WO2018102715A1 - Compositions et procédés permettant de traiter et/ou de réduire la dystrophie cornéenne - Google Patents

Compositions et procédés permettant de traiter et/ou de réduire la dystrophie cornéenne Download PDF

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WO2018102715A1
WO2018102715A1 PCT/US2017/064264 US2017064264W WO2018102715A1 WO 2018102715 A1 WO2018102715 A1 WO 2018102715A1 US 2017064264 W US2017064264 W US 2017064264W WO 2018102715 A1 WO2018102715 A1 WO 2018102715A1
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corneal
glutamine
endothelium
dystrophy
cornea
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JR. Francis PRICE
Matthew FENG
Zhang WENLIN
Joseph Bonanno
Hongde LI
Jason TENNESSEN
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Indiana University Research And Technology Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses

Definitions

  • Various aspects and embodiments disclosed herein relate generally to the modelling, treatment, prevention, and diagnosis of diseases characterized by the formation of corneal dystrophy.
  • Cornea Endothelium is a cell monolayer on the posterior surface of the cornea responsible for maintaining corneal transparency.
  • This abundant expression of Na + -K + - ATPase and numerous secondary membrane transporters facilitate an outward osmotic driving force that counteracts an inward imbibition pressure from corneal stromal glycosaminoglycans, thereby maintaining corneal hydration, thickness, and clarity.
  • Anterior chamber aqueous humor bathes its apical surface and provides all nutrients for the CE.
  • the robust transport activity places a heavy metabolic demand on the CE, which is consistent with a high mitochondrial density that is second in the human body only to photoreceptors.
  • the dependence on mitochondrial activity is consistent with reports of CE dysfunction associated with a number of mitochondrial disorders, such as Pearson Syndrome, Kearns-Sayre syndrome, and Leigh's syndrome.
  • Compromised CE loses its fluid transport efficacy, resulting in corneal edema and vision loss.
  • the CE does not proliferate or regenerate in vivo, and there is a continuous decrease in CE cell density throughout life. Therefore, cell protection is a high priority for patients with corneal endothelial dystrophies and/or for people undergoing intraocular surgeries, such as cataract surgery.
  • Endothelial corneal dystrophies are inherited disorders characterized by degenerated corneal endothelium and/or accelerated cell loss that may result in corneal edema, visual loss and require surgical transplantation.
  • SLC4A11 At least one gene that encodes a membrane transporter. Mutations in SLC4A11 may cause Congenital Hereditary Endothelial Dystrophy (CHED) and Harboyan Syndrome (endothelial dystrophy with perceptive deafness). See Vithana, E.N., et al. (2008), SLC4A11 mutations in Fuchs endothelial corneal dystrophy. HUMAN MOLECULAR GENETICS, 17: 656- 666.
  • CHED Congenital Hereditary Endothelial Dystrophy
  • Harboyan Syndrome endothelial dystrophy with perceptive deafness
  • SLC4A11 mutations are also present in several genes that cause age-related Fuchs Endothelial Corneal Dystrophy (FECD) and Peters anomaly (Corneal opacity resulted from dysgenesis of posterior layers of the cornea).
  • Fuchs Endothelial Corneal Dystrophy FECD
  • Peters anomaly Corneal opacity resulted from dysgenesis of posterior layers of the cornea.
  • SLC4A11 has been characterized as a novel H 3 :2H + co-transporter. See Zhang, W. et al. (2015), Human SLC4A11 Is a Novel NH3/H+ Co-transporter, THE JOURNAL OF BIOLOGICAL CHEMISTRY, 290: 16894-16905.
  • the CE may require high expression of an ammonia-linked transporter.
  • the existence of a specific Ammonia transporter suggests a highly active amino acid metabolism.
  • the amino acid glutamine can contribute up to 90% of ammonia generated in some cell types concentrating of glutamine in aqueous humor [glutamine] is about 0.63 mM similar to that of serum (0.61 mM).
  • Many stem cells and/or transformed cancer cells utilize glutamine as synthetic intermediates for cell growth. This study investigated whether the non-regenerating well-differentiated CE uses glutamine mainly for ATP generation to supply energy for its abundant Na -K -ATPase activity and support the physiological pump function necessary to maintaincorneal clarity.
  • Glutaminolysis via mitochondrial glutaminase is a highly dynamic and regulated metabolic pathway.
  • the substrate (glutamine) and/or products of its metabolism may provide short-term activity regulation while acidosis and oncogene s/proto- oncogenes such as c-Myc, p53 and p63 can affect long-term expressions.
  • a Slc4al l-/- mouse model of Congenital Hereditary Endothelial Dystrophy may exhibit altered ammonia handling resulting in the disruption of CE glutamine metabolism.
  • a first embodiment includes a method of protecting cornea endothelium, comprising the steps of: providing a patient diagnosed with corneal endothelial dystrophy, undergoing intraocular surgeries, and/or post-operative care, with a therapeutic regime that includes at least one therapeutically effective dose of at least one composition comprising glutamine, alpha-ketoglutarate, and/or any stabilized/direvatized forms thereof.
  • a second embodiment includes the method according to the first embodiment, wherein the at least one composition further comprises glucose.
  • a third embodiment includes a method of protecting or enhancing the viability of a corneal endothelium, comprising the steps of: contacting at least one portion of a corneal endothelium with a therapeutic regime that includes at least one therapeutically effective dose of at least one composition comprising glutamine, alpha-ketoglutarate, and/or any stabilized/direvatized forms thereof.
  • a fourth embodiment includes the method according to the third embodiment, wherein the at least one composition further comprises glucose.
  • a fifth embodiment includes the method according to any one of the third and the fourth embodiments, wherein the corneal endothelium is damaged or stressed.
  • a sixth embodiment includes the method according to any one of the third to the fifth embodiments, wherein the step of contacting is performed prior to transplantation, after transplantation, during transplantation, and/or during transportation of said coreal endothelium.
  • a seventh embodiment includes the method according to any one of the third to the sixth embodiments, wherein the corneal endothelium is damaged or stressed after trauma, and/or an injury comprising chemical injury, hypoxic injury, thermal/freezng injury, radiation injury, photoxic injury, reactive oxidation injury, and/or any other forms of injury occurred by localized nutritional dysfunction, corneal inlays, implants, and/or contact lenses.
  • An eighth embodiment includes a method of treating corneal endothelial dystrophies, comprising the steps of: treating a patient diagnosed with corneal endothelial dystrophies with at least one therapeutically effective composition that inhibits and/or reduces glutaminolysis.
  • a nineth embodiment includes the eighth embodiment, wherein the at least one therapeutically effective composition comprises at least one glutamine analogue, at least one glutaminase inhibitor, at least one gamma-glutamyl-transpeptidase (GGT) inhibitor, and/or at least one membrane glutamate excitatory amino acid transporter (EAAT) inhibitor.
  • the at least one therapeutically effective composition comprises at least one glutamine analogue, at least one glutaminase inhibitor, at least one gamma-glutamyl-transpeptidase (GGT) inhibitor, and/or at least one membrane glutamate excitatory amino acid transporter (EAAT) inhibitor.
  • a tenth embodiment includes the method according to any one of the eighth and the nineth embodiments, wherein the at least one glutaminase inhibitor comprises a glutaminase type 1 (GLS1) inhibitor.
  • the at least one glutaminase inhibitor comprises a glutaminase type 1 (GLS1) inhibitor.
  • An eleventh embodiment includes the method according to any one of the eighth to the tenth embodiments, wherein the at least one glutamine analogue comprises 6-Diazo-5-oxo- L-norleucine (DON).
  • DON 6-Diazo-5-oxo- L-norleucine
  • a twelfth embodiment includes the method according to any one of the eighth to the tenth embodiments, wherein the corneal endothelial dystrophy is at least one dystrophy selected from the group consisting of: Congenital Hereditary Endothelial Corneal Dystrophy, Fuchs Endothelial Corneal Dystrophy, Posterior Polymorphous Corneal Dystrophy, and/or Schnyder Crystalline Corneal Dystrophy.
  • a thirteenth embodiment includes a therapeutic composition for protecting cornea endothelium, comprising: at least one agent comprising glutamine, alpha-ketoglutarate, dimethyl alpha-ketoglutarate, and any stabilized/direvatized forms thereof; at least one pharmaceutically acceptable salt; and at least one physiological buffer.
  • a fourteenth embodiment includes a therapeutic composition for treating corneal endothelial dystrophies, comprising: at least one therapeutically effective agent that inhibits and/or reduces glutaminolysis; at least one pharmaceutically acceptable salt; and at least one physiological buffer.
  • a fifteenth embodiment includes the method of the fourteenth embodiment, wherein the at least one therapeutically effective agent comprises at least one glutamine analogue, at least one glutaminase inhibitor, at least one gamma-glutamyl-transpeptidase (GGT) inhibitor, and/or at least one membrane glutamate excitatory amino acid transporter (EAAT) inhibitor.
  • the at least one therapeutically effective agent comprises at least one glutamine analogue, at least one glutaminase inhibitor, at least one gamma-glutamyl-transpeptidase (GGT) inhibitor, and/or at least one membrane glutamate excitatory amino acid transporter (EAAT) inhibitor.
  • a sixteenth embodiment includes a kit comprising at least one composition according to any one of the twelfth to the fifteenth embodiments, and at least one container.
  • SEQ ID NO: 15 AGCAGATGCTGTTTCCAAACCAGG
  • SEQ ID NO 20 CAGGCATGGAGAAGTTGAGAACTG
  • SEQ ID NO 24 AGTCACTTCAATGGAGCACAGCTG
  • SEQ ID NO 21 TTATTGGAGGGTTGCTGCAAGCAC
  • FIG. 1 Expression of glutaminase, glutamate transporters and glutamine transporters in corneal endothelium.
  • A RT-PCR of human and mouse corneal endothelium tissue for glutaminase (GLS1, GLS2, GGT), glutamate dehydrogenase (GDH) and glutamate transporters (EAAT1-5);
  • B Immunofluorescence staining of human cornea section showing GLS1, GLS2 and GGT expression in cornea endothelium;
  • C RT-PCR of HCEC showing expression of GLS1, GLS2, GGT, EAAT1-3, ten glutamine transporters as well as SLC4A11.
  • D Immunofluorescence staining of HCEC showing cellular localization of GLS1 (mitochondria) and GGT (membrane).
  • FIG. 2. Glutamine contributes to TCA cycle in HCEC.
  • A Schematic illustration of proposed glutamine metabolism in corneal endothelium;
  • B Ammonia release is dose- dependent on glutamine concentration in HCEC culture;
  • C GC-MS results show -60% of carbons in TCA intermediates are from glutamine.
  • FIG. 3 Glutamine supplies energy for corneal endothelial pump function.
  • B Central corneal thickness (CCT) is better maintained with glutamine-supplemented perfusion. Left: time course of CCT change.
  • FIG. 4 Slc4aH ⁇ ⁇ mouse cornea endothelium shows sign of ammonia toxicity and altered glutaminolysis enzymes.
  • A Photography of Slc4all +/+ and Slc4aH ⁇ ⁇ mouse cornea, shows diffuse edema (increase of light reflection in clear stroma) in 12-week Slc4aH ⁇ ⁇ .
  • B H&E staining of 40-week Slc4all +/+ and Slc4aH ⁇ ⁇ mouse cornea section shows endothelial vacuolation and Descemet's membrane thickening.
  • C Nitrotyrosine immunostaining shows increased intensity in 40-week Slc4all " corneal endothelium, suggesting ammonia toxicity.
  • FIG. 5 Verification of glucose contribution to TCA cycle in HCEC.
  • A GC-MS results show -50% of carbons in TCA intermediates are from glucose.
  • B Percentage contribution of glucose into TCA intermediates does not change even in presence of sufficient glucose.
  • FIG. 6. (A) Picture of cornea mounted on to Barron Artificial Anterior chamber with Ringer's solution. (B) Alizarin Red staining visualization of rabbit corneal endothelium after deswelling experiment. Cell density was counted using these images. (C) Alizarin Red staining visualization of human corneal endothelium after deswelling experiment. Cell density was counted using these images.
  • FIG. 8 A diagram illustrating proposed mechanisms involved in human corneal endothelium.
  • the terms 'therapeutically effective dose,' 'therapeutically effective amounts,' and the like refers to a portion of a compound that has a net positive effect on the health and well being of a human or other animal.
  • Therapeutic effects may include an improvement in longevity, quality of life and the like these effects also may also include a reduced susceptibility to developing disease or deteriorating health or well being.
  • the effects may be immediate realized after a single dose and/or treatment or they may be cumulative realized after a series of doses and/or treatments.
  • Pharmaceutically acceptable salts include salts of compounds of the invention that are safe and effective for use in mammals and that possess a desired therapeutic activity.
  • Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention.
  • Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l, l'-methylene-bis
  • Certain compounds of the invention may form pharmaceutically acceptable salts with various amino acids.
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanol amine salts.
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanol amine salts.
  • “Pharmaceutically acceptable salts” as used herein unless defined otherwise refers to: Pharmaceutically acceptable salts, and common methodology for preparing them, are known in the art. See, e.g., P. Stahl, et al, HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2002); S.M. Berge, et al, "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977. [0086] Pharmaceutical formulation: The compounds of the invention and their salts may be formulated as pharmaceutical compositions for administration.
  • compositions and processes for making the same are known in the art for both humans and non- human mammals. See, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, (A. Gennaro, et al., eds., 19 th ed., Mack Publishing Co., 1995).
  • the pharmaceutical formulations of the present invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous) and rectal administration.
  • the formulations may be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient, i.e., the compound or salt of the present invention, with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier or, a finely divided solid carrier or both, and then, if necessary, forming the associated mixture into the desired formulation.
  • the pharmaceutical formulations of the present invention suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, and may also include an antioxidant, buffer, a bacteriostat and a solution which renders the composition isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may contain, for example, a suspending agent and a thickening agent.
  • the formulations may be presented in a single unit-dose or multi-dose containers, and may be stored in a lyophilized condition requiring the addition of a sterile liquid carrier prior to use.
  • phrases Pharmaceutically acceptable carrier unless stated or implied otherwise, is used herein to describe any ingredient other than the active component(s) that maybe included in a formulation.
  • the choice of carrier will to a large extent depend on factors such as the particular mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form.
  • a tablet may be made by compressing or moulding the active ingredient with the pharmaceutically acceptable carrier.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, in admixture with, for example, a binding agent, an inert diluent, a lubricating agent, a disintegrating and/or a surface active agent.
  • Moulded tablets may be prepared by moulding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient.
  • treating includes administering to a human or an animal patient at least one dose of a compounds, treating includes preventing or lessening the likelihood and or severity of a at least one disease as well as limiting the length of an illness or the severity of an illness it may or may not result in a cure of the disease.
  • inhibitortion or “inhibitory activity” each encompass whole or partial reduction of activity or effect of an enzyme or all and/or part of the pathway.
  • a “therapeutically effective amount” in general means the amount that, when administered to a subject or animal for treating a disease, is sufficient to affect the desired degree of treatment for the disease.
  • a “therapeutically effective agent” includes, but is not limited to, glutamine analogues, glutaminase (e.g., GLS 1 and/or GLS2) inhibitors, gamma-glutamyl- transpeptidase (GGT) inhibitors, and/or membrane glutamate excitatory amino acid transporter (EAAT) inhibitors.
  • glutamine analogues e.g., GLS 1 and/or GLS2
  • GTT gamma-glutamyl- transpeptidase
  • EAAT membrane glutamate excitatory amino acid transporter
  • a “selective" inhibitor is one that has at least 2, 5, 10, 20, 50, 100, or 200 fold greater inhibitory activity (for example, as determined by calculation of IC 50 , 3 ⁇ 4, or other measure of affinity or effect) for a particular isozyme of the inhibitor compared to other members of the family.
  • endothelial corneal dystrophy includes, but is not limited to, posterior corneal dystrophies.
  • Posterior corneal dystrophies include, but are not limted to, Congenital Hereditary Endothelial Corneal Dystrophy, Fuchs Endothelial Corneal Dystrophy, Posterior Polymorphous Corneal Dystrophy, and Schnyder Crystalline Corneal Dystrophy.
  • Human and/or mouse Corneal Endothelium expresses metabolic enzymes involved in glutaminolysis and glutamine and/or glutamate transporters. The expression of enzymes involved in glutaminolysis as well as glutamine and/or glutamate transporters in human and/or mouse CE tissue were examined. Conserved expressions of glutaminase (i.e, one of the enzymes involved in flutaminolysis) and glutamine and/or glutamate transporters were evident in both human and mouse CE.
  • both mitochondrial glutaminase, phosphate-activated glutaminase type 1 (GLS 1, kidney -type) and type 2 (GLS2, liver-type) were expressed in human and mouse CE and the expression of GLSl was higher than that of GLS2 in both human and mouse (FIG. 1A).
  • both human and mouse CE expressed gamma-glutamyl-transpeptidase (GGT), which had been shown to have a membrane glutaminase activity and to function along with membrane glutamate excitatory amino acid transporters (EAATs).
  • EAATs membrane glutamate excitatory amino acid transporters
  • HCEC immortalized human CE cell line
  • the expressions of GLSl, GLS2, and GGT were detected (FIGs. 1C and ID).
  • FIG. IB the expressions of GLSl, GLS2 and GGT in human CE tissue was verified by immunostaining healthy human donor cornea sections.
  • FIG. ID the expression of GLSl revealed mitochondrial localization on immunostaining of HCEC, whereas GGT showed membrane localization.
  • FIG. ID eleven putative glutamine transporters had been screened in HCEC.
  • HCEC expressed the following ten transporters: SLC1A5, Na + - glutamine/neutral amino acid antiport; SLC6A19, Na + - glutamine co-transport; SLC7A5, glutamine/ large neutral amino acid antiport; SLC7A8, glutamine/ small neutral amino acid antiport; SLC38A1, Na + -glutamine cotransport; SLC38A2, Na + -glutamine cotransport; SLC38A3, Na + -glutamine/H + antiport; SLC38A5, Na+-glutamine/H+ antiport; SLC38A7, Na + - glutamine/H + antiport; SLC38A8, Na + -Neutral Amino Acid Transporter.
  • Glutamine contributes to about half of the TCA cycle intermediate pool found in CE.
  • the inventors investigated whether CE utilizes aqueous humor glutamine via active uptake by glutamine transporters and the conversion of glutamine to glutamate through GGT/EAATs complex.
  • FIG. 2A glutamine and/or glutamate can be metabolized via the TCA cycle in mitochondria to produce energy.
  • glutamine-derived ammonia release in HCEC were examined.
  • FIG. 2B dose-dependent increases of ammonia production were observed when glutamine concentration increased, suggesting that CE may utilize glutamine efficiently.
  • HCEC cells were fed 4 mM of U- C5-glutamine in the presence of 2.5 g/L (13.9 mM) glucose, and gas chromatography mass spectrometry (GC/MS) was used to measure the TCA cycle intermediates derived from 13 C labeled glutamine.
  • GC/MS gas chromatography mass spectrometry
  • glutamine sourced carbons contribute to about 60% of the TCA cycle intermediate pool.
  • Glucose was another major source for TCA cycle intermediates in HCEC (FIG. 5).
  • 1 g/L (5.6 mM) and 2.5 g/L (13.9 mM) glucose did not change the percent distribution of TCA cycle intermediates (FIG. 5B), suggesting that HCECs may prefer metabolizing glutamine in the TCA cycle even in the presence of sufficient glucose.
  • Glutamine supplies ATP for the CE pump function.
  • the immortalized HCEC cell line it was observed that more than half of the TCA cycle intermediates originated from glutamine.
  • glutamine may have been used for ATP generation to supply energy for CE fluid transport.
  • the intracellular ATP content in HCEC was measured using conditional DMEM medium that indicated the following: 1) Glc/Gln: 5 g/L glucose + 4 mM glutamine; 2) -/Gin: 4 mM glutamine; 3) Glc/-: 5 g/L glucose; 4) -/-: neither glucose nor glutamine.
  • the concentration of ATP was the highest in Glc/Gln medium, and interestingly only slightly lower in -/Gin, but significantly lower in Glc/- and -/-.
  • the results indicate that glucose can sustain half of the CE energy supply, consistent with GC-MS data that more than half of TCA intermediates were labeled when cultured with U- 13 C6-glucose and unlabeled L-glutamine.
  • CE in vivo shows a similar active glutamine metabolism.
  • the effect of glutamine supplementation on perfused freshly dissected ex vivo rabbit corneas was examined. If glutamine is beneficial for energy production, the corneal thickness should be better maintained ⁇ i.e., thinner) over a prolonged perfusion period as a result of better pump activity.
  • central corneal thickness (CCT) was 2.5 ⁇ less in glutamine supplemented corneas after 3.5 h of perfusion.
  • glutamine supplementation facilitates a faster deswelling rate.
  • the Ringer's solution was collected and residual [glucose] and [glutamine] was measured by GC-MS.
  • the glucose and/or glutamine consumption rate per cell was estimated from the total glutamine/glucose consumed and total corneal endothelial cell count was determined using Alizarin Red staining.
  • 0.5- 0.7 pmol/cell/h glucose and 0.3-0.6 pmol/cell/hr glutamine was consumed (FIG. 3D).
  • Glutaminolysis has been shown to provide an essential component of CE metabolism that supports its crucial physiological pump function.
  • the inventors showed: 1) expression of GLS1 (kidney type), GLS2 (liver type) and GGT ( ⁇ -glutamyl-transpeptidase, membrane form) enzymes that can facilitate glutamine deamination; 2) the expression of a wide variety of Na + - glutamine, H + -glutamine transporters and glutamate transporters; 3) that glutamine contributed -60% of carbons in TCA cycle intermediates; and 4) that glutaminolysis sustained the ATP level in CE and in turn supported pump function.
  • Glutaminolysis metabolism in CE is conserved across all of the species investigated: rabbit, mouse and human.
  • CE Unlike tumor cells that manifest active glutaminolysis metabolism after metabolic reprogramming, or renal proximal tubule epithelium upregulation of glutaminolysis in response to acidosis, the active glutaminolysis in CE is present in normal corneal endothelium fulfilling its physiological function. Since CE does not proliferate in vivo, glutaminolysis is mainly dedicated to supporting energy metabolism. Previously, glucose was the only substrate thought as an energy source for CE. See Zurawski, C.A., et al. (1989), Glucose consumption in cultured corneal cells, CURRENT EYE RESEARCH, 8: 349-355.
  • CE pump function can be inhibited 33% by 6-Diazo-5-oxo-L-norleucine (DON), a glutamine analogue with a wide inhibitory effect on glutaminase and GGT.
  • DON 6-Diazo-5-oxo-L-norleucine
  • GGT GGT
  • DON irreversibly inhibits 95% of glutaminase activity and 90% of GGT activity, thereby inhibiting all major routes of glutamine metabolism in cells.
  • the glutaminolysis works cooperatively together with the membrane ammonia transporter SLC4A11 to maintain the energy supply for CE fluid transport, shedding some light on the pathophysiological changes in CE due to loss of SLC4A11 function.
  • Mutations in human Slc4al 1 gene are causative of CHED and Harboyan Syndrome, and are associated with FECD and Peters anomaly. All of the above endothelial dystrophies share common signs of corneal edema and endothelial degeneration, suggesting an essential functional role of SLC4A11 in maintaining corneal endothelium health and physiological function.
  • the model suggests ammonia transport function of SLC4A11 facilitates ammonia detoxification by removing NH 3 in corneal endothelium.
  • Loss of Slc4al l expression in the mouse leads to corneal endothelial dystrophy and secondary changes very similar to human CHED.
  • generalized ammonia toxicity in Slc4aH ⁇ ⁇ murine corneal endothelium was observed as revealed by nitrotyrosine staining.
  • expressions of three major enzymes catalyzing the first step of deamination in glutaminolysis Glsl, Gls2 and Ggt were altered in Slc4aH ⁇ ⁇ .
  • FECD is associated with multiple genes in which Slc4al l mutations are thought to contribute about 11% of FECD. Nevertheless, despite the diverse genetic background the expression of SLC4A11 mRNA in FECD is reduced. Given the upregulated GLS1 and downregulated GLS2 expression observed in Slc4all " CHED mouse CE, the expression of GLS1 and GLS2 in FECD patients' CE was investigated (FIG. 7). A similar trend of GLS1 upregulation and GLS2 downregulation in response to SLC4A11 downregulation in FECD patients was observed, but due to the diverse genetic background of FECD, the difference was not resolved with traditional statistical testing.
  • GSH oxidized glutathione
  • GSH is a thiol tripeptide (y-glutamyl- cysteinyl-glycine) that neutralizes oxygen free radicals and itself becomes oxidized glutathione disulfide (GSSG).
  • Extracellular GSSG can elevate intracellular GSH in CE measured by 35 S-GSSG radioactive tracing.
  • RNA extraction, PCR and qPCR and Nested PCR Total RNA, from human corneal endothelium tissue, mouse endothelium tissue, immortalized HCEC cell line, and FECD patient's corneal endothelium tissue were extracted and purified via RNeasy mini kit (#74104, Qiagen) with DNase digestion (#79254, Qiagen).
  • PCR, Nested PCR and real-time qPCR were performed with human and murine gene primers listed in Tables 1 and 2 respectively. Details can be found in supplemental experimental procedures.
  • Immortalized HCEC culture Immortalized HCEC were cultured at 37°C, 5% C02 in appropriate plates or flasks coated with undiluted FNC Coating Mix® (AthenaES). Complete medium (OptiMEM-I®; Invitrogen) contains 8% FBS (Hyclone Laboratories Inc.), EGF 5 ng/mL (Millipore), pituitary extract 100 ⁇ g/mL (Hyclone Laboratories), calcium chloride 200 mg/L, 0.08% chondroitin sulfate (Sigma-Aldrich), gentamicin 50 ⁇ g/mL, and antibiotic/antimycotic solution diluted 1 : 100 (Invitrogen).
  • Intracellular ATP assay HCEC were seeded at 1.5* 10 4 /ml in 12-well plate to sub- confluent, then put under serum-free conditional DMEM medium (glucose free, glutamine-free, pyruvate-free, Gibco #A1443001) with glucose and/or glutamine supplementation for 12 hours before measurement. ATP is extracted by boiling water method, and measured by luciferin- luciferase based ATP assay kit (A22066, Molecular Probes). See Yang, N.C., et al. (2002), A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP .
  • BR Base contains (mM): 148.5 Na + , 4 K + , 1.4 Ca2 + , 0.6 Mg2 + , 103.2 CI " , 28.5 HC0 3 " .
  • the addition of 5 mM glucose and/or 2 mM glutamine was made to BR base to get BR Glc/Gln, BR Glc/-, and BR -/Gin. All solutions were adjusted to pH 7.5 with IN HC1 and osmolarity 295 mOsm/L with mannitol.
  • rabbit or human cornea buttons with sclera skirt was mounted to a Barron® artificial anterior chamber (K20-2125, Katena Products Inc.) with corresponding Ringer's. Corneal thickness was monitored over time with Optical Coherent Tomography (OptoVue iVue SD-OCT). The corneal surface was kept moist with Artificial tears (Refresh®) after each measurement, and the cornea was kept in 37 °C incubator between measurements. Corneal Endothelial cells were stained with Alizarin red and counted after experiments for calculation of glutamine and/or glucose consumption rate. See supra Supplemental Experimental Procedures for more details.
  • RNA extraction Healthy human corneal endothelium: Human donor cornea were obtained from Indiana Lion Eye Bank in 4°C Optisol® GS medium. Corneal endothelium with Descemet's membrane was peeled off in Corneal Viewing Chamber (Stephens Instruments) using Submerged Cornea Using Backgrounds Away (SCUBA) technique. Peeled cornea endothelium sheet was processed immediately or stored in RNAlater® (AM7020, Ambion) at 4°C. Corneal endothelium sheet was rapidly frozen in liquid nitrogen and grinded following by RNeasy column (Qiagen) purification. Mouse corneal endothelium: Mouse cornea with sclera skirt was dissected from the globe.
  • Corneal endothelium with Descemet's membrane was peeled off followed by RNA extraction similar as described above.
  • FECD corneal endothelium Patients diseased cornea endothelium samples were collected during Decrements Membrane Endothelial Keratoplasty (DMEK) surgery, and shipped on ice overnight in Optisol® GS medium. Cornea endothelium sheet was put into RNAlater® in 4°C upon arrival or processed immediately. RNA extraction steps were the same as described above.
  • Immortalized HCEC cell line HCEC cells were culture in OptiMEM complete medium to confluent, RNA extraction steps were the same as described above. [00118] PCR and qPCR and Nested PCR.
  • RNA-to-cDNA Kit (Applied Biosy stems) at 10 ng RNA/ A L reverse transcription. Sequences of human and mouse gene primers used are listed in Supplemental Table 1 and Table 2 respectively. Conventional PCR was performed with MyCycler Thermal cycler (Bio-Rad) following the AmpliTaq® 360 DNA Polymerase protocol (Applied Biosystems). Real-time qPCR reactions were set up in triplicate using SYBR Green PCR Master Mix (Agilent Technologies). All assays used similar amplification efficiency, and a 2 _ ⁇ experimental design was used for relative quantification and normalized to ACTB (mouse) or GAPDH (human) for differential expression levels of target genes. Nested PCR was conducted for genes with no CT value in real-time qPCR. An additional 40 cycles of PCR are conducted on reverse transcription PCR product using the same gene primer.
  • Serum-free DMEM conditional medium was made using DMEM (glucose free, glutamine-free, pyruvate-free, GIB CO® #A1443001) supplemented with calcium chloride 200 mg/L, 0.08% chondroitin sulfate (Sigma-Aldrich), and the corresponding isotope-labeled glucose and/or glutamine.
  • DMEM glucose-free, glutamine-free, pyruvate-free, GIB CO® #A1443001
  • calcium chloride 200 mg/L 0.08% chondroitin sulfate (Sigma-Aldrich)
  • Fresh Rabbit Corneal Perfusion Freshly dissected rabbit cornea with conjunctiva and lids was atraumatically mounted on a plastic ring and pressed on a fixed hollow rod. Then lids and conjunctiva were everted over the globe and pulled down tightly to the rod and tied with a suture on mounting ring. After mounting, sclera posterior to the suture were removed with scalpel and scissors, as well as vitreous, ciliary body-iris, and lens to reveal the corneal endothelial surface. Then an anterior metal platform and a posterior plastic cap with openings connected to tubing were clamped against the mounting ring to form an artificial anterior chamber. The central tube provided inflow, and the two side tubes provided outflow.
  • the cornea chamber was fitted in a metal jacket with embedded electric heater and thermistor to maintain 37 °C.
  • Ringer's solutions contain (mM): 153.5 Na + , 4 K + , 1.4 Ca 2+ , 0.6 Mg 2+ , 113.2 CI " , 1 HP0 4 2" , 16.4 gluconate- and 28.5 HCO 3 " . All solutions were equilibrated with 5% C0 2 , pH adjusted to 7.5 and osmolarity adjusted to 295 mOsm/L with sucrose. 5 mM glucose and/or 1 mM glutamine were added. Perfusion solutions were maintained at 37°C in a water bath during experiment. [00123] Revised Corneal De-Swelling Experiments.
  • rabbit cornea rabbit eye balls stored on ice were shipped overnight from a regional supplier (Pel freez Biologicals). Corneal thickness was measured immediately before dissecting as data point of time zero by Optical Coherent Tomography with anterior segment attachment (OptoVue iVue SD-OCT). Then eye balls were assigned to experimental groups 1) Glc/Gln 2) Glc/- and 3) -/Gin and dissected to cornea button with sclera skirt, rinsed and mounted with corresponding Ringer's solution. Cornea with sclera skirt was mounted on to Barron® artificial anterior chamber (K20-2125, Katena Products, Inc.) and anterior chamber was filled with 350 ⁇ _, Ringer's solution corresponding to the assigned group.
  • Barron® artificial anterior chamber K20-2125, Katena Products, Inc.
  • Corneal thickness is monitored using OptoVue iVue SD- OCT with anterior segment attachment every 15 min till 150 min after mounting.
  • Artificial tears (Refresh®) is applied on epithelial surface every 15 min after OCT measurement, and Barron Chamber with mounted cornea is put in 37 °C incubator when not being measured.
  • Final fluid about 350 ⁇ .) after 150 min experiment in artificial anterior chamber is collected and prepared for GC-MS analysis for residual glutamine and glucose.
  • cornea button is stained with Alizarin Red for corneal endothelium visualization and counted under light microscope.
  • Total number of corneal endothelium cells per cornea was calculated as the product of corneal endothelium density and cornea dome surface area.
  • rabbit cornea diameter 13.2 mm and radius of curvature 7.26 mm were used for calculation.
  • Rabbit corneal endothelium glutamine and/or glucose consumption rate per cell was estimated with total glutamine/glucose consumed divided by total corneal endothelial cell count.
  • human donor cornea De-identified human donor cornea with sclera skirt was obtained from Indiana Lion Eye Bank, stored in 4 °C upon arrival until experiments. Cornea with sclera skirt was rinsed with ice-cold BR base solution to get rid of Optisol cornea storage medium, then soaked in ice-cold BR base solution for 1 h to let it swell. Then the human cornea was mounted similarly as rabbit cornea with 300 ⁇ _, BR Glc/Gln or BR Glc/-, and monitored for central corneal thickness with OCT every 15 min for 165min. Artificial tears were applied after each measurement, and Barron Chamber with mounted cornea was put in 37 °C incubator between measurement.
  • Final fluid (about 300 ⁇ ,) is collected and prepared for GC-MS analysis for residual glutamine and glucose.
  • total corneal endothelium cell count per cornea was estimated with Alizarin Red visualization of corneal endothelium under light microscope, and human cornea diameter 11.5 mm and radius of curvature 7.75 mm were used for calculation. Rabbit and human corneal endothelium density and total cell count were shown in Table 3.

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Abstract

Selon la présente invention, l'endothélium cornéen est la monocouche de cellules postérieures de la cornée qui maintient la clarté de la cornée par le biais de sa fonction de pompage. La présente invention porte sur le métabolisme de la glutamine qui joue un rôle crucial dans le maintien de l'alimentation en énergie pour la fonction de pompage physiologique de l'endothélium cornéen. Cette glutaminolyse est présente dans l'endothélium cornéen sain et conservée à travers les espèces. De plus, elle est perturbée lorsqu'il y a une perte de la fonction de transporteur du gène SLC4A11 liée à l'ammoniac, dans des conditions telles que la dystrophie endothéliale héréditaire congénitale (CHED pour Congenital Hereditary Endothelial Dystrophy) et la dystrophie cornéenne endothéliale de Fuch (FECD pour Fuch's Endothelial Corneal Dystrophy).
PCT/US2017/064264 2016-12-02 2017-12-01 Compositions et procédés permettant de traiter et/ou de réduire la dystrophie cornéenne WO2018102715A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100152130A1 (en) * 2002-08-01 2010-06-17 Eisai Inc. Treatment of cancer with glutamine
US20130315996A1 (en) * 2010-11-19 2013-11-28 Albert-Ludwigs-Universitaet Freiburg Bio-functionalized stimulus-responsive dissolvable peg-hydrogels
WO2015101958A2 (fr) * 2014-01-06 2015-07-09 Rhizen Pharmaceuticals Sa Nouveaux inhibiteurs de glutaminase
US20160287564A1 (en) * 2015-03-30 2016-10-06 Calithera Biosciences, Inc. Methods of administering glutaminase inhibitors

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Publication number Priority date Publication date Assignee Title
US20100152130A1 (en) * 2002-08-01 2010-06-17 Eisai Inc. Treatment of cancer with glutamine
US20130315996A1 (en) * 2010-11-19 2013-11-28 Albert-Ludwigs-Universitaet Freiburg Bio-functionalized stimulus-responsive dissolvable peg-hydrogels
WO2015101958A2 (fr) * 2014-01-06 2015-07-09 Rhizen Pharmaceuticals Sa Nouveaux inhibiteurs de glutaminase
US20160287564A1 (en) * 2015-03-30 2016-10-06 Calithera Biosciences, Inc. Methods of administering glutaminase inhibitors

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Title
GOROVITS ET AL.: "Glutamine Synthetase Protects Against Neuronal Degeneration in Injured Retinal Tissue", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 94, no. 13, 1 June 1997 (1997-06-01), pages 7024 - 7029, XP055490729 *
JUN ET AL.: "An alpha 2 Collagen VIII Transgenic Knock-In Mouse Model of Fuchs Endothelial Corneal Dystrophy Shows Early Endothelial Cell Unfolded Protein Response and Apoptosis", HUMAN MOLECULAR GENETICS, vol. 21, no. 2, 14 October 2011 (2011-10-14), pages 384 - 393, XP055490732 *
ZHANG ET AL.: "Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium", EBIOMEDICINE, vol. 16, 1 February 2017 (2017-02-01), pages 292 - 301, XP055490734 *

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