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WO2023194615A1 - Procédé pour déterminer le dosage d'insuline - Google Patents

Procédé pour déterminer le dosage d'insuline Download PDF

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Publication number
WO2023194615A1
WO2023194615A1 PCT/EP2023/059334 EP2023059334W WO2023194615A1 WO 2023194615 A1 WO2023194615 A1 WO 2023194615A1 EP 2023059334 W EP2023059334 W EP 2023059334W WO 2023194615 A1 WO2023194615 A1 WO 2023194615A1
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time
succinate
subject
level
isf
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Peter Meinardus Theodorus DEEN
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Need4health BV
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Need4health BV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the present disclosure relates to healthcare.
  • the disclosure relates to methods for determining insulin dosage, in view of insulin resistance and/or Insulin Sensitivity Factor (ISF) variability within a patient over time.
  • the method involves using an electrochemical biosensor.
  • Glucose derived from food, is supplied to our cells via our bloodstream in which glucose concentration is kept within the homeostatic range of 3.9-7.8 mM (not sober) by the action of the pancreatic hormones insulin and, to a lower extent, glucagon.
  • BG blood glucose
  • Insulin Upon uptake of a carbohydrate-containing meal, blood glucose (BG) level rises within the homeostatic range, which stimulates the pancreatic beta cell to release insulin. Insulin subsequently stimulates the liver and muscle to take up and convert glucose into glycogen, whereas adipose tissue is stimulated to store glucose in the form of fat. Due to these activities BG levels are attenuated. When BG levels near the lower homeostatic level, insulin levels drop and glucagon levels rise. Glucagon thus counteracts insulin, its release is inversely related to that of insulin, and therewith prevents development of hypoglycemia (BG ⁇ 4 mM).
  • BG ⁇ 4 mM hypoglycemia
  • Type-1 diabetes mellitus is an auto-inflammatory disease of the beta cells of the pancreas that can develop as early as with birth and for which there is no cure. It has a high prevalence (1 :300) and a high incidence (2.6:1000), which is still increasing. Due to this disease, patients are not able to secrete sufficient amounts of insulin leading to hyperglycemia. While hyperglycemia gives irritation, sweating, headaches and brain fog with T1 DM patients at the short-term, repetitive stages of hyperglycemia yields neuropathy, blindness, kidney failure, cardiovascular disease and amputations of extremities on the long term. It is the most common auto-inflammatory disease with >100.000 patients in the Netherlands and with estimated health care costs in the US of USD 1000 per person per year, counting up to a total US health care costs of 813 billion USD per year.
  • BG ⁇ 4 hypoglycemia
  • these modules are able to maintain BG within a narrow range (4-10 mM) for a large part of time by (1) maintaining a personalized insulin infusion level based on the amount needed to maintain a steady BG level during the last 3-10 hours, (2) adjusting subcutaneous insulin infusion to the trend of BG changes, and (3) incorporating daily repetitive changes via Al (e.g. walking every morning). Due to these changes, the golden standard of the diabetic status has changed from the extent of glycation of red blood cell haemoglobin (HbA1c), which is mostly a mean readout of time spent in hyperglycaemia during the last 3-4 months, to glucose variability (GV).
  • HbA1c red blood cell haemoglobin
  • DCCT Diabetes Control and Complication Trial
  • EDIC Epidemiology of Diabetes Interventions and Complications
  • the present invention aims to solve one or more of the above-mentioned or other problems.
  • the present inventor surprisingly found an improvement for healthcare, in particular that insulin resistance and/or Insulin Sensitivity Factor (ISF) variability over time within a patient can be followed through measurement over time of succinate level in a bodily fluid of said patient.
  • ISF Insulin Sensitivity Factor
  • the present inventor found that cells produce more succinate, resulting in a cumulative blood succinate level, consistent with an increased state of insulin resistance and/or Insulin Sensitivity Factor (ISF), for example in T1 DM patients. Following adaptation, which varies in time and between individuals, homeostatic steady state conditions re-establish and succinate production/blood levels normalize.
  • ISF Insulin Sensitivity Factor
  • the present inventor particularly envisages a biosensor, which allows for continuous succinate monitoring (CSM).
  • This sensor may comprise a succinate-(ubi)quinone (oxido) reductase (SQR/SQOR) enzyme complex, at least comprising the succinate dehydrogenase (SDH) subunits SDHA/SDHB, coated on an amperometric device, that can be brought into contact with the patient’s bodily fluid, for example a blood (plasma) sample, a tear sample, or an interstitial fluid sample.
  • SQR/SQOR succinate-(ubi)quinone (oxido) reductase
  • SDH succinate dehydrogenase
  • SDHA/SDHB succinate dehydrogenase
  • the A and B subunit of the succinate dehydrogenase (SDH) complex can be used to quantitatively determine blood succinate levels.
  • SDH succinate dehydrogenase
  • mitochondria the enzyme complex of SQR subunits A-D specifically recognize and convert succinate to fumarate (Hock et al, 2020).
  • the mammalian complex II encompassing succinate:ubiquinone oxidoreductase (SQR), and SQR homologs oxidize succinate to fumarate and consist of four subunits, SDHA-SDHB- SDHC-SDHD (Moosavi et al., 2019).
  • the catalytic domain (SDHA and SDHB) is extrinsic on the matrix side, while the anchor subunits (SDHC and SDHD) are intrinsic transmembrane proteins, allowing transfer of the electrons from succinate into the mitochondrial matrix to ubiquinone, located in the inner membrane.
  • the SDHA subunit may be a flavoprotein containing a covalently-bound FAD cofactor and the binding site for dicarboxylates (e.g.
  • SDHB may be an iron-sulphur cluster protein containing three Fe-S clusters.
  • SDHA and SDHB make up the catalytic domain and are historically-named succinate dehydrogenase (SDH). They extend out into the matrix and constitute the hydrophilic head. Specific features of the SDH domain useful to the sensor are:
  • SDHA FAD cofactor binding protein
  • Fe-S cluster containing protein SDHB
  • FGD fumarate reductases
  • QFR quinokfumarate reductases
  • the SQORs as a superfamily all have a flavoprotein and iron-sulphur protein with strong sequence (and where known, structural) homology.
  • the great majority of well-characterized SQORs also have transmembrane anchors related to SDHC and SDHD, but these have considerably more variation in sequence and structure.
  • SdhA/FrdA has soluble homologs, which catalyze a, p-dehydrogenase reactions.
  • the fumarate reductase (flavocytochrome c) from Shewanella frigidimarina and Shewanella putrefaciens, and l-aspartate oxidase from E. coli are examples of soluble SdhA/FrdA homologs.
  • the Shewanella enzyme consists of a single polypeptide chain harbouring a domain similar to the flavoprotein (FrdA/SdhA) fused to a tetra-heme cytochrome.
  • Fcc3 flavocytochrome c3 fumarate reductases
  • E. coli l-aspartate oxidase does not contain any peripheral domain to accommodate redox cofactors.
  • FAD is non-covalently bound in these soluble enzymes, while it is covalently bound in the SQORs of Classes A, B, C, or D.
  • the present disclosure relates to a method for determining Insulin Sensitivity Factor (ISF) and/or insulin dosage, e.g. for a subject in need thereof, the method comprising: a1) preferably determining (basal) Insulin Sensitivity Factor (ISF) (in a sample) of the subject, preferably in conditions wherein the subject is at least 1 , 2, 3, 4, 5, 6 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has not taken an insulin dosage (e.g.
  • ISF Insulin Sensitivity Factor
  • a meal bolus or correction bolus) for at least 4 hours has not eaten any food for the last 4 hours, is free from infection (and (major) emotional stress), and is at least 2 hours free from (strenuous) physical activity; a2) preferably determining (basal) succinate level in a sample of the subject, preferably in conditions wherein the subject is at least 1 , 2, 3, 4 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has slept as usual, refrained from food and caloric beverages for 16 hours, refrained from any (moderate) physical activity for the last 24 hours (or any vigorous physical activity for the last 48 hours), and remaining still when sampling; b) determining succinate level in a sample obtained at a first time from the subject; c) determining succinate level in a sample obtained at a second time from the subject, wherein the time period between the first time and the second time preferably covers conditions of having meal, physical activity and/or infection by the subject; d) determining glucose level in a
  • a succinate level increase between the first time and the second time by more than 35, 40, 45% indicates a decrease of (actual) ISF by more than 35, 40, 45% and at most 75, 80, 85% of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 35, 40, 45% indicates an increase of (actual) ISF by more than 55, 60, 65% and at most 400% of the subject between the first time and the second time;
  • succinate level increase between the first time and the second time by at least 5, 10, 15% and at most 15, 20, 25% indicates a decrease of (actual) ISF by at least 5, 10, 15% and at most 15, 20, 25% of the subject between the first time and the second time;
  • an identical or less than 5, 10, 15% deviation of succinate level between the first time and the second time indicates no or less than 5, 10, 15% change of (actual) ISF of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 15, 20, 25% indicates an increase of (actual) ISF by more than 35, 40, 45% and at most 95, 100% of the subject between the first time and the second time;
  • the present disclosure relates to a method for determining Insulin Sensitivity Factor (ISF) and/or insulin dosage for a subject in need thereof, the method comprising: a1) determining (basal) Insulin Sensitivity Factor (ISF) (in a sample) of the subject, preferably in conditions wherein the subject is at least 1, 2, 3, 4 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has not taken an insulin dosage (e.g.
  • a meal bolus or correction bolus) for at least 4 hours has not eaten any food for the last 4 hours, is free from infection (and (major) emotional stress), and is at least 2 hours free from (strenuous) physical activity ; a2) determining (basal) succinate level in a sample of the subject, preferably in conditions wherein the subject is at least 1, 2, 3, 4 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has slept as usual, refrained from food and caloric beverages for 16 hours, refrained from any (moderate) physical activity for the last 24 hours (or any vigorous physical activity for the last 48 hours), and remaining still when sampling; b) determining succinate level in a sample obtained at a first time from the subject; c) determining succinate level in a sample obtained at a second time from the subject, wherein the time period between the first time and the second time preferably covers conditions of having meal, physical activity and/or infection (and/or stress queue) by the subject; d) determining glucose
  • - a succinate level increase between the first time and the second time by more than 40% indicates a decrease of (actual) ISF by more than 40% and at most 80% of the subject between the first time and the second time;
  • - a succinate level increase between the first time and the second time by at least 10% and at most 40% indicates a decrease of (actual) ISF by at least 15% and at most 40% of the subject between the first time and the second time,
  • a succinate level decrease between the first time and the second time by more than 40% indicates an increase of (actual) ISF by more than 60% and at most 400% of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 10% and at most 40% indicates an increase of (actual) ISF by more than 15% and at most 60% of the subject between the first time and the second time; in case the basal succinate determined under a2) is more than 50, preferably between 50 and 100 pM:
  • a succinate level increase between the first time and the second time by more than 20% indicates a decrease of (actual) ISF by more than 20% and at most 60% of the subject between the first time and the second time;
  • succinate level increase between the first time and the second time by at least 10% and at most 20% indicates a decrease of (actual) ISF by at least 10% and at most 20% of the subject between the first time and the second time;
  • an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 10% change of (actual) ISF of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 20% indicates an increase of (actual) ISF by more than 40% and at most 100% of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 10% and at most 20% indicates an increase of (actual) ISF by more than 10% and at most 40% of the subject between the first time and the second time; f) preferably, administering an insulin dosage to the subject, wherein the dosage in insulin units is within a 20% margin of: (determined glucose level minus healthy reference glucose level) divided by (actual/indicated) ISF (ISF determined in e)).
  • the method can be used to determine the (actual) ISF of the subject, e.g. at a given time such as at the second time or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes thereof.
  • the method can determine a decrease/increase (or no change) of the (actual) ISF relative to the basal ISF and/or relative to the ISF that was (actual) at an earlier time such as at the first time.
  • the ISF at the first time may be known, e.g. prior determined such as based on performing the present method over time (and ultimately based on basal ISF) and/or may be determined by calculating the glucose lowering effect of an earlier insulin dosage and/or may be prior determined (e.g. by known means).
  • the ISF at the first time may be the same as basal ISF. From the (actual) ISF, the skilled person will know how to calculate and administer an appropriate insulin dosage.
  • ISF Insulin Sensitivity Factor
  • the Insulin Sensitive Factor is defined as the amount of blood glucose level (mmol/L) that is lowered by one unit of insulin.
  • the definition of one unit of insulin is historically set to be the amount of insulin required to lower the fasting blood sugar of a rabbit by 2.5 mmol/L. Now, it is known to be the equivalent to 0.0347mg of pure crystalline of insulin.
  • the basal ISF varies from person to person, typically is dependent on age, eating and exercise habits, and a subject’s (actual) ISF varies at different times of day or in different situations.
  • the (actual) ISF can be used to calculate the appropriate insulin dose needed to correct high blood glucose levels. For example, if the subject's target blood glucose level is 5.6 mmol/L and the subject’s current blood glucose level is 11.1 mmol/L, the subject would need two units of insulin to correct the blood glucose level (the difference between 11.1 mmol/L and 5.6 mmol/L, which is 5.5 mmol/L, divided by the ISF of in this case 2.8 mmol/L per unit of insulin).
  • the (basal) Insulin Sensitivity Factor (ISF) of the subject is determined, preferably in conditions wherein the subject is at least 1 , 2, 3, 4, 5, 6 hours free from having any meal(s), physical activity and/or infection, more preferably in conditions wherein the subject has not taken an insulin dosage (e.g. a meal bolus or correction bolus) for at least 4 hours, has not eaten any food for the last 4 hours, is free from infection (and (major) emotional stress), and is at least 2 hours free from (strenuous) physical activity.
  • ISF Insulin Sensitivity Factor
  • the (basal) succinate level of the subject is determined.
  • the basal ISF and/or basal succinate level is determined before the first time as referred to above.
  • the basal ISF and/or basal succinate level may be determined once, or periodically (e.g. every week, month or year, such as every 1-10 weeks, or every 1-12 months), whereas the (actual) ISF can be determined every 1 , 2, 3, 4, 5, 10, 15, 30 minutes or every 1 , 2, 3, 4, 5, 6 hours, such as every 1-60, preferably every 1-30 or 1-10 minutes.
  • Having a meal by a subject generally refers to the act of consuming food and/or beverages that provide the body with nutrients, such as carbohydrates, proteins, and fats, as well as vitamins and minerals.
  • Meal(s) can vary in size and composition depending on cultural, social, and individual factors.
  • a meal can relate to more than 100, 200, 300, 400, 500 kilocalories. From a medical perspective, having a meal often refers to consuming a significant amount of carbohydrates, which can cause a rise in blood glucose levels.
  • Physical activity by a subject generally refers to any bodily movement that results in energy expenditure, such as more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 calories per minute. This can include exercise, sports, leisure activities, or other forms of physical movement that are part of daily life, such as walking, climbing stairs, and doing household chores.
  • An infection of a subject occurs when microorganisms such as bacteria, viruses, fungi, or parasites invade and multiply within the body, causing harm and disrupting normal bodily functions. Infections can range from mild to severe and can affect various parts of the body, including the skin, respiratory system, gastrointestinal system, urinary system, and nervous system. Diagnosis and treatment of infections typically involve identifying the specific pathogen responsible for the infection and using appropriate medications, such as antibiotics or antiviral drugs, to eradicate the infection.
  • the following protocol may be used to set the basal ISF for a new patient (starting usage of a CL-CGM system): •
  • the total daily dose (TDD) is a priori roughly determined using the following formula: 0.6 unit rapid-acting insulin/kg body weight/day.
  • TDD total daily dose
  • the basal insulin dosage and thus the ISF is fine-tuned, which is often determined for three day periods of 8 hr in which meals are omitted (00.00-8.00; 8.00-16.00; 16.00-24.00).
  • different insulin infusion profiles can be set with the CL-CGM. If measured BG during the day is regularly at the lowest side of the set target range and there are frequent basal suspensions, the set prior rates are too high (and need to decreased by e.g. 0.025 u/hr).
  • the basal rate never or rarely gets suspended and BG during the day (and before a meal) is regularly at the higher side of the target BG range, the rates are likely too low (and need to be increased by e.g. 0.025 u/hr). Due to changes in IR and thus ISF, the set basal insulin dosing will still often not fully correct BG to the ideal target range. Using the ISF, this difference is then mostly corrected with the meal insulin bolus (with the CL-CGM devices, a ‘bolus wizard’ corrects the meal insulin bolus with the lack/surplus of insulin from the basal level).
  • the second time is preferably at least 1 , 5, 10 seconds, or at least 1, 5, 10 minutes, or at least 1 , 5, 10 hours, or at least 1 , 5, 10 days, or at least 1 , 5, 10 weeks after the first time (and/or between 1-10 seconds, or between 1-10 minutes, or between 1-10 hours, or between 1-10 days, or between 1-10 weeks after the first time).
  • the second time is preferably at most 1, 5, 10 minutes, or at most 1 , 5, 10 hours after the first time.
  • succinate level is also determined in a sample obtained at a third, fourth, fifth, and/or possible further times, wherein each subsequent time is preferably at least 1 , 5, 10 seconds, or at least 1, 5, 10 minutes, or at least 1, 5, 10 hours after the preceding time. At the same time or, alternatively, the subsequent time is preferably at most 1, 5, 10 minutes, or at most 1, 5, 10 hours after the preceding time. Accordingly, it can be determined if the succinate level determined for the subsequent time is lower, identical or higher than the succinate level determined for the preceding time, wherein
  • succinate level indicates a decrease in insulin resistance in the subject between the preceding time and the subsequent time
  • step d) glucose level is determined in a sample obtained from the subject.
  • the most common and reliable way to determine glucose levels in a blood sample is through a blood glucose test. For example, by continuous glucose monitoring: this involves wearing a small device called a glucose sensor which is placed upon or under the skin. The sensor continuously measures glucose levels in the interstitial fluid and transmits the data to a receiver or smartphone app.
  • the decrease or increase of (actual) ISF may be relative to the basal ISF, or relative to an earlier determined (actual) ISF, e.g. at a time at or before the first time, e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes before the first time.
  • an 60% increase means an increase from e.g. 100 to 160.
  • the insulin dosage administered in step f) typically is sufficient to restore a healthy blood glucose level, preferably for a period of at least 1, 2, 3 ,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 minutes.
  • a healthy reference glucose level typically is between 4-10 mmol/L preferably between 4-6.5 mmol/L. Of course, if the glucose level is below the healthy reference value, no insulin needs to be administered.
  • Step f) may be performed after the second time such as within 1, 2, 3 ,4, 5, 6, 7, 8, 9, 10 minutes thereof.
  • the insulin dosage in step f) is based on (actual/corrected) ISF, which is (more adequately) determined based on succinate level variation, i.e. ISF is corrected for any increases and decreases thereof over time, such as between the first and the second time, due to changes in succinate level.
  • insulin infusion by all other means of application of insulin dosing to T1 DM patients is foreseen, such as insulin (smart)pens.
  • the method according to the present disclosure may be performed over time, e.g. repeated at least every 1 , 2, 3, 4, 5, 6, 7, 8, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 60, 120 minutes.
  • ISF Insulin Sensitivity Factor
  • variant insulin resistance
  • Succinate is an important metabolite at the cross-road of several metabolic pathways.
  • Succinate(2-) is a dicarboxylic acid di-anion resulting from the removal of a proton from both of the carboxy groups of succinic acid:
  • Succinate levels can be determined by different methods available in the art.
  • a Succinate Colorimetric Assay Kit (Sigma-Aldrich®) is a highly sensitive assay for determining succinate levels in a variety of samples. Succinate concentration is determined by a coupled enzyme reaction, which results in a colorimetric (450 nm) product proportional to the succinate present.
  • an EnzyChromTM Succinate Assay Kit can be used for quantitative determination of succinate (succinic acid) in biological samples.
  • succinate is converted to pyruvate which reacts with specific reagents and dye to form a coloured product.
  • Succinate changes may also be used in connection to readouts of other body functions, such as brain activity (fMRI, EEG), heart activity (ECG), menstrual cycle/menopause/hot-cold environment (temperature).
  • determining succinate level is performed by using an electrochemical biosensor as described elsewhere herein, preferably a voltametric biosensor or an amperometric biosensor.
  • the sample that is obtained prior to and subject to performing the steps as according to the method of the present disclosure may be a body fluid sample, wherein the body fluid sample preferably is chosen from the group consisting of blood sample, blood plasma sample, blood serum sample, tear fluid sample, saliva fluid sample, sweat sample, urine sample and interstitial fluid sample.
  • the sample e.g. in steps b), c) and/or d)
  • the method according to the present disclosure is preferably applied ex vivo.
  • the present disclosure may exclude methods for treatment of the human or animal body by surgery or therapy and/or diagnostic methods practiced on the human or animal body.
  • the subject according to the present disclosure may be a mammalian subject, preferably a human subject.
  • the subject may have an insulin resistance-related condition, which refers to conditions that may cause or are caused and/or exaggerated by insulin resistance.
  • insulin resistance-related conditions as used herein may refer to conditions wherein the symptoms have been associated with insulin resistance.
  • Prevention/and or treatment of insulin resistance in insulin resistance-related conditions may relieve symptoms, mitigate progression of disease, and/or reverse disease. Prevention/and or treatment of insulin resistance in these conditions may also mitigate the chance of acquiring additional comorbidities associated with the insulin resistance-related condition.
  • Insulin resistance-related conditions disclosed herein include type 1 diabetes mellitus, type 2 diabetes mellitus, dyslipidemia, metabolic syndrome, insulin resistance in endocrine disease, Polycystic Ovary Syndrome (PCOS), Non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and cancer (treatment), preferably insulin resistance, type 1 diabetes, type 2 diabetes, and metabolic syndrome. Also encompassed may be cardiovascular diseases, neurological diseases, endocrine diseases.
  • the present method may be a computer-implemented method, wherein preferably insulin resistance variability is followed over time, and may be used as input for determining dosage of medication, e.g. insulin.
  • the present disclosure may provide for a computer-readable medium comprising instructions for performing the present method.
  • Type 1 diabetes mellitus is well-known to the skilled person. Type 1 diabetes mellitus is thought to result from an autoimmune destruction of pancreatic p-cells, and the predominant pathophysiology may be an almost absolute insulinopenia. Insulin resistance has also been described in type 1 diabetes mellitus and may be a potential target for intervention in addition to insulin therapy. Insulin resistance is often observed in subjects suffering type 1 diabetes mellitus during e.g. pubertal development, inter-current illness, female menstrual cycle changes, or during high-intensity exercise.
  • type 2 diabetes mellitus is well-known to the skilled person. Patients with type 2 diabetes mellitus are characterized by high blood sugar, insulin resistance, and relative high level or lack of insulin as compared to healthy subjects. These patients experience a high insulin resistance or usually have passed through an earlier stage of insulin resistance, although this earlier stage often goes undiagnosed. It is widely accepted that insulin resistance is a powerful predictor of future development of type 2 diabetes mellitus, and also is the main therapeutic target once high blood sugar is present.
  • dyslipidaemia is well-known to the skilled person.
  • Dyslipidaemia is characterized by abnormal amount of lipids in the blood.
  • most dyslipidaemias are hyperlipidaemias, or an elevation of lipids in the blood, which has a strong relation to cardiovascular disease.
  • Insulin resistance, and the compensatory hyperinsulinemia that results, has been linked to dyslipidaemia.
  • the characteristic dyslipidaemia of insulin resistance consists of elevated triglyceride and triglyceride-rich lipoprotein levels, low levels of high-density lipoprotein cholesterol (HDL), and increased concentrations of small low-density lipoprotein cholesterol (LDL).
  • HDL high-density lipoprotein cholesterol
  • LDL small low-density lipoprotein cholesterol
  • metabolic syndrome is well-known by the skilled person. Within the present disclosure, the term encompasses all conditions diagnosed as “metabolic syndrome” by an (authorized) medical practitioner. For example, metabolic syndrome may be diagnosed if a patient has at least two, or at least three of the following traits:
  • Large waist A waistline that measures at least 35 inches (89 centimetres) for women and 40 inches (102 centimetres) for men;
  • High triglyceride level 150 milligrams per decilitre (mg/dL), or 1.7 millimoles per litre (mmol/L), or higher of this type of fat found in blood;
  • Reduced "good” or HDL cholesterol Less than 40 mg/dL (1 .04 mmol/L) in men or less than 50 mg/dL (1.3 mmol/L) in women of high-density lipoprotein (HDL) cholesterol;
  • Increased blood pressure 130/85 millimetres of mercury (mm Hg) or higher; Elevated fasting blood sugar — 100 mg/dL (5.6 mmol/L) or higher.
  • Metabolic syndrome is an example of an insulin resistance-related condition. Most people who have metabolic syndrome have insulin resistance. Insulin resistance has been suggested as an important underlying cause of metabolic syndrome.
  • insulin resistance in endocrine disease refers to endocrine disorders associated with changes in sensitivity to insulin, often leading to high blood glucose. Examples are acromegaly, but also growth hormone deficiency, hypercortisolism in the course of Cushing's syndrome, hyper- or hypothyroidism, primary hyperparathyroidism, aldosteronism, pheochromocytoma, congenital hypertrophy of the adrenal glands, polycystic ovaries syndrome, hypogonadism, or other hormonally active neuroendocrine tumours.
  • High blood glucose in insulin resistance in endocrine disease is often reversible, and may be reversed by insulin-sensitizing agents.
  • PCOS Polycystic Ovary Syndrome
  • Non-alcoholic fatty liver disease refers to a group of conditions where there is accumulation of excess fat in the liver of people who drink little or no alcohol.
  • the most common form of NAFLD is called fatty liver.
  • NAFLD is strongly associated with insulin resistance and type 2 diabetes mellitus, therefore treatments of NAFLD may aim at lowering insulin resistance.
  • Non-alcoholic steatohepatitis refers to liver inflammation and damage caused by a build-up of fat in the liver. NASH is associated with a markedly increased risk of developing cirrhosis and hepatocellular carcinoma as well as other diseases not directly associated with liver damage, including increased risk of cardiovascular disease. An association between NASH and insulin resistance is well-known, and strategies to lower insulin resistance may decrease disease progression or symptoms in NASH.
  • cancer refers to or describes the physiological condition (e.g. in mammals) that is typically characterized by unregulated cell growth.
  • cancer examples include, but are not limited to, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, skin cancer, blood cancer, leukemia, melanoma, head and neck cancer, and brain cancer.
  • cancer is also referred to as malignant neoplasm.
  • determining succinate level is performed by using an electrochemical biosensor, preferably a voltametric biosensor or an amperometric biosensor.
  • the measured current is directly related to the conversion (oxidation or reduction, preferably oxidation) of the substrate in the sample by the enzyme coupled to the amperometric device and with the applied potential during the electrochemical reaction.
  • determining succinate level is performed by
  • step (ii) measuring current change, which corresponds to succinate level in the sample, wherein step (ii) is preferably performed at a constant voltage.
  • the present disclosure provides for a sensor device for determining succinate level in a sample, wherein the sensor device comprises a (working) electrode coated with an immobilized enzyme.
  • the sensor device or electrochemical biosensor may comprise a working electrode, a reference electrode and/or a counter electrode.
  • the voltametric biosensor or the amperometric biosensor comprises a (working) electrode coated with an immobilized enzyme.
  • said biosensor comprises a sample supply region.
  • the working electrode and/or the counter electrode may comprise or consist of carbon, optionally with ferricyanide mediator.
  • the reference electrode may comprise or consist of silver/silverchloride.
  • the current changes may be measured by way of differential pulse voltametric.
  • the method is preferably carried out at pH 6.5 to 8.5 at a 50-150, or 100-200 mV electrode potential.
  • the (immobilized) enzyme may be chosen from the group consisting of succinate dehydrogenase (SDH); succinate-coenzyme Q reductase (SQR); succinate: ubiquinone oxidoreductase; succinate:quinone oxidoreductase and/or succinate: (ubi)quinone reductase. quinol fumarate reductase (QFR);
  • the (immobilized) enzyme is (derived) from Escherichia coli.
  • the said succinate dehydrogenase may comprise or be a dimer of FAD cofactor binding (flavo)protein (SDHA) and Iron-sulphur (Fe-S) cluster containing protein (SDHB).
  • SDHA FAD cofactor binding
  • Fe-S Iron-sulphur
  • the SDH according to the present disclosure comprises
  • SDHA preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:1 and/or
  • SDHB preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:2 (Janssen et al, 1997).
  • the SQR according to the present disclosure comprises
  • DHSC preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:3 and/or
  • DHSD preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:4.
  • the QFR according to the present disclosure comprises
  • FRDA preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:5 and/or
  • FRDB preferably having at least 20%, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:6 (Janssen et al., 1997).
  • FRDC preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:7 and/or
  • FRDD preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:8.
  • SQR is complex II of the tricarboxylic acid[TCA]/krebs cycle/electron transport chain (ETC).
  • the sensor device or electrochemical biosensor according to the present disclosure may be combined with a sensor device or electrochemical biosensor for determining glucose level in a sample obtained from the subject.
  • This sensor device or electrochemical biosensor may comprise a (working) electrode coated with an immobilized enzyme, preferably glucose dehydrogenase or glucose oxygenase.
  • the sensor device or electrochemical biosensor e.g. a voltametric biosensor or an amperometric biosensor, may comprise a working electrode, a reference electrode and/or a counter electrode.
  • the voltametric biosensor or the amperometric biosensor comprises a (working) electrode coated with an immobilized enzyme, e.g. glucose dehydrogenase/oxygenase.
  • said biosensor comprises a sample supply region.
  • the present disclosure relates to a method for determining insulin resistance variability in a subject over time (and/or determining progression/reduction of insulin resistance in a subject over time), the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • a lower succinate level indicates a decrease in insulin resistance in the subject between the first time and the second time
  • a higher succinate level indicates an increase in insulin resistance in the subject between the first time and the second time.
  • the method may comprise: a) determining photon emission in the visible wave-lengths (400-800 nm) from (skin of) a subject at a first time from the subject; b) determining photon emission in the visible wave-lengths (400-800 nm) from (skin of) a subject at a second time from the subject; c) determining if the photon emission determined under b) is lower, identical or higher than the photon emission determined under a), wherein
  • a lower photon emission indicates a decrease in insulin resistance in the subject between the first time and the second time
  • the second time may be at least 1 , 5, 10 seconds, or at least 1 , 5, 10 minutes, or at least 1 , 5, 10 hours after the first time.
  • insulin resistance is well known by the skilled person. It is also well known that insulin resistance may vary within a patient over time. Within the present disclosure, the term encompasses all conditions diagnosed as ‘insulin resistance’ by an (authorized) medical practitioner.
  • insulin resistance as used herein preferably refers to peripheral insulin resistance and/or hepatic insulin resistance and/or (skeleto)muscular insulin resistance.
  • Insulin resistance may for example be diagnosed by the gold standard which is the "hyperinsulinemic euglycemic clamp" (DeFronzo et al, 1979). This method measures the amount of glucose necessary to compensate for an increased insulin level without causing hypoglycemia. The procedure may take two hours and typically involves the following steps: Through a peripheral vein, insulin is infused at 10-120 mU per m 2 per minute. In order to compensate for the insulin infusion, glucose 20% is infused to maintain blood sugar levels between 5 and 5.5 mmol/L. The rate of glucose infusion is determined by checking the blood sugar levels every five to ten minutes(Muniyappa et al, 2008). The rate of glucose infusion e.g.
  • insulin sensitivity determines insulin sensitivity. If high levels (7.5 mg/min or higher) are required, the patient is insulin sensitive. Low levels (4.0 mg/min or lower) indicate insulin resistance. Levels between 4.0 and 7.5 mg/min are not definitive, and suggest "impaired glucose tolerance," an early sign of insulin resistance.
  • HOMA or HOMA-IR insulin resistance
  • FSIVGTT minimal model analysis of frequently sampled intravenous glucose tolerance test
  • OGTT oral glucose tolerance test
  • HOMA or HOMA-IR homeostatic model assessment for insulin resistance
  • a HOMA(-IR) score that deviates from a reference range can indicate insulin resistance.
  • HOMA(-IR) has been widely applied in epidemiological studies and in experimental research.
  • HOMA(-IR) denotes a value which represents an estimation for insulin resistance, derived from dividing Insulin and Glucose levels in the blood of a person.
  • the HOMA(-IR) value can be calculated by the following equation: wherein H is the HOMA(-IR) value, Glucose represents fasting glucose levels in the blood expressed in mmol/L, Insulin represents fasting insulin levels in the blood expressed for example in mIU/L.
  • IU (relating to enzyme activity) is an abbreviation of International Units, also called enzyme units. The skilled person is familiar with the methods used to quantify these levels. Enzyme activity is the amount of substrate converted per unit of time. One IU equals the conversion of one mol of substrate per minute.
  • HOMA(-IR) For an individual with normal insulin sensitivity, HOMA(-IR) may equal 1.
  • the upper limit of normal HOMA(-IR) is frequently considered to be 2.0, although the normal HOMA(-IR) may be dependent on the characteristics of the population subgroup.
  • a healthy subject has a HOMA-IR value below 2.0, preferably a HOMA-IR value below 1.9, 1.8, or 1.7, more preferably a HOMA-IR value below 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 , and most preferred a HOMA-IR value below 1.0.
  • a fasting serum insulin level greater than 25 mIU/L or 174 pmol/L has also been considered as indicating insulin resistance.
  • insulin resistance may be measured by HOMA(-IR).
  • Cardiac pacemaker cells initiate and precisely regulate the spontaneous heart beat rhythm through modulation of an ion channel ensemble in sinoatrial nodal cells (SANC) through an intracellular Ca 2+ cycling clock.
  • SANC sinoatrial nodal cells
  • the SANC’s Ca 2+ clock is manifested by spontaneous, but precisely timed, rhythmic, submembrane, local Ca 2+ releases from sarcoplasmic reticulum (SR) that appear shortly before firing of the next action potential and which is orchestrated via intrinsic signalling mechanisms, such as phosphorylation cascades(Maltsev & Lakatta, 2007; Yaniv et al, 2015).
  • SR sarcoplasmic reticulum
  • the method may comprise: a) determining HRV of a subject at a first time from the subject; b) determining HRV of a subject at a second time from the subject; c) determining if the HRV determined under b) is lower, identical or higher than the HRV determined under a), wherein
  • the second time may be at least 1 , 5, 10 seconds, or at least 1 , 5, 10 minutes, or at least 1 , 5, 10 hours after the first time.
  • the present disclosure further provides for the use of succinate for determining Insulin Sensitivity Factor (ISF) and/or insulin resistance in a subject, preferably ISF variability and/or insulin resistance variability in a subject over time.
  • ISF Insulin Sensitivity Factor
  • the present disclosure provides for the use of succinate for determining an appropriate dosage of insulin to be administered to a subject in need of treatment with said insulin.
  • Also provided is a method for determining insulin resistance variability and/or succinate- induced insulin resistance variability for a subject comprising: a) determining (basal) succinate level in a sample of the subject, preferably in conditions wherein the subject is at least 1 , 2, 3, 4 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has slept as usual, refrained from food and caloric beverages for 16 hours, refrained from any (moderate) physical activity for the last 24 hours (or any vigorous physical activity for the last 48 hours), and remaining still when sampling; b) determining succinate level in a sample obtained at a first time from the subject; c) determining succinate level in a sample obtained at a second time from the subject, wherein the time period between the first time and the second time preferably covers conditions of having meal, physical activity and/or infection (and/or stress queue) by the subject; d) determining if the succinate level determined under c) is lower, identical or higher than the succinate level determined under b
  • a succinate level increase between the first time and the second time by more than 40% indicates a decrease of insulin resistance by more than 40% and at most 80% of the subject between the first time and the second time;
  • a succinate level increase between the first time and the second time by at least 10% and at most 40% indicates a decrease of insulin resistance by at least 15% and at most 40% of the subject between the first time and the second time
  • a succinate level decrease between the first time and the second time by more than 40% indicates an increase of insulin resistance by more than 60% and at most 400% of the subject between the first time and the second time;
  • a succinate level increase between the first time and the second time by more than 20% indicates a decrease of insulin resistance by more than 20% and at most 60% of the subject between the first time and the second time;
  • a succinate level increase between the first time and the second time by at least 10% and at most 20% indicates a decrease of insulin resistance by at least 10% and at most 20% of the subject between the first time and the second time;
  • an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 10% change of insulin resistance of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 20% indicates an increase of insulin resistance by more than 40% and at most 100% of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 10% and at most 20% indicates an increase of insulin resistance by more than 10% and at most 40% of the subject between the first time and the second time.
  • the above method is useful in monitoring attacks in auto-immune disease, guiding dosing/monitoring the efficacy/efficiency of therapies for disorders related to insulinresistance.
  • the insulin resistance at the first time may be prior determined or known.
  • the present disclosure relates to a method for determining disease severity and/or monitoring disease progression and/or disease treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • succinate level indicates a decrease in disease severity and/or decrease in disease progression and/or positive disease treatment efficacy (e.g. increase in disease treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in disease severity and/or disease progression and/or disease treatment efficacy in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in disease severity and/or increase in disease progression and/or negative disease treatment efficacy (e.g. decrease in disease treatment efficacy) in the subject between the first time and the second time.
  • the present disclosure relates to a method for determining burnout severity and/or monitoring burnout progression and/or burnout treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • succinate level indicates a decrease in burnout severity and/or decrease in burnout progression and/or positive burnout treatment efficacy (e.g. increase in burnout treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in burnout severity and/or burnout progression and/or burnout treatment efficacy in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in burnout severity and/or increase in burnout progression and/or negative burnout treatment efficacy (e.g. decrease in burnout treatment efficacy) in the subject between the first time and the second time.
  • Burnout is a recognized disorder. In May 2019, the World Health Organization (WHO) officially classified burnout as an occupational phenomenon in the International Classification of Diseases (ICD-11). According to the ICD-11 , burnout is defined as a syndrome resulting from chronic workplace stress that has not been successfully managed. The three key dimensions of burnout are:
  • burnout To diagnose burnout, a healthcare provider would typically assess a person's symptoms, duration, and impact on their daily functioning. Treatment for burnout may involve addressing the underlying workplace stressors, implementing self-care strategies, and seeking mental health support if needed. It is important to note that burnout is not the same as depression or anxiety, although it may coexist with these conditions.
  • the present disclosure relates to a method for determining cancer severity and/or monitoring cancer progression and/or cancer treatment efficacy and/or assessing subject’s resilience to cancer treatment (e.g. chemotherapy) in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • - a lower succinate level indicates a decrease in cancer severity and/or decrease in cancer progression and/or positive cancer treatment efficacy (e.g. increase in cancer treatment efficacy) and/or increase in resilience to cancer treatment in the subject between the first time and the second time;
  • an identical succinate level indicates no change in cancer severity and/or cancer progression and/or cancer treatment efficacy and/or resilience to cancer treatment in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in cancer severity and/or increase in cancer progression and/or negative cancer treatment efficacy (e.g. decrease in cancer treatment efficacy) and/or decrease in resilience to cancer treatment in the subject between the first time and the second time.
  • Cancer is a broad term that refers to a group of diseases characterized by the uncontrolled growth and spread of abnormal cells in the body. Normally, cells in the body divide and grow in a controlled way, but in cancer, this process is disrupted and cells can grow and divide uncontrollably, leading to the formation of a mass of abnormal cells called a tumor.
  • cancer there are many different types of cancer, each with its own characteristics and treatment options. Some common types of cancer include lung cancer, breast cancer, prostate cancer, and colorectal cancer, among others.
  • Resilience to cancer treatment refers to a patient's ability to tolerate and recover from the toxic effects of chemotherapy drugs.
  • Chemotherapy is a type of cancer treatment that uses powerful drugs to kill cancer cells, but it can also damage healthy cells and tissues in the body, leading to side effects such as fatigue, nausea, vomiting, hair loss, and an increased risk of infections.
  • Patients who are more resilient to chemotherapy may be able to tolerate higher doses or longer courses of chemotherapy, which can lead to better outcomes in terms of cancer control or survival.
  • Factors that may contribute to resilience to chemotherapy include a patient's age, overall health status, nutritional status, and the presence of other medical conditions.
  • the present disclosure relates to a method for determining myocardial infarction severity and/or monitoring myocardial infarction progression and/or myocardial infarction treatment efficacy and/or assessing subject’s resilience to myocardial infarction treatment in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein - a lower succinate level indicates a decrease in myocardial infarction severity and/or decrease in myocardial infarction progression and/or positive myocardial infarction treatment efficacy (e.g. increase in myocardial infarction treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in myocardial infarction severity and/or myocardial infarction progression and/or myocardial infarction treatment efficacy in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in myocardial infarction severity and/or increase in myocardial infarction progression and/or negative myocardial infarction treatment efficacy (e.g. decrease in myocardial infarction treatment efficacy) in the subject between the first time and the second time.
  • Myocardial infarction is a serious medical condition that occurs when blood flow to a part of the heart is blocked, usually by a blood clot. This can damage or destroy part of the heart muscle, which can lead to serious complications or even death.
  • coronary artery disease occurs when the arteries that supply blood to the heart become narrowed or blocked due to the buildup of plaque (a fatty substance) inside the artery walls.
  • Other risk factors for heart attack include high blood pressure, smoking, obesity, diabetes, and a family history of heart disease.
  • the present disclosure relates to a method for determining cerebrovascular accident (CVA) severity and/or monitoring cerebrovascular accident (CVA) progression and/or cerebrovascular accident (CVA) treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • CVA cerebrovascular accident
  • CVA cerebrovascular accident
  • CVA cerebrovascular accident
  • CVA cerebrovascular accident
  • CVA cerebrovascular accident
  • CVA cerebrovascular accident
  • CVA cerebrovascular accident
  • CVA cerebrovascular accident
  • an identical succinate level indicates no change in cerebrovascular accident (CVA) severity and/or cerebrovascular accident (CVA) progression and/or cerebrovascular accident (CVA) treatment efficacy in the subject between the first time and the second time; and/or - a higher succinate level indicates an increase in cerebrovascular accident (CVA) severity and/or increase in cerebrovascular accident (CVA) progression and/or negative cerebrovascular accident (CVA) treatment efficacy (e.g. decrease in cerebrovascular accident (CVA) treatment efficacy) in the subject between the first time and the second time.
  • a cerebrovascular accident is a serious medical condition that occurs when blood flow to the brain is interrupted or reduced, either by a blood clot or a ruptured blood vessel. This can damage or destroy brain cells, leading to long-term disability or even death.
  • Ischemic stroke occurs when a blood clot blocks a blood vessel in the brain
  • hemorrhagic stroke occurs when a blood vessel in the brain ruptures and bleeds into the surrounding brain tissue.
  • the present disclosure relates to a method for determining Multiple sclerosis (MS) severity and/or monitoring Multiple sclerosis (MS) progression and/or Multiple sclerosis (MS) treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • a lower succinate level indicates a decrease in Multiple sclerosis (MS) severity and/or decrease in Multiple sclerosis (MS) progression and/or positive Multiple sclerosis (MS) treatment efficacy (e.g. increase in Multiple sclerosis (MS) treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in Multiple sclerosis (MS) severity and/or Multiple sclerosis (MS) progression and/or Multiple sclerosis (MS) treatment efficacy in the subject between the first time and the second time; and/or
  • MS Multiple sclerosis
  • MS Multiple sclerosis
  • MS multiple sclerosis
  • MS multiple sclerosis
  • MS multiple sclerosis
  • MS negative Multiple sclerosis
  • treatment efficacy e.g. decrease in Multiple sclerosis (MS) treatment efficacy
  • MS Multiple sclerosis
  • CNS central nervous system
  • MS occurs when the immune system mistakenly attacks and damages the myelin sheath, a protective covering that surrounds nerve fibers in the CNS. This damage disrupts the normal flow of nerve impulses and can lead to a wide range of symptoms.
  • the present disclosure relates to a method for determining infection severity and/or monitoring infection progression and/or infection treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • a lower succinate level indicates a decrease in infection severity and/or decrease in infection progression and/or positive infection treatment efficacy (e.g. increase in infection treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in infection severity and/or infection progression and/or infection treatment efficacy in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in infection severity and/or increase in infection progression and/or negative infection treatment efficacy (e.g. decrease in infection treatment efficacy) in the subject between the first time and the second time.
  • An infection is a condition that occurs when harmful microorganisms, such as bacteria, viruses, fungi, or parasites, invade and multiply within the body.
  • harmful microorganisms such as bacteria, viruses, fungi, or parasites
  • COVID-19 it is caused by the SARS-CoV-2 virus, which can be transmitted through respiratory droplets from an infected person.
  • the present disclosure relates to a method for determining thyroid disease severity and/or monitoring thyroid disease progression and/or thyroid disease treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • a lower succinate level indicates a decrease in thyroid disease severity and/or decrease in thyroid disease progression and/or positive thyroid disease treatment efficacy (e.g. increase in thyroid disease treatment efficacy) in the subject between the first time and the second time;
  • succinate level indicates no change in thyroid disease severity and/or thyroid disease progression and/or thyroid disease treatment efficacy in the subject between the first time and the second time; and/or - a higher succinate level indicates an increase in thyroid disease severity and/or increase in thyroid disease progression and/or negative thyroid disease treatment efficacy (e.g. decrease in thyroid disease treatment efficacy) in the subject between the first time and the second time.
  • Thyroid disease is a medical condition that affects the function of the thyroid gland, a small butterfly-shaped gland located in the neck. The thyroid gland produces hormones that regulate metabolism, growth, and development throughout the body.
  • thyroid disease There are several different types of thyroid disease, including:
  • TSH serum thyroid-stimulating hormone
  • Hypothyroidism a condition in which the thyroid gland does not produce enough thyroid hormone, leading to a slower metabolism and a range of symptoms, such as fatigue, weight gain, constipation, depression, and dry skin.
  • Hyperthyroidism a condition in which the thyroid gland produces too much thyroid hormone, leading to a faster metabolism and a range of symptoms, such as weight loss, rapid heartbeat, anxiety, irritability, and heat intolerance.
  • Thyroid nodules small lumps or growths on the thyroid gland that can be benign or cancerous. Most thyroid nodules are non-cancerous and do not cause symptoms, but some may produce excess thyroid hormone or grow large enough to cause difficulty swallowing or breathing.
  • Thyroid cancer a rare form of cancer that develops in the cells of the thyroid gland. Most cases of thyroid cancer are treatable and have a good prognosis if detected early.
  • the present disclosure relates to a method for determining Parkinson’s disease severity and/or monitoring Parkinson’s disease progression and/or Parkinson’s disease treatment efficacy (e.g. uptake and/or treatment by levodopa) in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein - a lower succinate level indicates a decrease in Parkinson’s disease severity and/or decrease in Parkinson’s disease progression and/or positive Parkinson’s disease treatment efficacy (e.g. increase in Parkinson’s disease treatment efficacy) in the subject between the first time and the second time;
  • a lower succinate level indicates a decrease in Parkinson’s disease severity and/or decrease in Parkinson’s disease progression and/or positive Parkinson’s disease treatment efficacy
  • an identical succinate level indicates no change in Parkinson’s disease severity and/or Parkinson’s disease progression and/or Parkinson’s disease treatment efficacy in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in Parkinson’s disease severity and/or increase in Parkinson’s disease progression and/or negative Parkinson’s disease treatment efficacy (e.g. decrease in Parkinson’s disease treatment efficacy) in the subject between the first time and the second time.
  • Parkinson's disease is a chronic and progressive neurological disorder that affects movement and coordination. It is caused by the degeneration of dopamine-producing neurons in the brain, which leads to a decrease in dopamine levels. Dopamine is a neurotransmitter that helps to control movement and emotional response.
  • the present disclosure relates to a method for determining nausea severity and/or monitoring nausea progression and/or nausea treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • a lower succinate level indicates a decrease in nausea severity and/or decrease in nausea progression and/or positive nausea treatment efficacy (e.g. increase in nausea treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in nausea severity and/or nausea progression and/or nausea treatment efficacy in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in nausea severity and/or increase in nausea progression and/or negative nausea treatment efficacy (e.g. decrease in nausea treatment efficacy) in the subject between the first time and the second time.
  • Nausea is an unpleasant sensation of discomfort in the upper abdomen that is often accompanied by a feeling of the urge to vomit and thus may include vomiting. It is a common symptom that can be caused by a variety of factors, including: Stomach or gastrointestinal problems, such as gastroenteritis, food poisoning, or indigestion Motion sickness or vertigo
  • Medications such as chemotherapy drugs or opioids
  • the present disclosure relates to a method for determining gastroesophageal reflux disease (GERD) severity and/or monitoring GERD progression and/or GERD treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • GSD gastroesophageal reflux disease
  • a lower succinate level indicates a decrease in GERD severity and/or decrease in GERD progression and/or positive GERD treatment efficacy (e.g. increase in GERD treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in GERD severity and/or GERD progression and/or GERD treatment efficacy in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in GERD severity and/or increase in GERD progression and/or negative GERD treatment efficacy (e.g. decrease in GERD treatment efficacy) in the subject between the first time and the second time.
  • GERD GERD Symptoms of GERD are: a burning sensation in the chest (heartburn), regurgitation of food/sour liquid, upper abdominal chest pain, trouble swallowing, ongoing cough, new/worsening asthma, inflammation of vocal cords. These common symptoms can be caused by a variety of factors, including:
  • Hypersensitivity to food such as greasy/fat-laden food, tomatoes, citrus fruits, spicy food, chocolate, caffeinated beverages, food poisoning
  • Pregnancy particularly during the first trimester Medications, such as non-steroidal anti-inflammatory drugs (NSAIDs), antidepressants, chemotherapy drugs, opioids, calcium channel blockers
  • NSAIDs non-steroidal anti-inflammatory drugs
  • antidepressants such as antidepressants, chemotherapy drugs, opioids, calcium channel blockers
  • the present disclosure relates to a method for determining Chronic Fatigue Syndrome (CFS) severity and/or monitoring Chronic Fatigue Syndrome (CFS) progression and/or Chronic Fatigue Syndrome (CFS) treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • a lower succinate level indicates a decrease in Chronic Fatigue Syndrome (CFS) severity and/or decrease in Chronic Fatigue Syndrome (CFS) progression and/or positive Chronic Fatigue Syndrome (CFS) treatment efficacy (e.g. increase in Chronic Fatigue Syndrome (CFS) treatment efficacy) in the subject between the first time and the second time;
  • CFS Chronic Fatigue Syndrome
  • CFS Chronic Fatigue Syndrome
  • CFS Chronic Fatigue Syndrome
  • CFS Chronic Fatigue Syndrome
  • CFS Chronic Fatigue Syndrome
  • CFS chronic Fatigue Syndrome
  • CFS negative Chronic Fatigue Syndrome
  • CFS Chronic Fatigue Syndrome
  • ME myalgic encephalomyelitis
  • the present disclosure relates to a method for determining Primary mitochondrial disease (PMD) severity and/or monitoring Primary mitochondrial disease (PMD) progression and/or Primary mitochondrial disease (PMD) treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein - a lower succinate level indicates a decrease in Primary mitochondrial disease (PMD) severity and/or decrease in Primary mitochondrial disease (PMD) progression and/or positive Primary mitochondrial disease (PMD) treatment efficacy (e.g. increase in Primary mitochondrial disease (PMD) treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in Primary mitochondrial disease (PMD) severity and/or Primary mitochondrial disease (PMD) progression and/or Primary mitochondrial disease (PMD) treatment efficacy in the subject between the first time and the second time; and/or
  • a higher succinate level indicates an increase in Primary mitochondrial disease (PMD) severity and/or increase in Primary mitochondrial disease (PMD) progression and/or negative Primary mitochondrial disease (PMD) treatment efficacy (e.g. decrease in Primary mitochondrial disease (PMD) treatment efficacy) in the subject between the first time and the second time.
  • PMD Primary mitochondrial disease
  • PMD Primary mitochondrial disease
  • PMD Primary mitochondrial disease
  • PMD negative Primary mitochondrial disease
  • treatment efficacy e.g. decrease in Primary mitochondrial disease (PMD) treatment efficacy
  • PMD Primary mitochondrial disease
  • the present disclosure relates to a method for determining allergy severity and/or monitoring allergy progression and/or allergy treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • a lower succinate level indicates a decrease in allergy severity and/or decrease in allergy progression and/or positive allergy treatment efficacy (e.g. increase in allergy treatment efficacy) in the subject between the first time and the second time;
  • an identical succinate level indicates no change in allergy severity and/or allergy progression and/or allergy treatment efficacy in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in allergy severity and/or increase in allergy progression and/or negative allergy treatment efficacy (e.g. decrease in allergy treatment efficacy) in the subject between the first time and the second time.
  • An allergy is an overreaction of the body's immune system to a substance that is normally harmless, such as pollen, dust, certain foods, or medications.
  • a person with an allergy comes into contact with an allergen, their immune system produces an antibody called immunoglobulin E (IgE), which triggers the release of histamine and other chemicals that cause allergy symptoms.
  • IgE immunoglobulin E
  • the term allergy as used herein excludes gluten allergy.
  • the present disclosure relates to a method for determining withdrawal syndrome severity and/or monitoring withdrawal syndrome progression and/or withdrawal syndrome treatment efficacy and/or withdrawal syndrome treatment resilience in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • succinate level indicates a decrease in withdrawal syndrome severity and/or decrease in withdrawal syndrome progression and/or positive withdrawal syndrome treatment efficacy (e.g. increase in withdrawal syndrome treatment efficacy) and/or increased resilience to withdrawal syndrome treatment in the subject between the first time and the second time;
  • a higher succinate level indicates an increase in withdrawal syndrome severity and/or increase in withdrawal syndrome progression and/or negative withdrawal syndrome treatment efficacy (e.g. decrease in withdrawal syndrome treatment efficacy) and/or decreased resilience to withdrawal syndrome treatment in the subject between the first time and the second time.
  • Withdrawal syndrome also known as withdrawal disease, is a set of symptoms that can occur when a person stops using a substance or medication that they have been using regularly for a period of time. Withdrawal syndrome is a physical and/or psychological response to the absence of the substance, and the severity and duration of the symptoms can vary depending on the type of substance, the duration of use, and other individual factors. Patients show a large inter- and intra-individual variability in the resilience to withdrawal therapy.
  • the present disclosure relates to a method for monitoring health status/resilience (e.g. risk for disease development) in a subject over time, the method comprising: a) determining (basal) succinate level in a sample obtained at a first time from the subject; b) determining (basal) succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • an identical (basal) succinate level indicates no change in health status in the subject between the first time and the second time;
  • Health status refers to an individual's overall state of health, which is determined by various factors, such as physical, mental, and social well-being.
  • a person's health status can be influenced by a range of factors, including genetics, lifestyle choices, environmental factors, and access to healthcare services.
  • a sustained optimal health status leads to longevity
  • Some common indicators of health status include measures such as body mass index (BMI), blood pressure, cholesterol levels, and blood glucose levels. Other factors that can be used to assess an individual's health status include their overall physical fitness, mental health, and ability to perform daily activities without difficulty.
  • BMI body mass index
  • blood pressure blood pressure
  • cholesterol levels cholesterol levels
  • blood glucose levels Other factors that can be used to assess an individual's health status include their overall physical fitness, mental health, and ability to perform daily activities without difficulty.
  • the present disclosure relates to a method for monitoring labour fatigue in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • succinate level indicates no change in labour fatigue in the subject between the first time and the second time; and/or - a higher succinate level indicates an increase in labour fatigue in the subject between the first time and the second time.
  • Symptoms of labor fatigue may include reduced physical and cognitive performance, decreased concentration and attention, irritability, and mood swings. It can also lead to a higher risk of accidents and injuries in the workplace.
  • the sample that is obtained may be a body fluid sample, wherein the body fluid sample preferably is chosen from the group consisting of blood sample, blood plasma sample, blood serum sample, tear fluid sample, saliva fluid sample, sweat sample, urine sample and interstitial fluid sample.
  • the sample e.g. in steps b), c) and/or d)
  • the method according to the present disclosure is preferably applied ex vivo.
  • the present disclosure may exclude methods for treatment of the human or animal body by surgery or therapy and/or diagnostic methods practiced on the human or animal body.
  • the subject according to the present disclosure may be a mammalian subject, preferably a human subject.
  • identity refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" perse has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.
  • polypeptide or nucleotide sequence having at least, for example, 95% "identity" to a reference polypeptide or nucleotide sequence it is intended that the polypeptide or nucleotide sequence is identical to the reference sequence, except that there may be up to five point mutations per each 100 amino acids or nucleotides of the reference polypeptide or nucleotide sequence.
  • sequence identity refers to the sequence identity over the entire length of the sequence. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.
  • polypeptides having a different amino acid sequence can have the same activity. It is common general knowledge that it is often possible to substitute a certain amino acid by another one, without loss of activity of the polypeptide. For example, the following amino acids can often be exchanged for one another:
  • substitutions are those that are conservative, i.e., wherein the residue is replaced by another of the same general type.
  • the hydropathic index of amino acids may be considered. It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a polypeptide having similar biological activity.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those that are within ⁇ 1 are more preferred, and those within ⁇ 0.5 are even more preferred.
  • select amino acids may be substituted by other amino acids having a similar hydrophilicity, as set forth in U.S. Pat. No. 4,554,101.
  • substitution of amino acids whose hydrophilicity indices are within ⁇ 2 is preferred, those that are within ⁇ 1 are more preferred, and those within ⁇ 0.5 are even more preferred.
  • Method for determining insulin resistance variability in a subject over time comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein
  • a lower succinate level indicates a decrease in insulin resistance in the subject between the first time and the second time
  • the sample is a body fluid sample, wherein the body fluid sample preferably is chosen from the group consisting of blood sample, blood plasma sample, blood serum sample, tear fluid sample, saliva fluid sample, sweat sample, and interstitial fluid sample.
  • determining succinate level is performed by using an electrochemical biosensor, preferably a voltametric biosensor or an amperometric biosensor.
  • a sensor device for determining succinate level in a sample comprising an electrode coated with an immobilized enzyme, wherein the enzyme is succinate dehydrogenase (SDH), succinate-coenzyme Q reductase (SQR), quinol fumarate reductase (QFR), succinate:ubiquinone oxidoreductase, succinate:quinone oxidoreductase or succinate:(ubi)quinone reductase.
  • SDH succinate dehydrogenase
  • SQR succinate-coenzyme Q reductase
  • QFR quinol fumarate reductase
  • succinate dehydrogenase comprises or is a dimer of FAD cofactor binding (flavo)protein (SDHA) and Iron-sulphur (Fe-S) cluster containing protein (SDHB).
  • Figure 1 shows a simplified schematic of the working mechanism of an amperometric sensors, consisting of a working (WE), counter (CE), and reference (RE) electrodes.
  • a current between the working and counter electrode is generated by the redox reaction between the SDH/SQR complex oxidizing succinate to fumarate and the modified working electrode, while the opposite reaction takes place at the counter electrode.
  • a constant voltage, sufficiently high to initiate the reduction or oxidation of succinate, is applied between the working and reference electrode. This results in the generation of an electrochemical catalytic current between the working and counter electrode, which is proportional to the concentration of the biomarker, succinate.
  • Figure 2 shows Dose-response curve of human SUCNR1 to succinate, lnositol-3 phosphate formation by cells expressing human SLICNR1 in response to different concentrations of succinate.
  • AA Primary amino acid sequences of the E.coli strain K12 homologues of the SQR subunits SDHA (No. 1), SDHB (No 2), SDHC (No 3), SDHD (No 4) and the QFR subunits QFRA (No. 5), QFRB (No. 6), QFRC (No 7) and QFRD (No 8). Sequences were obtained from the uniprot blast server.
  • SEQ ID NO:1 SDHA_ECOLI; 588 AA
  • SEQ ID NO:2 SDHB_ECOLI 238 AA
  • SEQ ID NO:6 FRDB_ECOLI 244 A A
  • Example 1 illustrates the different embodiments of the invention.
  • basal succinate levels and insulin resistance can be expected to be low (10-20 uM for succinate).
  • triggers of metabolic imbalance are anticipated to increase blood succinate and insulin resistance levels.
  • Relatively strong triggers include high intensity exercise, pregnancy, menopause, alcohol abuse, food intolerances/allergies, infection, and inflammation.
  • Intermediate strong triggers include nervosity and female hormonal changes.
  • basal succinate levels are anticipated to be increased.
  • the triggers of metabolic imbalance indicated above are anticipated to lead to more moderately increased succinate and insulin resistance levels.
  • the succinate and insulin resistance values are indicated in Table 1. Succinate level relative to time point 1 was determined as disclosed herein on the same day for all subjects before (time point 1), on top (time point 2) and shortly after (time point 3) the trigger of metabolic imbalance. A healthy subject of 20-30 years old will have a low, basal level of succinate and insulin resistance before any trigger (i.e. time point 1 of subject no. 1). The time point-1 succinate level of a person with a particular condition (i.e. person 3 onwards) before the respective trigger is indicated relative to this basal level of the healthy subject of 20-30 years old. Succinate and insulin resistance of time points 2 and 3 of a particular subject are relative to time point 1 of that particular subject.
  • Insulin resistance level was inferred from combined glucose and insulin level measurement.
  • subjects are meant suffering from type-2 diabetes mellitus, dyslipidemia, metabolic syndrome, obesity, PCOS, NAFLD, NASH, COPD, intestinal bowel disease, cancer, or with endocrine/cardiovascular/neurological disease related insulin resistance or longstanding mental problems. Subjects indicated are:
  • the invention indeed can be used to determine insulin resistance and/or Insulin Sensitivity Factor (ISF) variability over time and hence allows to tailor insulin administration on insulin resistance at any given time.
  • ISF Insulin Sensitivity Factor
  • the increased BS will bind its receptor (SLICNR1) in the liver, inducing liver insulin resistance (IR).
  • SLICNR1 liver insulin resistance
  • the liver subsequently produces and releases more glucose (giving hyperglycaemia; increase CGM signal), which stimulates the pancreas to release insulin, therewith stimulating increased uptake of glucose by skeletal muscles.
  • CGM signal giving hyperglycaemia; increase CGM signal
  • the succinate stress response thus always PRECEDES the glucose response.
  • succinate With humans suffering from chronic acquired low grade inflammation/stress, such as with obesity, diabetes type-2 (T2DM), cardiovascular disease (CVD), NAFLD, COPD, intestinal bowel disease, depression, dementia, parkinsonism, hypothyroidy, or with genetic chronic low- grade inflammation/stress, such as cystic fibrosis, succinate is constitutively increased to about 60-80 uM. Their liver and kidneys are thus steady state insulin resistant. Upon a short-term stressor, the absolute elevation in succinate is anticipated to be the same, but the removal will be slow compared to above, and will not go lower than the previously (increased) steady state level.
  • the extent of the increase in succinate is determined by the (personal) intensity of the stressor and the duration of the change.
  • the experienced intensity of the stressor depends on the genetic package (‘talent’ to deal with it) and the times the person encountered the challenge before (experience). For example, a slight increase in CGM reading occurs when one is standing up from bed: getting from a laid down to standing position necessitates an increase in blood pressure, because our brains will otherwise not receive enough oxygen.
  • the change of standing up is the stressor, but as adaptation by vasoconstriction occurs fast (short duration) and we are used to stand up (experience), the stress/succinate-induced CGM change is relatively small (small increase in time and fast removal by a healthy liver and kidney). In contrast, when one performs high-intensity exercise when one nearly never performs sport or is frail, the stress/succinate-induced CGM change is high.
  • TIR time-in-range
  • BG blood glucose
  • TBR time-below-range
  • GV glucose variability
  • ALL conditions that may lead to hypoglycaemia with current CL-CGM devices are due to the following aspects:
  • Delay in reaction to BG changes of 50 minutes.
  • CL-CGM devices detect BG in the interstitial fluid, of which the glucose concentration has a delay of 10 minutes in relation to BG.
  • Insulin is infused subcutaneously, which takes 40 minutes before it is evenly diffused in blood (Phillip et al, 2023). The (ultra) rapid insulin used has an activity period of around 4 hours.
  • the risky conditions are characterised by a period of increased insulin resistance (e.g. high intensity exercise, stress, infection, period, carb meal) shortly followed by a period of increased insulin sensitivity (stop exercise, end of meal, sleeping) combined with and increased glucose uptake by liver and, with exercise, elevated glucose uptake by skeletal muscle (to fill up the used glycogen stores).
  • a period of increased insulin resistance e.g. high intensity exercise, stress, infection, period, carb meal
  • stop exercise end of meal, sleeping
  • CSM continuous succinate monitoring
  • ISF insulinsensitive factor
  • ICR insulin-carb ratio
  • the ISF is most relevant here.
  • the ISF is the reduction in blood glucose (in mM or, in mg/dl, x 18) for 1 unit of fast acting insulin.
  • the ISF is usually determined and set for the used CL-CGM device by the diabetes health care team.
  • the ISF is usually calculated on basis of how much insulin had to be infused during the last 3 days. This ISF, however, is depending on the IR, which varies numerous times in and between days. These changes occur when one e.g.
  • the ICR which indicates the amount of extra fast-acting insulin needed to be taken with a carb meal, however, also varies and is also relevant here.
  • due to long term unhealthy nutrition (carbs, allergens) many persons show an intestinal stress response to (unhealthy, sometimes personal) food components.
  • the intestine will produce different amounts of succinate, which are transferred via the portal vein to the liver, again leading to an increased liver IR and succinate/IR-related increases in BG.
  • CSM detects and accurately corrects the daily changes in stress-induced BG changes. There is no need for patients anymore to correct for the changing stress-induced IR.
  • T1 DM patients experience stress-induced increases in BG due to medication dose/timing, caffeine consumption, high-intensity exercise, illness, dawn effect, allergies, periods (menstrual cycle), celiac disease, smoking, puberty, sunburn, altitude, dehydration, cold outside temperature, worries, family and social pressure, recent hypoglycaemic encounters, and impaired awareness of hypoglycaemia. These all lead to increases in BS, for which items 1 and 2 above count.
  • T1 DM patients experience increases in BG with meal constitution and personal microbiome for which item 3 counts.
  • the ISF is thus directly related to succinate level. Integration of the CSM data with the CGM data will thus optimize insulin infusion at any time based on momentary info on changing ISF. With the to-be-developed algorithms, GV with all these daily activities is thus minimized leading to a higher TIR. This will not only be the case for T1 DM patients using (hybrid) CL-CGM systems, but also for patients suffering from gestational diabetes or type-2 diabetes using the CL-CGM system.
  • this K remains similar in the linear range of activation by succinate, being roughly between 5 and 50 uM. Below and above this range, the ISF will be more and less sensitive to changes in [succinate], respectively (and K will thus be higher and lower, respectively).
  • Figure 2 shows Dose-response curve of human SUCNR1 to succinate. I nositol-3 phosphate formation by cells expressing human SLICNR1 in response to different concentrations of succinate. Taken from Rexen-Ulven, Sci reports, 2018, Pubmed 29968758.
  • the training module is performed with 100 subjects to establish the relationship between succinate level and ISF (and hence insulin dosage). Basal succinate as well as the first and second time point are determined.
  • succinate level increase between the first time and the second time by at least 10% and at most 40% indicates a decrease of (actual) ISF by at least 15% and at most 40% of the subject between the first time and the second time
  • a succinate level decrease between the first time and the second time by more than 40% indicates an increase of (actual) ISF by more than 60% and at most 400% of the subject between the first time and the second time;
  • a succinate level increase between the first time and the second time by more than 20% indicates a decrease of (actual) ISF by more than 20% and at most 60% of the subject between the first time and the second time;
  • succinate level increase between the first time and the second time by at least 10% and at most 20% indicates a decrease of (actual) ISF by at least 10% and at most 20% of the subject between the first time and the second time;
  • an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 10% change of (actual) ISF of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 20% indicates an increase of (actual) ISF by more than 40% and at most 100% of the subject between the first time and the second time;
  • a succinate level decrease between the first time and the second time by more than 10% and at most 20% indicates an increase of (actual) ISF by more than 10% and at most 40% of the subject between the first time and the second time.
  • Example 3 10 Type 1 Diabetes Mellitus (T 1 DM) patients are recruited: (male/female) ⁇ 40 year with normal BMI using a commercial CL-CGM device targeting for a healthy blood glucose level of 6 mM.
  • insulin dosage is based on standard (basal) ISF.
  • insulin dosage is based on ISF corrected based on succinate level changes according to the invention (see Example 2).
  • insulin dosage is based on ISF that has been corrected based on succinate level changes but not according to the invention.
  • Insulin dosage is based on standard (basal) ISF
  • Insulin dosage is based on ISF corrected based on succinate level changes according to the invention (see Example 2).
  • Insulin dosage is based on ISF that has been corrected based on succinate level changes but not according to the invention (see Example 2).
  • T2DM/T1 DM patients are recruited, (higher burden, more complex (poly-pharmacy), less number of people, possibly more difficult to study as compared to Example 3; disease more severe/covers aspects of common T2DM and CVD): typically older adult T1 DM patient with minimally exercising and/or with high risk of hypoglycaemia, or for older adults with coexisting chronic illnesses or two or more instrumental ADL impairments or mild- to-moderate cognitive impairment. Patients are randomly assigned to one of two groups:
  • Insulin dosage is based on standard ISF
  • Insulin dosage is based on ISF corrected based on succinate level changes according to the invention.
  • Health is defined as the ability to adapt to these changing environmental stressors, deal with these allostatic loads and to maintain homeostasis and longevity is the resultant of it.
  • stressors are mainly (1) consumption of non-natural (processed) food/drinks, (2) surplus of food/drinks, (3) alcohol consumption, (4) air/water/food pollution, (5) social distance/stress, (6) relational/work stress, and (7) reduced energy usage (less exercise, experience of cold).
  • longevity is determined by genetics in combination with lifestyle.
  • a healthy lifestyle encompasses (1) consuming moderate volumes of nonprocessed (and high quality) food and drinks (intermittent fasting), (2) no alcohol/smoking, (3) recognition and removal of/escape from of mental stresses (stay away from energy-absorbing situations/enjoy energy-giving conditions, (4) regular exercise, (5) regularly experience cold (bath/swimming; walk in cold with moderate dressing).
  • Result 1 Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: fatigue, pain, lack of energy, fever, thirst, dry mouth, dyspnea, anxiety, worry, communication difficulty, polyneuropathy, myopathy, fog over the brain/delirium (low ability to concentrate, easily forgetting thing, weak memory, slow movements and thoughts), susceptible to infection, venous thromboembolism, development of post-traumatic stress syndrome, mechanical ventilation, high ICU/hospital lengths of stay, high mortality.
  • hypothesis measuring succinate will show (1) strongly increased levels compared to healthy peers, (2) discriminative power for the different forms of hypothyroidy, (3) a decrease with effective therapy (addressing the cause(s) of critical illness), and (4) may serve as an OEM in thyroid hormone dosing (as the extent of hypothyroidism shows will vary during/between days and between individuals).
  • basal succinate In maximally healthy individuals, basal succinate will be low and which, following a rise with an environmental queue, will fastly return to basal. This latter aspect is a feature of a high resilience for the experienced environmental queue.
  • People suffering from a chronic acquired or inherited disease characterized by chronic (low-grade inflammation (diabetes, obesitas, CVD, liver/intestinal disease, CFTR) will have a basally high succinate level, which, following a rise with an environmental queue, will slowly return to the elevated basal.
  • succinate As the relative increase in succinate is small, it goes with a low level of resilience.
  • Longevity/healthy ageing is determined by the ability to respond to changing environmental conditions, and which thus is in line with a sustained low level of succinate. Regularly monitoring one’s blood succinate level will provide insight on the moment, intensity, and thus identity, of his personal environmental queues. With knowledge on these queues, one can take steps to prevent them.
  • the dose is limited by the level of toxicity it realizes with the patient.
  • a too high level of toxicity leads to abrogation of the therapy and/or loss of compliance of the patient.
  • the extent of toxicity is determined by the resilience of the patient towards these strong stressors for our bodies and shows a strong inter-individual and intra-individual variability (between days, within a day).
  • Prehabilitation and post-habilitation therapy to cancer treatment succinate measurements will allow: (1) determination of the patient’s resilience, (2) monitoring of his/her change in resilience with therapy, (3) identify which therapy is most efficient for the particular patient to increase his/her resilience, and (4) determine to which intensity and duration the therapy can be given to benefit the patient.
  • succinate measurements will allow: (1) monitoring of the patient’s resilience level in time, (2) determine to which intensity and duration the treatment can be given in order not not becoming toxic [i.e. the maximal tolerated exposure, MTE, and personalized maximum tolerated dose, MTD], (3) optimized chemotherapeutic dosing in time irt to the resilience level of the patient.
  • Levodopa (or L-dopa) is a precursor of dopamine and is the golden standard treatment for parkinsonism. L-dopa, however, is partially broken down by the intestinal microbiome, which varies between patients, despite inhibition via carbidopa. Consequently, the dose that reaches the brain varies between persons. Lifestyle has a major role in the efficiency of L-dopa as a treatment for parkinsonism.
  • basal blood succinate will be a measure of the health status of the patient: healthy from 0-50 uM, diseased from 60 and higher. Blood succinate measured before and 60-120 minutes after a meal will reveal the healthiness status of his/her microbiome. measuring blood succinate will allow (1) monitoring of the health status of the patient with therapy, (2) optimized timing and dosing of levodopa +/- carbidopa/benserazide in relation to blood succinate at the start of and later with therapy (due to stress induced).
  • Parkinson’s patients treated with levodopa +/- carbidopa/benserazide are recruited. Patients are randomly assigned to group 1 or group 2.
  • basal blood succinate will be a measure of the health status of the patient: healthy from 0-50 uM, diseased from 60 and higher. Blood succinate measured before and 60-120 minutes after a meal will reveal the healthiness status of his/her microbiome. measuring blood succinate will allow optimized dosing of medication (oral or otherwise) to prevent side effects
  • CFS/ME chronic fatigue syndrome/myalgic encephalomyelitis
  • Results 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: itchy red rash (urticarial); red skin without a rash; swelling of the face, mouth (angioedema), feeling dizzy/lightheaded; sneezing or an itchy, runny or blocked nose; intestinal irritation, diarrhoea
  • benzodiazepines, opioids, anti-epileptics, and anti-inflammatory drugs are well known to cause addiction/dependency to these medications. While all of them have significant side effects and with several of them the original problem will come back after some time (as they do not address the cause, but treat a symptom), it is difficult for patients to taper them off, as the extent to which they can taper them off depends on de dose of reduction, which can often not be flexibly determined, AND the extent of stress/resilience the individual experiences at that moment.
  • Monitoring succinate as a readout of their momentary stress level and the response to the change of reducing the dose, which indicates their resilience, will allow flexible and scientifically-based personalized tapering medication.
  • Opioids sweating, diarrhoea, vomiting, abdominal cramps, chills, anxiety, insomnia, tremor, drowsiness, dizziness, low blood pressure
  • Anti-epileptics seizure re-occurrence, injury, loss of self-esteem, unemployment, inability to drive
  • NSAIDs swelling, pain, anxiety, agitation, nausea, headache, depression, blood clothing, increased risk of heart attack
  • Methotrexate joint stiffness, pain, swelling, fatigue, fever, disease flares
  • HRV range is limited by its dependence on heart rate (Monfredi & Lakatta, 2019)
  • Distal HRV measurements e.g. smartwatches at the wrist
  • Distal HRV measurements have a low signal-to-noise ratio as sensors close to the heart and it is stated that ‘It is of concern to incorporate such poor-quality data into daily clinical decision making. It is leading to a calculated $3.1 trillion of health care costs in the US’ (Stone et al, 2021).
  • HRV cardiovascular autonomic neuropathy
  • E.coli SQR operon is cloned into an expression vector and a cleavable his-tag is cloned at the N-terminus of SDHB as reported for the Thermophilus SQR (Kolaj-Robin et al, 2013).
  • This construct is transfected into bacterial strain DW35, which lacks both the SQR and QFR, due to which the bacteria have to use plasmid SQR to survive (Tornroth et al, 2002). Bacteria will be grown as described (Park et al, 1995).
  • Isolation of the SQR complex (being SDHA-SDHD) and SDH (SDHA and SDHB) is done using his-tag affinity chromatography as described (Kolaj-Robin et al., 2013). All steps will be done and materials and buffers will be pre-cooled at 2-3°C. Briefly, pelleted E.coli cells are lysed using lysozyme and spun down to remove cytosolic proteins. The proteins of the pelleted membrane fraction are solubilized in 0.05 mM Tris-HCI, pH 7.6, 300 mM NaCI, 10 mM imidazole and 5% Triton X-100 and non-solubilized proteins are removed by centrifugation.
  • the column-bound his-tagged SQR complex is treated with his-tagged rTEV protease (Sigma-Aldrich), eluted from the column, concentrated, aliquoted and snap-frozen.
  • his-tagged rTEV protease Sigma-Aldrich
  • the dimer is separated from the pelleted membrane fraction with sodium perchlorate as described (Morningstar et al, 1985). Following removal of the membrane fraction with centrifugation, the supernatant is subjected to the Nickel sepharose column as described above. The his-tagged and cleaved-off SDH will subsequently be isolated as described above.
  • E.coli SQR is isolated as described by Tornroth et al (Tornroth et al., 2002). Briefly, E.coli SQR is produced in DW35 bacteria from the pFAS plasmid, producing E.coli SQR directed from the fumarate reductase promoter. Cells are disrupted with an homogeniser, unbroken cells removed, and membrane fraction isolated following centrifugation at 120000g. The pellet fraction is dissolved in Thesit-containing buffer, loaded on a DEAE-Sepharose FF column and, after washing, the enzyme is eluted using a NaCI gradient and concentrated.
  • the collected SQR enzyme is further purified using a Poros 50HQ and Sephacryl S-300 gel filtration column, concentrated, and used to coat/couple on voltametric device or amperometric sensor.
  • the enzyme obtained is highly active with a succinate-quinone reductase kcat at 303 K in the range of 90-100/s using the Q1 homologue of ubiquinone as described.
  • malonate is added to 1 mM to reversible inhibit SDH activity as described (Stepanova et al., 2016). While stabilizing the complex, malonate is easily displaced by succinate, which allows direct activation upon succinate addition.
  • the purity, redox cofactor content and SDH activity can be determined.
  • SQR/SDH only needs succinate as a substrate and generates, like glucose dehydrogenase (GDH) used in CGM devices, an electron upon oxidation of succinate that can be quantitatively measured using amperometry, SQR/SDH is chosen as the protein complex to work out as a sensor for bodily fluid succinate detection.
  • GDH glucose dehydrogenase
  • fungi-derived FAD dependent GDHs are currently the most popular enzymes and are used in sensor strips in second-generation SMBG sensors (Okuda-Shimazaki etal, 2020).
  • fFADGDHs fungi-derived FAD dependent GDHs
  • BcGDH Burkholderia cepacia
  • EDT direct electron transfer
  • SQR/SDH does not need to be of human origin.
  • Eukaryotic or prokaryotic SQR/SDH a. Simplicity and yield: bacteria and yeast have the advantage of high expression of proteins due to their easy growth and ability to use versatile plasmids that allow controlled high expression of proteins. Moreover, bacteria express the SQR four subunits in one operon, which facilitates potentially- needed modification to optimize protein features. Therefore, expression in bacteria, or otherwise yeast, is desirable, independent on whether eukaryotic or prokaryotic SQR/SDH needs to be expressed.
  • Bacterial SQR/SDH useful eukaryotic (yeast, mammalian) proteins often need post-translational modifications for proper functioning, a feature that does not occur in prokaryotes.
  • the SQR/SDH proteins are proteins of mitochondria, which are evolutionarily derived from bacteria that started to ‘live’ in symbiosis with eukaryotic cells. As such, the four SQR subunits are not modified like eukaryotic proteins. Moreover, the SDHA and SDHB subunits, which form the functional unit of the SDH complex, are highly conserved between prokaryotes and eukaryotes, including mammalian mitochondria (Lemos et al, 2002; Maio et al, 2014). Thus, bacterial SQR/SDH can be used to form the succinate sensor, allowing expression in its own host.
  • SDH SDHA-SDHB
  • SQR the SDHA-SDHB two-subunit complex displayed succinate dehydrogenase activity with artificial electron acceptors (Moosavi et al., 2019) and is water-soluble, which facilitates its isolation.
  • the in vivo SQR complex consists of a trimer of the SDHA-D subunits (Kolaj-Robin et al., 2013; Yankovskaya et al, 2003).
  • in vivo conformations are usually more stable and it is difficult to tell in advance whether the dimer or the full complex has better electron guidance when coated on sensor material (see later), the bacterial SQR as well as SDH can be isolated and tested.
  • Blood plasma conditions as a selection criterion: the SQR activity of the different bacteria has adapted to the living conditions of the respective bacteria.
  • SDH of Archae and thermophilus bacteria are active in 1 M KCI and at 70°C, respectively, as these are the conditions these bacteria live in.
  • Thermophilus SQR is more stable than E.coli SQR and yields higher activities at different voltages (Melin et al, 2014)
  • its Km of 300 uM Kolaj-Robin et al., 2013
  • E.coli SQR/SDH As succinate detection of blood/interstitial fluid is needed, a bacterial SQR/SDH is needed that can sensitively detect succinate under blood-physiological conditions.
  • E.coli SQR/SDH is chosen, because it is most active at 37°C, has a succinate Km value of 70 uM (Kita et al, 1989), which is well in the range of plasma succinate levels, and is active at high oxygen concentrations.
  • the electric signal correlates with the substrate concentration, but this relationship is also affected by the sensor construction and the environment of the measurement.
  • the perfect biosensor characterized by a low limit of detection, wide linear range, high sensitivity, good selectivity and the absence of non-specific bindings, effective electron transfer between the enzyme and transducer, as well as high stability and an excellent understanding of the interactions between the enzyme modification components are beneficial.
  • the immobilization of an enzyme can have various effects on enzyme activity. It is valuable because this makes it possible to reuse an enzyme multiple times, thereby extending its life span as well as reducing its degradation. In addition, this immobilization influences the improvement of its pH and temperature stability.
  • the selectivity, specificity and activity can be improved after immobilization by changing the conformation of the enzyme. Therefore, the enzyme is spotted onto different nanoparticles.
  • many nanoparticles include the noble metals Au, Ag, Pt, Pd, and oxides (CuO, Cu2O, NiO, Fe2O3, as well as bimetallic systems (Au-Pt, Au-Pd, and Cu-Ag, have been used for glucose detection. Nevertheless, the major benefit of using gold is a higher glucose oxidation current than for other noble metals.
  • the main advantages of immobilization are multiple or repeatable usage of molecules and improving their stability. There are five most commonly applied methods: covalent bonding, adsorption, cross-linking, entrapment and self-assembled monolayers.
  • Cyclic voltammograms (redox activity) of, among others, E.coli SDH have been performed following adsorption of a protein film on pyrolytic graphite edge (PGE) or gold rotating disk electrodes (Ackrell et al, 1993; Pershad et al, 1999). Also Thermophilus SQR was adsorbed onto PGE rotating disk electrodes (Kolaj-Robin et al, 2011).
  • the isolated SQR/SDH complexes can be adsorbed onto single-walled carbon nanotubes (SWNT) as described (Melin et al., 2014). Briefly, a commercial glassy carbon disk electrode (3 mm diameter) can be modified by dropcasting it with a dispersion of SWNT in EtOH/H2O mixture. In total, 3 SWNT deposits can be made. Following SWNT annealing, the SDH/SQR proteins can be diluted to less than 0.05% detergent concentration and 4 ul of a 0.5 uM SQR/SDH solution can be O/N adsorbed to the working electrode surface at 4oC. After adsorption, the electrode will be rinsed to remove excess protein.
  • SWNT single-walled carbon nanotubes
  • Succinate concentrations can be determined in blood/interstitial fluid, which has a pH around 7.4, [Na] of around 140 mM, [K] of around 4 mM, [Cl] of around 100 mM.
  • Liquid chromatography tandem mass-spectrometry as e.g. performed in (Lamy et al, 2022).
  • T2DM type-2 diabetic
  • succinate levels in T2DM patient was significantly decreased from 60 to 46 uM (Ceperuelo- Mallafre et al., 2019).
  • the EC50 van de SLICNR1 is 28-56 uM and 99% responses is achieved at 200 uM or higher (He et all, 2004).
  • Circulating succinate correlates not only with body mass index (B I) but also with several markers of a worse metabolic profile, including insulin, glucose, homeostatic model assessment of insulin resistance, diastolic blood pressure, and triglycerides. Indeed, after adjusting for confounders, BMI and glucose were the main determinants of circulating succinate, suggesting that both obesity and diabetes have an impact on this metabolite. It was found that in patients with T2D and morbid obesity, in whom surgical weight loss intervention decreased circulating succinate levels. During these analyses, we noted that preoperative levels of succinate can be used as a determinant of diabetes remission after bariatric surgery and also to allocate these patients to the best-suited surgical procedure in terms of diabetes remission and complications.
  • succinate as a predictive marker, which previously had been inferred in other pathologies, including in critically injured patients and in patients with cancer, offers a new perspective of succinate as an important sensor of metabolic disturbances.
  • succinate might provide a broader view of homeostasis as an easily measurable blood based metabolic biomarker.
  • our data also showed that the nutritional modulation of plasma succinate that occurs under healthy conditions is blunted in patients with T2D and morbid obesity and is recovered after weight loss, which is reminiscent of the typical behaviour of some hormones in an obesogenic context.
  • Tornroth S Yankovskaya V, Cecchini G, Iwata S (2002) Purification, crystallisation and preliminary crystallographic studies of succinate:ubiquinone oxidoreductase from Escherichia coli. Biochim Biophys Acta 1553: 171-176 van Diepen JA, Robben JH, Hooiveld GJ, Carmone C, Alsady M, Boutens L, Bekkenkamp- Grovenstein M, Hijmans A, Engelke UFH, Wevers RA etal (2017) SUCNR1 -mediated chemotaxis of macrophages aggravates obesity-induced inflammation and diabetes.

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Abstract

La présente invention concerne un procédé pour déterminer le dosage d'insuline pour un sujet en ayant besoin, le procédé comprenant : a) déterminer le facteur de sensibilité à l'insuline (FSI) basal du sujet ; b) déterminer le niveau de succinate dans un échantillon prélevé une première fois sur le sujet ; c) déterminer le niveau de succinate dans un échantillon prélevé une deuxième fois sur le sujet ; d) déterminer le niveau de glucose dans un échantillon prélevé sur le sujet ; e) déterminer si le niveau de succinate déterminé à l'étape c) est inférieur, identique ou supérieur au niveau de succinate déterminé à l'étape b), afin de déterminer un dosage d'insuline approprié pour le sujet. Selon le procédé, la détermination du taux de succinate peut être effectuée à l'aide d'un capteur ampérométrique.
PCT/EP2023/059334 2022-04-07 2023-04-07 Procédé pour déterminer le dosage d'insuline Ceased WO2023194615A1 (fr)

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