WO2008149103A1 - Procédés d'évaluation et de traitement d'une insuffisance de multiples organes - Google Patents

Procédés d'évaluation et de traitement d'une insuffisance de multiples organes Download PDF

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WO2008149103A1
WO2008149103A1 PCT/GB2008/001943 GB2008001943W WO2008149103A1 WO 2008149103 A1 WO2008149103 A1 WO 2008149103A1 GB 2008001943 W GB2008001943 W GB 2008001943W WO 2008149103 A1 WO2008149103 A1 WO 2008149103A1
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mof
sample
compound
lymph
protein
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PCT/GB2008/001943
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English (en)
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Damian James Mole
Neil V. Mcferran
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University Court Of The University Of Edinburgh
The Queen's University Of Belfast
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    • 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
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol

Definitions

  • the present invention provides medicaments for the prevention and/or treatment of multiple-organ failure as well as methods for determining the multiple- organ failure status of a patient.
  • AP Severe acute pancreatitis
  • MOF multiple-organ failure
  • the mesenteric lymphatic system and gut-associated lymphoid tissue are secondary lymphoid tissues which are strategically placed to marshal the interface between host and environment in the gastrointestinal tract (12).
  • immature dendritic cells constitutively migrate from the periphery to draining lymph nodes (LNs) in the mesenteric lymphatic system, constantly trafficking debris from apoptotic and effete intestinal epithelial cells (13, 14) to maintain peripheral tolerance (15).
  • innate immune activation (16) results in chemokine-mediated DC recruitment to sites of injury and subsequent migration to the relevant draining nodes (17).
  • DC recruitment and mobilisation to mesenteric lymph nodes (MLNs) is rapid, and has been shown in airway mucosa to occur within one hour of bronchial antigen administration (17).
  • DCs regulate the activity of the effector arms of innate and adaptive immunity through several mechanisms, including the production of cytokines, especially IL- 12 p75, IL-27, IFN ⁇ , IL-4, IL-5, IL-IO and TGF ⁇ , and through the differential expression of co-stimulatory molecules, for example OX40L (18).
  • cytokines especially IL- 12 p75, IL-27, IFN ⁇ , IL-4, IL-5, IL-IO and TGF ⁇
  • co-stimulatory molecules for example OX40L (18).
  • Other small soluble molecules are involved, in particular the immunoregulatory catabolites of tryptophan formed via the kynurenine pathway, controlled by the inducible rate-limiting enzyme indoleamine-2,3-dioxygenase (IDO) (19-22).
  • Kynurenines which are utilised by murine placenta to prevent foetal rejection by the elimination of maternal T cells (22), are important for the induction and maintenance of peripheral tolerance by DCs (21), and have been recently shown to participate in suppurative granuloma formation (23).
  • IDO catalyses the conversion of tryptophan to N-formylkynurenine, which is converted to kynurenine and then 3-hydroxykynurenine by the specific, constitutively active enzyme kynurenine 3-monooxygenase (24).
  • Downstream metabolites of kynurenine are also generated without increased IDO activity if kynurenine is introduced directly as a substrate, with regulatory effects on the elimination and suppression of autoreactive T cell clones (24).
  • the present inventors have discovered that, experimental AP results in the presence of one or more cytotoxic factors transported in mesenteric lymph that are capable of causing lung injury, endothelial and gut epithelial cell death, neutrophil respiratory burst activation and decreased erythrocyte deformability. Furthermore, the hierarchical clustering of lymph-derived mass spectra generated by surface-enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF MS) predicted the onset of lymph cytotoxicity and its resolution to an inert state in survivors of AP. In particular, the inventors have shown that the lymph contained elevated concentrations of kynurenine and 3-hydroxykynurenine.
  • the object of the invention is therefore to develop medicaments for use in treating MOF and methods for assessing a subject's MOF status.
  • the present invention is based upon the observation that, following a multiple-organ failure (MOF) initiating event, certain compounds carried in biological fluids play an important role in the development or exacerbation of MOF. These proteinaceous compounds may be detected in biological fluids.
  • the precise proteinaceous content of any given biological fluid may vary depending on the patient's MOF status - i.e. whether the patient has recently been exposed to a MOF initiating event and is likely to develop MOF, is currently suffering from or experiencing MOF, has recovered/is recovering from MOF or has not been exposed to a MOF initiating event.
  • the present inventors have discovered that, among others, the proteinaceous compounds involved in the development of MOF include components of the kynurenine pathway.
  • MOF multiple-organ failure
  • a method of treating MOF comprising the step of administering a therapeutically effective amount of a compound capable of modulating the kynurenine pathway.
  • a compound capable of modulating the kynurenine pathway for treating MOF is provided.
  • the kynurenine pathway is responsible for the catabolism of tryptophan which is first converted to N-formylkynurenine by either of the enzymes tryptophan 2,3- dioxygenase or indolamine-2,3-dioxygenase (IDO). Tryptophan 2,3-dioxygenase is predominantly expressed in the liver while IDO occurs in many extrahepatic tissues, dendritic cells and macrophages.
  • N-formylkynurenine is in turn converted to kynurenine and then to 3 -hydroxy kynurenine by the specific and constitutively active enzyme kynurenine 3-monoxygenase.
  • the present invention is principally concerned with MOF having a "sterile" aetiology.
  • Sterile initiators of MOF (referred to hereinafter as “sterile events”) may broadly be defined as non-microbial initiators and may include, for example, diseases and/or pathological conditions, trauma, injury, surgery and haemorrhagic shock.
  • MOF stems from an uncontrolled and inappropriate inflammatory response and may affect organs somewhat remote from the initiating event. As such, MOF may involve the lung, kidney, liver, cardiovascular, haematopoietic and other organ systems.
  • the present invention may be considered to relate to pancreatitis-associated MOF.
  • the components of the kynurenine pathway identified as involved in MOF and having cytotoxic activity may include an end product of the kynurenine pathway or kynurenine pathway intermediates.
  • the medicaments described herein may comprise one or more compounds capable of modulating the kynurenine pathway.
  • the medicament comprises one or more compounds capable of inhibiting or preventing the production of certain kynurenine pathway components.
  • compounds of the present invention may modulate an enzyme or enzymes of the kynurenine pathway.
  • a compound of the present invention may inhibit a particular enzyme involved in tryptophan catabolism.
  • Enzyme inhibitors are well known to one of ordinary skill in the art. Enzyme inhibitors may be classified as, for example, specific, non-specific, reversible, nonreversible, competitive and non-competitive.
  • any compound capable of inhibiting any enzyme or enzymes of the kynurenine pathway may potentially be used in the preparation of a medicament for the treatment and/or prevention of MOF.
  • inhibitor it is meant that, when compared to an enzyme which has not been contacted and/or exposed to a compound which might inhibit its activity, the rate at which an enzyme catalyses the production of a particular compound from a particular substrate is reduced.
  • the compound capable of modulating the kynurenine pathway may be a specific or non-specific enzyme inhibitor.
  • non-specific refers to a compound, the effects of which are not restricted to a particular enzyme, or class of enzyme, but which generally affects the activity of substantially all enzymes.
  • specific inhibitor may be taken to refer to a compound which may inhibit a particular enzyme but which has no effect on the activity of another enzyme.
  • non-specific inhibitors may function as a result of the fact they denature by means of a change in temperature, pH or the like.
  • non-specific inhibitors may also irreversibly bind to and block the active site of an enzyme thus preventing it from functioning to catalyse the production of a particular compound.
  • Specific enzyme inhibitors may function by means of binding to a particular enzyme and causing either the active site to be blocked or the enzyme to undergo a conformational change such that it can no longer receive and catalyse the conversion of a particular substrate.
  • the term "competitive inhibitors" may be taken to comprise compounds which are capable of occupying the active site of a particular enzyme resulting in the exclusion of the correct substrate. In this way the rate at which a particular enzyme is able to catalyse the production of a particular compound is reduced. Such compounds may closely resemble the particular substrate of the enzyme.
  • compounds for use in modulating the kynurenine pathway may also include competitive inhibitors of each of the enzymes involved in the kynurenine pathway.
  • such compounds may structurally resemble the native (natural/correct) substrate of the enzyme.
  • a competitive inhibitor may comprise a peptide which, for example, binds to the active site or substrate binding domain of an enzyme to block substrate binding.
  • said compounds may be small organic molecules capable of modulating the function of an enzyme or enzymes of the kynurenine pathway.
  • the small organic molecule may be an analogue of any of the substrates of the enzymes utilised in such pathways.
  • the substrate analogue may be modified in such a way so as to either inhibit and/or prevent the progression of a particular enzymatic reaction in a particular pathway.
  • a compound for use in modulating the kynurenine pathway may interact with a particular enzyme or enzymes which in turn catalyse the production of a product which is incapable of being utilised by another enzyme of the kynurenine pathway. In this way further enzymatic reactions will not be possible and the pathway will be unable to progress.
  • non-competitive inhibitor may be taken to include those compounds which affect the rate at which an enzyme catalyses the production of a particular compound.
  • non-competitive inhibitors may interact with an enzyme at a position other that the active site of an enzyme. For example a non-competitive inhibitor may bind to an enzyme and induce a conformational change in the enzyme such that the binding site of the enzyme may no longer receive a substrate.
  • a small organic molecule may modulate an enzyme of the kynurenine pathway and thus the production of one or more of the kynurenine pathway products.
  • the enzymes Tryptophan 2,3- dioxygenase and/or indolamine-2,3-dioxygenase it may be possible to reduce or eliminate the production of N-formylkynurenine. This would have the effect of eliminating or substantially reducing the amount of N-formylkynurenine available for conversion into kynurenine. This may, in turn, reduce the production of those components involved in MOF.
  • the amount of 3-hydroxykynurenine produced may be eliminated or significantly reduced.
  • the present invention provides the use of an inhibitor of an enzyme involved in the kynurenine pathway for the preparation of a medicament for the treatment and/or prevention of MOF.
  • Suitable inhibitor compounds include 1-methyltryptophan (an IDO inhibitor) and 3,4-dichlorobenzoylalanine (PNU156561 : a K3M0 inhibitor).
  • IDO inhibitor 1,3-dichlorobenzoylalanine
  • PNU156561 3,4-dichlorobenzoylalanine
  • a more comprehensive list of kynurenine pathway modulators which may have therapeutic potential is provided below. Tryptophan 2,3-Dioxygenase inhibitors
  • the present invention provides the use of one or more of the abovementioned kynurenine pathway modulators for the preparation of a medicament for the treatment and/or prevention of MOF.
  • compounds for use in modulating the kynurenine pathway may include oligonucleotide sequences. Such compounds may modulate the expression and/or activity of enzymes of the kynurenine pathway.
  • oligonucleotide sequences may be complementary to the genes (or parts thereof) which encode the enzymes involved in the kynurenine pathway.
  • the oligonucleotide sequences may be sequences of nucleic acid, either DNA or RNA which interfere with the functional sequences encoding enzymes of the kynurenine pathway. Cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase Lorenzen MD et al. Genetics. 2002 Jan;160(l):225-34.
  • the oligonucleotide sequences may be sections of messenger or ribosomal RNA (rRNA) which interfere with the RNA transcripts of the functional enzyme genes.
  • rRNA messenger or ribosomal RNA
  • Such oligonucleotides are referred to in the art as interfering RNA (iRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), or antisense oligonucleotides.
  • Algorithms such as BIOPREDs/ may be used to computationally predict siRNA sequences that have an optimal knockdown effect for a given gene.
  • BIOPREDs may be used to computationally predict siRNA sequences that have an optimal knockdown effect for a given gene.
  • the present invention also relates to methods for analysing the MOF status of a subject so to ensure that the most appropriate treatment is given without delay.
  • a method for analysing the multiple organ failure status of a subject comprising the steps of:
  • component profile encompasses those molecules or compounds which comprise a biological fluid and may include, for example, proteins, peptides, amino acids, other small organic molecules and/or nucleic acids.
  • identifying the component profile may also be considered to mean assessing or analysing the constituents of the sample and encompasses identifying protein- expression profiles.
  • a method for analysing the multiple-organ failure status of a subject comprising the steps of: (a) providing a sample from a subject; and
  • biological fluid and “sample” encompass fluids obtained from the human or animal body and include, for example, whole blood, plasma, serum, urine, lymph and saliva.
  • sample used is whole blood, plasma or lymph.
  • a protein profile may also be referred to as a sample's "proteome”.
  • the method of determining the MOF status of a subject comprises analysing a sample, and in particular the protein content of a sample, by mass spectrometry. Fathman, C. G., Soares, L., Chan, S. M. and Utz, P. J.: 2005, 'An array of possibilities for the study of autoimmunity.' Nature 435, 605-11. Issaq, H.
  • the sample may be analysed by matrix-assisted laser desorption/ionization (MALDI-TOF) or surface-enhanced laser desorption/ionization (SELDI-TOF) mass spectrometry.
  • MALDI-TOF matrix-assisted laser desorption/ionization
  • SELDI-TOF surface-enhanced laser desorption/ionization
  • the sample may be analysed by chromatography techniques such as, for example High Performance Liquid Chromatography (HPLC) or the like.
  • HPLC High Performance Liquid Chromatography
  • the "read-out" for example the component or protein profile
  • the "read-out” is represented as a series of resolved peaks which reveal the level of expression and molecular weights of the various components of the sample (i.e., in the case of a protein profile, the level of expression and molecular weights of the proteins comprising the protein profile).
  • the detection parameters used such that only compounds falling within a particular range of molecular weights are included in the read-out.
  • the profile of those compounds for example proteins, having a molecular weight of about 100 to 200000 Da, preferably 1000 to 180000, more preferably 2000 to 170000 Da and still more preferably, 3000 to 150000 Da.
  • GC-MS gas chromatography-MS
  • mass spectrometry techniques allow the general analysis of the content of a particular sample
  • other techniques may be required to permit the identification of specific components.
  • tandem MS techniques for example MS-MS, and/or separate/resolve the proteins by liquid chromatography or 2D gel electrophoresis, subject certain resolved proteins to a digestion protocol (for example tryptic digestion) and further MS analysis (for example electrospray MS analysis).
  • MS-MS mass spectrometry techniques
  • one of skill in the art may compare the component (for example, protein) profile of the sample, with the component profile of a reference sample.
  • a "reference sample” may be considered as a sample derived from a subject of known MOF status - i.e. a reference subject. Additionally, or alternatively, the results obtained may be compared with a number of reference samples, each having been obtained from a subject of known MOF status. Preferably, the reference sample should be derived from the same biological fluid.
  • organ failure may be inferred from a clinical need for organ support, for example renal replacement therapy (for example by haemodialysis) or ventilatory support for respiratory failure, or vasopressor support for failure to maintain peripheral vascular resistance.
  • Certain combinatorial scoring systems for example the multiple organ dysfunction score (Marshall, J. C, Cook, D. J., Christou, N. V., Bernard, G. R., Sprung, C. L. and Sibbald, W.
  • a sample to be representative of a subject in the resolution phase may be derived from a patient known to have recovered from MOF.
  • a reference sample from a subject currently suffering from or experiencing MOF may be derived from a subject clinically diagnosed as having MOF (as above).
  • a sample to be representative of a subject who has not been exposed to an MOF initiating event may be obtained from a healthy individual.
  • a sample, representative of a subject who, at the time the sample is taken, is not critically ill but may go on to develop MOF, it may be possible to take regular biological fluid samples, for example blood samples, until such time as MOF develops or begins to resolve. In this way, once MOF has been confirmed, it would be possible to select a sample representative of a subject about to develop MOF.
  • MOF status and which are particularly useful for identifying those not critically ill but who may go on to develop MOF include, for example, clinical assessment by an experienced physician, serial markers of pulse, blood pressure, arterial blood gas analysis and urine output, serum biomarkers, for example procalcitonin and/or C-reactive protein.
  • the present invention provides a method of determining the multiple-organ failure status of a subject, said method comprising the steps of: (a) providing a sample from a subject;
  • the component profile is a protein-expression profile.
  • a number of reference samples - each derived from subjects of known MOF status are analysed for their component or protein profile and subjected to a hierarchical clustering system.
  • the hierarchical clustering may be achieved by using an algorithm such as that described by Eisen et al. (29).
  • Eisen et al. 29
  • By clustering the various reference samples it is possible to associate particular component or protein profiles (or proteomes) with particular MOF statuses. For example, when the component or protein profile of a particular sample is analysed, it may be compared with the clustered reference data, and the subject can be appropriately identified as having a particular MOF status.
  • component or protein profiles obtained from samples derived from different reference subjects will display considerable variation.
  • One of skill in the art will appreciate that by increasing the number of reference samples and subjecting the reference data to a hierarchical clustering protocol, it may be possible to provide an accurate assessment of a subject's MOF status.
  • the method may be substantially automated such that a computer assesses the component or protein profile of the sample and automatically allocates the most appropriate MOF status.
  • the computer may be preloaded with the component or protein profiles of one or more reference samples.
  • the reference samples may have been subjected to a hierarchical clustering protocol such that particular component or protein profiles are associated with particular MOF statuses.
  • the computer may assign that sample a particular MOF status.
  • the computer may simply compare the component or protein profile of the sample with the reference samples to find a close match, or, using a hierarchical clustering algorithm (such as that described above), allocate the component or protein profile of the sample to a particular cluster of reference samples.
  • the present invention provides a data set, comprising representative component or protein profile data, wherein the data represents the component or protein profiles of biological fluids obtained from subjects know to have one of the aforementioned MOF statuses.
  • the component or protein profile data is generated by mass spectrometry and represents the levels and molecular weights of the components or proteins comprising the protein profile.
  • the data set may be subjected to a hierarchical clustering protocol such that particular component or protein profiles are associated with particular MOF statuses.
  • a sterile MOF initiating event may lead to the generation of compounds which induce or exacerbate MOF.
  • These compounds are carried in biological fluids such as blood and lymph to various organs and/or organ systems and may induce or exacerbate MOF.
  • the compounds have an effect upon the cytotoxicity of the fluid and the cytotoxicity of the biological fluid is generally associated with the MOF status of the patient.
  • Biological fluids obtained from subjects suffering from or experiencing MOF may exhibit cytotoxic properties - i.e. when certain cells, for example endothelial cells such as Human umbilical vein endothelial cells (HUVEC) or epithelial cells such as HCTl 16 colonic epithelial cells, are brought into contact with the biological fluid, the cells die.
  • endothelial cells such as Human umbilical vein endothelial cells (HUVEC) or epithelial cells such as HCTl 16 colonic epithelial cells
  • the same fluid obtained from a patient who has not been exposed to a sterile MOF initiating event may not be cytotoxic (i.e. inert).
  • Biological fluids obtained from subjects recovering from MOF may exhibit low/no cytotoxicity.
  • Table 1 shows how the level of cytotoxicity of the sample may be equated to the MOF status of the subject.
  • cytotoxic indicates that, when brought into contact with certain cells, for example endothelial and/or epithelial cells, the biological fluid causes the cells to die.
  • An "inert” biological fluid is one which exhibits no cytotoxic effect.
  • post-cytotoxic refers to biological fluids derived from subjects who are critically ill but are beginning to convalesce (i.e. in the resolution phase). Subjects having biological fluid identified as “pre-cytotoxic”, although not critically ill at the time the sample is taken, are likely to develop MOF. By way of example and, in the case of a plasma sample, the method would require the analysis of the component or protein profile.
  • the component or protein profile of the plasma sample is analysed by SELDI-TOF mass spectrometry.
  • the resulting profile may be compared with one or more reference plasma sample(s) derived from a subject or subjects of known MOF status.
  • the collection of reference samples will have been subjected to a hierarchical clustering protocol so as to associate a particular component or protein profile with particular MOF status. In this way, it is possible to assess a subject's MOF status and administer the most appropriate treatment as soon as possible.
  • the method may comprise analysing a sample to identify a level of a kynurenine pathway component, for example kynurenine and/or 3-hydroxykynurenine.
  • a kynurenine pathway component for example kynurenine and/or 3-hydroxykynurenine.
  • the sample may be analysed by, for example, High Performance Liquid Chromatography (HPLC) or the like.
  • HPLC High Performance Liquid Chromatography
  • the identified level of the kynurenine pathway component is indicative of a subject's MOF status and, in one embodiment, the identified level of a kynurenine pathway component or components may be compared or analysed together with one or more reference sample(s) or standards derived from a subject or subjects of known MOF status.
  • a reference "standard” may be considered a particular level of a compound known to be associated with a particular disease, condition or subject status.
  • transferrin ii) haptoglobin; (iii) ⁇ l -protease inhibitor; and (iv) native apoAl.
  • the inventors ascertained that, as cytotoxicity develops, the levels of haptoglobin and transferrin decrease, the level of ⁇ l -protease inhibitor increases and the native apoAl is altered to a number of distinct isoforms with variable pis.
  • the present invention provides a method for determining the MOF status of a subject, said method comprising the steps of:
  • the method may include the further step of identifying a level of one or more of the kynurenine pathway components.
  • Altered isoforms of apoAl are identified as those having a pi value different to that attributed to native apoAl (pI5.5). Specifically, the altered isoforms may have pi values of about 3.2, 4.7 and 5.0.
  • immunological techniques such as ELISA may also be used.
  • an agent for example an antibody, known to specifically bind to these proteins may be used to detect their presence in a sample derived from a subject.
  • an agent for example an antibody
  • an antibody known to specifically bind to these proteins may be used to detect their presence in a sample derived from a subject.
  • the present invention provides the use of one or more compounds capable of modulating the kynurenine pathway and/or the level of transferrin, haptoglobin and/or ⁇ l -protease inhibitor, for the treatment and/or prevention of MOF.
  • a method of treating MOF comprising the step of administering a therapeutically effective amount of one or more compounds capable of modulating the kynurenine pathway and/or the level of transferrin, haptoglobin and/or ⁇ l -protease inhibitor.
  • a tenth aspect there is provided the use of one or more compounds capable of modulating the kynurenine pathway and/or the level of transferrin, haptoglobin and/or ⁇ l -protease inhibitor, for treating MOF.
  • the component profile is a protein-expression profile.
  • Figure 1 Cytotoxicity of post-nodal mesenteric lymph.
  • A Viability of HUVECs
  • Figure 2 AP lymph induces necrosis of HCTl 16 colonic epithelial cells.
  • A Light microscopy (10Ox original magnification) of HCTl 16 cells incubated for 18 hours with 10% v/v FCS or sham-operated lymph or AP lymph (as marked)
  • B Viability of cultures from Figure 2A measured by MTT assay.
  • C Western blot of culture lysates from Fig. 2A probed with polyclonal antibody to PARP. Lanes: FCS; sham-operated lymph; AP lymph (as marked). Intact PARP migrates at an estimated MW of 116kDa. Cleaved PARP is seen at an estimated MW of 85kDa in lane 3.
  • Figure 3 (A) Prediction of impending toxicity and confirmation of resolution by hierarchical clustering of SELDI-TOF mass spectra (anion exchange, pH 9) based on the Eisen algorithm (29). A binary value for each sample was allocated according to cytotoxicity to HUVECs. Lymph samples cluster into four groups: inert throughout (sham-operated animals); impending cytotoxicity (AP early in disease course); actual cytotoxicity (AP at height of disease); resolution to inertia (survivors of AP). Red (y) and blue (x) arrows indicate m/z species displayed in detail in Figure 3, B. (B) Representative sections of mass spectra used in the hierarchical clustering analysis in Figure 3A.
  • Figure 5 Serum cytokine and chemokine profile in response to intra-arterial LPS administration in established experimental AP.
  • A TNF ⁇ ;
  • B IFN ⁇ ;
  • C IL- l ⁇ ;
  • D IL-6;
  • E CINC-2 ⁇ ;
  • F IL-IO.
  • a minimum of 6 rats was used for each combination of
  • FIG. 6 Tryptophan catabolites mediate lymph cytotoxicity in experimental AP and plasma concentrations correlate with the extent of distant organ injury in human patients with AP.
  • A Concentrations of kynurenine and
  • C Viability (MTT assay) of HCTl 16 cells incubated for 18 hours with combinations of tryptophan, kynurenine and 3-hydroxykynurenine at the concentrations shown.
  • D Dose-response of 3-hydroxykynurenine cytotoxicity towards HCTl 16 cells in culture. For (C) and (D), bars represent the mean ⁇ 2SEM of three experiments.
  • E and F Kynurenine concentrations in peripheral blood plasma in a cohort of 34 patients with AP on admission (day 0), day 1 and day 7 after hospitalisation. 19 patients had a severe attack defined by the Atlanta criteria. 10 developed organ failure necessitating invasive organ support in the intensive care unit.
  • Kynurenine concentrations are plotted against the APACHE II score (E) and multiple organ dysfunction score (F) of each patient at the time of blood sampling.
  • G and H Peripheral plasma kynurenine concentrations plotted against the eventual requirement for haemodialysis (G) and intubation and mechanical ventilation (H).
  • Figure 7 proposed paradigm of the contribution of the kynurenine pathway to multiple-organ failure in severe acute pancreatitis.
  • An immunological "state of alarm” is triggered by tissue distraction in the pancreas.
  • Gut DC and macrophage activation occurs through danger signals, pattern recognition receptors and scavenger receptors, especially by self-derived molecules e.g. haptoglobin, oxLDL via SR-A and CD36 family.
  • Gut DCs migrate via afferent lymphatics to MLN.
  • MLN a contradictory combination of pro-inflammatory signals and lack of cognate microbial antigen, increased self or altered-self antigen and the presence of potentially autoreactive T cells leads to a necessary but potentially excessive suppression of immunity to self.
  • Cytotoxic by-products of the induction of anergy or suppression including cytotoxic kynurenine catabolites, overspill into the efferent mesenteric lymphatics (5) Cytotoxic mesenteric lymph enters the central venous circulation and arrives first at the lungs. Direct toxicity to endothelium with capillary leakage occurs. Erythrocyte injury affects the micro-circulation. Neutrophils are activated and effect further organ damage. Materials and methods. Reagents. All reagents were purchased from Sigma-Aldrich Company Ltd. (Gillingham, Dorset, UK) unless otherwise stated.
  • AP was induced in rats by the method of Schmidt et al (40). Briefly, under general anaesthesia (intraperitoneal ketamine 2.5 mg/kg and xylazine 1.2 mg/kg, post-operative buprenorphine 20 ⁇ g/kg i.v. 6 hourly), carotid artery and jugular vein catheters were placed, and at laparotomy a 10 minute, pressure-controlled retrograde biliopancreatic duct infusion (10 mM glycodeoxycholic acid, 500 mM glycyl-glycine, 4 mM CaCl 2 , pH 8.0) was given at 8.4 mL/kg/hour. I.v.
  • caerulein in 150 mM NaCl was given at 5 ⁇ g/kg/hour for 6 hours. Animals were resuscitated with i.v. 150 mM NaCl at 4 mL/kg/hr for 12 hours. Sham-operated rats underwent identical anaesthesia, surgery and resuscitation without glycodeoxycholic acid infusion and replacement of caerulein with 150 mM NaCl. AP with organ dysfunction was confirmed by sickness behaviour, pancreatic histology, elevated serum amylase >1000 IU, elevated haematocrit and metabolic acidosis on serial arterial blood gas analysis, as previously described (41). Mesenteric lymph duct ligation and lymph collection. Mesenteric lymph duct ligation and lymph collection was performed as previously described (10).
  • the mesenteric lymph duct was either divided between ligatures, or cannulated with heparin-flushed polypropylene tubing passing externally through the scruff. Continuously collected lymph was aliquoted hourly into sterile, heparin-rinsed polypropylene tubes onto ice. The hourly lymph volume was replaced with 150 mM NaCl i.v.. Lymph was centrifuged immediately (60Og, 4°C, 3 minutes) and the supernatant stored at -80°C for ⁇ 12 months.
  • HUVECs (Clonetics, San Diego, USA), or HCTl 16 cells (European Collection of Animal Cell Cultures, Salisbury, Wilts., UK) were seeded at 2 x 10 4 cells per well and grown for 24 hours in DMEM (95% air, 5% CO 2 , 100% humidity). Lymph or FCS was added at 10% v/v, or an equivalent volume of DMEM was used as a negative control. After 18 hours, cell viability was measured using the TOXl (MTT) assay kit, quantified by spectrophotometric absorbance at 570 nm (T- Max, Molecular Devices, Wokingham, Berks., UK).
  • TOXl TOXl
  • Neutrophil respiratory burst activation was measured as previously described (9). Briefly, rat neutrophils were prepared from whole blood by erythrocyte lysis in 0.84 mM NH 4 Cl and incubated (1 hour) with 5% v/v lymph (or HBSS as a negative control) and dihydrorhodamine-123 (Molecular Probes, Oregon, USA), prior to stimulation with 270 ng/mL PMA. The respiratory oxidative burst was quantified using a FACSscan flow cytometer and Cellquest software (Beckton Dickinson, San Jose, CA, USA). Daily calibration was performed using Calibrate beads (Pharmingen, USA). Neutrophils were identified using forward and side scatter analysis.
  • Ektacytometry was performed on a laser- assisted optical rotational cytometer (RR Mechatronics, Hoorn, Netherlands).
  • the elongation index, E.I. was calculated as (x - y)(x + y) '1 , where x and y represent the maximum longitudinal and transverse erythrocyte diameters.
  • Lymph aliquots were incubated on blood agar for 18 hours at 37°C prior to examination by eye. Endotoxin was assayed using the Limulus amoebocyte lysate assay (Pyrochrome® LAL Assay; Quadratech, Epsom, Surrey, UK). Heating lymph for 10 minutes in a block heater at 90°C prior to MTT assay was performed to evaluate the effect on cytotoxicity. Lymph pH was measured on solid state i-STAT® CG 8 + cartridges (i-STAT Corporation, East Windsor, NJ, USA).
  • TNF- ⁇ was measured by ELISA (QuantikineTM, R&D Systems, Minneapolis, MN, USA). Protease activity was measured using the EnzCheckTM Protease Activity kit, (E-6638, Molecular Probes, Eugene, OR, USA).
  • RNAse A 0.1 mg/ml
  • Nucleic acids were precipitated with 0.1 volume 3 M sodium acetate and 3 volumes cold ethanol followed by centrifugation (13,000g, 15 minutes). Samples were resuspended in 1OmM Tris-HCl, pH 7.5, ImM EDTA, and visualised on a 1.5% agarose gel containing ethidium bromide.
  • ProteinChipsTM (Ciphergen, Fremont, CA, USA) employing anion exchange (QlO) with elution at pH 9 and pH 6, weak cation exchange (CMlO) with elution at pH 7 and pH 4, immobilised metal affinity capture (IMAC30) using Cu 2+ and Ca 2+ bait, and a hydrophobic (H50) surface, were screened. QlO and IMAC30 Cu 2+ were used for comprehensive analysis.
  • Arrays were equilibrated with 150 ⁇ L binding buffer (5OmM Tris HCl pH 9 for QlO; 10OmM sodium phosphate, 50OmM sodium chloride, pH 7 for IMAC Cu 2+ ) for 5 minutes, twice, with shaking. Samples were diluted 10-fold in binding buffer and incubated on the array for 30 minutes prior to washing off non- interacting proteins with binding buffer. Energy-absorbing matrix (sinapinic acid 100 ⁇ g in 200 ⁇ L acetonitrile, 200 ⁇ L 1% trifluoroacetic acid) was applied twice. Chips were read on a CiphergenTM PBSII SELDI-TOF mass spectrometer, using ProteinChipTM Reader and Ciphergen Express software.
  • the spot protocol was: high mass 100,000 Da, optimised from 2,000 to 100,000 Da; laser intensity 220, adjusted between 180 and 240 as necessary; focus by optimisation centre; SELDI acquisition set from position 20 to position 80, delta 2, transients 5; with 2 warming shots not included in the analysis.
  • calibration was performed with protein standards: hirudin 7.0 kDa, cytochrome c 12.2 kDa, myoglobin 17.0 kDa, carbonic anhydrase 29.0 kDa, yeast enolase 46.7 kDa, albumin 66.4 kDa, and bovine IgG 147.3 kDa (Ciphergen, Fremont, CA, USA).
  • phase 1 300V, 2mA, 5 W, 0.01 hour
  • phase 2 increasing to 350OV, 2mA, 5W, over 1.5 hour
  • phase 3 3,500V, 2mA, 5W, 26.5 hours.
  • the second dimension was run by equilibrating strips for 15 min at 20 0 C in 50 mM Tris pH 8.8, 6M Urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, 10 mg/mL dithiothreitol, followed by 15 min with dithiothreitol replaced by 25 mg/mL iodoacetamide.
  • second dimension electrophoresis was performed at 30 mA for 4 hours at 2O 0 C. Gels were fixed and stained with colloidal Coomassie Blue for 4 hours prior to destaining in 25% ethanol and scanning.
  • Sequencing grade trypsin (Promega, Inc., Southampton, UK) with overnight incubation at 3O 0 C was used for in-gel digestion of spots visually selected on the basis of differential expression, followed by peptide extraction (5% v/v trifluoroacetic acid, 50% v/v acetonitrile 3 x 6 min with sonication at 2O 0 C), evaporation, rehydration, and desalting using Cl 8 reversed phase resin (ZipTip, Millipore (UK) Ltd., Watford, Herts., UK).
  • Extracted peptides were analysed by electrospray tandem mass spectrometry using a ThermoQuest LCQ Deca mass spectrometer, in nanospray mode, and MS/MS data collected using the Xcalibur software suite (Thermo Fisher Scientific Inc., Waltham, MA, USA). MS/MS ion search was performed using the MASCOT search service (Matrix Science, www.matrixscience.com). Immunohistochemistry.
  • Sections were stained on a Ventana NexES autostainer (Ventana Inc., Arlington, AZ, USA) using anti-CDl lc mAb (NCL-L-CDl lc-563, Novocastra, Newcastle-upon-Tyne, UK) at 1:100 dilution, visualised with diaminobenzoate conjugate (DAKO Ltd, Ely, UK), counterstained with hematoxylin. SearchlightTM multiplex ELISA.
  • Serum samples were outsourced to Pierce Biotechnology, Inc., Rockford, IL, USA for SearchlightTM Rat Cytokine/Chemokine Array multiplex analysis by ELISA using streptavidin/biotin-HRP conjugates visulaised with SupersignalTM Luminol Enhancer. 10 ⁇ L of serum, at 1 :5 dilution, was analysed by technicians blind to the sampling conditions. Luminescent images were digitally recorded within 10 minutes and analysed using Array VisionTM software (Imaging Research, Inc., GE Healthcare BioSciences Corp., Piscataway, NJ., USA). Kynurenine assay. This was a development of the HPLC method proposed by Yong and Lau (42).
  • peripheral blood was sampled from 34 patients with a diagnosis of AP based on a history of epigastric pain, vomiting and an elevated serum amylase of greater than 3 times normal. Patients were included if they had presented to hospital within 72 hours of the onset of symptoms. Peripheral venous blood was sampled on admission (day 0), 24 hours later (day 1) and 1 week later (day 7) and plasma stored at -8O 0 C for ⁇ 12 months. The APACHE II score and the MODS score at the time of venepuncture were recorded (43, 44). Organ failure requiring mechanical ventilation or haemodialysis was noted. Severity of AP was defined according to the Atlanta criteria (45).
  • the humoral component of mesenteric lymph in rats with AP contains factor(s) which cause extrapancreatic organ injury.
  • mesenteric lymph contains cytotoxic factor(s) during other sterile initiators of MOF (11)
  • AP lymph induces necrosis rather than apoptosis of HCTl 16 gut epithelial cells.
  • AP lymph damaged gut epithelial cells. Lymph aliquots identified as cytotoxic to HUVECs caused death of HCTl 16 colonic epithelial cells in vitro ( Figure 2, A and B).
  • Hierarchical clustering of SELDI-TOF mass spectra predicts the onset and resolution of mesenteric lymph cytotoxicity.
  • SELDI-TOF MS To determine the extent of alterations to the lymph humoral proteome during the development of cytotoxicity, we used high-throughput SELDI-TOF MS.
  • QlO anion exchange
  • ClO weak cation exchange
  • IMAC30 immobilised metal affinity capture
  • H50 hydrophobic
  • CDlIc + DCs migrate from the gut to draining mesenteric lymph nodes during experimental AP with disruption of nodal architecture.
  • MLNs are key sites of immunoregulation, and that increased DC migration from the gut lamina limbal to draining MLNs occurs during systemic inflammation (13, 30), we asked whether DC migration occurred synchronously with the development of AP lymph cytotoxicity.
  • CDl Ic + DCs were frequent in ileum lamina intestinal ( Figure 4, A) and scarce in the corresponding MLN ( Figure 4, B).
  • Experimental AP (18 hours after induction) resulted in fewer CDl Ic + cells in the lamina limbal N ( Figure 4, C), and increased CDl Ic + cells in the corresponding MLN ( Figure 4, D).
  • Kynurenine pathway intermediates contribute to MOF in severe AP in rats and humans. Having found that cytotoxicity of mesenteric lymph in AP resides in the soluble cell-free compartment, and that the cytotoxic factor(s) damage several cell types, we wished identify the effector agent(s).
  • endotoxin, bacterial contamination, pH, total protease activity and TNF ⁇ content could not account for the cytotoxicity of mesenteric lymph. This conclusion follows the observation that some inert lymph samples contained elevated levels of endotoxin, TNF ⁇ or protease activity, while some cytotoxic samples showed no evidence of endotoxin, TNF ⁇ or protease activity. No viable bacteria were detected in any sample. Heating of mesenteric lymph from AP rats for 10 minutes at 90 0 C did not abrogate cytotoxicity.
  • Figure 8 is a confocal microscope image of mesenteric lymph node from a rat with acute pancreatitis. It shows that the first (and rate-limiting) enzyme of the kynurenine pathway (indoleamine-2,3,dioxygenase, IDO) is present and expected to be upregulated in CDl Ic cells in the time frame of acute pancreatitis. This supports the hypothesis of enzymes in the kynurenine pathway being targets for intervention in acute organ failure. Discussion
  • MOF is the key determinant of mortality in patients with conditions such as severe AP. In one study, those patients in whom organ failure persisted beyond 48 hours experienced a mortality of 37%, compared to those with transient organ failure or no organ failure, in whom mortality was less than 3% (2). In a population-based study of death from AP, one third of all deaths from acute pancreatitis occurred before the patient reached hospital, and, where known, the median duration of symptoms was less than 24 hours (31 ). MOF in acute pancreatitis is therefore the main determinant of outcome and an early event.
  • GALT Gut-associated lymphoid tissue
  • the evolutionary advantage of the development of GALT is to provide a framework in which adaptive immune cells (mainly T and B cells) may co-localise with antigen- presenting cells, mostly DCs.
  • Afferent lymphatics deliver antigen and facilitate the migration of DCs to MLNs, draining into the marginal sinus immediately below the LN capsule.
  • experimental AP was associated with increased numbers of CDl Ic + cells in MLNs and decreased numbers of CDl Ic + cells in gut lamina limbal, suggesting DC migration in AP.
  • the MLN is separated into an outer cortex and an inner paracortex, the functional unit of which, and likely site of suppression or elimination of autoreactive T cell clones, is the paracortical cord, regarded as the space in which DCs encounter and signal the activation and proliferation of rare antigen-specific circulating T cells (for a review see (12)).
  • the paracortical cord regarded as the space in which DCs encounter and signal the activation and proliferation of rare antigen-specific circulating T cells (for a review see (12)).
  • architectural disruption of MLN in AP was observed towards the efferent regions within MLNs, at the putative sites of DC-T cell interaction, with relative cortical sparing.
  • Post-nodal mesenteric lymphatics coalesce in a racemose network in the root of the midgut mesentery before passing cephalad to join the central venous system at the origin of the left brachiocephalic vein.
  • the first capillary bed which returning lymph encounters is therefore in the lung, notably the most commonly injured extrapancreatic organ during AP.
  • 35 (88%) had pulmonary injury, compared to 14 (35%) with renal dysfunction (32).
  • Lung injury in AP is mediated at least in part by neutrophils (9), and the humoral component of cytotoxic AP lymph activated rat neutrophils.
  • Gut epithelial cell injury is a feature of AP, and the cytotoxic activity of AP lymph to HCTl 16 cells suggests that recirculating mesenteric lymph may propagate gut injury.
  • the effect of cytotoxic lymph on erythrocyte deformability implies that there is a non-specific damaging effect of lymph which does not require de novo gene transcription.
  • Reduced erythrocyte deformability with evidence of oxidative stress has been shown previously in AP (33, 34). 3-hydroxykynurenine, as a candidate cytotoxic factor proposed in this study, causes cell injury by oxidative damage (24).
  • LDL scavenger receptor A
  • SR-BI scavenger receptor A
  • SR-BI facilitates the selective transfer of cholesteryl esters from the hydrophobic core, but not the apolipoprotein at the surface, which is rejected back into the circulation (36). This mechanism potentially explains the altered apoAl isoforms present in cytotoxic lymph.
  • Cytotoxic lymph was also depleted of haptoglobin.
  • Haptoglobin scavenges free hemoglobin, preventing free hemoglobin from causing oxidative damage to tissues, and the haptoglobin-hemoglobin complex, bound by the scavenger receptor CD 163 on macrophages, is a further powerful initiator of innate immune responses and potential stimulant of DC maturation (35, 37).
  • DC-mediated immunoregulation depends on the microenvironment within the
  • MLN in particular the relative concentrations of cytokines, especially IL-IO.
  • High concentrations of IL-IO signal immature monocyte-derived DCs to induce a CD25 + CD4 + regulatory T cell phenotype, characterised by the secretion of further IL- 10 and capable of limiting the activation of other CD4 + and CD8 + T cells by mature DCs (38).
  • the diminished production of TNF ⁇ , IFN ⁇ , IL- l ⁇ and IL-6, the unchanged CINC2 ⁇ chemokine response, enhanced IL-10 production and absent IL-4 seen in established experimental AP in response to intra-arterial LPS in this study suggest that DCs orchestrate a suppressive or regulatory phenotype in AP. Munn et al.
  • kynurenine 3-monooxygenase which catalyses the formation of 3-hydroxykynurenine from kynurenine
  • kynurenine 3-monooxygenase which catalyses the formation of 3-hydroxykynurenine from kynurenine
  • others have shown that introduction of kynurenine directly as a substrate leads to the formation of 3-hydroxykynurenine and other immunoregulatory metabolites in the absence of signals required for the upregulation of IDO (24). Therefore, the measurement of kynurenine in peripheral plasma gives a qualitative, but not accurately quantitive assessment of the amount of 3-hydroxykynurenine present.
  • the clinical correlate is the potential to distinguish between patients who are, 1 ) unlikely ever to develop organ failure, 2) at the height of their illness, 3) have been critically ill but are in the resolution phase, and 4) not critically ill at the time of assessment but are likely to develop organ failure in the immediate future. If borne out by a larger study, this predictive capability would be of great value in the early management of severe AP in humans.

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Abstract

La présente invention concerne des médicaments pour la prévention et/ou le traitement d'une insuffisance de multiples organes, ainsi que des procédés pour déterminer l'état d'insuffisance de multiples organes d'un patient.
PCT/GB2008/001943 2007-06-06 2008-06-06 Procédés d'évaluation et de traitement d'une insuffisance de multiples organes WO2008149103A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2536278C1 (ru) * 2013-11-25 2014-12-20 Анна Сергеевна Семенова Способ прогнозирования осложнений в раннем периоде после операций шунтирования коронарных артерий в условиях искусственного кровообращения
RU2536279C1 (ru) * 2013-11-25 2014-12-20 Анна Сергеевна Семенова Способ прогнозирования осложнений после операций шунтирования коронарных артерий в условиях искусственного кровообращения
US9073875B2 (en) 2012-11-20 2015-07-07 Vertex Pharmaceuticals Incorporated Compounds useful as inhibitors of indoleamine 2,3-dioxygenase
CN113995854A (zh) * 2021-11-09 2022-02-01 中山大学 一种谷胱甘肽敏感的纳米载药系统及其制备方法和应用
WO2022185295A1 (fr) * 2021-03-03 2022-09-09 Qatar University Biomarqueurs pour prédiction de la durée de séjour en unité de soins intensifs pour des patients atteints de covid-19 sous ventilation mécanique

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007054348A1 (fr) * 2005-11-11 2007-05-18 Ernst-Moritz-Arndt-Universität Greifswald Nouveau medicament

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007054348A1 (fr) * 2005-11-11 2007-05-18 Ernst-Moritz-Arndt-Universität Greifswald Nouveau medicament

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9073875B2 (en) 2012-11-20 2015-07-07 Vertex Pharmaceuticals Incorporated Compounds useful as inhibitors of indoleamine 2,3-dioxygenase
US9499497B2 (en) 2012-11-20 2016-11-22 Vertex Pharmaceuticals Incorporated Compounds useful as inhibitors of indoleamine 2,3-dioxygenase
RU2536278C1 (ru) * 2013-11-25 2014-12-20 Анна Сергеевна Семенова Способ прогнозирования осложнений в раннем периоде после операций шунтирования коронарных артерий в условиях искусственного кровообращения
RU2536279C1 (ru) * 2013-11-25 2014-12-20 Анна Сергеевна Семенова Способ прогнозирования осложнений после операций шунтирования коронарных артерий в условиях искусственного кровообращения
WO2022185295A1 (fr) * 2021-03-03 2022-09-09 Qatar University Biomarqueurs pour prédiction de la durée de séjour en unité de soins intensifs pour des patients atteints de covid-19 sous ventilation mécanique
CN113995854A (zh) * 2021-11-09 2022-02-01 中山大学 一种谷胱甘肽敏感的纳米载药系统及其制备方法和应用

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