WO2019226750A1 - Procédés de traitement et de prévention d'une inflammation rénale par inhibition de la signalisation de l'udp-hexose - Google Patents

Procédés de traitement et de prévention d'une inflammation rénale par inhibition de la signalisation de l'udp-hexose Download PDF

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WO2019226750A1
WO2019226750A1 PCT/US2019/033486 US2019033486W WO2019226750A1 WO 2019226750 A1 WO2019226750 A1 WO 2019226750A1 US 2019033486 W US2019033486 W US 2019033486W WO 2019226750 A1 WO2019226750 A1 WO 2019226750A1
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Prior art keywords
udp
hexose
sample
subject
reagent
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PCT/US2019/033486
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English (en)
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Sylvie Breton
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Kantum Diagnostics, Inc.
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Publication of WO2019226750A1 publication Critical patent/WO2019226750A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07009—UTP-glucose-1-phosphate uridylyltransferase (2.7.7.9), i.e. UDP-glucose-pyrophosphorylase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • 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
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01017—Glucuronosyltransferase (2.4.1.17)

Definitions

  • the invention relates to methods of treating and preventing renal inflammation.
  • the invention provides compositions and methods for reducing the concentration of UDP-hexoses or altering the structure of UDP-hexoses so that they cannot bind to P2Y 14. In that manner, the invention reduces or eliminates binding of UDP-hexoses to P2Y 14 and prevents development of renal inflammation. Consequently, the methods allow prevention of kidney failure in patients suffering from either acute or chronic kidney conditions.
  • aspects of the invention include reducing the concentration of a UDP-hexose in a subject.
  • Reduction of UDP-hexose concentration may be accomplished by providing a reagent that promotes degradation of the UDP-hexose or conversion of the UDP-hexose to a compound that does not promote renal inflammation.
  • the reagent may be an enzyme that degrades or modifies the UDP-hexose.
  • the reagent may be a chelating agent, such as an agent that sequesters divalent cations to promote enzymatic degradation or modification of the UDP-hexose.
  • the reagent may be an antibody (polyclonal or monoclonal) or other protein that binds and sequesters the UDP- hexose(s).
  • the methods may include providing multiple reagents that promote degradation of the UDP-hexose or conversion of the UDP-hexose to a different compound or sequestration of the UDP-hexoses.
  • the multiple reagents may be provided simultaneously or sequentially.
  • the methods may include providing a reagent that reduces the concentration of a UDP- hexose in combination with another therapeutic agent that treats a disease, disorder, or condition that is associated with kidney failure, AKI, or CKD or may cause kidney failure, AKI, or CKD.
  • the methods may include measuring the concentration of UDP-hexose in a sample from the subject.
  • the methods may include making multiple measurements of the concentration of UDP-hexose. The measurements may be taken before or after providing a reagent that promotes degradation of the UDP-hexose or conversion of the UDP-hexose to a different compound or sequestration of the UDP-hexoses.
  • the methods may include taking at least one measurement before providing the reagent and taking at least one measurement after providing the reagent.
  • methods of the invention include altering the structure or availability of a UDP-hexose in a subject.
  • Alteration of the structure of the UDP-hexose may be accomplished by providing a reagent that changes the conformation of the UDP-hexose.
  • the reagent may be an antibody that binds to the UDP-hexose.
  • the reagent may change the conformation of the UDP-hexose.
  • the reagent may alter the availability of the UDP-hexose to serve as a reactant or to bind to other molecules.
  • the UDP- hexose may be any UDP-hexose associated with renal inflammation, such as those described above.
  • Kidney failure can result from a variety of primary conditions but is nearly always a consequence of renal inflammation.
  • renal inflammation contributes to the development and progression of acute kidney injury (AKI) and is also correlated with morbidity in patients with chronic kidney disease.
  • the invention recognizes that alleviating renal inflammation can prevent kidney failure even if the underlying cause of impaired kidney function cannot be remedied or is unknown.
  • P2Y 14 is a G protein-coupled receptor expressed on the surface of intercalated cells (ICs) in the collecting duct system of the kidney.
  • P2Y 14 binds uridine diphosphate glucose (UDP-glucose), an ester of pyrophosphoric acid with the nucleoside uridine, and other UDP-hexoses, such as UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP-glucosamine, and N-acetyl-UDP- galactosamine.
  • UDP-glucose uridine diphosphate glucose
  • UDP-glucuronic acid such as UDP-galactose
  • N-acetyl-UDP-glucosamine N-acetyl-UDP- galactosamine.
  • UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family, Trends Pharmacol Sci. 2003 Feb;24(2):52-5, DOI: l0.l0l6/S0l65-6l47(02)00038-X, the contents of which are incorporated herein by reference.
  • UDP-glucose is released into extracellular fluids from damaged cells and in a regulated manner from intact cells. Binding of UDP-glucose to P2Y 14 triggers ICs to produce chemokines that lead to infiltration of neutrophils into the renal medulla.
  • the invention recognizes that renal inflammation in a subject can be mitigated by decreasing the availability of UDP-hexoses to bind to P2Y 14.
  • the invention provides methods that reduce the effective levels of UDP-hexoses, thereby attenuating P2Y 14 signaling.
  • Certain embodiments of the invention entail degrading UDP-hexoses, converting them into other compounds that are unable to activate P2Y 14, or otherwise rendering them unavailable to bind to P2Y 14.
  • Other embodiments involve changing the structure of UDP-hexoses to inhibit their ability to bind to P2Y 14.
  • the invention provides methods of reducing the concentration of one or more UDP- hexoses, such as UDP-glucose, UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP- glucosamine, and N-acetyl-UDP-galactosamine, that stimulate activity of P2Y14 in subject.
  • UDP-hexoses are synthesized by uridylyltransferases that reversibly transfer uridine
  • UDP-hexoses are broken down by enzymes that remove UMP moieties from the hexose.
  • enzymes include UDP-glucose pyrophosphatase, also called UGPPase, described in, for example, Entrez Gene ID no. 256281, GenBank ID no. BC041584, and RefSeq ID no.
  • UDP-hexose metabolism Other enzymes involved in UDP-hexose metabolism include UDP— glucose— hexose- 1 -phosphate uridylyltransferases, UDP— N- acetylgalactosamine diphosphorylases, UDP— N-acetylglucos amine diphosphorylases, UTP— monosaccharide- 1 -phosphate uridylyltransferase, and UTP— xylose- l-phosphate
  • Uridylyltransferases may be suitable for this purpose because they catalyze both addition and removal of UMP depending on environmental conditions, and an excess of UDP-hexose can shift the reaction in favor of UMP removal.
  • Another suitable reagent is an agent that chelates divalent cations.
  • Many of the aforementioned enzymes require divalent cations, such as Mg 2+ or Zn 2+ , for catalytic activity. Consequently, chelators can attenuate synthesis of UDP-hexoses by preventing cations from serving as cofactors in the enzymatic reactions.
  • chelating agents include, without limitation, ZX1 ((2-((Bis(pyridin-2-ylmethyl)amino)methylamino)benzenesulfonic acid; Pan, et al., Neuron 2011, 71, 1116-1126.), ZX1E, TPA (Tris[(2-pyridyl)methyl]amine), phanquinone (4,7-phenanthroline-5,6-dione), clioquinol (PN Gerolymatos SA), chloroquinol, penicillamine, trientine, N,N'-diethyldithiocarbamate (DDC), 2,3,2'-tetraamine (2,3,2'-tet), neocuproine, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), l,lO-phenanthroline (PHE), tetraethylenepentamine (TEPA), triethylene tetraamine
  • EDTA ethylene glycol
  • EGTA bis aminoethyl ether tetra acetic acid
  • nitrilotriacetic acid N,N-bis(2-hydroxyethyl)glycine (bicine); 0,0'-bis(2-aminophenyl ethylene glycol)
  • BAPTA ethylenediamine-N,N,N',N'-tetraacetic acid
  • CyDTA 2-diamino cyclohexane- ethylenediamine-N,N,N',N'-tetraacetic acid
  • DPTA-OH 2-diamino-2-hydroxy-propane- ethylenediamine-N,N,N', N'-tetraacetic acid
  • EDDP ethylene-diamine-N,N'-dipropionic acid dihydrochloride
  • EDDP ethylenediamine-N,N'-bis(methylenephosphonic acid
  • EDTPO N,N'-bis(2-hydroxybenzyl)ethylene diamine-N,N'-diacetic acid
  • HDTA 1,6- hexamethylenediamine-N,N,N', N'-tetraacetic acid
  • HEDTA N-(2- hydroxyethyl)iminodiacetic acid
  • IDA iminodiacetic acid
  • IDA iminodiacetic acid
  • NMTA nitriltriacetic acid
  • NTP nitrilotripropionic acid
  • NTPO nitrilotris (methylenephosphonic acid) trisodium salt (NTPO), triethylenetetramine- N,N,N',N'',N''-hexaacetic acid (TTHA), bathocuproine, bathophenanthroline, TETA, citric acid, salicylic acid, and malic acid, and analogue
  • Another class of reagents that can reduce the effective concentration of a UDP-hexose includes agents that bind to a UDP-hexose and prevent it from activating P2Y 14. Any suitable binding agent may be used.
  • the binding agent may be an antibody.
  • antibody encompasses complete antibody molecules, fragments of antibody molecules, and antibody- derived molecules that specifically bind an antigen.
  • an antibody may be a complete immunoglobulin, antigen-binding fragment (Fab), Fab2, variable domain (Fv), single chain variable fragment (scFv), third-generation (3G) antibody.
  • the antibodies may be natural monoclonal antibodies or synthetic antibodies, such as recombinant antibodies, non-immunoglobulin derived synthetic antibodies, or affimer proteins.
  • Methods of making monoclonal antibodies are known in the art and described in, for example, Antibodies: A Laboratory Manual, Second edition, edited by Greenfield, Cold Spring Harbor Laboratory Press (2014) ISBN 978-1-936113-81-1.
  • Methods of making synthetic antibodies are described in, for example, US 2014/0221253; US 2016/0237142; and Miersch and Sidhu, Synthetic antibodies: concepts, potential and practical considerations, Methods. 2012 Aug;57(4):486-98. doi:
  • the invention also provides methods of altering the structure of one or more UDP- hexoses, such as UDP-glucose, UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP- glucosamine, and N-acetyl-UDP-galactosamine, that stimulate activity of P2Y14 in subject.
  • the structure of the UDP-hexose may be altered by providing a reagent that binds to the UDP-hexose to alter its conformation. Any suitable reagent may be used.
  • the reagent may be an antibody, an enzyme (including a modified version of an enzyme), a chelator, an ion, or a small molecule.
  • Kidney failure may result from, or be a stage of, acute kidney injury (AKI) or chronic kidney disease (CKD).
  • AKI is an abrupt loss of kidney function that develops within 7 days.
  • CKD is the gradual loss of kidney function over a period of months or years.
  • AKI may be assessed by any suitable standard.
  • Several standards for acute kidney injury are known in the art, such as the criteria provided by the Acute Kidney Injury Network (AKIN); Kidney Disease Improving Global Outcomes (KDIGO); and Risk, Injury, Failure, Loss, and End-stage Kidney (RIFLE).
  • AKI may be categorized or staged according to the AKI, KDIGO, or RIFLE criteria. For example, a subject may be deemed to have stage 1, stage 2, or stage 3 AKI, or a subject may be deemed to have risk, injury, failure, or loss.
  • the standard may apply to an adult, pediatric, newborn, neonatal, infant, child, adolescent, pre-teen, teenage, or elderly subject.
  • Standards typically include measurements of serum creatinine (SCr) concentrations, urine output, or glomerular filtration rate (GFR). Standards may include multiple parameters, e.g., combinations of the aforementioned standards.
  • a subject may be deemed to have AKI, or a stage or category thereof, when she has abnormally high SCr concentration, abnormally low urine output, abnormally low GFR, or any combination thereof.
  • Standards may be absolute, e.g., they may require a value above or below a defined threshold value. Alternatively, standards may be relative, e.g., they may require an increase or decrease relative to a baseline value. Standards for different parameters, e.g., abnormally high SCr concentration abnormally low urine output, or abnormally low GFR, may independently be absolute or relative.
  • Standards for acute kidney injury may include a temporal component.
  • a subject may be deemed to have AKI when an elevated SCr concentration is measured at some interval following a preceding event.
  • the preceding event may be cardiac surgery, cardiac arrest, admission to a hospital, clinic, medical facility, or any unit thereof.
  • the interval may be 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours.
  • a subject may be deemed to have AKI when urine output is measured across some interval, such as 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours.
  • a standard for GFR associated with AKI may be a GFR of less than 35 ml/min per 1.73 mm .
  • a standard for GFR associated with AKI may be a decrease of at least at least 25% relative to a baseline, a decrease of at least at least 50% relative to a baseline, or a decrease of at least at least 75% relative to a baseline.
  • CKD may be secondary to, or associated with, another disease, disorder, or condition.
  • AKI may result from, or be associated with diabetes, high blood pressure, nephrotic syndrome, glomerulonephritis, or polycystic kidney disease.
  • the invention provides methods of providing a reagent to a subject.
  • Providing the reagent to the subject may include administering it to the subject.
  • the reagent may be administered by any suitable means.
  • the reagent may be administered orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, intravenously, subcutaneously, nasally, pulmonarily, or with or on an implantable medical device (e.g., stent or drug-eluting stent or balloon equivalents).
  • an implantable medical device e.g., stent or drug-eluting stent or balloon equivalents.
  • the reagent may be provided at any suitable dosage.
  • the reagent may be provided at from 0.001 mg/kg body weight to 5 g/kg body weight.
  • the dosage range is from 0.001 mg/kg body weight to 1 g/kg body weight, from 0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.001 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25 mg/kg body weight, from 0.001 mg/kg body weight to 10 mg/kg body weight, from 0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001 mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg body weight to 0.1 mg/kg body weight, or from 0.001 mg/kg body weight to 0.005 mg/kg body weight.
  • the dosage range is from 0.1 g/kg body weight to 5 g/kg body weight, from 0.5 g/kg body weight to 5 g/kg body weight, from 1 g/kg body weight to 5 g/kg body weight, from 1.5 g/kg body weight to 5 g/kg body weight, from 2 g/kg body weight to 5 g/kg body weight, from 2.5 g/kg body weight to 5 g/kg body weight, from 3 g/kg body weight to 5 g/kg body weight, from 3.5 g/kg body weight to 5 g/kg body weight, from 4 g/kg body weight to 5 g/kg body weight, or from 4.5 g/kg body weight to 5 g/kg body weight.
  • doses may be administered at a single time, at multiple times, or continuously, for example, by continuous intravenous infusion. Multiple doses may be administered at any interval. For example, the interval between doses may be about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 1 day, about 2 days, about 3 days, or more.
  • Effective doses may be estimated from dose-response relationships derived from in vitro or animal model test bioassays or systems or from clinical trials of the P2Y 14 antagonist. The dosage should not be so large as to cause unacceptable adverse side effects.
  • the reagent may be provided in combination with another therapeutic agent that treats or prevents kidney failure, CKD, AKI, or an underlying cause of any of the aforementioned conditions.
  • the reagent may be provided in combination with one or more of an angiotensin converting enzyme inhibitor (ACEI), angiotensin II receptor antagonist (ARB), cyclophosphamide, diuretic, such as furosemide, inotrope, such as dobutamine, intravenous fluid, phosphate binder, phosphodiesterase-5 inhibitor, steroid, vasopressors, such as norepinephrine, or zinc.
  • ACEI angiotensin converting enzyme inhibitor
  • ARB angiotensin II receptor antagonist
  • cyclophosphamide diuretic, such as furosemide, inotrope, such as dobutamine, intravenous fluid, phosphate binder, phosphodiesterase-5 inhibitor, steroid, vasopressors, such as norepinephrine,
  • UDP-glucose levels may also be measured using the protocols described in Barrett et ah, Molec. Pharmacol., 2013, 84, 41-49, the contents of which are incorporated herein by reference in their entirety. Alternatively, UDP-glucose levels may be measured by using an anti-UDP-glucose antibody.
  • the assay may include pre-processing steps to remove proteins that interact with NADH production and/or endogenous NADH from the sample.
  • High levels e.g., > 2 mM
  • a control reaction lacking exogenous enzyme may be performed to measure the amount of pre-existing NADH in the sample, and this value can be subtracted from the value obtained from a reaction that receives exogenous enzyme.
  • the liquid sample is buffered to pH 8-9, for example, pH 8.0.
  • the enzyme UDP-glucose dehydrogenase is added to the reaction along with the co-factor NAD + .
  • UDP-glucose is converted to UDP-glucuronic acid, and a stoichiometric amount of NAD + is converted to NADH. NADH is then measured, and its concentration is used to deduce the starting UDP-glucose concentration.
  • the amount of substrate and/or the reaction rate may be optimized so that the reaction occurs substantially in a substantially linear portion of the Michaelis-Menten graph.
  • excess NAD + is added to the reaction, along with enzyme in excess, such that UDP-glucose is limiting.
  • NAD + can be added to a concentration of 2 mM per well, and 0.04 units of enzyme added per well to achieve an excess of both.
  • One unit of enzyme is the amount of UDP-glucose dehydrogenase required to oxidize 1.0 pmole of UDP-glucose to UDP-glucuronic acid per minute at pH 8.7 at 25°C.
  • the complete reaction curve can be determined for each sample and the data fit to a non-linear rate equation (e.g.,“progress-curve analysis”). This is particularly useful when the slope of the linear region of the Michaelis-Menten kinetics curve for a desired enzyme is very steep (e.g., when the initial rate is too fast to measure accurately) or when an excess of substrate (e.g., NAD + ) is used in the reaction mix
  • the methods may include lateral flow assays adapted for use in the detection of NADH or UDP.
  • Such lateral flow assays permit the flow of a liquid sample, applied to the sample application zone, to deliver the sample/reactants to a test region (e.g., a reaction zone) of the lateral strip or device, and then the sample with a generated byproduct is delivered to a detection zone, which provides a readout (e.g., visual, optical, fluorescent, etc.).
  • an assay may use reduction of nitro blue tetrazolium (NBT) by NADH to generate a colored product at a test region. As samples with generated NADH flow over a region with NBT (no color), the NBT is reduced to the blue form, which is visible on a strip
  • a reductase may be immobilized on the dipstick or test strip.
  • the reductase may be a diaphorase, and it may be immobilized via adsorption or via immunocapture. As the NADH-containing solution flows through the region with the reductase enzyme, the NADH is oxidized and would reduce the NBT to the colored precipitate NBTH.
  • the level of NADH or UDP in a sample is detected by a lateral flow assay test (LFA), or strip test.
  • LFAs detect the presence or absence of an analyte, e.g.
  • NADH or UDP in a liquid sample.
  • a spatial separation is defined in the strips between the sample application zone and detection region.
  • Most conventional lateral flow strips are designed for test samples that are readily available in large quantities (e.g., urine).
  • Lateral flow immunoassays are described below, but lateral flow assays may also be adapted for the measurement of an analyte without the use of antibody. Both lateral flow immunoassays (e.g., using a UDP-glucose antibody) and lateral flow analyte assays (e.g., detection of NADH to measure UDP-glucose levels) are contemplated for use herein.
  • test sample flows along a solid substrate via capillary action. After the sample is applied to the lateral flow strip, it encounters a test region where an enzymatic reaction coupled to NADH or UDP production occurs and continues to a region comprising a detection reagent that permits detection of NADH or UDP.
  • the liquid may go through one or more different regions on the lateral flow strip following the test region and prior to the detection region.
  • LFAs are adapted to operate along a single axis to suit the test strip format or a dipstick format. Typically, LFAs proceed from sample application to readout without additional steps by the user, so sample application generally leads to an assay result with the further user input.
  • lateral flow configurations may include one or more steps by the user after sample application, e.g., insertion into a detector device (e.g., a luminometer, fluorescence detector, etc.) or addition of another reagent.
  • Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens or analytes from fluid samples such as urine, blood, water samples etc. Strip tests are also known as "dipstick tests," the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFA strip tests are easy to use, require minimum training and can easily be included as components of point- of-care test (POCT) diagnostics to be used on site in the field.
  • POCT point- of-care test
  • a typical test strip may comprise one or more of following components: (1) sample application zone comprising e.g., an absorbent pad (i.e., the matrix or material) onto which the test sample is applied; (2) test region comprising immobilized enzyme; (3) a test results area comprising a detection reagent or reaction membrane - such as a hydrophobic nitrocellulose or cellulose acetate membrane onto which, for example, a detection reagent is immobilized in a line across the membrane as a capture zone or test line (a control zone may also be present, containing NADH or another reducing agent, for example, that reduces NBT to generate a blue color) or an antibody reagent; and (4) optional wick or waste reservoir - a further absorbent pad designed to draw the sample across the detection reagent zone or reaction membrane by capillary action and collect it.
  • lateral flow strips as described herein may further comprise one or more of the following: a region comprising a strong base or a region comprising immobilized NAD + nucleosidas
  • the components of the strip may be fixed to an inert backing material and may be presented in a simple dipstick format or within a plastic casing with a sample port and reaction window showing the test readout/capture and control zones.
  • the test may incorporate a second, coated line which contains an antibody or other reagent that picks up free readout substrate (e.g., free latex or gold particles) in order to confirm the test has operated correctly.
  • free readout substrate e.g., free latex or gold particles
  • the reaction to generate a stoichiometric amount of NADH from the reaction of UDP-glucose with UDP-glucose dehydrogenase is incubated for a matter of minutes, e.g., 5 or 10 minutes, in the liquid assay format in order to generate sufficient amounts of NADH for detection.
  • This extended time is not as readily achieved in the dipstick or lateral flow format.
  • options to overcome this include performing the first enzymatic reaction in an assay well for a prescribed period of time before inserting a dipstick or applying sample to a test strip.
  • a urine dipstick is a colorimetric chemical assay comprising a reagent stick-pad.
  • the dipstick is typically immersed in a fresh urine specimen and then withdrawn.
  • the urine sample may be applied directly to the sample application zone by the subject (e.g., analogous to a pregnancy test). After predetermined times the colors of the reagent pad are compared to standardized reference charts.
  • the urine dipstick offers an inexpensive and fast method to perform screening urinalyses, which helps in identifying the presence of various diseases or health problems.
  • a urine dipstick provides a simple and clear diagnostic guideline and may be used in the methods and kits as described herein.
  • one aspect of the present technology relates to a method for detecting NADH or UDP using a device, such as a dipstick, as described herein.
  • a centrifugation or filtration step to render a clear sample may be applied so as to avoid pigment or other entities from fouling the optical readout.
  • the lateral flow strip may also comprise a control that gives a signal to the user that the assay is performing properly.
  • the control zone may contain an immobilized receptive material that is generally capable of forming a chemical and/or physical bond with probes or with the receptive material immobilized on the probes.
  • receptive materials include, but are not limited to, antigens, haptens, antibodies, protein A or G, avidin, streptavidin, secondary antibodies, and complexes thereof.
  • control zone receptive material may also include a polyelectrolyte that may bind to uncaptured probes. Because the receptive material at the control zone is only specific for probes, a signal forms regardless of whether the analyte is present.
  • the control zone may be positioned at any location along the test strip, but is preferably positioned downstream from the detection zone.
  • control line may include a line of NBT spatially downstream of the test line and immediately downstream of a line or zone of dried reducing agent. Flow of sample past the test line will liberate the reducing agent and carry it to the control line of NBT, which will be reduced to generate a control line indicating the sample reactants have successfully reacted at that point.
  • the intensity of any signals produced at the region comprising a detection reagent may be measured with e.g., an optical reader.
  • the actual configuration and structure of the optical reader may generally vary as is readily understood by those skilled in the art.
  • optical detection techniques include, but are not limited to, luminescence (e.g., fluorescence, phosphorescence, etc.), absorbance (e.g., fluorescent or non- fluorescent), diffraction, etc.
  • Further optical methods include but are not limited to, measurement of light scattering or simple reflectance, e.g., using a luminometer or photomultiplier tube;
  • radioactivity e.g., using a Geiger counter
  • electrical conductivity or dielectric capacitance e.g., electrical conductivity or dielectric capacitance
  • electroactive agents such as indium, bismuth, gallium or tellurium ions.
  • the methods may include lateral flow immunoassays (LFIAs), in which antibodies that bind a target analyte are used in a competitive or sandwich immunoassay adapted to the lateral flow format.
  • LFIAs lateral flow immunoassays
  • Conventional sandwich LFIAs are similar to sandwich ELIS As.
  • the sample first encounters and mobilizes colored particles which are labeled with antibodies raised to the target antigen.
  • the test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen.
  • the test line will show as a colored band in positive samples, resulting from the accumulation or capture of antibody-bearing colored particles.
  • the lateral flow immunoassay may be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof.
  • Suitable detectable substances may include, for instance, luminescent compounds (e.g., fluorescent, phosphorescent, etc.);
  • radioactive compounds include visual compounds (e.g., colored dye or metallic substance, such as gold); liposomes or other vesicles containing signal-producing substances; enzymes and/or substrates, and so forth.
  • visual compounds e.g., colored dye or metallic substance, such as gold
  • liposomes or other vesicles containing signal-producing substances e.g., liposomes or other vesicles containing signal-producing substances
  • enzymes and/or substrates e.g., enzymes and/or substrates, and so forth.
  • Other suitable detectable substances are described in U.S. Pat. Nos. 5,670,381 and 5,252,459, which are incorporated herein in their entirety by reference. If the detectable substance is colored, the ideal electromagnetic radiation is light of a complementary wavelength. For instance, blue detection probes strongly absorb red light.
  • the detectable substance may be a luminescent compound that produces an optically detectable signal.
  • suitable fluorescent molecules may include, but are not limited to, fluorescein, europium chelates, phycobiliprotein, rhodamine, and their derivatives and analogs.
  • Other suitable fluorescent compounds are semiconductor nanocrystals commonly referred to as "quantum dots.”
  • the detection agent is a particle.
  • particles useful in the methods, assays and kits described herein include, but are not limited to, colloidal gold particles; colloidal sulfur particles; colloidal selenium particles; colloidal barium sulfate particles; colloidal iron sulfate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrous) oxide particles; colloidal metal sulfide particles; colloidal lead selenide particles; colloidal cadmium selenide particles; colloidal metal phosphate particles; colloidal metal ferrite particles; any of the above-mentioned colloidal particles coated with organic or inorganic layers; protein or peptide molecules; liposomes; or organic polymer latex particles, such as polystyrene latex beads.
  • suitable phosphorescent compounds include metal complexes of one or more metals, such as ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, iron, chromium, tungsten, zinc, and so forth.
  • metal complex may contain one or more ligands that facilitate the solubility of the complex in an aqueous or non- aqueous environment.
  • ligands include, but are not limited to, pyridine; pyrazine; isonicotinamide; imidazole; bipyridine; terpyridine;
  • Such ligands may be, for instance, substituted with alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing groups, phosphorus containing groups, and the carboxylate ester of N-hydroxy-succinimide.
  • Porphyrins and porphine metal complexes possess pyrrole groups coupled together with methylene bridges to form cyclic structures with metal chelating inner cavities. Many of these molecules exhibit strong phosphorescence properties at room temperature in suitable solvents (e.g., water) and an oxygen-free environment.
  • suitable porphyrin complexes that are capable of exhibiting phosphorescent properties include, but are not limited to, platinum (II) coproporphyrin- 1 and II, palladium (II) coproporphyrin, ruthenium coproporphyrin, zinc(II)- coproporphyrin-I, derivatives thereof, and so forth.
  • Bipyridine metal complexes may also be utilized as phosphorescent compounds.
  • suitable bipyridine complexes include, but are not limited to, bis [(4,4'- carbomethoxy)-2,2'-bipyridine]2-[3-(4-methyl-2,2'-bipyridine-4-yl)propyl]-l,3-dioxolane ruthenium (II); bis(2,2'bipyridine)[4-(butan-l-al)-4'-methyl-2,2'-bi-pyridine]ruthenium (II); bis(2,2'-bipyridine)[4-(4'-methyl-2,2'-bipyridine-4'-yl)-butyric acid] ruthenium (II);
  • multiwell plates may include, in addition to test wells for assaying an unknown test sample, control wells including, e.g., blanks lacking enzyme or other reagents, to permit, among other things, the determination of background levels of, e.g., intermediate or surrogate indicator NADH.
  • Other controls may include, e.g., positive control wells including a known amount of UDP-glucose; a set of separate positive control wells may include varying known amounts of UDP-glucose to establish a standard curve, e.g., over one or a plurality of orders of magnitude, that is read by the device and used to calculate amounts of UDP-glucose in the unknown test samples.
  • the reference level of UDP-hexose may be defined by a statistic describing the distribution of levels in normal healthy subjects.
  • the reference level may be an average level of UDP-hexose in a sample from a normal healthy subject or a population of normal healthy subjects.
  • the reference level of UDP-hexose may be an average level of UDP- hexose in a sample from a subject who has not had kidney failure, AKI, or CKD.
  • the reference level may be a level of UDP-hexose in a sample of the same subject measured at an earlier time point.
  • the reference level may be at least one standard deviation, at least two standard deviations, or at least three standard deviations above a level of UDP-hexose in a sample obtained from the same subject at an earlier time point. Any level above the reference level may be deemed to be significantly different from the level in the earlier sample.
  • the reference level may be adjusted to account for variables such as sample type, gender, age, weight, and ethnicity. Thus, reference levels accounting for these and other variables may provide added accuracy for the methods described herein.
  • the assay/method comprises or consists essentially of a system for determining (e.g. transforming and measuring) the level of UDP-hexose as described herein and comparing it to a reference level. If the comparison system, which may be a computer implemented system, indicates that the amount of the measured level of UDP-hexose is statistically higher than that of the reference amount, the subject from which the sample is collected may be identified as having renal inflammation.
  • a system comprising: (a) at least one memory containing at least one computer program adapted to control the operation of the computer system to implement a method that includes (i) a determination module configured to measure the level of UDP-hexose in a test sample obtained from a subject; (ii) a storage module configured to store output data from the determination module; (iii) a computing module adapted to identify from the output data whether the measured level of UDP-hexose in the test sample obtained from the subject is higher, by a statistically significant amount, than a reference level, and to provide a retrieved content; (iv) a display module for displaying for retrieved content (e.g., the amount of the measured level of UDP-hexose , or whether the measured level of UDP- hexose is higher than the reference level); and (b) at least one processor for executing the computer program.
  • a determination module configured to measure the level of UDP-hexose in a test sample obtained from
  • the computer-readable storage media may be any available tangible media that can be accessed by a computer.
  • Computer readable storage media includes volatile and nonvolatile, removable and non-removable tangible media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data.
  • Computer readable storage media includes, but is not limited to, RAM (random access memory), ROM (read only memory), EPROM (erasable programmable read only memory), EEPROM (electrically erasable programmable read only memory), flash memory or other memory technology, CD-ROM (compact disc read only memory), DVDs (digital versatile disks) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non-volatile memory, and any other tangible medium which can be used to store the desired information and which can accessed by a computer including and any suitable combination of the foregoing.
  • RAM random access memory
  • ROM read only memory
  • EPROM erasable programmable read only memory
  • EEPROM electrically erasable programmable read only memory
  • flash memory or other memory technology CD-ROM (compact disc read only memory), DVDs (digital versatile disks) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non-vol
  • the computer-readable media may be transportable such that the instructions stored thereon may be loaded onto any computer resource to implement the aspects of the technology discussed herein.
  • the instructions stored on the computer-readable medium, described above are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions may be embodied as any type of computer code (e.g., software or microcode) that can be employed to program a computer to implement aspects of the technology described herein.
  • the computer executable instructions may be written in a suitable computer language or combination of several languages.
  • the functional modules of certain embodiments may include at minimum a determination module, a storage module, a computing module, and a display module.
  • the functional modules may be executed on one, or multiple, computers, or by using one, or multiple, computer networks.
  • the determination module has computer executable instructions to provide e.g., levels of expression products, etc. in computer readable form.
  • the determination module may comprise any system for detecting a signal resulting from the detection of UDP-glucose in a biological sample.
  • such systems may include an instrument, e.g., a plate reader for measuring absorbance.
  • such systems may include an instrument, e.g., such as the instrument sold under the trade name Cell Biosciences NANOPRO 1000TM System (Protein Simple; Santa Clara, CA), for quantitative measurement of proteins.
  • the storage module is adapted or configured for having recorded thereon, for example, sample name, patient name, and numerical value of the level of ETDP-glucose.
  • Such information may be provided in digital form that may be transmitted and read electronically, e.g., via the Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication.
  • Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising expression level information.
  • the storage module stores the output data from the determination module.
  • the storage module stores the reference information such as levels of UDP-hexose in healthy subjects.
  • the storage module stores the information such as levels of UDP-hexose measured from the same subject in earlier time points.
  • the computing module may use a variety of available software programs and formats for computing the levels of UDP-hexose. Such algorithms are well established in the art. A skilled artisan is readily able to determine the appropriate algorithms based on the size and quality of the sample and type of data.
  • the data analysis may be implemented in the computing module.
  • the computing module further comprises a comparison module, which compares the level of UDP-hexose in the test sample obtained from a subject as described herein with the reference level. For example, when the level of UDP-hexose in the test sample obtained from a subject is measured, a comparison module may compare or match the output data, e.g. with the reference level. In certain embodiments, the reference level has been pre-stored in the storage module.
  • the comparison module may determine whether the level of UDP-hexose in the test sample obtained from a subject is higher than the reference level to a statistically significant degree.
  • the comparison module may be configured using existing commercially- available or freely-available software for comparison purpose, and may be optimized for particular data comparisons that are conducted.
  • the World Wide Web server supports a TCP/IP protocol.
  • Local networks such as this are sometimes referred to as "Intranets.”
  • An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., the GenBank or Swiss Pro World Wide Web site).
  • users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web browsers and Web servers.
  • the computing and/or comparison module provides a computer readable comparison result that can be processed in computer readable form by predefined criteria, or criteria defined by a user, to provide content based in part on the comparison result that may be stored and output as requested by a user using an output module, e.g., a display module.
  • an output module e.g., a display module.
  • the content displayed on the display module may be the relative levels of UDP-hexose in the test sample obtained from a subject as compared to a reference level. In certain embodiments, the content displayed on the display module may indicate whether the levels of UDP-hexose are found to be statistically significantly higher in the test sample obtained from a subject as compared to a reference level. In some embodiments, the content displayed on the display module may show the levels of UDP-hexose from the subject measured at multiple time points, e.g., in the form of a graph. In some embodiments, the content displayed on the display module may indicate whether the subject has renal inflammation. In certain embodiments, the content displayed on the display module may indicate whether the subject is in need of a treatment for renal inflammation.
  • a World Wide Web browser is used for providing a user interface for display of the content based on the computing/comparison result. It should be understood that other modules may be adapted to have a web browser interface. Through the Web browser, a user can construct requests for retrieving data from the computing/comparison module. Thus, the user will typically point and click to user interface elements such as buttons, pull down menus, scroll bars and the like conventionally employed in graphical user interfaces.
  • Measurable markers of renal function are well known in the medical and veterinary literature and to those of skill in the art, and include, but are not limited to, blood urea nitrogen or "BUN" levels (both static measurements and measurements of rates of increase or decrease in BUN levels), serum creatinine levels (both static measurements and measurements of rates of increase or decrease in serum creatinine levels), measurements of the BUN/creatinine ratio (static measurements of measurements of the rate of change of the BUN/creatinine ratio), urine/plasma ratios for creatinine, urine/plasma ratios for urea, glomerular filtration rates (GFR), serum concentrations of sodium (Na+) or potassium (K+), urine osmolarity, daily urine output, urine protein/creatinine ratio, albuminuria, and the like.
  • BUN blood urea nitrogen or "BUN” levels
  • serum creatinine levels both static measurements and measurements of rates of increase or decrease in serum creatinine levels
  • measurements of the BUN/creatinine ratio static measurements of measurements

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Abstract

L'invention concerne des procédés de traitement et de prévention d'une inflammation rénale par réduction de la concentration ou par modification de la structure d'une ou de plusieurs hexoses UDP, telles que l'UDP-glucose, l'UDP-galactose, l'acide UDP-glucuronique, la N-acétyl-UDP-glucosamine et la N-acétyl-UDP-galactosamine, chez un sujet.
PCT/US2019/033486 2018-05-23 2019-05-22 Procédés de traitement et de prévention d'une inflammation rénale par inhibition de la signalisation de l'udp-hexose WO2019226750A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022047243A1 (fr) 2020-08-27 2022-03-03 Enosi Life Sciences Corp. Méthodes et compositions pour traiter des maladies auto-immunes et un cancer
WO2023168426A1 (fr) 2022-03-03 2023-09-07 Enosi Therapeutics Corporation Compositions et cellules contenant des mélanges de protéines de fusion oligo-trap (ofps) et leurs utilisations

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US20160274131A1 (en) * 2013-11-07 2016-09-22 The General Hospital Corporation Compositions and methods for detecting and/or treating inflammation

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022047243A1 (fr) 2020-08-27 2022-03-03 Enosi Life Sciences Corp. Méthodes et compositions pour traiter des maladies auto-immunes et un cancer
WO2023168426A1 (fr) 2022-03-03 2023-09-07 Enosi Therapeutics Corporation Compositions et cellules contenant des mélanges de protéines de fusion oligo-trap (ofps) et leurs utilisations

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