WO2017052455A1 - Fragments amylase pour la régulation de la glycémie - Google Patents

Fragments amylase pour la régulation de la glycémie Download PDF

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Publication number
WO2017052455A1
WO2017052455A1 PCT/SE2016/050886 SE2016050886W WO2017052455A1 WO 2017052455 A1 WO2017052455 A1 WO 2017052455A1 SE 2016050886 W SE2016050886 W SE 2016050886W WO 2017052455 A1 WO2017052455 A1 WO 2017052455A1
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Prior art keywords
amylase
composition
composition according
trypsin
porcine
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PCT/SE2016/050886
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English (en)
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Stefan Pierzynowski
Kateryna GONCHAROVA
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Anara Ab
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the field of means and methods for controlling blood glucose in a subject, in particular with regard to treatment of hyperglycemic conditions, conditions involving hyperinsulinemia and conditions involving impaired insulin sensitivity.
  • Insulin misregulation in adolescence has a considerable impact on metabolic processes in adults.
  • High carbohydrate consumption and as well as high protein diet results especially when combined with physical inactivity in the overproduction of insulin, leading to the development of insulin resistance, undesirable anabolic activity (including weight gain and obesity), metabolic syndrome and ultimately type 2 diabetes.
  • Existing mainstream treatments for type 2 diabetes include exogenous insulin administration, GLP-1 receptor agonists, metformin, sulfonylureas, meglitinides, DPP-4 inhibitors, thiazolidinediones and SGLT2 inhibitors.
  • Other treatments include exercise and dietary management. None of the existing treatments is without drawbacks and the treatments are not fully efficacious in a substantial number of patients.
  • Glucose intolerance associated with a delayed insulin release has been observed during exocrine pancreatic insufficiency (i.e. in a condition characterized by lack on pancreatic enzyme secretion) in humans and pigs (Knop, et al. 2007; Lozinska, et al. 2013). So far, such physiological changes are recognized as a pancreatogenic type of diabetes (3c type) and suggested to occur together with exocrine pancreatic insufficiency or pancreas injury (Ewald and Hardt 2013). In humans, the influence of pancreatic enzymes replacement therapy on glucose
  • pancreatic enzymes present in the gut lumen improved direct blood glucose utilization (without reinforcing insulin release) and improved growth performance of the pigs (Lozinska, et al. 2013). Furthermore, utility of amylase in the treatment of diabetes has been mentioned in patent literature in WO2006136161 and GB2468629.
  • An object of the present invention is to provide improved methods and means for blood glucose control, in particular with regard to insulin sensitivity. Another object of the present invention is to provide improved methods and means for the treatment of hyperglycemic conditions, conditions involving hyperinsulinemia and for conditions involving impaired insulin sensitivity.
  • amylase refers to an enzyme having alpha-amylase activity (EC 3.2.1.1).
  • treatment in the present context refers to treatments resulting in a beneficial effect on a subject afflicted with the condition to be treated, including any degree of alleviation, including minor alleviation, substantial alleviation, major alleviation as well as cure.
  • degree of alleviation is at least a minor alleviation.
  • prevention in the present context refers to preventive measures resulting in any degree of reduction in the likelihood of developing the condition to be prevented, including a minor, substantial or major reduction in likelihood of developing the condition as well as total prevention.
  • the degree of likelihood reduction is at least a minor reduction.
  • sequence identity expressed in percentage is defined as the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the comparison window is the entire length of the sequence being referred to.
  • GenBank accession numbers cited herein refer to entries in the most recent version of said database at the date of filing of the present application.
  • FIG 1 illustrates effect of amylase and amylase fragments on intravenous glucose tolerance test (IVGTT) glucose clearance. Fragmented amylase is clearly more efficacious than intact amylase.
  • FIG. 2 illustrates effect of amylase and amylase fragments on insulin secretion in IVGTT. Fragmented amylase is clearly more efficacious than intact amylase.
  • FIG. 3 illustrates effect of amylase and amylase fragments on C-peptide in IVGTT.
  • Fragmented amylase is clearly more efficacious than intact amylase.
  • the present invention relates to the following items.
  • the subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.
  • composition comprising one or more effective fragments of an amylase
  • the one or more fragments have a greater biological effect in increasing glucose clearance in a mammal after oral administration, compared to intact amylase from which the one or more fragments are derived,
  • composition according to item 1 for use as a medicament.
  • composition according to item 1 for use as a dietary supplement.
  • composition according to any of the preceding items, wherein the composition comprises an effective fragmented amylase.
  • composition according to any of the preceding items, wherein the composition comprises an effective protease-fragmented amylase.
  • composition according to any of the preceding items, wherein the composition comprises an effective trypsin-digested amylase.
  • amylase is selected from a fungal amylase, a mammalian amylase, a vertebrate amylase, an invertebrate amylase, a bacterial amylase or a plant amylase.
  • amylase is selected from a human amylase, a porcine amylase, a fungal amylase, an Aspergillus amylase, preferably an Aspergillus oryzae-a mylase, more preferably a human amylase, most preferably a human salivary amylase.
  • composition according to any of the preceding items, wherein the composition comprises an Aspergillus oryzae-a mylase digested with a porcine trypsin.
  • composition according to any of the preceding items, wherein the composition comprises an Aspergillus oryzae-a mylase according to SEQ ID NO: 1 digested with a porcine trypsin.
  • composition according to any of the preceding items wherein the composition comprises trypsin-digested human amylase, preferably human salivary amylase.
  • composition according to any of the preceding items wherein the composition comprises human amylase, preferably human salivary amylase, digested with porcine trypsin.
  • composition according to any of the preceding items, wherein the composition comprises trypsin-digested porcine amylase.
  • composition according to any of the preceding items wherein the composition comprises porcine amylase digested with porcine trypsin. 15. The composition according to any of the preceding items, wherein the composition comprises one or more of the peptides according to any of SEQ ID NOs: 2-29.
  • composition according to any of the preceding items, wherein the composition further comprises a protease.
  • composition according to any of the preceding items, wherein the composition further comprises a trypsin, preferably a porcine or human trypsin.
  • composition according to any of the preceding items, wherein the composition is pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • composition according to any of the preceding items, wherein the composition is included in a medical food further comprising a calorie source.
  • composition according to any of the preceding items, wherein the composition is formulated for release in the duodenum.
  • hyperglycemic conditions conditions involving hyperinsulinemia
  • composition according to item 21, wherein the condition is selected from the list consisting of type 1 diabetes, type 2 diabetes, type 3 diabetes, metabolic syndrome, prediabetes, obesity, Alzheimer's disease, Cushing's syndrome.
  • the inventors have studied the role of digestive enzymes, produced by the exocrine pancreas, in blood glucose regulation.
  • the inventors have surprisingly found that one or more fragments of amylase (e.g. amylase digested with a proteolytic enzyme such as trypsin) are significantly more effective in regulating blood glucose than the amylase from which the one or more fragments are derived from in its intact (non-fragmented) state (See Examples 1 and 2).
  • a composition comprising one or more effective fragments of an amylase, for use as a medicament or a dietary supplement.
  • the first aspect also includes provision of a method of treatment comprising administering one or more effective fragments of an amylase as a medicament or a dietary supplement to a subject in need thereof.
  • the first aspect includes provision of a use of one or more effective fragments of an amylase in the manufacture of a medicament or a dietary supplement.
  • the composition is for use as a medicament.
  • the one or more fragments have a greater biological effect in increasing glucose clearance in a mammal after oral administration, compared to intact amylase from which the one or more fragments are derived.
  • the term "effective" in this context also entails that the one or more fragments have a greater biological effect in increasing insulin sensitivity in a mammal after oral
  • the composition contains an effective amount of the effective amylase fragments.
  • Amylase from which the one or more fragments are derived refers to an amylase comprising the corresponding primary structure(s) as the primary structure of the one or more effective fragments.
  • the one or more fragments and the intact amylase may or may not share a common origin or method of manufacture.
  • the difference can be detected with generally accepted statistical significance (e.g. p ⁇ 0.05).
  • the amplitude of the difference may be that the biological effect of the one or more effective fragments is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, or 300% higher than that of the intact amylase.
  • the one or more fragments are preferably regarded as effective when they have a statistically significantly greater effect on glucose clearance in an intravenous glucose tolerance test compared to intact amylase from which the one or more fragments are derived, wherein the one or more fragments are administered orally lh before the glucose injection. Structural features of effective fragments
  • the one or more fragments of an amylase may be derived from a non-recombinant amylase (or a part thereof) subjected to a fragmentation treatment, a recombinant amylase (or a part thereof) subjected to a fragmentation treatment or may be prepared recombinantly or via chemical synthesis as ready-made fragments.
  • composition may comprise an effective protease-fragmented amylase.
  • the composition comprises an effective trypsin-digested amylase.
  • the composition comprises an effective fragmented amylase, i.e. the complete set of fragmentation products of an amylase subjected to a fragmentation treatment.
  • composition may comprise one or more fragments of an Aspergillus oryzae-a mylase digested with porcine trypsin, preferably the entire collection of fragments resulting from porcine trypsin digestion of an Aspergillus oryzoe-amylase.
  • the Aspergillus oryzoe-amylase comprises the sequence according to GenBank accession no. XP_003189619 (SEQ ID NO:l).
  • the composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 of the peptides according to SEQ ID NOs: 2-29 which represent a computer simulation of trypsin digestion of an Aspergillus oryzoe-amylase of XP_003189619.
  • the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 of the peptides according to SEQ ID Nos: 2- 29 which are at least 5, 6, 7, 8, 9, 10 or 12 amino-acids long.
  • Table 7 Peptides resulting from simulation of trypsin digestion of an Aspergillus oryzae- amylase of XP_003189619
  • the fragmentation treatment discussed above may be a chemical treatment or an enzymatic treatment.
  • the fragmentation treatment is a treatment with a protease enzyme.
  • the protease enzyme may a trypsin or an enzyme with a similar activity, however a large number of different proteases and various chemical treatments also can be used to generate effective fragments. Any of the proteases and chemical treatments listed below in Table 4 may be useful in generating effective fragments.
  • the skilled reader is directed to the ExPASy website "PeptideCutter" (http://web.expasy.org/peptide_cutter/)
  • a protease enzyme in a fragmentation treatment is trypsin or an enzyme with similar activity, most preferably porcine trypsin.
  • the amylase of the first aspect of the invention may be selected from a fungal amylase (such as an Aspergillus amylase), a mammalian amylase, a vertebrate amylase, an invertebrate amylase, a bacterial amylase or a plant amylase.
  • a fungal amylase such as an Aspergillus amylase
  • a mammalian amylase such as an Aspergillus amylase
  • a vertebrate amylase such as an Aspergillus amylase
  • an invertebrate amylase such as a bacterial amylase
  • bacterial amylase such as Bacillus amylase
  • amylase enzyme may exist in several isoforms or homologues.
  • the amylase excreted in saliva is not identical (albeit highly similar) as the enzyme excreted by the pancreas.
  • said mammalian amylase may be a salivary amylase or a pancreatic amylase, preferably a salivary amylase.
  • Aspergillus oryzae in turn has three highly similar but not identical amylases.
  • the amylase may preferably be an Aspergillus oryzoe-a mylase (such as GenBank accession no XP_003189619), more preferably a porcine amylase (such as GenBank accession no NP_999360, SEQ I D NO: 30), and most preferably human salivary a-amylase 1 (such as GenBank accession no NP_001008219, SEQ I D NO: 31), or human pancreatic a-amylase (such as GenBank accession no NP_000690, SEQ ID NO: 34).
  • GenBank accession no XP_003189619 an Aspergillus oryzoe-a mylase
  • a porcine amylase such as GenBank accession no NP_999360, SEQ I D NO: 30
  • human salivary a-amylase 1 such as GenBank accession no NP_001008219, SEQ I D NO: 31
  • human pancreatic a-amylase such as GenBank
  • the amylase preferably has a degree of sequence identity (see Definitions) with a human amylase (preferably according to GenBank accession no NP_001008219, SEQ I D NO: 31).
  • the sequence identity may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.
  • amylases include porcine amylase (GenBank accession no NP_999360) with 86% identity, a Vulconisoeto distributo DSM14429 amylase (GenBank accession no ADN51908, SEQ ID NO: 32) with 67% identity, a Desulfurococcus fermentans DSM16532 amylase (GenBank accession no AFL66315, SEQ ID NO: 33) with 50% identity and an Aspergillus oryzae amylase (such as GenBank accession no XP_003189619, SEQ ID NO: 1) with 28% identity.
  • Alpha-amylase 15 15 1% 7.4 50% AFL66315.1 33
  • composition may further comprise a protease, preferably a trypsin, more preferably porcine or human trypsin.
  • a protease preferably a trypsin, more preferably porcine or human trypsin.
  • the composition may have any formulation, and can also be included in foodstuffs.
  • the composition may be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • the composition may be a dietary supplement or a nutraceutical.
  • the composition may further comprise a calorie source and may be a medical food, a functional food or a part thereof.
  • the calorie source may comprise a protein, a carbohydrate and/or a lipid.
  • the calorie source may contain at least 20 kcal, preferably at least 50 kcal, most preferably at least 100 kcal, per serving.
  • the composition may be formulated as a powder, a tablet, a capsule, a liquid, as a part of a liquid food item, or as a part of a solid food item.
  • the composition may be as an enterically coated formulation.
  • the composition is formulated for release in the duodenum, to avoid exposing the amylase fragments to conditions in the stomach that might degrade efficacy. Suitable delayed-release
  • composition of the present invention increases the capacity for glucose clearance (see Example 1), with apparently lower insulin release needed to achieve the glucose clearance (see Example 2), the composition of the first aspect is useful for treatment for a number of pathological conditions.
  • the composition according to the first aspect may be for use in the treatment or prevention of a condition selected from the list consisting of hyperglycemic conditions, conditions involving hyperinsulinemia and conditions involving impaired insulin sensitivity.
  • the subject in the method of treatment of the first aspect may be in need of treatment of a condition selected from the list consisting of hyperglycemic conditions, conditions involving hyperinsulinemia and conditions involving impaired insulin sensitivity.
  • the medicament manufactured according to the first aspect may be for the treatment of a condition selected from the list consisting of hyperglycemic conditions, conditions involving hyperinsulinemia and conditions involving impaired insulin sensitivity.
  • AD Alzheimer's disease
  • the link between insulin resistance and Alzheimer's disease is important for prevention and for treatment of AD as well. Problems regulating blood sugar may impact cognitive function at any age. For Alzheimer's, it's not just people with Type 2 diabetes. Even people with mild or moderate insulin resistance who don't have Type 2 diabetes might have an increased risk for Alzheimer's disease because they're showing many of the same sorts of brain and memory relationships" (Auriel et al. supra).
  • the improved insulin sensitivity achieved by fragmented amylase as shown herein provides a novel therapeutic approach for the treatment of Alzheimer's disease.
  • the above-mentioned condition to be prevented or treated may be a condition selected from the list consisting of type 1 diabetes, type 2 diabetes, type 3 diabetes, metabolic syndrome, prediabetes, obesity, Alzheimer's disease, Cushing's syndrome.
  • Example 1 Effect of different supplements on glucose utilization during IVGTT
  • Intravenous glucose tolerance test was chosen as convenient and appropriate for testing glucose-stimulated insulin response and capacity for blood glucose clearance.
  • the main advantages of IVGTT are that it can reveal the loss of beta-cell glucose sensitivity and responsiveness at an early stage of metabolic syndrome and type 2 diabetes development by detecting slower blood glucose utilization. Young healthy pigs were used as the experimental model because of their physiological similarity to humans, good reproducibility, and ability to be trained, which decreases stress under experimental conditions.
  • amylase-frag herein amylase-frag herein
  • amylase or amylase-frag affected blood glucose clearance, amylase caused lower blood glucose level at 5 minutes, compared to control values (p ⁇ 0.001).
  • amylase-frag exhibit significantly more potent lowering effect since postloading values at 5 and 15 for glucose were significantly lower for amylase-frag as compared to amylase alone and to controls (p ⁇ 0.001).
  • AUCgiucose is informative of the sum of glucose utilization during IVGTT in pigs after the administration of different treatments (Table 1, Fig. 1). There was a significant influence on the total blood glucose utilization of both amylase and amylase-frag, but the effect of amylase-frag was significantly greater than that of amylase.
  • Example 2 Effect of different supplements on insulin / C-peptide response during IVGTT
  • Amylase and amylase-frag oral administration caused lower plasma insulin level at 5 and 15 minutes, following glucose injection, as compared to controls values (p ⁇ 0.001).
  • Amylase-frag loading had a significantly greater lowering effect as compared to controls and pure amylase loading.
  • C-peptide was performed to verify glucose-stimulated insulin response in the pig's plasma after amylase and amylase-frag supplementation.
  • the plasma C-peptide level after oral administration of amylase and amylase-frag was still significantly lower than in the control samples at 5 and 15 minutes (p ⁇ 0.001), however amylase-frag administration induced the most potent and significantly stronger effect on C-peptide (Table 3, Fig 3).
  • Insulin sensitivity index S2 was also higher for samples, taken after amylase supplementation, compared to control feeding. S2 was proposed to be relevant for pigs as an accurate representative insulin sensitivity index based on IVGTT. This data indicates that the administration of pancreatic- like amylase but most probably it fragments requires less insulin release and perhaps limit glucose uptake. Table 1. Glucose concentration in blood, mmol/L
  • Control 1 ⁇ 0.01 ⁇ 0.02a ⁇ 2.05 a ⁇ 3.02 a ⁇ 0.11 ⁇ 0.05 ⁇ 0.01 ⁇ 0.01 ⁇ 0.01 ⁇ 0.03 ⁇ 0.01 885.93+34.09 a
  • Control 2 ⁇ 0.02 ⁇ 0.01a ⁇ 3.433 ⁇ 5.553 ⁇ 0.08 ⁇ 0.23 ⁇ 0.02 ⁇ 0.01 ⁇ 0.03 ⁇ 0.01 ⁇ 0.03 835.56 ⁇ 23.76 a
  • Amylase 0.15 0.05 10.09 12.04 2.03 1.20 0.03 0.04 0.02 0.02 0.03
  • Amylase enzyme (Sigma-Aldrich: Amylase A9857 from Aspergillus oryzae; was tested in as intact enzyme and after fragmentation (with trypsin) as oral supplements. The dose of the investigated microbial enzyme were correlated to the 4 pancrelipase capsules (Creon ® 10,000, Abbott Healthcare Products Ltd, Southampton, United Kingdom) (Fedkiv, et al. 2009; Rengman, et al. 2009) and based on the corresponding amylolytic activity.
  • Amylase activity was analysed using ethylidene-pNP-G7 (4,6-ethylidene-p-nitrophenyl-alpha, D- maltoheptaoside) as the substrate, according to the manufacturer's instruction (Infinity Amylase Liquid Stable Reagent; Thermo Electron, Victoria, Australia). The specific activity in 1 mg of individual enzymes were measured at 37 ⁇ C and compared with the activity of 1 pancrelipase capsule and the appropriate amount (in milligrams) of the investigated enzymes matching corresponding enzymatic activities in 4 capsules of commercial pancrelipase were used as a single dose.
  • Amylase fragments (also termed Amylase-frag herein) were obtained after adding to amylase solution of 30 ml 2 ml of trypsin (NovoNordisk, Denmark) solution lmg/ml. The solution was placed on water batch 37 C and stirred for 15 minutes. Amylase and amylase-frag solutions was loaded orally through the syringe within 2 minutes and immediately after that pig was watered with another 40ml of tap water to rinse mouth and throat from enzymes. As a control vehiculum (30 ml of water with 3 tablets of sweetener + trypsin 2 mg) was tested by itself without amylase.
  • IVGTT Intravenous glucose tolerance test
  • the intravenous glucose tolerance test (IVGTT) was performed on pigs that had fasted overnight for about 20 hours, and 1 hour prior to glucose injection, the oral administration of the: 1/ Amylase or 2/ Amylase-Frag, or 3/ vehiculum (Control 1 Group) or 4/ vehiculum + trypsin (Control 2 Group) were performed.
  • Glucose loading was carried out with a sterile 50% D-glucose solution (0.5 g/kg body weight) infused via the venous catheter during 30 seconds. Immediately after infusion of glucose, the catheter was flushed with 5ml of 0,9% sterile saline solution.
  • Blood samples for the glucose tolerance test were drawn at -1 (presented as time point 0'), 5, 15, 30, 45, 60, 120 minutes after infusion. Blood was collected into 5 ml syringes containing EDTA (0.20 mg) and a protease inhibitor, aprotinin (1000 klU, Bayer, Leverkusen, Germany), as described previously (Rantzer, et al. 1995). The blood samples were immediately cooled on ice and then centrifuged at 3.000 ⁇ g for 15 min at 4°C. Plasma was separated and stored at -20°C until analyses.
  • Glucose was measured in the fresh blood samples using a glucose-meter with test strips (Accu-Chek s Aviva, Roche Diagnostics, Mannheim, Germany). Plasma insulin and C-peptide levels were measured using porcine insulin or C-peptide ELISA kits, respectively (Mercodia, Uppsala, Sweden), according to the manufacturer's protocols with a lower detection limit of 0.2 pmol/L for insulin and 2 pmol/L for C-peptide.
  • AUC incremental area under the curve
  • Insulin sensitivity was calculated as a surrogate index S2, using the classic glucose disappearance rate (k) related to released insulin concentration during 0-30minutes, 30*k/o- 3oAUCinsuiin.
  • the area under the curve (o-3oAUCinsuiin) of insulin concentration from 0 to 30 min was calculated with a trapezoidal rule.
  • the units of the new index are l/min*pM (Christoffersen, et al. 2009).

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Abstract

L'invention concerne une composition comprenant un ou plusieurs fragments efficaces d'une amylase, destinée à être utilisée en tant que médicament ou complément alimentaire, en particulier pour le traitement d'états hyperglycémiques, d'états impliquant l'hyperinsulinémie et d'états impliquant la sensibilité altérée à l'insuline, tel que le diabète. La composition peut comprendre l'amylase d'aspergillus oryzae digérée avec de la trypsine.
PCT/SE2016/050886 2015-09-23 2016-09-21 Fragments amylase pour la régulation de la glycémie WO2017052455A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0261213B1 (fr) 1986-03-07 1992-08-05 Eurand International S.P.A. Composition servant a preparer des medicaments a liberation prolongee a administrer oralement
WO2006136161A2 (fr) 2005-06-24 2006-12-28 Novozymes A/S Amylases a usage pharmaceutique
GB2468629A (en) 2008-06-08 2010-09-15 Tarig Sayed Mustafa Arbab Therapeutic uses of a composition comprising an amylase, a lipase and a protease
WO2013021377A1 (fr) * 2011-08-08 2013-02-14 Two To Biotech Ltd. Nouveaux peptides, compositions les comprenant et leurs utilisations dans des méthodes pour le traitement de troubles métaboliques, cardiaques et immunitaires
US20130273026A1 (en) * 2012-04-12 2013-10-17 Integrative Enzymatics, Inc. Composition and method for modulating inflammatory molecules with amylase
US8562981B2 (en) 2007-02-20 2013-10-22 Aptalis Pharma Limited Stable digestive enzyme compositions

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0261213B1 (fr) 1986-03-07 1992-08-05 Eurand International S.P.A. Composition servant a preparer des medicaments a liberation prolongee a administrer oralement
WO2006136161A2 (fr) 2005-06-24 2006-12-28 Novozymes A/S Amylases a usage pharmaceutique
US8562981B2 (en) 2007-02-20 2013-10-22 Aptalis Pharma Limited Stable digestive enzyme compositions
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