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EP-4735894-A1 - ASSAY FOR MONITORING CROHN'S DISEASE AND MITOCHONDRIAL DYSFUNCTION

EP4735894A1EP 4735894 A1EP4735894 A1EP 4735894A1EP-4735894-A1

Abstract

The present application provides an ultrasensitive colorimetric assay as well as an early biomarker for patient monitoring and medical treatment of patients suffering from a possible mitochondrial dysfunction, inflammatory bowel disease, particularly Crohn's disease. The ultrasensitive colorimetric assay measures the level of L-citrulline in a plasma or serum sample; and when the level of L-citrulline in plasma or serum decreases or falls even below 30 µmol L-citrulline, this indicates a mitochondrial cell disorder caused by a relapse or an increase in intestinal inflammation due to a flare of Crohn's disease. A method of detecting and treating the mitochondrial dysfunction is also provided.

Inventors

  • GROEN, HANS-JUERGEN
  • ARMBRUSTER, FRANZ PAUL

Assignees

  • Immundiagnostik AG

Dates

Publication Date
20260506
Application Date
20240628

Claims (14)

  1. 1. A method for monitoring patients suffering from Inflammatory Bowel Disease, in particular Morbus Crohn, which is characterized by the steps: - (i) obtaining an in-vitro sample of plasma or serum from the patient; (ii) measuring the concentration of L-citrulline in said plasma or serum sample; (iii) comparing the concentration of L-citrulline found in the plasma or serum sample with a previously measured concentration of L-citrulline in the patient’s plasma or serum, wherein any decreased concentration of L-citrulline indicates a mitochondrial cell disorder and a relapse or increase in Crohn’s disease.
  2. 2. The method of claim 1 , wherein a measured L-citrulline concentration below 30 pmol/L in the patient’s plasma or serum indicates mitochondrial dysfunction and inflammation.
  3. 3. The method of claim 1 or claim 2, wherein a decreased L-citrulline concentration below 30 pmol/L in the patient’s plasma or serum indicates a need to support mitochondrial function by providing the patient’s diet with an alternative energy source, additional antioxidants, nutritional supplements and vitamins.
  4. 4. The method of claim 1 or claim 2, wherein a measured L-citrulline concentration below 30 pmol in serum or plasma of a patient with Crohn’s disease indicates a relapse or increased inflammation due to this disease.
  5. 5. The method according to any preceding claims 1 to 4, wherein the L-citrulline concentration in the patient’s plasma or serum is determined using tandem mass spectrometry, amino acid profiling, and/or immunoassay.
  6. 6. The method according to any preceding claims 1 to 4, wherein the L-citrulline concentration in the patient’s plasma or serum is determined by a colour reaction.
  7. 7. The method according to claim 6, wherein the plasma or serum sample is subjected to an enzymatic treatment with urease prior to the colour reaction.
  8. 8. The method according to claim 6 or claim 7, further comprising a deproteination treatment before the colour reaction.
  9. 9. A method of monitoring patients suffering from Morbus Crohn or inflammatory bowel disease comprising the steps of: - (i) obtaining an in-vitro sample of plasma or serum taken from a patient; (ii) subjecting the sample to enzymatic treatment with urease to render it substantially urea free; (iii) subjecting the sample to a deproteination treatment to obtain a substantially protein-free liquid sample; (iv) sequentially adding (A) an aqueous solution containing diacetyl monoxime (DAMO) and thiosemicarbazide (TSC) and (B) an acidic solution containing a catalytically active iron or manganese redox agent; and (v) heating the sample mixture to above 90 degrees Celsius to form triazine chromophores having peak absorbance in the range of 530 to 540 nm; and (vi) measuring the colour intensity at 530 or 540 nm in comparison with a series of L-citrulline standards in a salt solution to determine the concentration of L-citrulline in the plasma or serum sample; and (vi) comparing the concentration of L-citrulline found in said plasma or serum sample with a previously measured concentration of L-citrulline in the patient’s plasma or serum, wherein any decreased concentration of L-citrulline indicates a mitochondrial disorder and relapse or an increase in Crohn’s disease.
  10. 10. The method of claim 9, wherein solution A contains diacetyl monoxime and thiosemicarbazide in a molar ratio of 30 to 70, preferably 40 to 50.
  11. 11 . The method of claim 9 or claim 10, wherein solution B contains 3 M phosphoric acid, 6 M H2SO4, and 0.1 to 5 mM NH4Fe(SO4)2, preferably 2 mM NH4Fe(SO4)2.
  12. 12. The method of any claim 9 to 11 , solution A and solution B are mixed in a ratio of 1 : 2 , 5.
  13. 13. The method of any claim 9 to 12, wherein the volume of the plasma or serum sample to the volume of the urease solution is greater than 20:1 .
  14. 14. A kit of parts for photometric testing and monitoring of patients suffering from inflammatory bowel disease, comprising:- an aqueous urease concentrate for treating and removing any substantial urea from the plasma or serum sample without diluting the sample by more than 5 percent, a concentrate solution of trichloroacetic acid for deproteinization of the sample without diluting the sample by more than 10 percent, a solution (A), containing diacetyl monoxime and thiosemicarbazide, and an acidic solution (B) containing a catalytically active iron- or manganese-containing redox agent suitable for the synthesis of triazine chromophores of L-citrulline having a peak absorption in the range of 530 to 560 nm, and one or more aqueous standard solutions of L-citrulline, suitable for colorimetric determination of L-citrulline in plasma or serum in the concentration range from 0.5 to 100 pmol/L.

Description

ASSAY FOR MONITORING CROHN'S DISEASE AND MITOCHONDRIAL DYSFUNCTION FIELD OF THE INVENTION [001] The present invention relates to a colorimetric assay of citrulline, particularly a colorimetric assay for monitoring Crohn’s disease, mitochondrial dysfunction and concurrent inflammations. DETAILED DESCRIPTION OF THE INVENTION [002] The diagnosis of inflammatory bowel disease (IBD) includes conditions that result from inflammation of the digestive tract, causing abdominal pain and symptoms. Crohn's disease (CD) is a form of IBD that can affect any part of the gastrointestinal tract, with the terminal ileum of the small intestine being the most affected area. Symptoms include abdominal pain, diarrhoea, fever, weight loss, an increased risk of colon and small bowel cancer, and, in chronic cases, bowel obstruction. Due to the location of the inflammation, Crohn's disease can lead to vitamin deficiencies, including vitamin B12, folic acid, and iron deficiency anaemia., Inflammation in the duodenum and jejunum can also affect the absorption of other nutrients, and if the stomach is affected, intrinsic factor production may be reduced. In addition, the disease can lead to an increased risk of gallstones due to reduced resorption of bile acid in the ileum and excretion of bile in the faeces. [003] Crohn's disease can affect more than just the gastrointestinal tract. It is also associated with neurological complications (reported in up to 15%) such as seizures, stroke, and depression, or a rheumatological condition called seronegative spondyloarthropathy, which causes inflammation of the joints and/or muscle attachments (enthesitis), leading to symptoms such as joint pain, stiffness, and reduced mobility. They can affect large weightbearing joints (hip, knee) and small joints in the hands and feet, and even the spine, leading to ankylosing spondylitis. [004] Crohn's disease can also affect the blood, endocrine system, and skin. It increases the risk of blood clots and autoimmune haemolytic anaemia, which can cause fatigue and a pale appearance. Common skin manifestations include erythema nodosum, which appears as raised, tender red nodules, and pyoderma gangrenosum, which is typically a painful ulcerative nodule. Additionally, Crohn’s disease also increases the risk of osteoporosis, or thinning of the bones, or can cause clubbing, a deformity of the fingertips. Crohn’s disease can also cause inflammation of the uvea (the inner portion of the eye), leading to blurred vision, eye pain, and if left untreated, vision loss. [005] The cause of Crohn's disease is unknown, but genetics play a role. It is a chronic disease in which the body's immune system attacks the gastrointestinal tract, possibly targeting microbial antigens. Although Crohn's disease is immune-related, it is not an autoimmune disease. Smokers are more likely to develop Crohn's disease than non-smokers. It is usually diagnosed in the teens and twenties but can occur at any age. Symptoms of Crohn’s disease often can often go undiagnosed for years before a diagnosis of the disease is made. There is currently no cure for the disease, and treatment options focus on managing the symptoms, preventing flares, and maintaining remission. A corticosteroid may be used for a short time to quickly improve symptoms, along with another medication such as methotrexate or thiopurine. Half of people with the disease will need surgery at some point in the next ten years. The state of the art represents a problem, and there is an urgent need for a method and an in-vitro test to enable the monitoring of patients suffering from Morbus Crohn. BRIEF DESCRIPTION OF THE INVENTION [006] The present disclosure provides a method for monitoring patients suffering from Inflammatory Bowel Disease, in particular Morbus Crohn, characterised by the steps: - (i) obtaining an in-vitro sample of plasma or serum from the patient; (ii) measuring the concentration of L-citrulline in said plasma or serum sample; and (iii) comparing the concentration of L-citrulline found in the plasma or serum sample with a previously measured concentration of L-citrulline in the patient’s plasma or serum, wherein any decreased concentration of L-citrulline is indicative of increased mitochondrial dysfunction preceding or accompanying a relapse or increase in Crohn’s disease. [007] In an embodiment of the invention, a measured L-citrulline concentration below 30 pmol/L in the patient’s plasma or serum is indicative of mitochondrial dysfunction and inflammation. A decreased L-citrulline concentration below 30 pmol/L in the patient’s plasma or serum is indicative of a deficiency and a need to support mitochondrial function by providing the patient with an alternative energy source, additional antioxidants, nutritional supplements and vitamins. [008] In a preferred embodiment of the invention, a measured L-citrulline concentration below 30 pmol in the plasma or serum of a patient suffering from Morbus Crohn indicate