal Growth Medium (Cell Applications Incorporated) in an incubator at a temperature of 37°C and having an atmosphere of 5% 02 and 95% C02. When HAoEC were about 90% confluent, medium was refreshed.
At timepoints 0,1 , 3, 6, 10, and 24 hours samples of culture medium and the HAoEC cells were taken and frozen at -80 'C until measurements of lipid profiles were executed.
The results for the cells are shown in Figure 1 and the results for the cell culture medium are shown in Figure 3.
Example 2
Example 1 was repeated, but additionally 20 μg/ml anti-TNFa antibody was added to the cells. The results for the cells are shown in Figure 2. Lipid profiles were determined by means of the following LCMS method:
To a cell suspension of 100.000 HAoEC in 100 μΙ, 10 μΙ Internal standard (STD) (C17 ceramide) and 750μΙ MeOH/CHCI3 (2/1 ) (MeOH stands for methanol) were added and this was vigorously shaken for 10 minutes. Subsequently, the mixture was incubated at 48 °C for 1 hour. Then 75 μΙ 1 N KOH in MeOH were added and mixed. This mixture was incubated at 37°C. After 1 hour the pH was adjusted to pH ~ 5 by addition of 10% acetic acid.
Then, 750 μΙ of CHCI3 were added and mix with the sample. Samples were centrifuged at2600 rpm at 4<C for 3minutes. The organic phase was taken and reduced to dryness. After sonication for 3 minutes and centrifuging at 800g for 10 minutes the residue was reconstituted in a eluent mixture of 80% B and 20% A (see below).
LC-Conditions. Gradient elution according to the following table:
Eluent A 5 mM ammonium acetate in methanol/H20 (1 :1 ) containing 1 % acetic acid and 0.25% tetrahydrofurane
Eluent B 5 mM ammonium acetate in methanol containing 1 % acetic acid and
0.25% tetrahydrofuran
Gradient 0-0.1 min 1 % B isocratic, in 1 .9 min to 100% B and stays at 100% B for 6 min. Total analysis time is 12.5 minutes. The flow rate is 700 μΙ_Ληίη.
Column 2.1 x 100 mm, 1 .8 μηι Zorbax SB-C18 - or equivalent - operating at 50 'C. Injection Injection volume: 1 -2 μΙ_.
MS ionisation conditions.
ESI(+), MRM mode, fragmentor optimized for each compound (120 V); EMV +400 V, 300 'C gas temperature, flow 5 L N2/min, 45 psi, sheath gas temperature 250 'C, sheath gas flow 10 L N2/min, 3.5 kV cap, 0.5 kV nozzle, dwell time 5 ms. Two time segments are used: 0-2.5 minutes for sphingosine and sphingosine-1 -phosphate and 2.5-12.5 minutes for the ceramides. MS MRM conditions, tandem MS in the MRM mode:
Time segment 0-2.5 min:
Sphingosine; 300.2 ^ 282.2; EC 5 V
Sphingosine-1 -phosphate; 380.1 -» 264.2; EC 20 V
Time segment 2.5-12.5 min:
C12; 482.4 264.2; EC 15 V C16; 538.4 264.2; EC 20 V
C17 (IS); 552.0 264.2; EC 20 V
Clan ; 564.4 264.2; EC 20 V
C20; 594.5 -» 264.2; EC 20 V
C22; 622.5 -» 264.2; EC 20 V
C24:1 ; 648.5 -» 264.2; EC 20 V
C24; 650.6 -» 264.2; EC 25 V
Fig. 1 describes the lipid profile of the HAoEC cells in time, wherein TNFa was added; it specifically describes the concentrations of several ceramides: sphingosine with fatty acid side chains of 16 (C1 6), 1 8 (C18), 20 (C20), 22 (C22) or 24 (C24) C-atoms in time. In case of the sphingosine with fatty acid side chains of 24 C-atoms a sphingosine with an unsaturated fatty acid side chain (C24:1 ) and a sphingosine with a saturated fatty acid side chain (C24) were measured.
Fig. 2 describes the lipid profile of the HAoEC cells cells in time, wherein TNFa and anti-, TNFa antibody were added; it specifically describes the concentrations of several ceramides: sphingosine with fatty acid side chains of 16 (C16), 18 (C18), 20 (C20), 22 (C22) or 24 (C24) C-atoms in time. In case of the sphingosine with fatty acid side chains of 24 C-atoms a sphingosine with an unsaturated fatty acid side chain (C24:1 ) and a sphingosine with a saturated fatty acid side chain were measured.
Fig. 3 describes the lipid profile of the cell culture medium of the HAoEC cells in time, wherein TNFa was added; it specifically describes the concentrations of several ceramides: sphingosine with fatty acid side chains of 16 (C16), 18 (C18), 20 (C20), 22 (C22) or 24 (C24) C-atoms in time. In case of the sphingosine with fatty acid side chains of 24 C-atoms a sphingosine with an unsaturated fatty acid side chain (C24:1 ) and a sphingosine with a saturated fatty acid side chain were measured. Fig. 4 shows the lipid profile of primary human fibroblast-like synoviocytes after 1 hour of stimulation by TNFa (20ng/ml) (TNFa 1 H), after stimulation by TNFa followed by 4 hours of treatment by aspirin (TNFa + Asp 4H) or without stimulation or treatment (control). The comparison between Ex. 1 and Ex. 2 understood from Fig. 1 and 2 shows that the differences in lipid profiles can be used to determine the inhibiting effect of the cytokine pathway activity of a component (in this case anti-TNFa antibody).
Accumulation of lipids in the culture medium over time can be seen from Figure 3. Example 3
Primary Human Fibroblast-Like Synoviocytes (HFLS) of a healthy individual were cultured in 6 well culture plates using Synoviocytes Growth Medium (Cell Applications Incorporated) in an incubator at a temperature of 37 <C and having an atmosphere of 5% 02 and 95% C02. When HFLS were about 90% confluent, medium was refreshed. HFLS were either untreated (control), treated with TNFoc (20ng/ml) for one hour (TNFa 1 H), or treated with TNFa for one hour with subsequent addition of the antiinflammatory compound aspirin to the wells containing already TNFa (TNFa + Asp 4H). Aspirin was used in a concentration in the cell culture medium of 50μg/ml. A sample was taken after 4 hours exposure to the anti-inflammatory compound aspirin. Cells were taken and frozen at -80 °C until measurements of lipid profiles were executed. The lipid profiles were measured as described in the above examples.
Results are shown in Figure 4, wherein sphing stands for sphingosine. Similar to examples 1 and 2 above, C16 stands for a ceramide: sphingosine with fatty acid side chains of 16 C-atoms, C18 stands for a ceramide: sphingosine with fatty acid side chains of having 18 C-atoms, etc.
As can be seen in Figure 4, stimulation of HFLS with TNFa causes an increase in the concentrations of the individual lipids as compared to the concentrations before stimulation (control). By adding aspirin, the concentration of the individual lipids is decreased to the concentration before stimulation (control) or even below that concentration. The decrease of the concentration of the individual lipids shows that aspirin is potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity, such as (acute) inflammation. This is in line with the well-known fact that aspirin is a good drug for the treatment of acute inflammation
This example therefore shows that the method of the invention is suitable for finding potentially suitable pharmaceutically active ingredients for the treatment of a disease associated with increased or decreased cytokine pathway activity. The method of the invention provides an easy way to determine whether or not a component may be suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity. It does not require extensive calculations; rather, by looking at the behaviour of the individual lipids (the lipid profile), it can be seen instantaneously whether or not the component has an effect. If the component has an effect on one individual lipid, it is already a potential candidate, whereas in a method that only looks at the sum of the lipids, such candidate could easily be missed. The method of the invention therefore provides a very sensitive method for identifying active pharmaceutical ingredient potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity.

Claims

Method for finding an active pharmaceutical ingredient potentially suitable for the treatment of a disease associated with increased or decreased cytokine pathway activity, comprising the steps of
A1 ) providing a first lipid profile by
a) providing a cell culture medium comprising cells having cytokine receptors, b) adding a pro-inflammatory cytokine to the cell culture medium
c) adding a component that is potentially suitable for the prevention, treatment or co-treatment of a disease associated with increased or decreased cytokine pathway activity to the cell culture medium and
d) determining the lipid profiles of the cells and/or of the cell culture medium to obtain the first lipid profile,
A2) providing a second lipid profile by performing step A1 ) except for sub-step c) to obtain the second lipid profile,
B) comparing the first lipid profile to the second lipid profile and
C) identifying the component that was added in the method that shows a difference between the first lipid profile and the second lipid profile as the active pharmaceutical ingredient.
Method according to claim 1 , further comprising the steps of:
A3) providing a third lipid profile by performing step A1 ) except for sub-step b) to obtain the third lipid profile,
A4) providing a fourth lipid profile by performing step A1 ) except for sub-steps b) and c) to obtain the fourth profile,
D) comparing the third lipid profile to the fourth lipid profile and
E) identifying the component that was added in the method that shows a difference between the third lipid profile and the fourth lipid profile as the active pharmaceutical ingredient.
Method according to claim 1 or 2, wherein the pro-inflammatory cytokine is tumor necrosis factor a (TNFa).
Method according to any one of claims 1 -3, wherein the cells are human aorta endothelial cells (HAOEC).
Method according to any one of claims 1 -4, wherein the lipids of the lipid profiles that are compared are chosen from the group of ceramides, for example a ceramide that is a sphingosine with fatty acid chains of from 16 to 24 C-atoms, sphingosine and sphingosine-1 -phosphate.
PCT/EP2012/058217 2011-05-04 2012-05-04 Method for finding active pharmaceutical ingredients for the treatment of diseases associated with increased or decreased cytokine pathway activity WO2012150325A1 (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2004037205A2 (en) * 2002-10-24 2004-05-06 Abbott Biotechnology Ltd. LOW DOSE METHODS FOR TREATING DISORDERS IN WHICH TNFα ACTIVITY IS DETRIMENTAL
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