WO2008041849A2 - Procédés et moyens pour déterminer la satiété sur la salive - Google Patents

Procédés et moyens pour déterminer la satiété sur la salive Download PDF

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WO2008041849A2
WO2008041849A2 PCT/NL2007/050482 NL2007050482W WO2008041849A2 WO 2008041849 A2 WO2008041849 A2 WO 2008041849A2 NL 2007050482 W NL2007050482 W NL 2007050482W WO 2008041849 A2 WO2008041849 A2 WO 2008041849A2
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satiety
peptide
saliva
peptides
biomarkers
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PCT/NL2007/050482
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English (en)
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WO2008041849A8 (fr
WO2008041849A3 (fr
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Leunis Forrinus Harthoorn
Raymond Godfried Schipper
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Stichting Top-Instituut Food And Nutrition
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Priority claimed from EP07115641A external-priority patent/EP2033656A1/fr
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Publication of WO2008041849A2 publication Critical patent/WO2008041849A2/fr
Publication of WO2008041849A3 publication Critical patent/WO2008041849A3/fr
Publication of WO2008041849A8 publication Critical patent/WO2008041849A8/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

Definitions

  • the current invention relates to field of analytical biochemistry and diagnostics for bio markers, in particular peptide bio markers in saliva for determining a state of satiety in a mammal.
  • Food consumption in mammals is under control of a complex interplay between mechanisms of the central nervous system and the peripheral body.
  • the brain senses peripheral signals from the gastrointestinal tract, pancreas, and adipose tissue that reflect the body's nutritional state, and consequently employs several pathways to anticipate and adjust food intake and energy expenditure (1,2).
  • mammalian eating behavior is a complex interplay between hunger or appetite and satiety.
  • the opposite of hunger and appetite is satiety, which is the physiological and psychological experience of "fullness" that comes after eating and/or drinking.
  • biomarkers provide information about the metabolic physiology and nutritional state of the individual, and may be used as profiling tool for understanding the regulation of food intake and energy balance in relation to overweight and obesity (5,6).
  • biomarkers have to meet criteria such as validity and reproducibility in measuring them; dependent on the context of the study and research, medical or diagnostic question. Biomarkers should be measurable in a cost effective, easy and rapid way.
  • biomarker profiling in an ample, sensitive, and noninvasive fashion will have the advantage over its measurement in blood because of unlimited opportunities of collection and amount of volumes, and by avoiding discomfort of the human subject.
  • biomarker discovery has become a focus of proteome studies designed to evaluate body fluids as potentially rich sources of diagnostic markers.
  • saliva is secreted from exocrine glands and is suffused with lots of peptides and proteins.
  • the salivary glands can be classified into serous, mixed and mucous glands.
  • the only serous glands are the parotid glands, which produce serous secretions only.
  • the submandibular glands are denominated as mixed glands, producing mixed secretions that contain both serous and mucous secretory products.
  • the sublingual glands produce mucus secretions.
  • human saliva has multiple essential functions in relation to oral and general health (7,8), as well as to food processing and digestion that takes place in the upper parts of the gastrointestinal tract (9).
  • salivary glands Local synthesis and secretion of substances by the salivary glands is, thus, essential in maintaining a healthy oral environment and breakdown of for instance food starches.
  • saliva and in particular parotid saliva and also gingival crevicular fluid from the toot/gum margin represents an ultrafiltrate of the blood and contains many blood-born factors, it is a potential source of novel diagnostic markers and targets (10-12).
  • This technology combines matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to surface chromatography. It utilizes sample chips which display various kinds of chemically enriched and active surfaces that bind protein molecules based on established principles such as ion exchange chromatography, metal ion affinity, or hydrophobic affinity. The subset of proteins retained to a certain surface can be varied by changing the properties (e.g. pH, salt concentration) of the binding buffer and the stringency of the washing steps for removal of unbound or weakly bound molecules and salts, which can interfere with the analysis. Protein detection is then accomplished by TOF-MS.
  • TOF-MS matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry
  • SELDI-TOF-MS is more sensitive and requires only minute amounts of sample compared to other proteomic techniques.
  • the technology has been used successfully in the field of diagnostic proteomics to detect several disease-associated proteins and protein expression patterns in a variety of biological tissues and body fluids (22,23 and in US2004/0033613).
  • salivary markers to diagnose diseases, (18,19) the current inventors recently have demonstrated (24) that SELDI-TOF-MS is a high throughput and extremely sensitive proteomic approach that provides quantitative broad range expression profiling of peptides and proteins and allows detection of specific biomarkers in human saliva.
  • biomarkers that can be used to directly monitor perceived satiety in mammalian saliva.
  • Many biomolecules, peptides and peptide hormones are known in the art that influence appetite or satiety and that can be measured in plasma. It is known from Aydin et al. (2005, Peptides Vol. 26: 647-652) that the peptides leptin and ghrelin can be measured in saliva using ELISA or RIA assays and that levels measured in saliva seem to correlate with plasma levels.
  • Aydin et al. (2005) is silent on a direct correlation between the level of specific biomarkers in saliva, in particular stimulated saliva or parotid saliva, and the perceived satiety status in a subject.
  • the current invention discloses methods and means that allow detection of biomarkers in saliva that have a direct correlation with perceived satiety in a mammalian subject, hence providing the means for a method for rapid and reliable screening the level of satiety in subject, or changes in the level of satiety, as well as satiety inducing properties of compounds, compositions, food products and nutraceuticals.
  • SELDI-TOF-MS Surface-enhanced laser desorption/ionization time-of-flight
  • salivary peptides are provided, the level of which is indicative of the satiety status of a mammalian subject.
  • biomarkers can be used in a method to determine the satiety status of a mammalian subject, the method comprising the step of determining in a sample of saliva from the subject the level of one or more biomarkers (i.e. of one or more peptides) for satiety.
  • the peptide level can then be used to infer (i.e. determine) the satiety status of the mammalian subject and/or to infer (i.e. determine) the effect of compounds or compositions (such as food, feed, food supplements, food ingredients, etc.) on the satiety status of the subject.
  • the invention also pertains to means for an immunological test, i.e. a test based on antibody mediated detection in saliva of the biomarkers of the invention that correlate with perceived satiety in mammals.
  • an immunological test i.e. a test based on antibody mediated detection in saliva of the biomarkers of the invention that correlate with perceived satiety in mammals.
  • the invention discloses solid carriers comprising one or more antibodies specific for the biomarkers according to the invention, which can be applied to determine the level of satiety in a mammal at any given moment or the change in satiety over time and/or between two or more conditions, for instance before and after consuming a substance of any particular food, food product / ingredient or diet.
  • the methods and means of the invention can be applied to study satiety in the context of overweight and obesity, screening the satiety inducing properties of food products or components and ingredients therein, and screening of compounds, compositions, food products, medicaments or medical treatments that induce satiety in mammals and may be used to develop diets, food products, nutraceuticals and methods to treat overweight and/or obesity.
  • “Satiety” or “satiety status” is defined as the state of inhibition over further eating that follows the end of an eating episode and refers to the feeling of "fullness'Or “desire to eat” or “desire to drink”.
  • the degree of “satiety” may vary between hunger and appetite on the one side, and satiety, i.e. the physiological and psychological experience of "fullness” that comes after eating and/or drinking, on the opposite side. Hence, hunger and satiety form the respective low and high ends of the “satiety scale”.
  • “Modulating satiety” may involve either inducing or reducing satiety, the latter corresponding to a feeling of hunger or appetite.
  • “Satiation” is defined as a process which develops during eating and which brings an eating episode to an end. "Satiation” or “enhanced satiation” refers therefore to earlier meal termination due to an earlier feeling of fullness. In the context of the invention, “satiety” and “satiation” are used interchangeably unless indicated otherwise.
  • Protein or “peptide” are amino acid chains found naturally in mammalian subjects, e.g. in saliva. Throughout the description, the terms “peptides” and “peptide bio- markers” are used interchangeably, and are meant to include peptide modifications, such as glycosylations, phosphorylations, O-octanoylations and/or O-decanoylations.
  • peptide includes proteins and peptide hormone.
  • “Mammalian subject” includes human beings, but also comprises various animals, such as bovine, canines, cats, horses, sheep, goats, pigs and rodents and the like.
  • the term “salivary peptides” or “salivary proteins” includes peptides naturally present in saliva, such as gastrointestinal and/or pancreatic hormone peptides and/or other salivary peptides (not falling within the group of gastrointestinal and/or pancreatic hormone peptides).
  • substantially identical include any protein or peptide which comprises at least 70, 80, 90, 95, 98, 99% or more amino acid sequence identity to one or more of the peptides of SEQ ID NO: 1-96.
  • Sequence alignment can be carrier out using for example the programs “ Needle” or “Water” of EmbossWin (e.g. version 2.10.0), using default parameters.
  • Needle uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Needle is preferably used when aligning sequences of similar lengths.
  • the current invention provides a method for determining the satiety status of a mammalian subject comprising the step of (a) determining in a sample of saliva from the subject the level of one or more bio markers (peptides); and (b) inferring from the peptide level the satiety status of the subject.
  • the satiety status of a human subject may be defined as set out in the examples section by answering questions concerning satiety, desire to eat, appetite, fullness, hunger, thirst, general well being, eating behavior, food preference or selection etc.
  • satiety may be measured by monitoring ethology: the eating behavior or pattern, food searching behavior, food intake, food preferences etc.
  • the method of the invention comprises the use of the biomarkers selected from the group of biomarkers having a molecular mass as determined by SELDI-TOF-MS and that correlate with satiety as provided in Table A and Table 4 (see Examples) and/or the detection and preferably quantification of the salivary level of one or more peptides selected from the group consisting of SEQ ID NO: 1 - 96 or variants thereof or fragments of any of these.
  • Variants include peptides which are essentially similar in amino acid sequence to SEQ ID NO: 1- 96, but which differ by one or more amino acid insertions, deletions and/or replacements with respect to SEQ ID NO: 1-96 and which thus have a certain minimum percentage amino acid sequence identity to the peptides of SEQ ID NO: 1-96, as described further below.
  • the method for determining the level of satiety in a mammalian subject comprises the use of one or more biomarker(s) that are members of the group of gastrointestinal and/or pancreatic peptide hormone(s).
  • Gastrointestinal and pancreatic peptide hormones that are particularly suitable to be applied in the method of the invention comprise the following group: insulin, insulin ⁇ -chain, insulin ⁇ -chain, insulin-like peptides 1-3, somatostatin- 14 and -28, gastrin, cholecystokinin (CCK), secretin, motilin, vasoactive intestinal peptide (VIP), gastric inhibitory polypeptide, enteroglucagon, peptide YY (P-YY), pancreatic hormone, pancreatic icosapeptide, ghrelin, glucagon, glucagon-like peptide 1 (GLP-I), glucagon-like peptide 2 (GLP-2), glucose-dependent insulino tropic polypeptide (GIP) and amylin.
  • insulin insulin ⁇ -chain
  • insulin ⁇ -chain insulin-like peptides 1-3, somatostatin- 14 and -28
  • gastrin cholecysto
  • Leptin a peptide hormone synthesized and secreted by fat tissue
  • the biomarkers that are most preferred for carrying out the method of the invention are the peptides that show a high level of correlation with satiety: insulin-like peptide A chain, insulin B chain, ghrelin and glucagon.
  • the biomarker peptides of the invention may also comprise modified forms of the peptides that are glycosylated, phosphorylated or exhibit other biological modifications.
  • the method of the invention may comprise and/or may be further enhanced by determining the level of additional salivary peptides that are not gastrointestinal and/or pancreatic peptide hormones.
  • salivary peptides belonging to groups such as de fens ins, somatomedins, histatins and leptin are suitable for use in the method of the invention, either per se or more preferably in combination with gastrointestinal and/or pancreatic peptide hormones.
  • the most preferred are somatomedin B, neutrophil defensin-2, histatin-2 and beta-defensin 126, the levels of which can be readily determined in saliva and that show a high correlation to perceived satiety.
  • the method of the invention may preferably be carried out on saliva obtained from mammalian subjects, most preferably on human subjects although the method may also be adapted for use in various animals, bovine, canines, cats, horses, sheep, goats, pigs and rodents.
  • the method of the invention may be readily carried out on whole saliva which can be easily sampled, but is more preferably carried out on parotid saliva or stimulated saliva that is rich in parotid saliva, in order to obtain optimal results for determining satiety.
  • the method of the invention comprises the detection of at least one biomarker that may increase or decrease upon food intake of the mammal after a period of fasting / food deprivation or after a change in diet / food supply.
  • the method of the invention comprises the sequential or simultaneous detection of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bio markers that increase or decrease with changes in the level of satiety in the mammal, in order to increase the sensitivity and consistency/reproducibility of the method.
  • the method of the invention comprises the detection of 2 to 6 satiety biomarkers selected from those of Table A, Table 4 and 5 and SEQ ID NO: 1-96 (and variants thereof and fragments of any of these), with at least 1 up-regulated biomarker and 1 down-regulated biomarker in saliva samples, as an optimal number yielding sufficient sensitivity and practicality.
  • the biomarkers according to the invention that correlate with satiety may correlate positively or negatively, i.e. may increase or decrease in saliva with induction of experiencing satiety in the subject.
  • the above method may comprise multiple sampling and measuring, e.g. before and/or after food intake, to track any changes in the levels of satiety peptides and/or to determine the effect of a compound or composition on satiety.
  • the method involves a single measurement, comparing the figures thus obtained with a database of peptides.
  • the method comprises the sequential detection of peptide levels in saliva obtained from the subject under two different conditions.
  • the two different conditions and comparison of the two obtained mass spectra from saliva samples may for instance reflect the peptide spectrum before and after uptake of a substance, a composition, a food product, a food ingredient, a medicament or before and after the subject has undergone a certain treatment, all of which may alter the perceived level of satiety of the subject.
  • a method for determining the effect of a compound or composition on the satiety status of a mammalian subject comprising administering said compound or composition to a subject and determining the satiety status of said subject using a method as described above, i.e. by determining the level of one or more bio markers in the saliva of the subject at one or more timepoints, inferring the satiety status of the subject from the biomarker level and further inferring the effect of the compound or composition on satiety therefrom (such as satiety inducing or satiety suppressing effects compared to subjects having received a control compound or composition).
  • the compound or composition may comprise or consist of a food, feed, food supplement or a food- or feed ingredient, such as a substantially pure proteins or peptides from food or feed sources such as milk or dairy products, meat, etc., a protein hydrolysate from food or feed sources, such as eggs or parts thereof, whey, etc., one or more vitamins, a whole meal, a meal replacer, a drink, a carbohydrate, a fatty acid, a probiotic and/or prebiotic, a mixture of any of these and/or other components.
  • the compound or composition may also consist of or comprise a pharmaceutical or biologically active compound, a neutraceutical, etc. In relation to proteins or peptides tested for their effect on satiety, these are not, however, the biomarkers according to the invention themselves, but food or feed derived proteins, plant derived proteins, non- human animal derived proteins, etc..
  • the change in the level of satiety biomarkers in saliva may be determined as the fold increase/decrease, for example after food intake and induction of satiety, or alternatively as the relative increase or decrease as compared to peptides in the saliva sample that remain relatively constant and/or are unaffected by satiety or food intake.
  • Satiety biomarkers may also be measured as increases/decreases in absolute level (i.e. concentration) in saliva.
  • the satiety biomarkers of the invention may also be correlated to one another; i.e. the increase of one or more satiety biomarkers in saliva may be determined by a correlation to a relative decrease in one or more (other) satiety biomarkers, or as the ratio of two or more biomarkers.
  • a relative decrease in ghrelin and/or glucagon levels in saliva may be detected with a relative increase in levels of PYY in the saliva.
  • the step of determining the relative or absolute amounts of one or more bio markers in a sample of saliva from the subject may be carried out by obtaining a Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (SELDI-TOF MS) spectrum from the sample.
  • the step of determining the level of satiety bio markers in saliva may be carried out by applying an antibody-based assay, i.e. an immuno-assay.
  • biomarkers of the invention in particular the preferred gastrointestinal and pancreatic peptide hormones, monoclonal antibodies are commercially available from various suppliers all of which may be successfully applied, taking into account the manufacturers recommendations for use.
  • Suitable immuno-assays for determining the level of a satiety biomarker in saliva comprise various antibody-based assays known in the art and for instance described in Current Protocols in Immunology (26). Suitable assays comprise ELISA, RIA, protein arrays, gel-shift and immunoprecipitation assays.
  • the method of the invention is carried out using a solid carrier comprising one antibody specific for one or more biomarker(s) of the invention, such as those of Table A, Table 4 and SEQ ID NO: 1-96.
  • a solid carrier for carrying out the method of the invention comprises one or more antibodies or functional fragments thereof specific for gastrointestinal and/or pancreatic peptide hormones, and/or for non-gastrointestinal and/or pancreatic peptides, such as salivary peptides selected from the group consisting of defenses, somatomedins, histatins and leptin.
  • the current invention provides a solid carrier, preferably in the form of a (micro)-array, protein chip, test-dipstick or test-tube, suitable for carrying out the method of the invention, the carrier comprising one or more monoclonal or polyclonal antibodies or functional fragments thereof such as Fab, Fab2, scFv and VHH, that are specific for a satiety biomarker present in saliva.
  • the solid carrier of the invention comprises antibodies with specificity for several target peptides in distinct areas on the carrier in order to facilitate the sequential or simultaneous detection of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkers that increase or decrease with changes in the level of satiety in the mammal.
  • the solid carrier of the invention preferably comprises antibodies or functional antibody fragments are specific for a gastrointestinal and/or pancreatic peptide hormone, more preferably those peptides selected from the following group: insulin, insulin ⁇ -chain, insulin ⁇ -chain, insulin-like peptides 1-3, somatostatin- 14 and -28, gastrin, cholecystokinin (CCK), secretin, motilin, vasoactive intestinal peptide (VIP), gastric inhibitory polypeptide, enteroglucagon, peptide YY (PYY), pancreatic hormone, pancreatic icosapeptide, ghrelin, glucagon, glucagon-like peptide 1 (GLP-I), glucagon-like peptide 2 (GLP-2), glucose-dependent insulinotropic polypeptide (GIP) and amylin.
  • a gastrointestinal and/or pancreatic peptide hormone more preferably those peptides selected from the following group: insulin, insulin
  • the solid carrier of the invention comprises antibodies or functional fragments thereof specific for insulin like peptide 3 A chain, insulin B chain, ghrelin and/or glucagons, which are biomarkers that show a very tight correlation with the level of satiety perceived by the subject.
  • the solid carrier of the invention may also comprise antibodies or functional fragments thereof specific for salivary peptides that are not gastrointestinal and/or pancreatic peptide hormones.
  • antibodies against peptides belonging to groups such as defensins, somatomedins, histatins, amylin and/or leptin are suitable for use in the method of the invention, either per se or more preferably in combination with antibodies against gastrointestinal and/or pancreatic peptide hormones.
  • antibodies or functional fragments thereof specific for somatomedin B, neutrophil defensin-2, histatin-2 and/or beta-defensin 126 are preferred, the levels of which can be readily determined in saliva and that show a high correlation to perceived satiety.
  • the current invention provides a method for identifying a bio marker correlating with a level of satiety in a mammal comprising the step of i) subjecting a sample of saliva from the mammal to SELDI-TOF mass spectrometry, ii) correlating the peaks / molecular masses in the obtained spectrum with a state of satiety in the mammal and iii) determining the peptide identity of the satiety correlated peaks in the SELDI-TOF mass spectrum.
  • the method comprises the sequential detection of bio marker levels in saliva obtained from the subject under two different conditions.
  • the two different conditions and comparison of the two obtained mass spectra from saliva samples may for instance reflect the biomarker spectrum before and after uptake of a substance, a composition, a food product, a medicament or before and after the subject has undergone a certain treatment, all of which may alter the perceived level of satiety of the subject.
  • the biomarkers and/or peptides identified by this method and that correlate either positively or negatively with increased satiety may be suitably applied in the method of the invention for determining the level of satiety in a subject.
  • the identified satiety bio markers may be applied to design a solid carrier comprising antibodies or functional fragments thereof that are specific for the satiety biomarker identified, and which may be applied in carrying out the method of the invention.
  • the method of the invention for determining the satiety status of a mammalian subject is applicable for the screening and the development of novel food products and diets that induce desired levels of satiety.
  • the method of the invention may also be applied for screening compounds, compositions, food ingredients / nutrients, food products and nutraceuticals for their capability of inducing or reducing satiety in a mammal.
  • the invention may be used to study satiety inducing capabilities of various combinations of substances and food products. Satiety level perceived by the subject under study reflects the end result of the satiety inducing properties of multiple components in a composition, food product or diet.
  • the method of the invention allows for an easy, cost effective and rapid screening of satiety levels or change in satiety levels in a subject without having to collect blood samples at multiple times, which is unpleasant for the subject under study, more laborious and expensive.
  • blood sampling is known to cause a mild stress response in certain individuals.
  • a stress response may influence the biomarker levels under study, which is highly disadvantageous and may be circumvented by the method and means provided by the current invention.
  • the biomarker peptides are preferably those screened peptides found in the SELDI- TOF MS screening method.
  • the peptides are preferably selected from the group consisting of gastrointestinal and pancreatic peptide hormone(s) and salivary peptides.
  • Peptides according to the invention preferably concern salivary proteins and peptides ranging from 2 to 100 kDa, more preferably low-mass salivary proteins/peptides ( ⁇ 10 kDa).
  • the peptides are more selected from the group of peptides presented in table A below, in which 30 peptides show to have a high level of correlation with satiety.
  • the satiety-modulating effect of these peptides is reflected in the SELDI-TOF-MS satiety correlation coefficient
  • the numbers in table 1 are derived from SELDI-TOF-MS analysis on human saliva.
  • peptides either as a satiety-reducing or satiety- inducing agent can be directly derived from table 1 :
  • the sign of the correlation coefficient r satl ety corresponds to its satiety- or hunger-inducing effect: a negative value corresponds to a satiety- reducing effect; a positive value is associated with a satiety-inducing effect.
  • SPRY domain-containing SOCS box protein 1 tissue-type plasmonigen activator chain A testis expressed sequence 264 protein transcription cofactor vestigial-like protein 4
  • Gastrointestinal and pancreatic peptide hormones that are particularly suitable to be applied in the context of the invention comprise the following: insulin, insulin ⁇ - chain, insulin ⁇ -chain, insulin-like peptides 1-3, somatostatin- 14 and -28, gastrin, cholecystokinin (CCK), secretin, motilin, vasoactive intestinal peptide (VIP), gastric inhibitory polypeptide, enteroglucagon, peptide YY (P-YY), pancreatic hormone, pancreatic icosapeptide, ghrelin, glucagon, glucagon-like peptide 1 (GLP-I), glucagon- like peptide 2 (GLP-2), glicentin, glucose-dependent insulino tropic polypeptide (GIP) and amylin.
  • insulin insulin ⁇ - chain
  • insulin ⁇ -chain insulin-like peptides 1-3, somatostatin- 14 and -28
  • gastrin chole
  • Salivary peptides preferably comprise defensins, somatomedins, histatins and leptins. These peptides are preferably applied in combination with one or more of the aforementioned gastrointestinal and/or pancreatic peptide hormone(s).
  • the most preferred salivary peptides are somatomedin B, neutrophil defensin-2, histatin-2 and beta-defensin 126, the levels of which can be readily determined in saliva and that show a high correlation to perceived satiety.
  • leptin a peptide hormone synthesized and secreted by fat tissue, preferably in combination with the above mentioned group of gastrointestinal and pancreatic peptide hormones.
  • biomarker proteins or peptides according to the invention comprise or consist of those provided in Table A, 4 and Table 5, and in SEQ ID NO: 1-96. Encompassed herein are also variants and fragments of the proteins/peptides or of the variants, as defined, and/or fragments of any of these. Variation in amino acid sequence may exist between different mammalian species. For detecting and optionally quantifying the biomarker in a certain species undue experimentation is needed. One may detect and optionally quantify the whole protein or protein fragments. Preferably however fresh saliva is used comprising substantially whole proteins. In one embodiment the saliva is preferably not the morning saliva taken after an overnight fast (i.e. a period of 6, 7, 8 or more hours without food intake), but preferably saliva before and/or after food and/or drink intake, such as 1, 2, 3, 4 hours or more after a meal intake and/or consumption.
  • the particularly preferred peptides comprise insulin-like peptide A chain, insulin B chain, ghrelin and glucagons, or similar forms thereof, in accordance with the above- given sequence identity.
  • the term "satiety-modulating" is understood to comprise either “satiety-inducing” and “satiety-reducing".
  • the actual effect is dependent on the compound or composition tested ('test' compound/composition) and can be compared to a suitable control compound or composition.
  • the effect of a "test” compound or composition on satiety can be determined by scoring for example the period of fullness until the next meal. A satiety inducing effect is seen if the period of the feeling of fullness is significantly longer than in the controls, such as for example at least 1%, 2%, 3%, 5%, 10%, 20% longer, or more.
  • the satiety inducing effect can be determined by measuring the amount of food consumed (until a pleasant feeling of fullness is reached) during a meal that is taken after a certain period (e.g 4 hours) following use of the test composition.
  • a certain period e.g 4 hours
  • Control compounds or compositions can be any compound or composition for which a satiety score is determined.
  • an effect of a compound or composition on satiation can be determined by e.g. scoring the time point of meal termination.
  • a satiation inducing effect is seen if the amount of food or consumed calories at meal termination is significantly less than in the controls, such as for example at least 1%, 2%, 3%, 4%, 5%, 10% 20%, or more.
  • the composition can also score the body weight reduction or the body weight change compared to a control diet.
  • Body weight of a subject being administered regular amounts of the test compositions e.g. once daily, twice daily, or more
  • VAS Visual Analogue Scale
  • the composition may be a pharmaceutical preparation. If so, it is intended for oral administration. However, it is preferred that the composition is a nutritional food or feed product, or a food or feed supplement. It may be in liquid, semi-solid or solid form.
  • Figure 1 PC (principal component) scores for the time points for the 2 D model for PCl and PC2.
  • the scores of the time points appear to cluster in those before lunch, those immediately after lunch and a set consisting of the remaining time points.
  • Twenty-nine coded dots are components significantly related to satiety. Their interrelationships show that 38% of the variation was accounted for in one dimension.
  • the served lunch meals were between 2500 and 3300 kJ. Water was usually available ad libitum. Every 15 min subjects donated stimulated saliva, rich in parotid saliva, by chewing on a 25-cm2 piece of parafilm (Parafilm® M) for up to 1 min and spitting into a 12 ml polystyrene tube (Greiner Bio- One, Kremsm ⁇ nster, Austria). The saliva was immediately placed into a freezer at - 40 0 C.
  • VAS visual analogue line scales
  • SELDI-TOF-MS analysis was performed when testing of subjects was finished and all samples were collected.
  • the SELDI-TOF-MS technology (Ciphergen Biosystems, Fremont, CA, USA) consisted of three major components: the ProteinChip array, a mass spectrometer, and the data analysis software. All common chemicals and reagents were of analytical grade and were obtained from Merck (Darmstadt, Germany), Sigma Aldrich (St Louis. MI), and ACROS Organics (Geel, Belgium), unless stated otherwise. Protein chips, buffers, 3,5-dimethoxy-4-hydroxycinnamic acid, and calibrants were purchased from Ciphergen Biosystems (Ciphergen Biosystems, Fremont, CA).
  • Ammonium acetate was purchased from ICN Biomedicals (Aurora, OH).
  • IMAC ProteinChip arrays were used and spots were first loaded with 50 ⁇ l 0.1 M copper sulphate by vigorous shaking at room temperature for 10 min. After a short wash with water the chip surface was neutralised using 150 ⁇ l sodium acetate buffer (pH 4.0) followed by a short wash with water and preincubation with binding buffer (0.1 M Tris-HCl, pH 7.4, containing 0.1% Triton X-IOO, and 100 mM or 500 mM NaCl). Saliva samples were thawed, centrifuged for 2 min at 1000 g at 4°C, and stored on ice.
  • Matrix was applied twice (0.8 ⁇ l each time and 1 minute apart), and allowed to air dry again prior to reading on a ProteinChip Reader HC instrument (Ciphergen Biosystems, Fremont, CA). The following settings were used: detector sensitivity 9; detector voltage 2900; positions 20 to 80 were read with an increment of 10 (resulting in 7 different sampling positions); 50 laser shots were collected on each position (total shots collected and averaged: 350/sample); two warming shots were fired at each position, which were not included in the collection; lag time focus of 241 ns. Laser intensity was optimised for each chip type. Calibration was done with a mixture of proteins with masses ranging from 7 to 30 kDa.
  • the study was aimed at gaining insight into the human saliva proteome composition and its changes over time in response to food consumption.
  • saliva proteome composition was quantitatively analyzed for peptide and protein masses by SELDI-TOF-MS, and relative mass intensities of these spectra were related to BMI and subjectively rated scores for satiety across time. This allowed quantification and identification of various expressed peptides as salivary biomarkers associated with food consumption, perceived satiety, and body mass.
  • Example 2 Identification of ghrelin, glucagons and peptide YY in saliva as satiety biomarkers
  • PYY is a naturally occurring human hormone produced by produced in the small intestine and colon, in particular by specialized endocrine cells (L-cells) in the gut in proportion.
  • the GI hormone peptide YY belongs to the pancreatic polypeptide (PP) family along with PP and neuropeptide Y (NPY). These peptides mediate their effects through the NPY receptors of which there are several subtypes (Yl, Y2, Y4, and Y5).
  • PP pancreatic polypeptide
  • NPY neuropeptide Y
  • PYY exists in two endogenous forms: PYY(I -36) and PYY(3-36). The latter is produced by the action of the enzyme dipeptidyl peptidase-IV (DPP-IV).
  • PYY(l-36) binds to and activates at least three Y receptor subtypes (Yl, Y2, and Y5), whereas PYY(3-36) is more selective for Y2 receptor (Y2R).
  • the hypothalamic arcuate nucleus a key brain area regulating appetite, has access to nutrients and hormones within the peripheral circulation.
  • NPY neurons within the arcuate nucleus express the Y2R.
  • PYY(3-36) concentrations rise.
  • Ghrelin is a hormone produced by P/Dl cells lining the fundus of the human stomach that stimulate appetite. Ghrelin levels increase before meals and decrease after meals. It is considered the counterpart of the hormone leptin, produced by adipose tissue, which reduces appetite when present at higher levels. Obestatin is a hormone that was found, in late 2005, to decrease appetite. Both obestatin and ghrelin are encoded by the same gene; the gene's product breaks apart to yield the two peptide hormones. Receptors for ghrelin are expressed by neurons in the arcuate nucleus and the ventromedial hypothalamus.
  • the ghrelin receptor is a G-protein coupled membrane receptor, formerly known as the GHS receptor (growth hormone secretagogue receptor).
  • GHS receptor growth hormone secretagogue receptor
  • Ghrelin is also made by a small population of neurons in the arcuate nucleus and plays a role in neuro trophy, particularly in the hippocampus, and is essential for cognitive adaption to changing environments and the process of learning. Ghrelin exists in the body as an inactive and an active octanoylated form.
  • Glucagon is a 29-amino acid polypeptide acting as an important hormone in carbohydrate metabolism and facilitates the body to maintain proper blood sugar levels.
  • the polypeptide has a molecular weight of 3485 daltons.
  • the hormone is synthesized and secreted from alpha cells ( ⁇ -cells) of the islet of Langerhans, which are located in the endocrine portion of the pancreas.
  • Glucagon helps maintain the level of glucose in the blood by binding to glucagon receptors on hepatocytes, causing the liver to release glucose through glycogenolysis. As these stores become depleted, glucagon stimulates the liver to synthesize glucose by gluconeogenesis, that is released into the bloodstream. Both of these mechanisms lead to glucose release by the liver, preventing the development of hypoglycemia.
  • SELDI-TOF-MS analysis on mammalian saliva samples allows for quantification of differentially expressed and/or produced peptides and proteins that are highly relevant and useful as biomarkers associated with perceived satiety in a subject.
  • a selection of food-intake related biomarker peptides and proteins in human saliva is applied in this invention in non-invasive practical methods and means to study satiety in the context of food products, diets, and medical, pharmaceutical and nutraceutical prevention, intervention and treatment of overweight and/or obesity.
  • Insulin ⁇ chain G IVEQCCTSICSLYQLENYCN SEQ ID NO : 63
  • FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO : 64) Insulin-like 3A chain
  • KAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDF (SEQ ID NO : 75) CCK-39 YIQQARKAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDF
  • VIP vasoactive intestinal peptide
  • IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO : 82) pancreatic hormone
  • HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO : 88) glicentin RSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQD FVQWLMNTKRNRNNIA (SEQ ID NO : 89) gastric inhibitory polypeptide / glucose-dependent insulinotropic polypeptide (GIP) YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ
  • Beta-defensin 126 NWYVKKCLNDVGICKKKCKPEEMHVKNGWAMCGKQRDCCVPAD

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Abstract

L'invention concerne un procédé pour déterminer l'état de satiété d'un sujet mammifère comprenant l'étape de détermination dans un échantillon de salive du sujet du niveau d'un ou de plusieurs biomarqueurs. L'invention concerne également des biomarqueurs appropriés pour être utilisés dans le procédé de l'invention, en particulier dans des biomarqueurs d'hormone peptidique gastro-intestinale ou pancréatique et/ou des biomarqueurs salivaires corrélés directement à la satiété perçue dans un sujet de mammifère. L'invention propose également des supports solides comprenant un ou plusieurs anticorps spécifiques pour une hormone peptidique gastro-intestinale et/ou pancréatique et/ou des peptides salivaires appropriés pour réaliser le procédé de l'invention.
PCT/NL2007/050482 2006-10-04 2007-10-04 Procédés et moyens pour déterminer la satiété sur la salive WO2008041849A2 (fr)

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EP07115641A EP2033656A1 (fr) 2007-09-04 2007-09-04 Compositions pour la modulation de la satiété par application orale
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010120374A2 (fr) 2009-04-17 2010-10-21 New York University Peptides ciblant les récepteurs de la famille du tnf et contrant l'action du tnf, compositions, méthodes et utilisations afférentes
WO2015073878A1 (fr) * 2013-11-15 2015-05-21 Ur Diet, Llc Rétroaction biologique de la satiété en temps réel

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AALTO YAN ET AL: "Does bombesin-like peptide mediate radiation-induced anorexia and satiety?" ACTA ONCOLOGICA (STOCKHOLM), vol. 38, no. 8, 1999, pages 1099-1102, XP002476673 ISSN: 0284-186X *
AYDIN S ET AL: "A comparison of leptin and ghrelin levels in plasma and saliva of young healthy subjects" PEPTIDES, ELSEVIER, AMSTERDAM, US, vol. 26, no. 4, April 2005 (2005-04), pages 647-652, XP004773455 ISSN: 0196-9781 *
DATABASE UniProt [Online] 1 January 1998 (1998-01-01), "FMRFamide-related peptides precursor [Contains: Neuropeptide SF (NPSF); Neuropeptide FF (NPFF); Neuropeptide AF (NPAF)]." XP002476676 retrieved from EBI accession no. UNIPROT:O15130 Database accession no. O15130 *
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SUNTER DAVID ET AL: "Intracerebroventricular injection of neuropeptide FF, an opioid modulating neuropeptide, acutely reduces food intake and stimulates water intake in the rat" NEUROSCIENCE LETTERS, vol. 313, no. 3, 9 November 2001 (2001-11-09), pages 145-148, XP002476674 ISSN: 0304-3940 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010120374A2 (fr) 2009-04-17 2010-10-21 New York University Peptides ciblant les récepteurs de la famille du tnf et contrant l'action du tnf, compositions, méthodes et utilisations afférentes
EP2419121B1 (fr) * 2009-04-17 2018-07-18 New York University Peptides ciblant les récepteurs de la famille du tnf et contrant l'action du tnf, compositions, méthodes et utilisations afférentes
WO2015073878A1 (fr) * 2013-11-15 2015-05-21 Ur Diet, Llc Rétroaction biologique de la satiété en temps réel

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