WO2018005762A2 - Ascorbic acid-amino acid conjugates and their use in regulation of fluid outflow - Google Patents

Ascorbic acid-amino acid conjugates and their use in regulation of fluid outflow Download PDF

Info

Publication number
WO2018005762A2
WO2018005762A2 PCT/US2017/039940 US2017039940W WO2018005762A2 WO 2018005762 A2 WO2018005762 A2 WO 2018005762A2 US 2017039940 W US2017039940 W US 2017039940W WO 2018005762 A2 WO2018005762 A2 WO 2018005762A2
Authority
WO
WIPO (PCT)
Prior art keywords
side chain
amino acid
ascorbic acid
trabecular meshwork
subject
Prior art date
Application number
PCT/US2017/039940
Other languages
French (fr)
Other versions
WO2018005762A3 (en
Inventor
Richard Allen HILL
Richard Robert HILL
Original Assignee
Orasis Medical, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Orasis Medical, Inc. filed Critical Orasis Medical, Inc.
Priority to EP17821235.3A priority Critical patent/EP3478259A4/en
Publication of WO2018005762A2 publication Critical patent/WO2018005762A2/en
Publication of WO2018005762A3 publication Critical patent/WO2018005762A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/62—Three oxygen atoms, e.g. ascorbic 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • 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
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C07K5/06095—Arg-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C07K5/06113—Asp- or Asn-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06156—Dipeptides with the first amino acid being heterocyclic and Trp-amino acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp

Definitions

  • Glaucoma is a leading cause of blindness.
  • glaucoma As "a group of diseases with certain features including an intraocular pressure that is too high for the continued health of the eye.” Intraocular pressure remains the main treatable aspect of glaucoma therapy. If untreated, glaucoma can lead to destruction of the optic nerve and blindness.
  • a clear fluid called aqueous humor is constantly formed by the ciliary bodies and secreted into the posterior chamber. This fluid passes over the lens and enters the anterior chamber. Aqueous humor passes out the anterior chamber of the eye at approximately the same rate at which it is produced through one of two routes.
  • Glaucoma can result from the disordered drainage of the aqueous humor from the anterior chamber 40 (FIG. 1A) of the eye, across the trabecular meshwork 56 (FIG. 1A) and into Schlemm's Canal 54 (FIG. 1A).
  • the juxacanilicular trabecular meshwork is the site of the increased resistance to outflow of aqueous.
  • the precise mechanism of aqueous transport is poorly understood. It is known that the process, in a normal eye, is energy independent and self-regulating, such that the pressure of the eye remains between 10-21 mm Hg.
  • the outflow rate from the anterior chamber of the eye generally matches the production rate of aqueous humor in the ciliary bodies of the eye 74 (FIG. IB)
  • aqueous humor flows through the trabecular meshwork 56, into Schlemm's canal 54, exiting Schlemm's canal by the intra-scleral aqueous collector channels 70.
  • the collector channels terminate on the surface of the eye, forming aqueous veins of Ascher, which then drain into the venous system.
  • aqueous humor crosses the endothelial cells of Schlemm's canal. In this manner, trabecular meshwork cells and Schlemm's canal endothelial cells are thought to comprise the cells of the primary outflow pathway of the eye.
  • the trabecular meshwork and Schlemm's canal are located at the junction between the iris 46 and the sclera 72.
  • the cornea 50, lens 35, and pupil 44 are also shown.
  • the trabecular meshwork is wedge shaped in structure and runs around the entire circumference of the eye, forming a three-dimensional sieve structure.
  • the trabecular meshwork is formed of collagen beams aligned with a monolayer of cells called the trabecular cells, which produce an extracellular substance which fills the spaces between collagen beams.
  • the trabecular meshwork is suspended between the corneal endothelium and the ciliary body face, and is comprised of a series of parallel layers of thin, flat, branching, and interlocking bands termed trabeculae.
  • the inner portion of the trabecular meshwork (closest to the iris root and ciliary body 74) is called the uveal meshwork, whereas the portion closest to the sclera and cornea is called the corneoscleral trabecular meshwork.
  • the uveal meshwork trabeculae measure approximately 4 ⁇ in diameter, consist of a single layer of cells surrounding a collagen core, and are arranged in interconnected layers. The spaces between these trabeculae are irregular and range from about 25 ⁇ to about 75 ⁇ in size.
  • the trabeculae of the corneoscleral meshwork resemble broad, flat endothelial sheets about 3 ⁇ thick and up to about 20 ⁇ long.
  • the spaces between these trabeculae are smaller than in the uveal meshwork and more convoluted.
  • Schlemm's canal the spaces between the trabeculae decrease to about 2 ⁇ .
  • JCM juxtacanalicular meshwork
  • glaucoma There are a number of different forms of glaucoma, including open- angle and closed-angle glaucoma, as well as steroid induced glaucoma.
  • the most common form of glaucoma is open-angle, which results from increased resistance in the outflow pathway through the trabecular meshwork.
  • the mechanism by which the outflow pathway becomes blocked or inadequate is poorly understood, but the result is an increase in pressure within the eye, which compresses the axons in the optic nerve and can compromise vascular supply to the nerve. Over time, this can result in partial or total blindness.
  • the trabecular meshwork is not physically obstructed, but no longer efficiently transports fluid between the anterior chamber and the scleral drainage veins.
  • Medications for the treatment of glaucoma include prostaglandin analogs, which increase fluid percolation between muscle fibers of the ciliary body, and miotics, which are administered as drops and cause contraction of the pupil of the eye by tightening the muscle fibers of the iris to increase the rate at which the aqueous humor leaves the eye.
  • Epinephrine drops have also been successful in reducing intraocular pressure, but have significant side effects.
  • Other medications are employed, such as ⁇ -adrenergic blocking agents, as drops, or carbonic anhydrase inhibitors as pills, which reduce the production of fluid.
  • Surgical solutions include applying a laser to multiple spots along the trabecular meshwork, which is thought to change the extracellular material and enhance outflow.
  • Other solutions attempt to increase the permeability of the trabecular meshwork or widen Schlemm's canal.
  • Another surgical procedure is a trabeculectomy, wherein an incision is made in the conjunctiva to form a hole in the sclera for aqueous fluid to flow through. This is performed through an open surgical procedure and includes long term risk of infection or injury to the eye.
  • MIGS Micro Invasive Glaucoma Surgeries
  • a need remains for a way of safely, lastingly, and effectively treating the cause of open-angle glaucoma.
  • Current medical and surgical treatment options often lose their efficacy with time.
  • surgical treatments have associated risks of infection or injury to the eye, and current medical solutions often come with significant side effects either affecting vision, the structures of the eye, or with systemic side effects.
  • a need also exists for a treatment of glaucoma which addresses the underlying pathology in the aqueous humor outflow system and leads to a return of drainage as seen in non- glaucomatous eyes.
  • This disclosure provides, in certain embodiments, methods and compositions for treating glaucoma. More particularly, the treatment of glaucoma may involve restoring the filtration capability of the trabecular meshwork of the eye.
  • the disclosure is based on the discovery of ascorbic acid-amino acid conjugates in trabecular meshwork tissue, as analyzed by neutral loss mass spectrometry. This provides a potential mechanism for regulation of intraocular pressure.
  • Molecules of the invention include improved ascorbic acid-mono amino acid conjugates having Formula I and ascorbic acid-di amino acid conjugates having Formula II:
  • SCi and SC2 are the same or different amino acid side chains
  • Ri, R 2 , R3, and R5 are independently selected from -H, a carbonyl group selected from -CHO and -COX, wherein X is a Ci- 6 branched or unbranched alkyl, and an acetyl group selected from CH3CO- and CH3(CH 2 ) n CO-, wherein n is 1-6;
  • R 4 and R5 are independently selected from -H, -CH 3 , -CH2CH3, and -CF3;
  • R7 is H or CH3
  • One or more C-H in Formulas I and II can be replaced with C-F, C-Cl, C-Br, or C-I.
  • SCi of Formula I is selected from the group consisting of: an L-DOPA side chain, a Tyr side chain, an Arg side chain, and a Sec side chain.
  • SCi and SC 2 of Formula II are, respectively, (i) an Asn side chain and an Arg side chain, (ii) an Arg side chain and an Asn side chain, (iii) a Cys side chain and an Asp side chain, (iv) an Asp side chain and a Cys side chain, (v) a His side chain and a Cys side chain, (vi) a Cys side chain and a His side chain, (vii) a Glu side chain and an Ala side chain, (viii) a Lys side chain and an Ala side chain, (ix) a Phe side chain and an Ala side chain, (x) a Sec side chain and an Ala side chain, (xi) a Ser side chain and an Asn side chain, (xii) a Gly side chain.
  • SCi is not a tyrosine side chain. In some embodiments,
  • SC 2 is not a tyrosine side chain.
  • SCi is not a levodopa (L-DOPA) side chain.
  • SC2 is not an L-DOPA side chain.
  • Embodiments of the invention provide an isolated trabecular meshwork cell comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
  • compositions comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
  • the composition comprises at least one conjugate of Formula I and at least one conjugate of Formula II.
  • the composition comprises an isolated trabecular meshwork cell comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
  • the composition of the invention is a pharmaceutical composition.
  • compositions comprising conjugates of the invention can be racemic mixtures or can be enantiomerically and/or diastereomerically pure or enriched.
  • Embodiments of the invention additionally provide a kit comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II, a kit comprising a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II, and a kit comprising an isolated trabecular meshwork cell comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II or a composition thereof.
  • the invention provides a method of reducing intraocular pressure
  • IOP insulin pressure
  • the method comprising administering to the subject one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II or a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
  • the invention provides a method of treating high IOP in a
  • the method comprising administering to the subject one or more ascorbic acid- amino acid conjugates of Formula I and/or Formula II or a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
  • the invention provides a method of treating glaucoma in a
  • the method comprising administering to the subject one or more ascorbic acid- amino acid conjugates of Formula I and/or Formula II or a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
  • the glaucoma is open-angle glaucoma.
  • administration of the ascorbic acid-amino acid conjugates is topical, intraocular, systemic, or via a drug delivery device.
  • a drug delivery device In a particular
  • administration is via eye drops.
  • administration is oral, preferably via tablet or capsule.
  • Embodiments of the invention further provide a method of increasing aqueous outflow through trabecular meshwork tissue, the method comprising exposing the trabecular meshwork tissue to one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II, optionally in a composition. In one embodiment, the method is performed ex vivo.
  • Embodiments of the invention additionally provide a method of screening a candidate ascorbic acid-amino acid conjugate for its ability to modify fluid outflow through trabecular meshwork tissue, the method comprising: measuring fluid outflow through trabecular meshwork tissue before and after exposing the trabecular meshwork tissue to the candidate ascorbic acid-amino acid conjugate; wherein a change in fluid outflow after exposure to the candidate ascorbic acid-amino acid conjugate indicates that the ascorbic acid-amino acid conjugate is able to modify fluid outflow.
  • the candidate ascorbic acid-amino acid conjugate has Formula I or Formula II.
  • the method is performed ex vivo or in vivo in an animal model.
  • Formula I and/or Formula II to reduce IOP in a subject in need thereof, or to treat high IOP in a subject, or to treat glaucoma in a subject.
  • the ascorbic acid-amino acid conjugate(s) is formulated for topical, intraocular, or systemic delivery, or for delivery via a drug delivery device.
  • Another embodiment of the invention is the use of an isolated trabecular
  • meshwork cell comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II to reduce IOP in a subject in need thereof, or to treat high IOP in a subject, or to treat glaucoma in a subject.
  • FIGS. 1A-1B show schematic diagrams of the eye.
  • FIG. 1A shows a cross- sectional illustration of the anterior portion of the eye.
  • FIG. IB shows a cross-sectional illustration of the irido-corneal angle of the eye.
  • FIG. 2A-2C show schematics of particular structures within the eye.
  • FIG. 2A shows a schematic of the trabecular meshwork and Schlemm's canal.
  • FIG. 2B and FIG. 2C show schematics of the membrane of an endothelial cell in the juxtacanalicular lining of Schlemm's canal.
  • FIG. 3A-3F show the structure of ascorbic acid (FIG. 3A) and structures of ascorbic acid conjugates with tyrosine (FIG. 3B), L-DOPA (FIG. 3C), arginine- asparagine (FIG. 3D), histidine-cysteine (FIG. 3E), and cysteine-aspartic acid (FIG. 3F).
  • FIG. 4 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-tyrosine conjugate.
  • the mass spectrum shows a second fragmentation of ions containing a neutral loss of a group with a mass of 176 m/z, indicating the loss of ascorbate.
  • the peak at 164.0 m/z is consistent with the loss of C9HioN0 2 + from the larger molecule CisHnNOs.
  • FIG. 5 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-tyrosine conjugate.
  • FIG. 6 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-L-DOPA conjugate.
  • the mass spectrum shows a second fragmentation of ions containing a neutral loss of a group with a mass of 176 m/z, indicating the loss of ascorbate.
  • the peak at 180.1 m/z is consistent with the loss of C9HioN0 3 + from the larger molecule C15H17NO9.
  • FIG. 7 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-L-DOPA conjugate.
  • FIG. 8 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate- arginine-asparagine conjugate.
  • FIG. 9 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-histidine-cysteine conjugate.
  • FIG. 10 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-aspartate-cysteine conjugate.
  • FIG. 11 shows raw mass spec data from a trabecular meshwork sample
  • Masses of interest are at 340.4 m/z and 356.4 m/z. These protonated molecules are mass+1 of the expected species, 339.4 g/mol and 355.6 g/mol, respectively.
  • FIG. 12 shows a synthesis scheme for ascorbic acid-amino acid conjugates.
  • FIG. 12A shows synthesis of Asc-Tyr (Compound 1) and Asc-L-DOPA (Compound 2) conjugates. Reactants are as follows: A) BOMCl, Et 3 N, DCM; B) Compound 7, EDCI, Oxyma, MeCN; C) HC1, HHP.
  • BOM benzyloxymethyl
  • Boc t- butoxycarbonyl
  • t-Bu tert-butyl
  • TBS tert-butyldimethylsilyl
  • BOMCl benzyl chlorom ethyl ether
  • Et 3 N triethylamine
  • DCM dichloromethane
  • EDCI l-Ethyl-3- (3-dimethylaminopropyl)carbodiimide
  • Oxyma ethyl (hydroxyimino)cyanoacetate
  • MeCN acetonitrile
  • HFIP l, l,l,3,3,3-hexafluoro-2-propanol.
  • Reactants are as follows: A) Compound 7, EDCI, Oxyma, MeCN; B) DBU, MeCN; C) Boc-Cys(Trt)-OH, EDCI, Oxyma, MeCN; D) HCl, HFIP; E) Boc-Asp(OtBu)-OH, EDCI, Oxyma, MeCN; F) Boc-His(Trt)-OH, EDCI, Oxyma, MeCN; G) Boc-Asn(Trt)-OH, EDCI, Oxyma, MeCN; H) Boc-Arg(Pbf)-OH, EDCI, Oxyma, MeCN.
  • ascorbic acid as a conjugate with amino acids, including tyrosine, aspartic acid-cysteine, histidine-cysteine, asparagine-arginine, or L-DOPA, in samples of eye tissue.
  • amino acids including tyrosine, aspartic acid-cysteine, histidine-cysteine, asparagine-arginine, or L-DOPA
  • molecules comprising one or more amino acids associated with ascorbic acid, or a derivative or salt thereof are present in normal eye tissue, specifically in the endothelial layer 324 of Schlemm's canal 366 (FIG. 2A) and/or the trabecular meshwork cells 320 (FIG. 2A), and may play a role in the maintenance of normal intraocular pressure by regulating drainage of the aqueous humor through the membranes of the JCM cells as part of an osmotic drive.
  • the trabecular meshwork 320 is separated from
  • FIG. 2B the cell membrane 340 of an endothelial cell is seen with lipophilic regions 342 and hydrophilic regions 344.
  • FIG. 2C shows the endothelial cell membrane 380 with direction of aqueous humor travel indicated by the arrow facing Schlemm's canal 366.
  • a proposed structure in the cell membrane of the endothelial layer 380 is shown as an arrangement of pores or "vents" 368, which bridge the cell membrane 380 and serve to transport aqueous humor across the membrane. After traversing the opposing membrane, the aqueous humor is released into Schlemm's canal.
  • Amino acid molecules comprising an ascorbic acid or ascorbic acid derivative head may be produced by the specialized cells of the JCM and transported to the cell membranes, where they most likely bond via coulombic or ionic bonds to one or more transmembrane proteins.
  • the ascorbic acid or ascorbic acid derivative head may also be bound to an -OH group or other functional group of an amino acid, such as tyrosine or L-DOPA, already incorporated in a protein.
  • a transmembrane cylinder pore/vent
  • the bonds between polar moieties are increasingly favored over bonds with water molecules, and the flow diminishes, until equilibrium is reached.
  • the equilibrium may change based on various factors, such as the rate of production of aqueous humor, but will be self-regulating to maintain a desired pressure.
  • the above-described conjugates, spanning the cell membrane, may result in a self-regulating osmotic drive for water transport out of the anterior chamber of the eye into Schlemm's canal.
  • the ascorbic acid moieties When the pressure is balanced, the ascorbic acid moieties will generally bond with each other and water molecules from the aqueous humor will transport between the hydrophilic heads relatively slowly at a steady state rate. However, even small increases or decreases in pressure may cause the establishment of a new equilibrium flow rate.
  • Open angle glaucoma may result from a failure in the osmotic drive described above. Because relatively normal concentrations of ascorbic acid have been observed in eye tissue of glaucoma patients, absorption, transport, and ingestion of ascorbic acid are not likely causes of the failure and, furthermore, would be expected to cause systemic problems related to vitamin C deficiency, rather than isolated intraocular pressure elevations. In some patients, failure of normal conjugation of ascorbic acid with an amino acid may result from enzyme deficiency, decreased enzyme activity, or other disturbance. These enzymes may be specific to the cells of the eye or JCM or may exist in other places, in which case the patient may have other manifestations in addition to glaucoma, and therapeutic molecules may treat those manifestations as well.
  • Embodiments of the present invention provide novel compounds and compositions comprising ascorbic acid-amino acid conjugates for regulating intraocular pressure by affecting the aqueous humor outflow system of the eye.
  • the ascorbate-amino acid conjugates of the invention can be used treat and/or prevent glaucoma.
  • SI Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth.
  • Amino acids have the general structure NH2CHRCOOH, wherein R is a side chain specific to each amino acid.
  • An "amino acid” as used herein includes the 20 standard amino acids encoded by the universal genetic code (i.e., glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid) and non-standard amino acids, including derivatives of standard amino acids, such as analogs, and synthetic amino acids.
  • the universal genetic code i.e., glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine,
  • Non-limiting examples of nonstandard amino acids include aceglutamide, N-acetylaspartic acid, acetyl carnitine, acetylcysteine, N-acetylglutamic acid, acetylleucine, N-acetyl serine, S-adenosyl-L- homocysteine, ⁇ -alanine, alanosine, alloisoleucine, a-aminobutyric acid, ⁇ -aminobutyric acid (GABA), 2-aminoisobutyric acid, argitine, aspartame, carbocisteine, 4- chlorophenylalanine, citrulline, ⁇ -cyclohexyl alanine, dehydroalanine, dibromotyrosine, levodopa (L-DOPA), 4-fluorophenylalanine, N-formylmethionine, glutaurine, glycocyamine, 4-homoarginine, homoc
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length can be linear or branched, it can comprise modified amino acids and non-amino acids can interrupt it.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polypeptides can occur as single chains or associated chains.
  • a "polynucleotide” or “nucleic acid” or “nucleic acid molecule” or “nucleic acid sequence” refers to a polymer of nucleotides of any length, and includes DNA and RNA.
  • the polynucleotides can be isolated or recombinant.
  • the polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • a "vector” is a construct that is capable of delivering and, in some
  • vectors include viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
  • an "isolated" molecule e.g., an isolated polypeptide or an isolated
  • polynucleotide is one that is in a form not found in nature, including those which have been purified.
  • isolated as used herein with respect to nucleic acids, such as DNA or RN A, refers to molecules separated from other DNAs or RNAs that are present in the natural source of the macromolecule.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments, and would not be found in the natural state.
  • isolated as used herein to refer to polypeptides and/or proteins includes those that are isolated from other cellular proteins, and is meant to encompass both purified and recombinant polypeptides. In some embodiments, an isolated molecule is substantially pure.
  • the term “substantially pure” refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.
  • the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragments) thereof, are normally associated in nature.
  • an "isolated” cell is one that is not incorporated into an organism.
  • An isolated cell can be part of a multicellular tissue or culture. As is apparent to those of skill in the art, a non-naturally occurring
  • polynucleotide, peptide, polypeptide, protein, antibody or fragments) thereof does not require “isolation” to distinguish it from its naturally occurring counterpart.
  • a "label” is a detectable compound that can be conjugated directly or
  • the label can be detectable on its own (e.g., radioisotope labels or fluorescent labels) or can catalyze chemical alteration of a substrate compound or composition that is detectable (e.g., an enzymatic label).
  • inhibitor refers to any statistically significant decrease in biological activity, including full blocking of the activity.
  • inhibitor can refer to a decrease of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in biological activity.
  • Active agents include protective agents and diagnostic agents.
  • An active agent preferably means the conjugates of the present invention, but can also include any substance disclosed in at least one of: The Merck Index, 15th Edition (2013); Pei-Show Juo, Concise Dictionary of Biomedicine and Molecular Biology, (2001); U.S. Pharmacopeia Dictionary of US AN & International Drug Names (2014); and Physician's Desk Reference, 70th Edition (2016). See also Stedman's Medical Dictionary, 28th Edition (2013). The identity of the "active agent" will be apparent from the context.
  • Solvate refers to a compound or salt thereof that further includes a
  • the solvate is a hydrate.
  • pharmaceutically acceptable salt refers to salts that retain the biological effectiveness and properties of a molecule, and which are not undesirable for use in a pharmaceutical composition.
  • the ascorbic acid-amino acid conjugates of the invention are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups, or groups similar thereto.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, as described, for example, in WO 87/05297.
  • composition refers to a preparation in which the active agent is in an effective form, i.e., has biological activity, and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.
  • a composition can be sterile and can comprise a
  • pharmaceutically acceptable carrier such as physiological saline.
  • compositions can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. human albumin), a preservative (e.g. benzyl alcohol), an absorption promoter to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.
  • a buffer e.g. acetate, phosphate or citrate buffer
  • a surfactant e.g. polysorbate
  • a stabilizing agent e.g. human albumin
  • a preservative e.g. benzyl alcohol
  • an absorption promoter to enhance bioavailability e.g. benzyl alcohol
  • a "subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, sports animals, and laboratory animals including, e.g., humans, non-human primates, canines, felines, porcines, bovines, equines, rodents, including rats and mice, rabbits, etc.
  • an “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • a “therapeutically effective amount” refers to that amount (at dosages and for periods of time necessary) of an active agent which, when administered to a subject in need thereof, is sufficient to effect treatment.
  • the amount that constitutes a “therapeutically effective amount” will vary depending on the active agent being administered, the condition or disease and its severity, and the weight, age, etc. of the subject to be treated.
  • Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder.
  • a subject is successfully "treated” for a disease or disorder according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.
  • Prevent or “prevention” refer to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder.
  • those in need of prevention include those prone to have or susceptible to the disorder.
  • a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.
  • the invention provides non-naturally occurring compounds comprising an ascorbic acid moiety conjugated to an amino acid moiety.
  • L-ascorbic acid has Formula III:
  • the ascorbic acid moiety is not limited with respect to its form, and any known ascorbic acid, ascorbate, or ascorbate derivative can be used.
  • any pharmaceutically acceptable salt, hydrate, or other solvate of ascorbic acid is suitable for use in the compounds of the invention.
  • the invention provides conjugates in which ascorbic acid or a derivative thereof is covalently attached via an ester linkage at C 6 to the carboxy end of an amino acid or dipeptide.
  • the ester linkage is at C 2 or C 3 or C 5 of ascorbic acid.
  • ascorbic acid and an amino acid are covalently attached via a linker.
  • the linker can be any divalent linker known to those of skill in the art.
  • Non-limiting examples include aliphatic linkers of between 1-10 carbon atoms, poly(ethylene glycol) (PEG), poly(vinyl pyrrolidone) (PVP), poly(vinyl alcohol), poly(malic acid), N-(2-hydroxypropyl)methacrylamide (UPMA) copolymer, dextrin, hydroxy ethyl starch (HES), and polysialic acid.
  • PEG poly(ethylene glycol)
  • PVP poly(vinyl pyrrolidone)
  • UPMA N-(2-hydroxypropyl)methacrylamide copolymer
  • dextrin dextrin
  • HES hydroxy ethyl starch
  • the amino acid moiety can be any standard or non-standard amino acid(s) or derivative(s) thereof.
  • the amino acid can be a charged amino acid, such as lysine, arginine, histidine, aspartic acid, glutamic acid, or a derivative thereof.
  • the amino acid can be a polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, or a derivative thereof.
  • Conjugates of the invention can comprise more than one amino acid.
  • the conjugate comprises two, three, four, five, or six amino acids.
  • the amino acid moiety comprises two or more amino acids, they are preferably linked by a peptide bond.
  • the amino acids can be the same or different from one another.
  • Non-liming examples of certain ascorbic acid (Asc)-amino acid conjugates of the invention include variants of:
  • the conjugate is synthesized as a prodrug, wherein, upon administration to a subject, the prodrug is cleaved to release the conjugate.
  • a prodrug of an ascorbate conjugate can be a prodrug that decreases the hydrophilic character of the conjugate to enhance ocular penetration. See, e.g., Sloan, Kenneth B. Prodrugs: topical and ocular drug delivery . New York: Marcel Dekker, 1992.
  • At least one hydroxyl group of the amino acid moiety is modified to a substituted or unsubstituted carbonyl or acetyl group. These modifications stabilize the conjugates for formulation and administration, and can decrease toxic side effects in a subject to whom they are administered.
  • Molecules of the invention which increase transport of aqueous humor, can be modified in an effort to increase the ability of the molecule to enter the eye.
  • Examples may include, but are not limited to, the addition of cleavable ester groups or other leaving groups, or the addition of groups that are a substrate for native enzymes or metabolic pathways in the eye.
  • the amino acid(s) can optionally be modified or protected with a variety of protecting groups.
  • the protecting group can be any chemical moiety capable of addition to and, optionally, removal from a functional group on an amino acid ⁇ e.g., the N- terminus, the C-terminus, or a functional group associated with the side chain of an amino acid within the conjugate) to allow for chemical manipulation of the amino acid(s).
  • Embodiments of the invention include isolated trabecular meshwork cells comprising one or more ascorbic acid-amino acid conjugates of the invention ("modified trabecular meshwork cells").
  • the trabecular meshwork cells are comprised in a pharmaceutical composition configured for intraocular delivery or delivery to a region of the trabecular meshwork.
  • the trabecular meshwork cells may be cultured cells from suspension, cell, or organ cultures.
  • Conjugates of the invention can be prepared by methods known to the skilled artisan.
  • conjugates of the invention can be prepared according to methods adapted from Zhao et al , Eur. J. Med. Chem. 82:314-323 (2014) (Bom protection); Hong et al., Chem. Commun. 50: 1 1649-1 1652 (2014) (DOPA protection); Palladino et al , Org. Lett. 14:6346-6349 (2012) (HCl/HFIP deprotection); Kartha et al , Carbohydr. Res. 339:729-732 (2014) (MeOH/MeONa); and Sheppeck et al, Let. Lett. 41 :5329-5333 (2000) (thiol deprotection) (FIG. 12).
  • Step 1 To a round-bottom flask (RBF), 1 equiv. ascorbic acid, 55 equiv. dichloromethane (DCM), and 4 equiv. triethylamine (Et 3 ) are added. After cooling the solution to 0 °C, 3 equiv. benzyl chloromethyl ether (BOMCl) is added and the solution is allowed to warm to room temperature. When the reaction is complete (by TLC analysis), the crude mixture is extracted with DCM against 1 N HC1. The combined organic layers are dried with MgS0 4 and concentrated in vacuo. Purification by column
  • Step 2 To a RBF, 1.0-1.5 equiv. di-BOM ascorbate, 1.0-1.5 equiv. of an amino acid of interest, 1.0-1.5 equiv. ethyl(hydroxyamino)cyanoacetate (Oyxma), and 1.0-1.5 equiv. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) are added.
  • the RFB is cooled to 0 °C and 50-80 equiv. of either N,N- dimethylformamide (DMF) or acetonitrile (ACN) is added.
  • DMF N,N- dimethylformamide
  • ACN acetonitrile
  • Step 3 To a RBF, 1.0 equiv. ascorbate ester, 0.5-2.0 equiv. base (sodium
  • Step 4 To a RBF, 1.0-1.5 equiv. free-amine ascorbate, 1.0-1.5 equiv. of a second amino acid of interest, 1.0-1.5 equiv. Oyxma, and 1.0-1.5 equiv. EDCI are added.
  • the RBF is cooled to 0 °C and 50-80 equiv. of either DMF or ACN is added.
  • the reaction is complete (by TLC analysis), the crude mixture is extracted with EtOAc against 1 N HC1. The combined organic layers are dried with MgS0 4 and concentrated in vacuo. Purification by column chromatography gives the di-peptide derivative of ascorbate in 30-80% yield.
  • Step 5 To a RBF, 1.0 equiv. dipeptide ascorbate, 30-100 equiv. acid
  • Conjugates of the invention can be combined with a carrier or vehicle to provide a composition comprising an ascorbic acid-amino acid conjugate.
  • the composition comprises two or more different ascorbic acid-amino acid conjugates.
  • composition can be a pharmaceutical composition, comprising a
  • aqueous and non-aqueous carriers examples include water, salt solutions (such as sodium chloride and potassium chloride), ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), polyethers (such as polyethylene glycol), polyvinyls (such as polyvinyl alcohol and povidone), cellulose derivatives (such as methylcellulose and hydroxypropyl methylcellulose), petroleum derivatives (such as mineral oil and white petrolatum), animal fats (such as lanolin), polymers of acrylic acid (such as carboxypolymethylene gel), vegetable fats (such as peanut oil and olive oil), organic esters, such as ethyl oleate, polysaccharides (such as dextrans),
  • salt solutions such as sodium chloride and potassium chloride
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • polyethers such as polyethylene glycol
  • polyvinyls such as polyvinyl alcohol
  • glycosaminoglycans such as sodium hyaluronate
  • suitable mixtures thereof such as sodium hyaluronate
  • a suitable pharmaceutical composition can comprise one or more buffers (e.g. acetate, phosphate, citrate), surfactants (e.g. polysorbate), stabilizing agents (e.g. human albumin), and/or salts (e.g., acid addition salts, base addition salts) etc.
  • buffers e.g. acetate, phosphate, citrate
  • surfactants e.g. polysorbate
  • stabilizing agents e.g. human albumin
  • salts e.g., acid addition salts, base addition salts
  • microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like.
  • Isotonic agents such as sugars, sodium chloride, and the like, can also be added into the compositions.
  • prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • a pharmaceutical composition provided herein can also include a
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite, and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite, and the like
  • oil- soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytol
  • the pH of the composition will vary depending on the route of administration.
  • a topical solution containing the active agent can be adjusted to a pH of about 5 to about 11.
  • the pH can be about 6.5, about 7, about 7.5, about 8, or about 8.5.
  • the pH is about 7.4.
  • the invention provides a method of decreasing intraocular pressure by administering to a subject in need thereof one or more ascorbic acid-amino acid conjugates of the invention.
  • Embodiments of the invention further provide a method of treating or
  • Embodiments of the invention also provide a method of treating or preventing glaucoma by administering to a subject in need thereof one or more ascorbic acid-amino acid conjugates of the invention.
  • the glaucoma is open-angle glaucoma.
  • the treatment of glaucoma is tailored to the individual patient. Multiple variations and combinations of ascorbic acid-amino acid conjugates of the invention, in different concentrations, can be administered to different patients.
  • Methods of treatment may include the measurement of IOP prior to administration of the therapeutic agent, selection of one or more conjugates based on the desired reduction in IOP or target pressure, and administration of the selected conjugate(s).
  • IOP can be monitored during therapy, and different conjugates or combinations of conjugates can be selected and administered to maintain a desired pressure; for example, between 10 and 20 mm Hg, or between about 15 to about 18 mm Hg.
  • Embodiments of the invention provide a method of increasing aqueous
  • Embodiments of the invention additionally provide a method of screening a candidate ascorbic acid-amino acid conjugate for its ability to modify fluid outflow through trabecular meshwork tissue. Methods of the invention can be performed in vivo in the eye of a subject, or ex vivo.
  • the ascorbic acid-amino acid conjugate(s) can be introduced into trabecular meshwork cells ex vivo to provide modified trabecular meshwork cells comprising one or more ascorbic acid-amino acid conjugates of the invention.
  • the modified trabecular meshwork cells can be introduced onto the trabecular meshwork or into the anterior chamber of the eye of a subject.
  • Modified trabecular meshwork cells can be administered to a subject to lower intraocular pressure, to treat or prevent glaucoma, and/or to treat or prevent a disorder associated with high intraocular pressure.
  • the trabecular meshwork cells can be applied directly onto the trabecular meshwork.
  • the modified trabecular meshwork cells are administered intracamerally by passing a blade, needle, applicator, or delivery system through the cornea.
  • the trabecular meshwork cells are released into the anterior chamber of the eye, where the aqueous drainage will carry them into the trabecular meshwork. See e.g., Yue et al., Graefes Arch. Clin. Exp. Opthalmol. 226:262-268 (1988); Russell et al, Invest. Opthalmol. Vis. Sci. 49: 629-635 (2008); Gasiorowski et al., Exp. Eye Res.
  • the modified trabecular meshwork cells are administered in free solution.
  • the modified trabecular meshwork cells can be administered under direct or indirect visualization.
  • the trabecular meshwork can be contained in a paste-like erodible carrier under direct viewing by gonioscopy.
  • the trabecular meshwork cells can be xenogenic, allogenic, or autogenic.
  • the trabecular meshwork cells comprising ascorbic acid-amino acid conjugates of the invention can be from the subject to whom they are administered (i.e., homologous). However, because the eye is a relatively immune-privileged site, the modified trabecular meshwork cells can be heterologous, without the need for tissue typing or
  • the trabecular meshwork cells are grown in vitro, e.g., in tissue culture, suspension culture, organ culture, etc. In other embodiments, the trabecular meshwork cells are harvested from eye bank tissue.
  • conjugates of the invention can be utilized to treat other disorders in which fluid outflow regulation is dysfunctional, for instance, in an artificial kidney or in hydrocephalus.
  • Another method of the embodiments of the invention involves administering to a subject in need thereof an enzyme or enzymatic fragment having the activity of forming a conjugate comprising ascorbic acid and an amino acid or dipeptide.
  • a further method of the embodiments of the invention involves administering to a subject in need thereof an enzyme or enzymatic fragment having the activity of linking an ascorbic acid- amino acid conjugate to a transmembrane protein, which transmembrane protein is capable of forming a transmembrane pore.
  • the enzyme or enzymatic fragment can be a kinase that is capable of phosphorylating ascorbate, for example, phospholipase D, which can be used to synthesize 6-phosphatidyl-L-ascorbic acid (Nagao et al, Lipids 26:390-94 (1991)).
  • the enzyme or enzymatic fragment can be an esterase.
  • Suitable enzymes and enzymatic fragments may include phosphatases, phosphodiesterases, nucleases, proteases, transferases, ribosomal and non- ribosomal synthetases, and other enzymes that catalyze condensation reactions ⁇ e.g., amide- and ester-forming condensation enzymes), etc.
  • a further method of the embodiments of the invention involves administering to a subject in need thereof a nucleic acid encoding the enzyme or enzymatic fragment discussed above.
  • a composition comprising the nucleic acid can consist essentially of the nucleic acid or a vector comprising the nucleic acid, optionally in an acceptable carrier.
  • Viral vectors include retroviruses, lentiviruses, other RNA viruses, such as poliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV 40, vaccinia, and other DNA viruses. Replication-defective murine retroviral or lenti viral vectors are widely utilized gene transfer vectors.
  • the nucleic acid can be introduced through the use of fusogenic lipid vesicles, such as liposomes or other vesicles for membrane fusion.
  • a carrier harboring a nucleic acid of interest can be conveniently introduced into the eye or into body fluids or the bloodstream.
  • the carrier can be site specifically directed to the target organ or tissue in the body.
  • Cell or tissue specific DNA-carrying liposomes for example, can be used and the foreign nucleic acid carried by the liposome absorbed by those specific cells.
  • Gene transfer may also involve the use of lipid-based molecules which are not liposomes.
  • lipofectins and cytofectins are lipid-based molecules containing positive ions that bind to negatively charged nucleic acids and form a complex that can ferry the nucleic acid across a cell membrane.
  • Delivery of gene therapy may also be accomplished via cationic polymers.
  • Certain cationic polymers spontaneously bind to and condense nucleic acids such as DNA into nanoparticles.
  • proteins, peptides, or derivatives thereof have been used.
  • Synthetic cationic polymers such as polyethylenimine (PEI), polylysine (PLL) etc. condense DNA and are useful delivery vehicles. Dendrimers can also be used.
  • Many useful polymers contain both chargeable amino groups, to allow for ionic interaction with the negatively charged DNA phosphate, and a degradable region, such as a hydrolyzable ester linkage. Examples include poly(alpha-(4-aminobutyl)-L-glycolic acid), network poly(amino ester), and poly (beta- amino esters). These complexation agents can protect nucleic acids against degradation, e.g., by nucleases, serum components, etc., and create a less negative surface charge, which may facilitate passage through hydrophobic membranes (e.g., cytoplasmic, lysosomal, endosomal, nuclear) of the cell. Certain complexation agents facilitate intracellular trafficking events such as endosomal escape, cytoplasmic transport, and nuclear entry, and can dissociate from the nucleic acid.
  • hydrophobic membranes e.g., cytoplasmic, lysosomal, endosomal, nuclear
  • composition can comprise a drug-release regulating
  • the composition can comprise a cell that expresses the nucleic acid.
  • such cells secrete the enzyme or enzymatic fragment encoded by the nucleic acid into the extracellular space.
  • embodiments of the invention including topical, intraocular, systemic, cellular, and via a delivery device.
  • compositions comprising conjugates of the invention can be administered topically to the eye via drops, spray, gel, cream, ointment, or eye wash.
  • the active agent is in a liquid or gel suspension.
  • the active agent can be in liposomes.
  • compositions comprising conjugates of the invention can be administered by intraocular injection, performed periodically.
  • the active agent can be administered via subconjunctival injection, in others, through intracameral (anterior chamber), intravitreal, or subscleral injection.
  • the composition can be delivered directly to Schlemm's canal via catheter or implanted shunt.
  • the active agent can be administered systemically, via direct intra-operative instillation of a gel, cream, or liquid suspension.
  • the active agent can be administered, for example, by sustained release implants and microspheres for intracameral or anterior vitreal placement within a biodegradable or bioerodible polymer that releases a therapeutic amount of the active agent over a period of time ranging up to a year or more.
  • the active agent can be administered by an implanted drug delivery system which releases a therapeutically effective amount of the conjugates over time. Implantation of the drug delivery system may be surgical or via injection.
  • the active agent is delivered by iontophoresis.
  • the active agent is delivered by ultrasound.
  • the active agent can be administered via the mucosal route, for example, using nasal drops of a liquid formulation.
  • the active agent can also be delivered via a nasal spray or aerosol suspension of respirable particles, which the subject inhales.
  • the therapeutic molecule is absorbed into the bloodstream via the lungs and subsequently contacts the ocular tissues in a pharmaceutically effective amount.
  • the respirable particles are a liquid or solid, with a particle size sufficiently small to pass through the mouth and larynx upon inhalation; in general, particles ranging from about 1 to 10 microns, but more preferably 1-5 microns, in size are considered respirable.
  • the active agent is delivered orally, in the form of a tablet, lozenge, aqueous or oily suspension, dispersible powder or granules, emulsion, hard or soft capsule, syrup, or elixirs.
  • Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active agent in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
  • ascorbic acid-amino acid conjugates of the invention are delivered orally via an enteric coated capsule or via a capsule, wherein the oral formulation is configured for absorption in the small intestine, such that the active agent is shielded from the low pH of stomach acid.
  • An additional means of systemic administration is in the form of a suppository comprising the active agent, such that a therapeutically effective amount of the active agent reaches the eyes or other target site via systemic absorption and circulation.
  • the active agent can be infused into the tear film via a pump-catheter system.
  • Another embodiment involves the active agent contained within a continuous or selective-release device, for example, membranes such as, but not limited to, those employed in the OcusertTM System (Alza Corp., Palo Alto, Calif).
  • the active agent can be contained within, carried by, or attached to contact lenses, which are placed on the eye.
  • Another embodiment of the invention involves the active agent contained within a swab or sponge, which is applied to the ocular surface.
  • composition comprising ascorbic acid-amino acid conjugates can be administered as a single dose or multiple doses.
  • the composition can be administered as many times as needed to achieve a targeted endpoint, such as a particular IOP.
  • Administration intervals may vary.
  • topical or systemic administration is 1, 2, 3, or 4 times per day, or once every 2, 3, 4, 5 or 6 days, once per week, twice per week, every other week, twice per month, or once per month.
  • intraocular delivery is performed once every 1, 2, 3, 4, 5, 6, or 9 months, once per year, or once every 18 months, or once every 2, 3, 4, or 5 years. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).
  • Conjugates of the invention can be administered in combination therapy
  • a method of the invention can comprise administering a second active agent.
  • the second active agent can be administered in the same or a different composition as the ascorbic acid-amino acid conjugate(s) of the invention.
  • the second active agent is selected from the group consisting of a prostaglandin analog, a beta blocker, an alpha agonist, and a carbonic anhydrase inhibitor.
  • the second active agent is an enzyme or enzymatically active fragment capable of catalyzing the conjugation of ascorbic acid to an amino acid. In one embodiment, the second active agent increases the activity of an enzyme or enzymatically active fragment capable of catalyzing the conjugation of ascorbic acid to an amino acid. In one embodiment, the second active agent is an enzyme or enzymatically active fragment capable of catalyzing the linking of an ascorbic acid conjugate to a transmembrane protein. In one embodiment, the enzyme or enzymatically active fragment has kinase activity. In one embodiment, the enzyme or enzymatically active fragment has esterase activity. In one embodiment, the second active agent is a nucleic acid encoding the enzyme or enzymatically active fragment.
  • kits comprising one or more ascorbic acid-amino acid conjugates and/or compositions comprising them, as provided herein, and instructions for use.
  • the kit can further contain at least one additional reagent.
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • kits that comprise one or more ascorbic acid-amino acid conjugates and/or compositions comprising them, which can be used to perform the methods described herein.
  • a kit comprises at least one type of ascorbic acid-amino acid conjugate of the invention in one or more containers.
  • the kits contain all of the components necessary and/or sufficient to perform a screening assay, including all controls, directions for performing assays, and, optionally, any necessary software for analysis and presentation of results.
  • the disclosed ascorbic acid- amino acid conjugates can be readily incorporated into one of the established kit formats which are well known in the art.
  • Embodiments of the present invention can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain liposome constructs of the present disclosure and methods for using liposome constructs of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.
  • tissue culture media and refrigerated at 2-8° C until initial extraction was performed.
  • the samples were extracted as follows: the tissue culture media was rinsed off with balanced salt solution and the trabecular meshwork was stripped off using microsurgical instruments and a dissecting microscope. The tissue was transferred to a 13 X 100 mm glass test tube. The eye tissue was ground with the end of a glass stirring rod. Tissue was noted to be very fibrous and resistant to disruption.
  • 1.0 mL of HPLC grade methanol was added to the tissue and mixed. The mixture was then sonicated in 37° C water bath for 1 hour. Next, 1.0 ml of HPLC grade chloroform was added and sonicated for another 20 minutes. The mixture was then centrifuged at 2500 rpm for 5 minutes. Next, the top layer was transferred to a clean vial and sealed. The chloroform-methanol mixture was evaporated to dryness and the resultant material reconstituted with 100 ⁇ L of HPLC mobile phase B.
  • conjugate having an exact mass of 446.18 and a molecular weight of 446.41 produces an asparagine-arginine group after fragmenting at the ester linkage (observed as the acylium ion) with a mass of 271 m/z, or a fragment resulting from cleaving off the guanidinium imine (CH2NC( 1 ⁇ 4) H + ) and having a mass of 73 m/z.
  • the conjugate structure may additionally or alternatively have an ascorbic acid- asparagine-arginine configuration.
  • the conjugate structure may additionally or alternatively have an ascorbic acid-cysteine- histidine configuration.
  • Fragmentation as shown in FIG. 10 of an ascorbic acid-cysteine-aspartic acid conjugate (Ci 3 Hi8N20ioS) having an exact mass of 394.07 and a molecular weight of 394.35 produces an aspartic acid-cysteine group after fragmenting at an ester linkage (observed as the acylium ion) with a mass of 219 m/z. Cleavage of the CO2H or the CH2CO2H from the conjugate produces fragments having a mass of 349 m/z or 335 m/z, respectively.
  • the conjugate structure may additionally or alternatively have an ascorbate- aspartic acid-cysteine configuration.
  • Positive ions 358.4 amu and 374.4 amu were approximately 18 amu apart from positive ions 340.4 amu and 356.4 amu.
  • Positive ions 340.4 amu and 356.4 amu had molecular weights of 181.2 amu and 196.3 amu, respectively, which suggests the loss of an H2O or ( H 4 ) + group.
  • the molecular weight of the positive 356.4 amu ion was 355.4 amu.
  • the molecular weight of the negative 337.3 amu ion was 338.3 amu.
  • the molecular weight of the negative 369.0 amu ion was 370.0 amu.
  • the therapeutic efficacy of ascorbic acid conjugates of the invention is tested using fresh donor eye tissue from normal eyes or eyes with glaucoma.
  • the anterior segment of the eye with a scleral rim of about 3-4 mm is resected.
  • the uveal tissue is removed from the internal surface of the eye.
  • the remaining anterior eye tissue is clamped to a holding device. Fluid is perfused into the bare anterior chamber. This process removes other means of outflow from the anterior chamber; therefore, the only remaining outflow tract is through the trabecular meshwork, Schlemm's canal, and the intra-scleral aqueous collector channels.
  • a candidate composition comprising one or more ascorbic acid conjugates of the invention is infused into the anterior chamber, allowed to incorporate into the cell membranes.
  • Pressure decay curves and/or flow rates are measured using standard methods, such as a pressure transducer, tonography, fluorophotometry, and/or constant-pressure perfusion, to determine whether the candidate composition comprising one or more ascorbic acid conjugates of the invention has a favorable effect on facility of outflow.
  • a mathematical model is used to measure the effectiveness of the candidate compound by studying changes in facility of outflow before and after the compound is introduced.
  • the Goldman equation is used, for example, to measure facility of outflow, which should increase as the function of the trabecular meshwork increases.
  • the model has also been used to evaluate changes in outflow with the addition of drugs such as dexamethasone (Clark et al, Invest. Ophthalmol. Vis. Sci. 36:478-489 (1995)) and to evaluate the efficacy of surgical treatment (e.g., stents) for the trabecular meshwork.
  • drugs such as dexamethasone (Clark et al, Invest. Ophthalmol. Vis. Sci. 36:478-489 (1995)) and to evaluate the efficacy of surgical treatment (e.g., stents) for the trabecular meshwork.
  • a non-human primate glaucoma model see, e.g., Rasmussen et al, J.
  • Glaucoma 14:311-314 (2005) can be used to evaluate the efficacy of the ascorbic acid- amino acid conjugates of the invention.
  • Intraocular pressure (IOP) and tonography (outflow measure) are measured at the initiation and terminus of the study using known methods.
  • Test animals are divided into Groups 1, 2, 3, and 4.
  • eye drops comprising an ascorbic acid-amino acid conjugate of the invention in a pharmaceutical buffer of pH 7.4 are administered to one eye of the test animals; the contralateral eye acts as control and is administered vehicle alone. Drops are administered three times daily at doses shown in Table 1.
  • IOP intraocular pressure
  • the patient is treated with a loading dose of eye drops 2-3 times per day
  • the IOP is reduced and the medication is delivered twice daily.
  • the drops are tapered to the minimum dosing regimen required to maintain a therapeutic effect.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Provided are ascorbic acid-amino acid conjugates and compositions for use in methods of lowering intraocular pressure and/or treating glaucoma by restoring the filtration capabilities of the endothelial lining of Schlemm's canal.

Description

ASCORBIC ACID-AMINO ACID CONJUGATES
AND THEIR USE IN REGULATION OF FLUID OUTFLOW
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S. provisional application
No. 62/356,432, filed on June 29, 2016, and incorporated herein in its entirety for all purposes.
BACKGROUND
[0002] Glaucoma is a leading cause of blindness. The American Academy of
Ophthalmology now defines glaucoma as "a group of diseases with certain features including an intraocular pressure that is too high for the continued health of the eye." Intraocular pressure remains the main treatable aspect of glaucoma therapy. If untreated, glaucoma can lead to destruction of the optic nerve and blindness. A clear fluid called aqueous humor is constantly formed by the ciliary bodies and secreted into the posterior chamber. This fluid passes over the lens and enters the anterior chamber. Aqueous humor passes out the anterior chamber of the eye at approximately the same rate at which it is produced through one of two routes. Approximately 10% of the fluid percolates between muscle fibers of the ciliary body, and approximately 90% of the fluid is removed via the "canalicular route," through a filter-like mass of tissue called the trabecular meshwork and Schlemm's canal, and then enters the scleral venous network, as described in greater detail below.
[0003] Glaucoma can result from the disordered drainage of the aqueous humor from the anterior chamber 40 (FIG. 1A) of the eye, across the trabecular meshwork 56 (FIG. 1A) and into Schlemm's Canal 54 (FIG. 1A). The juxacanilicular trabecular meshwork is the site of the increased resistance to outflow of aqueous. However, the precise mechanism of aqueous transport is poorly understood. It is known that the process, in a normal eye, is energy independent and self-regulating, such that the pressure of the eye remains between 10-21 mm Hg. The outflow rate from the anterior chamber of the eye generally matches the production rate of aqueous humor in the ciliary bodies of the eye 74 (FIG. IB)
[0004] In the normal eye (FIG. 1A, IB), aqueous humor flows through the trabecular meshwork 56, into Schlemm's canal 54, exiting Schlemm's canal by the intra-scleral aqueous collector channels 70. The collector channels terminate on the surface of the eye, forming aqueous veins of Ascher, which then drain into the venous system. After passing through the trabecular meshwork, aqueous humor crosses the endothelial cells of Schlemm's canal. In this manner, trabecular meshwork cells and Schlemm's canal endothelial cells are thought to comprise the cells of the primary outflow pathway of the eye.
[0005] The trabecular meshwork and Schlemm's canal are located at the junction between the iris 46 and the sclera 72. The cornea 50, lens 35, and pupil 44 are also shown. The trabecular meshwork is wedge shaped in structure and runs around the entire circumference of the eye, forming a three-dimensional sieve structure. The trabecular meshwork is formed of collagen beams aligned with a monolayer of cells called the trabecular cells, which produce an extracellular substance which fills the spaces between collagen beams. The trabecular meshwork is suspended between the corneal endothelium and the ciliary body face, and is comprised of a series of parallel layers of thin, flat, branching, and interlocking bands termed trabeculae. The inner portion of the trabecular meshwork (closest to the iris root and ciliary body 74) is called the uveal meshwork, whereas the portion closest to the sclera and cornea is called the corneoscleral trabecular meshwork.
[0006] The uveal meshwork trabeculae measure approximately 4 μπι in diameter, consist of a single layer of cells surrounding a collagen core, and are arranged in interconnected layers. The spaces between these trabeculae are irregular and range from about 25 μπι to about 75 μπι in size.
[0007] The trabeculae of the corneoscleral meshwork resemble broad, flat endothelial sheets about 3 μπι thick and up to about 20 μπι long. The spaces between these trabeculae are smaller than in the uveal meshwork and more convoluted. As the lamellae approach Schlemm's canal, the spaces between the trabeculae decrease to about 2 μπι. The resistance to aqueous humor outflow through the trabecular meshwork has been reported to reside primarily in the juxtacanalicular meshwork (JCM). At this site, two cell types are found: trabecular meshwork cells and endothelial cells of the inner wall of Schlemm's canal. Treatments, both medical and surgical, have attempted to reduce intraocular pressure by increasing the permeability of the trabecular meshwork, creating new outflow pathways, or widening Schlemm's canal. However, these do not adequately address the JCM as the primary source of resistance to outflow. [0008] In contrast to the current level of knowledge regarding cellular processes responsible for aqueous humor production by the ciliary body, relatively little is known about the cellular mechanisms in the trabecular meshwork that determine the rate of aqueous outflow. Pinocytotic vesicles have been observed in the JCM and the inner wall of Schlemm's canal. The function of these vesicles remains unknown, but some investigators have suggested that the bulk flow of aqueous humor through the meshwork cannot be accounted for by flow through the intercellular spaces, and that these vesicles play a central role in outflow regulation. Management of outflow by regulation of ion channels in the cell membranes of the JCM and lining of Schlemm's canal has been proposed. However, it is proposed that a different mechanism, an osmotic drive, is responsible for the regulation of outflow of aqueous humor through the JCM. This osmotic drive is self-regulating, such that changes in intraocular pressure lead to corresponding changes in the rate of outflow so that a relatively constant pressure is maintained.
[0009] There are a number of different forms of glaucoma, including open- angle and closed-angle glaucoma, as well as steroid induced glaucoma. The most common form of glaucoma is open-angle, which results from increased resistance in the outflow pathway through the trabecular meshwork. As discussed above, the mechanism by which the outflow pathway becomes blocked or inadequate is poorly understood, but the result is an increase in pressure within the eye, which compresses the axons in the optic nerve and can compromise vascular supply to the nerve. Over time, this can result in partial or total blindness. The trabecular meshwork is not physically obstructed, but no longer efficiently transports fluid between the anterior chamber and the scleral drainage veins.
[0010] Current treatment of glaucoma is either medical, surgical, or both.
Medications for the treatment of glaucoma include prostaglandin analogs, which increase fluid percolation between muscle fibers of the ciliary body, and miotics, which are administered as drops and cause contraction of the pupil of the eye by tightening the muscle fibers of the iris to increase the rate at which the aqueous humor leaves the eye. Epinephrine drops have also been successful in reducing intraocular pressure, but have significant side effects. Other medications are employed, such as β-adrenergic blocking agents, as drops, or carbonic anhydrase inhibitors as pills, which reduce the production of fluid. [0011] Surgical solutions include applying a laser to multiple spots along the trabecular meshwork, which is thought to change the extracellular material and enhance outflow. Approximately 80% respond initially to this treatment, but, unfortunately, 50% have increased pressure within five years. Other solutions attempt to increase the permeability of the trabecular meshwork or widen Schlemm's canal. Another surgical procedure is a trabeculectomy, wherein an incision is made in the conjunctiva to form a hole in the sclera for aqueous fluid to flow through. This is performed through an open surgical procedure and includes long term risk of infection or injury to the eye.
Frequently the hole closes up over time with consequent increase in pressure. A variety of large apparatuses have been suggested, such as implantation of an aqueous shunt which produces an equatorial filtering bleb. Recently, Micro Invasive Glaucoma Surgeries (MIGS) have been described which drain across the trabecular network, draining to Schlemm's canal or through the eye way onto the surface of the sclera. Alternatively, some treatments have targeted the pores between endothelial cells lining Schlemm's canal.
[0012] A need remains for a way of safely, lastingly, and effectively treating the cause of open-angle glaucoma. Current medical and surgical treatment options often lose their efficacy with time. Furthermore, surgical treatments have associated risks of infection or injury to the eye, and current medical solutions often come with significant side effects either affecting vision, the structures of the eye, or with systemic side effects. A need also exists for a treatment of glaucoma which addresses the underlying pathology in the aqueous humor outflow system and leads to a return of drainage as seen in non- glaucomatous eyes. There exists a need, as well, for improved models of testing drugs ex vivo for use in this ophthalmic application.
SUMMARY OF THE EMBODPMENTS OF THE INVENTION
[0013] Some of the main aspects of the embodiments of the present invention are summarized below. Additional aspects are described in the Detailed Description of Embodiments of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention. [0014] This disclosure provides, in certain embodiments, methods and compositions for treating glaucoma. More particularly, the treatment of glaucoma may involve restoring the filtration capability of the trabecular meshwork of the eye. The disclosure is based on the discovery of ascorbic acid-amino acid conjugates in trabecular meshwork tissue, as analyzed by neutral loss mass spectrometry. This provides a potential mechanism for regulation of intraocular pressure.
[0015] Molecules of the invention include improved ascorbic acid-mono amino acid conjugates having Formula I and ascorbic acid-di amino acid conjugates having Formula II:
(I)
Figure imgf000006_0001
(Π)
Figure imgf000006_0002
wherein SCi and SC2 are the same or different amino acid side chains;
wherein Ri, R2, R3, and R5 are independently selected from -H, a carbonyl group selected from -CHO and -COX, wherein X is a Ci-6 branched or unbranched alkyl, and an acetyl group selected from CH3CO- and CH3(CH2)nCO-, wherein n is 1-6;
wherein R4 and R5 are independently selected from -H, -CH3, -CH2CH3, and -CF3; and
wherein R7 is H or CH3;
with the proviso that at least one of R1-R7 is not H.
[0016] One or more C-H in Formulas I and II can be replaced with C-F, C-Cl, C-Br, or C-I.
[0017] In some embodiments, SCi of Formula I is selected from the group consisting of: an L-DOPA side chain, a Tyr side chain, an Arg side chain, and a Sec side chain. In some embodiments, SCi and SC2 of Formula II are, respectively, (i) an Asn side chain and an Arg side chain, (ii) an Arg side chain and an Asn side chain, (iii) a Cys side chain and an Asp side chain, (iv) an Asp side chain and a Cys side chain, (v) a His side chain and a Cys side chain, (vi) a Cys side chain and a His side chain, (vii) a Glu side chain and an Ala side chain, (viii) a Lys side chain and an Ala side chain, (ix) a Phe side chain and an Ala side chain, (x) a Sec side chain and an Ala side chain, (xi) a Ser side chain and an Asn side chain, (xii) a Gly side chain and a Cys side chain, (xiii) an L-DOPA side chain and a Cys side chain, (xiv) a Ser side chain and a Gin side chain, (xv) a Tip side chain and a Gin side chain, (xvi) an Ala side chain and a Glu side chain, (xvii) a Cys side chain and a Gly side chain, (xviii) a Ser side chain and a Gly side chain, (xix) a Val side chain and a Gly side chain, (xx) a Cys side chain and an L-DOPA side chain, (xxi) an Ala side chain and a Lys side chain, (xxii) a Tip side chain and a Met side chain, (xxiii) an Ala side chain and a Phe side chain, (xxiv) an Ala side chain and a Sec side chain, (xxv) an Asn side chain and a Ser side chain, (xxvi) a Gin side chain and Ser side chain, (xxvii) a Gly side chain and a Ser side chain, (xxviii) a Gin side chain and a Tip side chain, (xxix) a Met side chain and Tip side chain, or (xxx) a Gly side chain and a Val side chain.
[0018] In some embodiments, SCi is not a tyrosine side chain. In some embodiments,
SC2 is not a tyrosine side chain. In some embodiments, SCi is not a levodopa (L-DOPA) side chain. In some embodiments, SC2 is not an L-DOPA side chain.
[0019] Embodiments of the invention provide an isolated trabecular meshwork cell comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
[0020] Also provided is a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II. In one embodiment, the composition comprises at least one conjugate of Formula I and at least one conjugate of Formula II. In one embodiment, the composition comprises an isolated trabecular meshwork cell comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II. In some embodiments, the composition of the invention is a pharmaceutical composition.
[0021] Compounds of the invention disclosed herein include all stereoisomers,
including diastereomers and enantiomers, regardless of the stereochemistry shown in representative structures. Compositions comprising conjugates of the invention can be racemic mixtures or can be enantiomerically and/or diastereomerically pure or enriched. [0022] Embodiments of the invention additionally provide a kit comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II, a kit comprising a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II, and a kit comprising an isolated trabecular meshwork cell comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II or a composition thereof.
[0023] In one aspect, the invention provides a method of reducing intraocular
pressure (IOP) in a subject in need thereof, the method comprising administering to the subject one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II or a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
[0024] In one aspect, the invention provides a method of treating high IOP in a
subject, the method comprising administering to the subject one or more ascorbic acid- amino acid conjugates of Formula I and/or Formula II or a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II.
[0025] In one aspect, the invention provides a method of treating glaucoma in a
subject, the method comprising administering to the subject one or more ascorbic acid- amino acid conjugates of Formula I and/or Formula II or a composition comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II. In some embodiments, the glaucoma is open-angle glaucoma.
[0026] In one embodiment, administration of the ascorbic acid-amino acid conjugates is topical, intraocular, systemic, or via a drug delivery device. In a particular
embodiment, administration is via eye drops. In another embodiment, administration is oral, preferably via tablet or capsule.
[0027] Embodiments of the invention further provide a method of increasing aqueous outflow through trabecular meshwork tissue, the method comprising exposing the trabecular meshwork tissue to one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II, optionally in a composition. In one embodiment, the method is performed ex vivo.
[0028] Embodiments of the invention additionally provide a method of screening a candidate ascorbic acid-amino acid conjugate for its ability to modify fluid outflow through trabecular meshwork tissue, the method comprising: measuring fluid outflow through trabecular meshwork tissue before and after exposing the trabecular meshwork tissue to the candidate ascorbic acid-amino acid conjugate; wherein a change in fluid outflow after exposure to the candidate ascorbic acid-amino acid conjugate indicates that the ascorbic acid-amino acid conjugate is able to modify fluid outflow. In some embodiments, the candidate ascorbic acid-amino acid conjugate has Formula I or Formula II. In certain embodiments, the method is performed ex vivo or in vivo in an animal model.
[0029] Also provided is a use of one or more ascorbic acid-amino acid conjugates of
Formula I and/or Formula II to reduce IOP in a subject in need thereof, or to treat high IOP in a subject, or to treat glaucoma in a subject. In some embodiments, the ascorbic acid-amino acid conjugate(s) is formulated for topical, intraocular, or systemic delivery, or for delivery via a drug delivery device.
[0030] Another embodiment of the invention is the use of an isolated trabecular
meshwork cell comprising one or more ascorbic acid-amino acid conjugates of Formula I and/or Formula II to reduce IOP in a subject in need thereof, or to treat high IOP in a subject, or to treat glaucoma in a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIGS. 1A-1B show schematic diagrams of the eye. FIG. 1A shows a cross- sectional illustration of the anterior portion of the eye. FIG. IB shows a cross-sectional illustration of the irido-corneal angle of the eye.
[0032] FIG. 2A-2C show schematics of particular structures within the eye. FIG. 2A shows a schematic of the trabecular meshwork and Schlemm's canal. FIG. 2B and FIG. 2C show schematics of the membrane of an endothelial cell in the juxtacanalicular lining of Schlemm's canal.
[0033] FIG. 3A-3F show the structure of ascorbic acid (FIG. 3A) and structures of ascorbic acid conjugates with tyrosine (FIG. 3B), L-DOPA (FIG. 3C), arginine- asparagine (FIG. 3D), histidine-cysteine (FIG. 3E), and cysteine-aspartic acid (FIG. 3F).
[0034] FIG. 4 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-tyrosine conjugate. The mass spectrum shows a second fragmentation of ions containing a neutral loss of a group with a mass of 176 m/z, indicating the loss of ascorbate. The peak at 164.0 m/z is consistent with the loss of C9HioN02 + from the larger molecule CisHnNOs. [0035] FIG. 5 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-tyrosine conjugate.
[0036] FIG. 6 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-L-DOPA conjugate. The mass spectrum shows a second fragmentation of ions containing a neutral loss of a group with a mass of 176 m/z, indicating the loss of ascorbate. The peak at 180.1 m/z is consistent with the loss of C9HioN03 + from the larger molecule C15H17NO9.
[0037] FIG. 7 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-L-DOPA conjugate.
[0038] FIG. 8 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate- arginine-asparagine conjugate.
[0039] FIG. 9 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-histidine-cysteine conjugate.
[0040] FIG. 10 shows a liquid chromatography-tandem mass spectrum of a trabecular meshwork sample extract and a chemical formula showing a proposed fragmentation of an ascorbate-aspartate-cysteine conjugate.
[0041] FIG. 11 shows raw mass spec data from a trabecular meshwork sample
extract. Masses of interest are at 340.4 m/z and 356.4 m/z. These protonated molecules are mass+1 of the expected species, 339.4 g/mol and 355.6 g/mol, respectively.
[0042] FIG. 12 shows a synthesis scheme for ascorbic acid-amino acid conjugates.
FIG. 12A shows synthesis of Asc-Tyr (Compound 1) and Asc-L-DOPA (Compound 2) conjugates. Reactants are as follows: A) BOMCl, Et3N, DCM; B) Compound 7, EDCI, Oxyma, MeCN; C) HC1, HHP. Abbreviations: BOM = benzyloxymethyl; Boc = t- butoxycarbonyl; t-Bu = tert-butyl; TBS = tert-butyldimethylsilyl; BOMCl = benzyl chlorom ethyl ether; Et3N = triethylamine; DCM = dichloromethane; EDCI = l-Ethyl-3- (3-dimethylaminopropyl)carbodiimide; Oxyma = ethyl (hydroxyimino)cyanoacetate; MeCN = acetonitrile; HFIP = l, l,l,3,3,3-hexafluoro-2-propanol. FIG. 12B shows synthesis of Asc-Glu-Cys (Compound 3a), Asc-Cys-Glu (Compound 3b), Asc-His-Cys (Compound 4a), Asc-Cys-His (Compound 4b), Asc-Arg-Asn (Compound 5a), and Asc- Asn-Arg (Compound 5b). Reactants are as follows: A) Compound 7, EDCI, Oxyma, MeCN; B) DBU, MeCN; C) Boc-Cys(Trt)-OH, EDCI, Oxyma, MeCN; D) HCl, HFIP; E) Boc-Asp(OtBu)-OH, EDCI, Oxyma, MeCN; F) Boc-His(Trt)-OH, EDCI, Oxyma, MeCN; G) Boc-Asn(Trt)-OH, EDCI, Oxyma, MeCN; H) Boc-Arg(Pbf)-OH, EDCI, Oxyma, MeCN. Abbreviations: Fmoc = 9-fluorenylmethoxycarbonyl; t-Bu = tert-butyl; BOM = benzyl oxymethyl; Boc = t-butoxycarbonyl; t-Bu = tert-butyl; Trt = triphenylmethyl; Pbf = 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; EDCI = 1 -Ethyl -3-(3- dimethylaminopropyl)carbodiimide; Oxyma = ethyl (hydroxyimino)cyanoacetate; MeCN = acetonitrile; DBU = l,8-diazabicyclo[5.4.0]undec-7-ene; Boc-Cys(Trt)-OH = N-{tert- Butoxycarbonyl^-trityl-L-cysteine; HFIP = l, l,l,3,3,3-hexafluoro-2-propanol; Boc- Asp(OtBu)-OH = a-fert-Butyl-N-Boc-L-aspartate; Boc-His(Trt)-OH = Na-Boc-N(im)- trityl-L-histidine; Boc-Asn(Trt)-OH = Na-Boc-N trityl-L-asparagine; Boc-Arg(Pbf)-OH = Na-Boc-Nco-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine; Na-Boc- Nco-Pbf-L-arginine. FIG. 12C shows the structures of the compounds synthesized in FIG. 12B.
DETAILED DESCRIPTION OF THE EMB ODFMENT S OF THE INVENTION
[0043] It is known that the levels of L-ascorbic acid in the aqueous humor (1.06
mmol/L; Arshinoff S. A., et al. "Ophthalmology", chapter 4.20.2, published by Mosby International Ltd., 1999) are about 20 times higher (Brubaker R. F. et al. "Investigative Ophthalmology & Visual Science", June 2000, vol. 41, No. 7, pp. 1681) than those present in the blood circulation (20-70 μιηοΐ/ΐ, Geigy Scientific Tables, vol. 3, page 132, 8th edition 1985, published by Ciba Geigy). In the case of the retina, the levels of L- ascorbic acid in the eye are actually 100 times higher than those present in the blood circulation.
[0044] Studies investigating the levels of ascorbic acid in the glaucomic eye (Pei-fei
Lee, MD et al., "Aqueous Humor Ascorbate Concentration and Open-Angle Glaucoma," Arch Ophthalmol. 1977; 95(2):308-310) and assessing the use of dietary antioxidants in preventing glaucoma (Jae H. Kang et al., "Antioxidant Intake and Primary Open-Angle Glaucoma: A Prospective Study," Am. J. Epidemiol. (2003) 158 (4): 337-346) show that the level of ascorbic acid did not appear to be predictably reduced in the glaucomic eye, nor does antioxidant use prevent or treat glaucoma. Treatments directed at use of ascorbic acid supplements have been proposed, theorizing that the antioxidant properties may play a role in maintaining reduced intraocular pressure (US Patent Application Publication No. 2006/0004089).
[0045] However, previously there has not been a satisfactory explanation for the
increased levels of L-ascorbic acid in the eye or an explanation of the role that it plays in maintaining normal function of the eye.
[0046] We describe herein the detection of ascorbic acid as a conjugate with amino acids, including tyrosine, aspartic acid-cysteine, histidine-cysteine, asparagine-arginine, or L-DOPA, in samples of eye tissue. Without wishing to be bound by theory, it appears that molecules comprising one or more amino acids associated with ascorbic acid, or a derivative or salt thereof, are present in normal eye tissue, specifically in the endothelial layer 324 of Schlemm's canal 366 (FIG. 2A) and/or the trabecular meshwork cells 320 (FIG. 2A), and may play a role in the maintenance of normal intraocular pressure by regulating drainage of the aqueous humor through the membranes of the JCM cells as part of an osmotic drive.
[0047] As seen in FIG. 2A, the trabecular meshwork 320 is separated from
Schlemm's canal 366 by a single layer of endothelial cells 324. Once the aqueous humor passes through the endothelial layer, it drains into Schlemm's canal and then into the scleral venous system by way of bridging vessels 370. In FIG. 2B, the cell membrane 340 of an endothelial cell is seen with lipophilic regions 342 and hydrophilic regions 344. FIG. 2C shows the endothelial cell membrane 380 with direction of aqueous humor travel indicated by the arrow facing Schlemm's canal 366. A proposed structure in the cell membrane of the endothelial layer 380 is shown as an arrangement of pores or "vents" 368, which bridge the cell membrane 380 and serve to transport aqueous humor across the membrane. After traversing the opposing membrane, the aqueous humor is released into Schlemm's canal.
[0048] Amino acid molecules comprising an ascorbic acid or ascorbic acid derivative head may be produced by the specialized cells of the JCM and transported to the cell membranes, where they most likely bond via coulombic or ionic bonds to one or more transmembrane proteins. Alternatively, the ascorbic acid or ascorbic acid derivative head may also be bound to an -OH group or other functional group of an amino acid, such as tyrosine or L-DOPA, already incorporated in a protein. A transmembrane cylinder (pore/vent) may be formed with enough protein molecules such that a hydrophilic interior is formed for water and solute transport. When intraocular pressure is low, the ascorbic acid heads form hydrogen bonds to each other, and as the intraocular pressure rises, the cellular osmolality drops and cell volume increases, placing the cell membrane on stretch. This allows the water molecules to compete increasingly effectively at the hydrogen binding sites, thereby allowing water and solute to pass through the channel. The increasing pressure from surrounding aqueous fluid may also play a mechanical role in distorting the cell membrane, thereby contributing to the dissociation of bonds between polar moieties and the consequent permeability to water molecules. Mechanical forces may also initiate pinocytotic vesicle formation that has been observed in the JCM and the inner wall of Schlemm's canal. As the intraocular pressure diminishes in response to increased flow, the bonds between polar moieties are increasingly favored over bonds with water molecules, and the flow diminishes, until equilibrium is reached. The equilibrium may change based on various factors, such as the rate of production of aqueous humor, but will be self-regulating to maintain a desired pressure.
[0049] The above-described conjugates, spanning the cell membrane, may result in a self-regulating osmotic drive for water transport out of the anterior chamber of the eye into Schlemm's canal. When the pressure is balanced, the ascorbic acid moieties will generally bond with each other and water molecules from the aqueous humor will transport between the hydrophilic heads relatively slowly at a steady state rate. However, even small increases or decreases in pressure may cause the establishment of a new equilibrium flow rate.
[0050] Open angle glaucoma may result from a failure in the osmotic drive described above. Because relatively normal concentrations of ascorbic acid have been observed in eye tissue of glaucoma patients, absorption, transport, and ingestion of ascorbic acid are not likely causes of the failure and, furthermore, would be expected to cause systemic problems related to vitamin C deficiency, rather than isolated intraocular pressure elevations. In some patients, failure of normal conjugation of ascorbic acid with an amino acid may result from enzyme deficiency, decreased enzyme activity, or other disturbance. These enzymes may be specific to the cells of the eye or JCM or may exist in other places, in which case the patient may have other manifestations in addition to glaucoma, and therapeutic molecules may treat those manifestations as well. In some patients, other pathologies may also result in the inability of the osmotic drive to assemble within the cell membrane. [0051] Embodiments of the present invention provide novel compounds and compositions comprising ascorbic acid-amino acid conjugates for regulating intraocular pressure by affecting the aqueous humor outflow system of the eye. In a particular embodiment, the ascorbate-amino acid conjugates of the invention can be used treat and/or prevent glaucoma.
[0052] The practice of the embodiments of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutical and protein chemistry, formulation science, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Handbook of
Pharmaceutical Excipients (7th ed., Rowe et al. eds., 2012); Martin 's Physical Pharmacy and Pharmaceutical Sciences (6th ed., Sinko, 2010); Remington: The Science and Practice of Pharmacy (21st ed., Univ. Sci. Philadelphia ed., 2005); Current Protocols in Molecular Biology (Ausubel et al. eds., 2016); Molecular Cloning: A Laboratory Manual (4th ed., Green and Sambrook eds., 2012); Lewin 's Genes XI (11th ed., Krebs et al. eds., 2012); DNA Cloning: A Practical Approach, Volumes I and II (2ά ed., Glover and Hames eds., 1995); Protein Engineering: A Practical Approach (1st ed., Rees et al. eds. 1993); Culture Of Animal Cells (6th ed. Freshney, 2010); Antibodies: A Laboratory Manual (2nd ed., Greenfield ed., 2013); Antibody Engineering (2d ed., Borrebaeck ed., 1995).
[0053] In order that the embodiments of the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, Dictionary of Pharmaceutical Medicine (3rd ed. Nahler and Mollet eds., 2013); The Dictionary of Cell and Molecular Biology (5th ed. J.M. Lackie ed., 2013), Oxford Dictionary of Biochemistry and Molecular Biology (2d ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of
Biomedicine and Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skill with general definitions of some terms used herein.
[0054] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. Definitions
[0055] As used in this specification and the appended claims, the singular forms "a,"
"an," and "the" include plural referents, unless the context clearly dictates otherwise. The terms "a" (or "an") as well as the terms "one or more" and "at least one" can be used interchangeably.
[0056] Furthermore, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
[0057] Wherever embodiments are described with the language "comprising,"
otherwise analogous embodiments described in terms of "consisting of and/or
"consisting essentially of are included.
[0058] Units, prefixes, and symbols are denoted in their Systeme International de
Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth.
[0059] "About" when used to modify a numerical value means ± 10% of the stated value.
[0060] Unless otherwise indicated, nucleic acid sequences are written left to right in
5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation. Nucleotides are referred to by their commonly accepted single-letter codes. Amino acids are referred to by their commonly known three-letter symbols or by the one- letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature
Commission.
[0061] Amino acids have the general structure NH2CHRCOOH, wherein R is a side chain specific to each amino acid. An "amino acid" as used herein, includes the 20 standard amino acids encoded by the universal genetic code (i.e., glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid) and non-standard amino acids, including derivatives of standard amino acids, such as analogs, and synthetic amino acids. Non-limiting examples of nonstandard amino acids include aceglutamide, N-acetylaspartic acid, acetyl carnitine, acetylcysteine, N-acetylglutamic acid, acetylleucine, N-acetyl serine, S-adenosyl-L- homocysteine, β-alanine, alanosine, alloisoleucine, a-aminobutyric acid, γ-aminobutyric acid (GABA), 2-aminoisobutyric acid, argitine, aspartame, carbocisteine, 4- chlorophenylalanine, citrulline, β-cyclohexyl alanine, dehydroalanine, dibromotyrosine, levodopa (L-DOPA), 4-fluorophenylalanine, N-formylmethionine, glutaurine, glycocyamine, 4-homoarginine, homocysteine, homoserine, hydroxyproline, 5- hydroxylysine, lisinopril, N-a-methylarginine, N-methyl-D-aspartic acid or N-methyl-L- glutamic acid, N-methylglycine, 3-methylhistidine, N-methylisoleucine, 3-(l- naphthyl)alanine, 3-(2-naphthyl)alanine, norleucine, ornithine, phenyl glycine, O- phosphoserine, pipecolinic acid, piperazic acid, 3-pyridylalanine, pyrroglutamine, pyrrolysine, sarcosine, selenocysteine (Sec), and valanine. Unless otherwise stated, the term "amino acid" and the names of individual amino acids include the L and the D enantiomers.
[0062] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids and non-amino acids can interrupt it. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. In certain
embodiments, the polypeptides can occur as single chains or associated chains.
[0063] A "polynucleotide" or "nucleic acid" or "nucleic acid molecule" or "nucleic acid sequence" refers to a polymer of nucleotides of any length, and includes DNA and RNA. The polynucleotides can be isolated or recombinant. The polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
[0064] A "vector" is a construct that is capable of delivering and, in some
embodiments expressing, one or more sequences of interest in a cell. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
[0065] An "isolated" molecule, e.g., an isolated polypeptide or an isolated
polynucleotide, is one that is in a form not found in nature, including those which have been purified. The term "isolated" as used herein with respect to nucleic acids, such as DNA or RN A, refers to molecules separated from other DNAs or RNAs that are present in the natural source of the macromolecule. The term "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments, and would not be found in the natural state. The term "isolated" as used herein to refer to polypeptides and/or proteins includes those that are isolated from other cellular proteins, and is meant to encompass both purified and recombinant polypeptides. In some embodiments, an isolated molecule is substantially pure. As used herein, the term "substantially pure" refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%. In other embodiments, the term "isolated" means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragments) thereof, are normally associated in nature. For example, an "isolated" cell is one that is not incorporated into an organism. An isolated cell can be part of a multicellular tissue or culture. As is apparent to those of skill in the art, a non-naturally occurring
polynucleotide, peptide, polypeptide, protein, antibody or fragments) thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart.
[0066] A "label" is a detectable compound that can be conjugated directly or
indirectly to a molecule, so as to generate a "labeled" molecule. The label can be detectable on its own (e.g., radioisotope labels or fluorescent labels) or can catalyze chemical alteration of a substrate compound or composition that is detectable (e.g., an enzymatic label).
[0067] The terms "inhibit," "block," and "suppress" are used interchangeably and refer to any statistically significant decrease in biological activity, including full blocking of the activity. For example, "inhibition" can refer to a decrease of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in biological activity.
[0068] The terms "active agent," "therapeutic agent," and "drug" are used
interchangeably to refer to any substance, other than food, used in the prevention, diagnosis, alleviation, treatment, or cure of a disease. Active agents include protective agents and diagnostic agents. An active agent preferably means the conjugates of the present invention, but can also include any substance disclosed in at least one of: The Merck Index, 15th Edition (2013); Pei-Show Juo, Concise Dictionary of Biomedicine and Molecular Biology, (2001); U.S. Pharmacopeia Dictionary of US AN & International Drug Names (2014); and Physician's Desk Reference, 70th Edition (2016). See also Stedman's Medical Dictionary, 28th Edition (2013). The identity of the "active agent" will be apparent from the context.
[0069] "Solvate" refers to a compound or salt thereof that further includes a
stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.
[0070] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of a molecule, and which are not undesirable for use in a pharmaceutical composition. In many cases, the ascorbic acid-amino acid conjugates of the invention are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups, or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, as described, for example, in WO 87/05297.
[0071] The term "pharmaceutical composition" refers to a preparation in which the active agent is in an effective form, i.e., has biological activity, and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such a composition can be sterile and can comprise a
pharmaceutically acceptable carrier, such as physiological saline. Suitable
pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. human albumin), a preservative (e.g. benzyl alcohol), an absorption promoter to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.
[0072] A "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and laboratory animals including, e.g., humans, non-human primates, canines, felines, porcines, bovines, equines, rodents, including rats and mice, rabbits, etc.
[0073] An "effective amount" of an active agent is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose. For example, a "therapeutically effective amount" refers to that amount (at dosages and for periods of time necessary) of an active agent which, when administered to a subject in need thereof, is sufficient to effect treatment. The amount that constitutes a "therapeutically effective amount" will vary depending on the active agent being administered, the condition or disease and its severity, and the weight, age, etc. of the subject to be treated.
[0074] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully "treated" for a disease or disorder according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder. [0075] "Prevent" or "prevention" refer to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of prevention include those prone to have or susceptible to the disorder. In certain embodiments, a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.
Ascorbic Acid-Amino Acid Conjugates
[0076] The invention provides non-naturally occurring compounds comprising an ascorbic acid moiety conjugated to an amino acid moiety.
[0077] L-ascorbic acid has Formula III:
Figure imgf000020_0001
The ascorbic acid moiety is not limited with respect to its form, and any known ascorbic acid, ascorbate, or ascorbate derivative can be used. For example, any pharmaceutically acceptable salt, hydrate, or other solvate of ascorbic acid is suitable for use in the compounds of the invention.
[0078] In one embodiment, the invention provides conjugates in which ascorbic acid or a derivative thereof is covalently attached via an ester linkage at C6 to the carboxy end of an amino acid or dipeptide. In other embodiments, the ester linkage is at C2 or C3 or C5 of ascorbic acid. In some embodiments, ascorbic acid and an amino acid are covalently attached via a linker. The linker can be any divalent linker known to those of skill in the art. Non-limiting examples include aliphatic linkers of between 1-10 carbon atoms, poly(ethylene glycol) (PEG), poly(vinyl pyrrolidone) (PVP), poly(vinyl alcohol), poly(malic acid), N-(2-hydroxypropyl)methacrylamide (UPMA) copolymer, dextrin, hydroxy ethyl starch (HES), and polysialic acid.
[0079] [0080] The amino acid moiety can be any standard or non-standard amino acid(s) or derivative(s) thereof. The amino acid can be a charged amino acid, such as lysine, arginine, histidine, aspartic acid, glutamic acid, or a derivative thereof. The amino acid can be a polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, or a derivative thereof.
[0081] Conjugates of the invention can comprise more than one amino acid. In some embodiments, the conjugate comprises two, three, four, five, or six amino acids. When the amino acid moiety comprises two or more amino acids, they are preferably linked by a peptide bond. The amino acids can be the same or different from one another.
[0082] Non-liming examples of certain ascorbic acid (Asc)-amino acid conjugates of the invention include variants of:
Arg-Asn-Asc
Asn-Arg-Asc
Asp-Cys-Asc
Cys-Asp-Asc
Cys-His-Asc
L-DOPA-Asc
His-Cys-Asc
Tyr-Asc
Arg-Asc
Sec-Asc
Ala-Glu-Asc
Ala-Lys-Asc
Ala-Phe-Asc
Ala-Sec-Asc
Asn-Ser-Asc
Cys-Gly-Asc
Cys-L-DOPA-Asc
Gln-Ser-Asc
Gln-Trp-Asc
Glu-Ala-Asc
Gly-Cys-Asc
Gly-Ser-Asc Gly-Val-Asc
L-DOPA -Cys-Asc
Lys-Ala- -Asc
Met-Trp- -Asc
Phe-Ala- -Asc
Sec-Ala- -Asc
Ser-Asn- -Asc
Ser-Gln- Asc
Ser-Gly- Asc
Trp-Gln- -Asc
Trp-Met- -Asc
Val-Gly- -Asc
[0083] In some embodiments, the conjugate is synthesized as a prodrug, wherein, upon administration to a subject, the prodrug is cleaved to release the conjugate. For example, a prodrug of an ascorbate conjugate can be a prodrug that decreases the hydrophilic character of the conjugate to enhance ocular penetration. See, e.g., Sloan, Kenneth B. Prodrugs: topical and ocular drug delivery . New York: Marcel Dekker, 1992.
[0084] Preferably, at least one hydroxyl group of the amino acid moiety is modified to a substituted or unsubstituted carbonyl or acetyl group. These modifications stabilize the conjugates for formulation and administration, and can decrease toxic side effects in a subject to whom they are administered.
[0085] Molecules of the invention, which increase transport of aqueous humor, can be modified in an effort to increase the ability of the molecule to enter the eye. Examples may include, but are not limited to, the addition of cleavable ester groups or other leaving groups, or the addition of groups that are a substrate for native enzymes or metabolic pathways in the eye.
[0086] The amino acid(s) can optionally be modified or protected with a variety of protecting groups. The protecting group can be any chemical moiety capable of addition to and, optionally, removal from a functional group on an amino acid {e.g., the N- terminus, the C-terminus, or a functional group associated with the side chain of an amino acid within the conjugate) to allow for chemical manipulation of the amino acid(s). [0087] Embodiments of the invention include isolated trabecular meshwork cells comprising one or more ascorbic acid-amino acid conjugates of the invention ("modified trabecular meshwork cells"). In one embodiment, the trabecular meshwork cells are comprised in a pharmaceutical composition configured for intraocular delivery or delivery to a region of the trabecular meshwork. In some embodiments, the trabecular meshwork cells may be cultured cells from suspension, cell, or organ cultures.
Synthesis of Ascorbic Acid- Amino Acid Conjugates
[0088] Conjugates of the invention can be prepared by methods known to the skilled artisan. As one example, conjugates of the invention can be prepared according to methods adapted from Zhao et al , Eur. J. Med. Chem. 82:314-323 (2014) (Bom protection); Hong et al., Chem. Commun. 50: 1 1649-1 1652 (2014) (DOPA protection); Palladino et al , Org. Lett. 14:6346-6349 (2012) (HCl/HFIP deprotection); Kartha et al , Carbohydr. Res. 339:729-732 (2014) (MeOH/MeONa); and Sheppeck et al, Let. Lett. 41 :5329-5333 (2000) (thiol deprotection) (FIG. 12).
[0089] An exemplary reaction scheme and protocol follow. In the structures shown, * denotes a chiral center with either stereochemistry possible; Fmoc = 9- fluorenylmethoxycarbonyl; Boc = tert-butoxycarbonyl.
Figure imgf000023_0001
[0090] Step 1 : To a round-bottom flask (RBF), 1 equiv. ascorbic acid, 55 equiv. dichloromethane (DCM), and 4 equiv. triethylamine (Et3 ) are added. After cooling the solution to 0 °C, 3 equiv. benzyl chloromethyl ether (BOMCl) is added and the solution is allowed to warm to room temperature. When the reaction is complete (by TLC analysis), the crude mixture is extracted with DCM against 1 N HC1. The combined organic layers are dried with MgS04 and concentrated in vacuo. Purification by column
chromatography gives the di-benzyloxymethyl (di-BOM) derivative of ascorbic acid in 70-83% yield.
Figure imgf000023_0002
For all structures,.
[0091] Step 2: To a RBF, 1.0-1.5 equiv. di-BOM ascorbate, 1.0-1.5 equiv. of an amino acid of interest, 1.0-1.5 equiv. ethyl(hydroxyamino)cyanoacetate (Oyxma), and 1.0-1.5 equiv. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) are added. The RFB is cooled to 0 °C and 50-80 equiv. of either N,N- dimethylformamide (DMF) or acetonitrile (ACN) is added. When the reaction is complete (by TLC analysis), the crude mixture is extracted with ethyl acetate (EtOAc) against 1 N HC1. The combined organic layers are dried with MgS04 and concentrated in vacuo. Purification by column chromatography gives the esterified derivative of ascorbate in 50- 85% yield.
Figure imgf000024_0001
[0092] Step 3 : To a RBF, 1.0 equiv. ascorbate ester, 0.5-2.0 equiv. base (sodium
methoxide, l,8-diazabicyclo[5.4.0]undec-7-ene, or tetrabutyl ammonium fluoride hydrate), and, optionally, up to 10 equiv. of a scavenger (iPrOH, MeOH, or n-CsHnSH) are added. The RBF is cooled to 0 °C and 130-1300 equiv. tetrahydrofuran is added. When the reaction is complete (by TLC analysis), the crude mixture is concentrated in vacuo. Purification by column chromatography gives the free-amine derivative of ascorbate in 50-100% yield.
Figure imgf000024_0002
[0093] Step 4: To a RBF, 1.0-1.5 equiv. free-amine ascorbate, 1.0-1.5 equiv. of a second amino acid of interest, 1.0-1.5 equiv. Oyxma, and 1.0-1.5 equiv. EDCI are added. The RBF is cooled to 0 °C and 50-80 equiv. of either DMF or ACN is added. When the reaction is complete (by TLC analysis), the crude mixture is extracted with EtOAc against 1 N HC1. The combined organic layers are dried with MgS04 and concentrated in vacuo. Purification by column chromatography gives the di-peptide derivative of ascorbate in 30-80% yield.
Figure imgf000025_0001
[0094] Step 5: To a RBF, 1.0 equiv. dipeptide ascorbate, 30-100 equiv. acid
(trifluoroacetic acid or 12M HC1), 100-3000 equiv. solvent (DCM or 1,1, 1,3,3,3- hexafluoro-2-propanol) are added. When the reaction is complete (by mass spectroscopy analysis), the crude mixture is concentrated in vacuo. Purification by column chromatography gives the deprotected ascorbate in 30-75% yield. Monopeptides of ascorbate can be deprotected in a similar fashion.
Compositions
[0095] Conjugates of the invention can be combined with a carrier or vehicle to provide a composition comprising an ascorbic acid-amino acid conjugate. In some embodiments, the composition comprises two or more different ascorbic acid-amino acid conjugates.
[0096] The composition can be a pharmaceutical composition, comprising a
pharmaceutically acceptable carrier, for example, saline. In some embodiments, the carrier is sterile. Examples of suitable aqueous and non-aqueous carriers that can be employed in the compositions provided herein include water, salt solutions (such as sodium chloride and potassium chloride), ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), polyethers (such as polyethylene glycol), polyvinyls (such as polyvinyl alcohol and povidone), cellulose derivatives (such as methylcellulose and hydroxypropyl methylcellulose), petroleum derivatives (such as mineral oil and white petrolatum), animal fats (such as lanolin), polymers of acrylic acid (such as carboxypolymethylene gel), vegetable fats (such as peanut oil and olive oil), organic esters, such as ethyl oleate, polysaccharides (such as dextrans),
glycosaminoglycans (such as sodium hyaluronate), and suitable mixtures thereof.
[0097] Usually, a suitable pharmaceutical composition can comprise one or more buffers (e.g. acetate, phosphate, citrate), surfactants (e.g. polysorbate), stabilizing agents (e.g. human albumin), and/or salts (e.g., acid addition salts, base addition salts) etc. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined and other well-known variables. [0098] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of
microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, can also be added into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
[0099] Proper fluidity can be maintained, for example, by the use of coating
materials, such as lecithin, by the maintenance of a certain particle size in the case of dispersions, and by the use of surfactants.
[00100] A pharmaceutical composition provided herein can also include a
pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite, and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[00101] The pH of the composition will vary depending on the route of administration.
A topical solution containing the active agent can be adjusted to a pH of about 5 to about 11. In some embodiments, the pH can be about 6.5, about 7, about 7.5, about 8, or about 8.5. In one embodiment, the pH is about 7.4.
Methods of Use
[00102] The ascorbic acid-amino acid conjugates of the invention are useful in
numerous methods related to fluid flow. For example, the invention provides a method of decreasing intraocular pressure by administering to a subject in need thereof one or more ascorbic acid-amino acid conjugates of the invention.
[00103] Embodiments of the invention further provide a method of treating or
preventing a disorder associated with high intraocular pressure or ocular hypertension by administering to a subject in need thereof one or more ascorbic acid-amino acid conjugates of the invention. In one embodiment, "high intraocular pressure" is IOP about 22 mm Hg or greater, as measured by tonometry. [00104] Embodiments of the invention also provide a method of treating or preventing glaucoma by administering to a subject in need thereof one or more ascorbic acid-amino acid conjugates of the invention. In one embodiment, the glaucoma is open-angle glaucoma. In some embodiments, the treatment of glaucoma is tailored to the individual patient. Multiple variations and combinations of ascorbic acid-amino acid conjugates of the invention, in different concentrations, can be administered to different patients.
Methods of treatment may include the measurement of IOP prior to administration of the therapeutic agent, selection of one or more conjugates based on the desired reduction in IOP or target pressure, and administration of the selected conjugate(s). IOP can be monitored during therapy, and different conjugates or combinations of conjugates can be selected and administered to maintain a desired pressure; for example, between 10 and 20 mm Hg, or between about 15 to about 18 mm Hg.
[00105] Embodiments of the invention provide a method of increasing aqueous
outflow through trabecular meshwork tissue by exposing the trabecular meshwork tissue to one or more ascorbic acid-amino acid conjugates of the invention. Embodiments of the invention additionally provide a method of screening a candidate ascorbic acid-amino acid conjugate for its ability to modify fluid outflow through trabecular meshwork tissue. Methods of the invention can be performed in vivo in the eye of a subject, or ex vivo.
[00106] In one embodiment of the present invention, the ascorbic acid-amino acid conjugate(s) can be introduced into trabecular meshwork cells ex vivo to provide modified trabecular meshwork cells comprising one or more ascorbic acid-amino acid conjugates of the invention. The modified trabecular meshwork cells can be introduced onto the trabecular meshwork or into the anterior chamber of the eye of a subject. Modified trabecular meshwork cells can be administered to a subject to lower intraocular pressure, to treat or prevent glaucoma, and/or to treat or prevent a disorder associated with high intraocular pressure.
[00107] In one embodiment, the trabecular meshwork cells can be applied directly onto the trabecular meshwork. In one embodiment, the modified trabecular meshwork cells are administered intracamerally by passing a blade, needle, applicator, or delivery system through the cornea. The trabecular meshwork cells are released into the anterior chamber of the eye, where the aqueous drainage will carry them into the trabecular meshwork. See e.g., Yue et al., Graefes Arch. Clin. Exp. Opthalmol. 226:262-268 (1988); Russell et al, Invest. Opthalmol. Vis. Sci. 49: 629-635 (2008); Gasiorowski et al., Exp. Eye Res. 88:671-675 (2009). Optionally, the modified trabecular meshwork cells are administered in free solution. The modified trabecular meshwork cells can be administered under direct or indirect visualization. As a non-limiting example, the trabecular meshwork can be contained in a paste-like erodible carrier under direct viewing by gonioscopy.
[00108] The trabecular meshwork cells can be xenogenic, allogenic, or autogenic. The trabecular meshwork cells comprising ascorbic acid-amino acid conjugates of the invention can be from the subject to whom they are administered (i.e., homologous). However, because the eye is a relatively immune-privileged site, the modified trabecular meshwork cells can be heterologous, without the need for tissue typing or
immunosuppression.
[00109] In some embodiments, the trabecular meshwork cells are grown in vitro, e.g., in tissue culture, suspension culture, organ culture, etc. In other embodiments, the trabecular meshwork cells are harvested from eye bank tissue.
[00110] In addition to uses in an ocular context, administration of ascorbic acid
conjugates of the invention can be utilized to treat other disorders in which fluid outflow regulation is dysfunctional, for instance, in an artificial kidney or in hydrocephalus.
[00111] Another method of the embodiments of the invention involves administering to a subject in need thereof an enzyme or enzymatic fragment having the activity of forming a conjugate comprising ascorbic acid and an amino acid or dipeptide. A further method of the embodiments of the invention involves administering to a subject in need thereof an enzyme or enzymatic fragment having the activity of linking an ascorbic acid- amino acid conjugate to a transmembrane protein, which transmembrane protein is capable of forming a transmembrane pore. In one embodiment, the enzyme or enzymatic fragment can be a kinase that is capable of phosphorylating ascorbate, for example, phospholipase D, which can be used to synthesize 6-phosphatidyl-L-ascorbic acid (Nagao et al, Lipids 26:390-94 (1991)). In one embodiment, the enzyme or enzymatic fragment can be an esterase. Other suitable enzymes and enzymatic fragments may include phosphatases, phosphodiesterases, nucleases, proteases, transferases, ribosomal and non- ribosomal synthetases, and other enzymes that catalyze condensation reactions {e.g., amide- and ester-forming condensation enzymes), etc.
[00112] A further method of the embodiments of the invention involves administering to a subject in need thereof a nucleic acid encoding the enzyme or enzymatic fragment discussed above. A composition comprising the nucleic acid can consist essentially of the nucleic acid or a vector comprising the nucleic acid, optionally in an acceptable carrier. Viral vectors include retroviruses, lentiviruses, other RNA viruses, such as poliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV 40, vaccinia, and other DNA viruses. Replication-defective murine retroviral or lenti viral vectors are widely utilized gene transfer vectors.
[00113] The nucleic acid can be introduced through the use of fusogenic lipid vesicles, such as liposomes or other vesicles for membrane fusion. A carrier harboring a nucleic acid of interest can be conveniently introduced into the eye or into body fluids or the bloodstream. The carrier can be site specifically directed to the target organ or tissue in the body. Cell or tissue specific DNA-carrying liposomes, for example, can be used and the foreign nucleic acid carried by the liposome absorbed by those specific cells. Gene transfer may also involve the use of lipid-based molecules which are not liposomes. For example, lipofectins and cytofectins are lipid-based molecules containing positive ions that bind to negatively charged nucleic acids and form a complex that can ferry the nucleic acid across a cell membrane. Delivery of gene therapy may also be accomplished via cationic polymers. Certain cationic polymers spontaneously bind to and condense nucleic acids such as DNA into nanoparticles. For example, naturally occurring proteins, peptides, or derivatives thereof have been used. Synthetic cationic polymers such as polyethylenimine (PEI), polylysine (PLL) etc. condense DNA and are useful delivery vehicles. Dendrimers can also be used. Many useful polymers contain both chargeable amino groups, to allow for ionic interaction with the negatively charged DNA phosphate, and a degradable region, such as a hydrolyzable ester linkage. Examples include poly(alpha-(4-aminobutyl)-L-glycolic acid), network poly(amino ester), and poly (beta- amino esters). These complexation agents can protect nucleic acids against degradation, e.g., by nucleases, serum components, etc., and create a less negative surface charge, which may facilitate passage through hydrophobic membranes (e.g., cytoplasmic, lysosomal, endosomal, nuclear) of the cell. Certain complexation agents facilitate intracellular trafficking events such as endosomal escape, cytoplasmic transport, and nuclear entry, and can dissociate from the nucleic acid.
[00114] Alternatively, the composition can comprise a drug-release regulating
component, such as a polymer matrix with which the nucleic acid or vector is physically associated; e.g., with which it is mixed or within which it is encapsulated or embedded. The vector can be a plasmid, virus, or other vector. Alternatively, the composition can comprise a cell that expresses the nucleic acid. Preferably, such cells secrete the enzyme or enzymatic fragment encoded by the nucleic acid into the extracellular space.
[00115] Various routes of administration can be employed in the methods of the
embodiments of the invention, including topical, intraocular, systemic, cellular, and via a delivery device.
[00116] Compositions comprising conjugates of the invention can be administered topically to the eye via drops, spray, gel, cream, ointment, or eye wash. In some embodiments, the active agent is in a liquid or gel suspension. The active agent can be in liposomes.
[00117] Compositions comprising conjugates of the invention can be administered by intraocular injection, performed periodically. In some embodiments, the active agent can be administered via subconjunctival injection, in others, through intracameral (anterior chamber), intravitreal, or subscleral injection. The composition can be delivered directly to Schlemm's canal via catheter or implanted shunt.
[00118] The active agent can be administered systemically, via direct intra-operative instillation of a gel, cream, or liquid suspension. In some embodiments, the active agent can be administered, for example, by sustained release implants and microspheres for intracameral or anterior vitreal placement within a biodegradable or bioerodible polymer that releases a therapeutic amount of the active agent over a period of time ranging up to a year or more. Additionally, in some embodiments, the active agent can be administered by an implanted drug delivery system which releases a therapeutically effective amount of the conjugates over time. Implantation of the drug delivery system may be surgical or via injection. In some embodiments, the active agent is delivered by iontophoresis. In some embodiments, the active agent is delivered by ultrasound.
[00119] The active agent can be administered via the mucosal route, for example, using nasal drops of a liquid formulation. The active agent can also be delivered via a nasal spray or aerosol suspension of respirable particles, which the subject inhales. The therapeutic molecule is absorbed into the bloodstream via the lungs and subsequently contacts the ocular tissues in a pharmaceutically effective amount. The respirable particles are a liquid or solid, with a particle size sufficiently small to pass through the mouth and larynx upon inhalation; in general, particles ranging from about 1 to 10 microns, but more preferably 1-5 microns, in size are considered respirable. [00120] In a particular embodiment, the active agent is delivered orally, in the form of a tablet, lozenge, aqueous or oily suspension, dispersible powder or granules, emulsion, hard or soft capsule, syrup, or elixirs. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active agent in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. In one preferred embodiment, ascorbic acid-amino acid conjugates of the invention are delivered orally via an enteric coated capsule or via a capsule, wherein the oral formulation is configured for absorption in the small intestine, such that the active agent is shielded from the low pH of stomach acid.
[00121] An additional means of systemic administration is in the form of a suppository comprising the active agent, such that a therapeutically effective amount of the active agent reaches the eyes or other target site via systemic absorption and circulation.
[00122] The active agent can be infused into the tear film via a pump-catheter system.
Another embodiment involves the active agent contained within a continuous or selective-release device, for example, membranes such as, but not limited to, those employed in the Ocusert™ System (Alza Corp., Palo Alto, Calif). As an additional embodiment, the active agent can be contained within, carried by, or attached to contact lenses, which are placed on the eye. Another embodiment of the invention involves the active agent contained within a swab or sponge, which is applied to the ocular surface.
[00123] The composition comprising ascorbic acid-amino acid conjugates can be administered as a single dose or multiple doses. The composition can be administered as many times as needed to achieve a targeted endpoint, such as a particular IOP.
Administration intervals may vary. In some embodiments, topical or systemic administration is 1, 2, 3, or 4 times per day, or once every 2, 3, 4, 5 or 6 days, once per week, twice per week, every other week, twice per month, or once per month. In some embodiments, intraocular delivery is performed once every 1, 2, 3, 4, 5, 6, or 9 months, once per year, or once every 18 months, or once every 2, 3, 4, or 5 years. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).
[00124] Conjugates of the invention can be administered in combination therapy
and/or combined with other agents. Accordingly, a method of the invention can comprise administering a second active agent. The second active agent can be administered in the same or a different composition as the ascorbic acid-amino acid conjugate(s) of the invention. In one embodiment, the second active agent is selected from the group consisting of a prostaglandin analog, a beta blocker, an alpha agonist, and a carbonic anhydrase inhibitor.
[00125] In one embodiment, the second active agent is an enzyme or enzymatically active fragment capable of catalyzing the conjugation of ascorbic acid to an amino acid. In one embodiment, the second active agent increases the activity of an enzyme or enzymatically active fragment capable of catalyzing the conjugation of ascorbic acid to an amino acid. In one embodiment, the second active agent is an enzyme or enzymatically active fragment capable of catalyzing the linking of an ascorbic acid conjugate to a transmembrane protein. In one embodiment, the enzyme or enzymatically active fragment has kinase activity. In one embodiment, the enzyme or enzymatically active fragment has esterase activity. In one embodiment, the second active agent is a nucleic acid encoding the enzyme or enzymatically active fragment.
Kits
[00126] Also within the scope of the embodiments of the invention are kits comprising one or more ascorbic acid-amino acid conjugates and/or compositions comprising them, as provided herein, and instructions for use. The kit can further contain at least one additional reagent. Kits typically include a label indicating the intended use of the contents of the kit. The term "label" includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
[00127] Embodiments of the invention further provide kits that comprise one or more ascorbic acid-amino acid conjugates and/or compositions comprising them, which can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one type of ascorbic acid-amino acid conjugate of the invention in one or more containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to perform a screening assay, including all controls, directions for performing assays, and, optionally, any necessary software for analysis and presentation of results. One skilled in the art will readily recognize that the disclosed ascorbic acid- amino acid conjugates can be readily incorporated into one of the established kit formats which are well known in the art.
EXAMPLES
[00128] Embodiments of the present invention can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain liposome constructs of the present disclosure and methods for using liposome constructs of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.
Example 1. Isolation of Ascorbic Acid Compounds from Trabecular Meshwork Tissue
[00129] We designed an experiment to identify ascorbic acid conjugates in isolated trabecular meshwork tissue samples from non-glaucomatous donors. Tandem mass spectrometry (MS/MS) and liquid chromatography-tandem mass spectrometry
(LC/MS/MS) techniques were developed using 6-O-Palmitoyl-L- Ascorbic acid as surrogate for the detection of ascorbic acid conjugates.
[00130] Corneal transplant tissue with scleral rims from 8 eyeballs were stored in
tissue culture media and refrigerated at 2-8° C until initial extraction was performed. The samples were extracted as follows: the tissue culture media was rinsed off with balanced salt solution and the trabecular meshwork was stripped off using microsurgical instruments and a dissecting microscope. The tissue was transferred to a 13 X 100 mm glass test tube. The eye tissue was ground with the end of a glass stirring rod. Tissue was noted to be very fibrous and resistant to disruption. 1.0 mL of HPLC grade methanol was added to the tissue and mixed. The mixture was then sonicated in 37° C water bath for 1 hour. Next, 1.0 ml of HPLC grade chloroform was added and sonicated for another 20 minutes. The mixture was then centrifuged at 2500 rpm for 5 minutes. Next, the top layer was transferred to a clean vial and sealed. The chloroform-methanol mixture was evaporated to dryness and the resultant material reconstituted with 100 μL of HPLC mobile phase B.
[00131] Samples were screened for mechanistic ester linked ascorbate structures
through precursor ion scanning techniques. To provide screening procedures for the possible discovery of these molecules in eye tissue slices, standard molecules were studied to optimize their detection through neutral loss MS/MS detection from a reversed phase HPLC separation. These neutral loss detection experiments were centered around the loss of 176. 1 atomic mass units (amu) for ascorbic acid. The mass offset of 176.1 amu was determined from the structures of ascorbate conjugates for tyrosine (FIG. 3B), L-DOPA (FIG. 3C), arginine-asparagine (FIG. 3D), histidine-cysteine (FIG. 3E), and cysteine-aspartic acid (FIG. 3F). Ascorbic acid (FIG. 3A) has the chemical formula C6H806 with an exact mass of 176.03 and a molecular weight of 176. 12. Indeed 176.1 is the common mass lost among the compounds as described below.
[00132] Fragmentation as shown in FIG. 4 of an ascorbic acid-tyrosine conjugate
(Ci5Hi7N08) having an exact mass of 339. 10 and a molecular weight of 339.30 produces a tyrosine group (C HioN02 +) having an exact mass of 164.07, and an ascorbic acid molecule (C6H7O6) having an exact mass of 175.02. Alternatively, fragmentation as shown in FIG. 5 produces an ascorbic acid (peak at 175.4 m/z), a quinone methide (CH2C6H40H+) having a mass of 107.3 m/z, and an C2H5NO+ fragment (peak at 57.3 m/z).
[00133] Fragmentation as shown in FIG. 6 of an ascorbic acid-L-DOPA conjugate
(C15H17NO9) having an exact mass of 355.09 and a molecular weight of 355.30 produces an L-DOPA group (C9HioN03 +) having an exact mass of 180.07, and an ascorbic acid molecule (C6H7O6) having an exact mass 175.02. Alternatively, fragmentation as shown in FIG. 7 produces a CH3C6H3(OH)2 fragment having a mass of 123 m/z, while cleavage of an ammonia produces a C15H15O9 fragment having a mass of 338 m/z.
[00134] Fragmentation as shown in FIG. 8 of an ascorbic-arginine-asparagine
conjugate
Figure imgf000034_0001
having an exact mass of 446.18 and a molecular weight of 446.41 produces an asparagine-arginine group after fragmenting at the ester linkage (observed as the acylium ion) with a mass of 271 m/z, or a fragment resulting from cleaving off the guanidinium imine (CH2NC( ¼) H+) and having a mass of 73 m/z. The conjugate structure may additionally or alternatively have an ascorbic acid- asparagine-arginine configuration.
[00135] Fragmentation as shown in FIG. 9 of an ascorbic acid-histidine-cysteine
conjugate (C15H20N4O8S) having an exact mass of 416.10 and a molecular weight of 416.41 produces a histidine-cysteine fragment after cleavage of the ester linkage
(observed as the acylium ion) with a mass of 241 m/z, or a fragment resulting from benzylic cleavage of the imidazole group (C3H3N2CH2+) having a mass of 81 m/z. The conjugate structure may additionally or alternatively have an ascorbic acid-cysteine- histidine configuration.
[00136] Fragmentation as shown in FIG. 10 of an ascorbic acid-cysteine-aspartic acid conjugate (Ci3Hi8N20ioS) having an exact mass of 394.07 and a molecular weight of 394.35 produces an aspartic acid-cysteine group after fragmenting at an ester linkage (observed as the acylium ion) with a mass of 219 m/z. Cleavage of the CO2H or the CH2CO2H from the conjugate produces fragments having a mass of 349 m/z or 335 m/z, respectively. The conjugate structure may additionally or alternatively have an ascorbate- aspartic acid-cysteine configuration.
[00137] Samples were analyzed via LC/+ L 176.1 amu scan (FIG. 11). The
calculated ligand molecular weights for positive ions 358.4 amu and 374.4 amu were 199.1 amu and 215.1 amu, respectively. These values were approximately 16 amu apart, which suggests a difference in structure of an OH group.
[00138] Positive ions 358.4 amu and 374.4 amu were approximately 18 amu apart from positive ions 340.4 amu and 356.4 amu. Positive ions 340.4 amu and 356.4 amu had molecular weights of 181.2 amu and 196.3 amu, respectively, which suggests the loss of an H2O or ( H4)+ group.
[00139] The calculated ligand molecular weights for negative ions 337.3 amu and 369 amu were 180.4 amu and 212.4 amu, respectively. These values were 32 amu apart, suggesting a difference in structure of two OH groups.
[00140] From these data observations, we determined that the molecular weight of the positive 340.4 amu ion was 339.4 amu. Accordingly, the ligand molecular weight was calculated by subtracting the L 176 amu ion of ascorbic acid from the 339.3 amu molecular weight, which yielded a ligand ion weight of 163.1 amu. Adding 18.0 amu
(H20) to restore the carboxylic acid moiety gave the full ligand weight of 181.2 amu.
[00141] The molecular weight of the positive 356.4 amu ion was 355.4 amu.
Subtracting the NL 176 amu ion of ascorbic acid from the 355.4 amu molecular weight yielded a ligand ion weight of 179.4 amu. Adding 18.0 amu (H20), to restore the carboxylic acid moiety, gave the full ligand weight of 197.4 amu.
[00142] The molecular weight of the negative 337.3 amu ion was 338.3 amu.
Subtracting the NL 176 amu ion of ascorbic acid from the 338.3 amu molecular weight yielded a ligand molecular weight of 162.3 amu. Adding 18.0 amu (H2O), to restore the carboxylic acid moiety, gave the full ligand weight of 180.3 amu.
[00143] The molecular weight of the negative 369.0 amu ion was 370.0 amu.
Subtracting the NL 176 amu ion of ascorbic acid from the 370.0 amu molecular weight yielded a ligand molecular weight of 194.0 amu. Adding 18.0 amu (H2O), to restore the carboxylic acid moiety, gave the full ligand weight of 212.0 amu.
[00144] Ascorbic acid conjugates and fragments of tyrosine and L-DOPA were
assayed under LC/MS/MS to corroborate the ligand identities by monitoring the fragmentation of 340.4 amu and 356.4 amu.
Example 2. Preparation of Ascorbic Acid-Asparagine Conjugate
[00145] In the reaction described below, Fmoc-Asn-Trt-OH = Na-(9-
Fluorenylmethoxycarbonyl)-Ny-trityl-L-asparagine; BOM = benzyl oxym ethyl; Oxyma = ethyl (hydroxyimino)cyanoacetate; EDCI = N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride; Fmoc = 9-fluorenylmethoxycarbonyl; ACN = acetonitrile; TLC = thin layer chromatography; and EtOAc = ethyl acetate.
Figure imgf000036_0001
[00146] To a flame-dried dram vial with stir bar, di-BOM ascorbate (420 mg, 1.0 mmol, 1.5 equiv), Fmoc-Asn-Trt-OH (400 mg, 0.67 mmol, 1.0 equiv), Oyxma (1 14 mg, 0.80 mmol, 1.2 equiv) and EDCI (193 mg, 1.0 mmol, 1.5 equiv) were added. The vial was evacuated and backfilled with argon, then cooled to 0 °C and ACN (2.6 mL) was added. When the reaction was complete (by TLC analysis, ca. 5 hours), the crude mixture was diluted with 8 mL EtOAc , 4 mL brine, and 4 mL 1 N HC1. The organic layer was removed and the aqueous layer extracted against EtOAc (8 mL x 2). The combined organic layers were dried with MgS04 and concentrated in vacuo. Purification by column chromatography gave the esterified derivative of ascorbate in 70% yield (530 mg).
Example 3. Effects of Ascorbic Acid Conjugates on Fluid Outflow Ex Vivo
[00147] The therapeutic efficacy of ascorbic acid conjugates of the invention is tested using fresh donor eye tissue from normal eyes or eyes with glaucoma. The anterior segment of the eye with a scleral rim of about 3-4 mm is resected. The uveal tissue is removed from the internal surface of the eye. The remaining anterior eye tissue is clamped to a holding device. Fluid is perfused into the bare anterior chamber. This process removes other means of outflow from the anterior chamber; therefore, the only remaining outflow tract is through the trabecular meshwork, Schlemm's canal, and the intra-scleral aqueous collector channels. A candidate composition comprising one or more ascorbic acid conjugates of the invention is infused into the anterior chamber, allowed to incorporate into the cell membranes. Pressure decay curves and/or flow rates are measured using standard methods, such as a pressure transducer, tonography, fluorophotometry, and/or constant-pressure perfusion, to determine whether the candidate composition comprising one or more ascorbic acid conjugates of the invention has a favorable effect on facility of outflow.
[00148] A mathematical model is used to measure the effectiveness of the candidate compound by studying changes in facility of outflow before and after the compound is introduced. The Goldman equation is used, for example, to measure facility of outflow, which should increase as the function of the trabecular meshwork increases.
Goldmann equation: Po = (F/C) + Pv
where Po is observed intraocular pressure, F is the rate of aqueous formation, C is the facility of outflow, and Pv is the episcleral venous pressure. In ex vivo testing of ascorbic acid conjugates, Pv is zero, as there are no aqueous veins, and C is made up of trabecular meshwork resistance and additional contributions, such as resistance within the intrascleral aqueous humor collector channels. These additional factors remain constant for a particular eye. Thus, changes in trabecular function in the living, perfused trabecular tissue are deduced based on changes in observed pressure/flow responses. [00149] An ex vivo anterior segment perfusion culture device, which functions as detailed above, was first described by Johnson and Tschumper {Invest. Ophthalmol. Vis. Sci. 28:945-953 (1987)). The model has also been used to evaluate changes in outflow with the addition of drugs such as dexamethasone (Clark et al, Invest. Ophthalmol. Vis. Sci. 36:478-489 (1995)) and to evaluate the efficacy of surgical treatment (e.g., stents) for the trabecular meshwork.
Example 4. Effects of Ascorbic Acid Conjugates on Fluid Outflow In Vivo
[00150] A non-human primate glaucoma model (see, e.g., Rasmussen et al, J.
Glaucoma 14:311-314 (2005)) can be used to evaluate the efficacy of the ascorbic acid- amino acid conjugates of the invention.
[00151] Prior to placement in the study, animals undergo clinical ophthalmic
examination (slit-lamp biomicroscopy and indirect ophthalmoscopy). Ocular findings are scored according to a modified McDonald-Shadduck Scoring System (McDonald et al. "Eye Irritation," in Advances in Modern Toxicology: Dermatoxicology, at 579-582 (Marzulli et al. Eds., 1977)).
[00152] Intraocular pressure (IOP) and tonography (outflow measure) are measured at the initiation and terminus of the study using known methods. Test animals are divided into Groups 1, 2, 3, and 4. On Days 0-13, eye drops comprising an ascorbic acid-amino acid conjugate of the invention in a pharmaceutical buffer of pH 7.4 are administered to one eye of the test animals; the contralateral eye acts as control and is administered vehicle alone. Drops are administered three times daily at doses shown in Table 1.
TABLE 1. Study Design
Figure imgf000038_0001
[00153] Animals undergo daily health observations, including observation of general appearance and behavior, and measurement of body weight. At the terminus of the study, eye pressure and tonography are repeated, and observed changes are noted. Example 5. Treatment of Glaucoma Using Ascorbic Acid-Amino Acid Conjugate
[00154] A 75-year-old patient presents with intraocular pressure (IOP) of 26 mm Hg in both eyes. This represents 4 standard deviations (2.5 mmHg) above average IOP (16 mm Hg). The filtering angles are inspected by gonioscopy and found to be open. Inspection of the nasal and temporal portions of the eye where aqueous veins are most prominent shows the structures to be intact. The optic nerves show only moderate damage. This patient would not have very low target IOPs and normalization of IOP would represent adequate response to therapy.
[00155] The patient is treated with a loading dose of eye drops 2-3 times per day
comprising up to 10 mg ascorbic acid-amino acid conjugate of the invention in a vehicle or delivery system. After 2 weeks on the treatment regimen, the IOP is reduced and the medication is delivered twice daily. Once the IOP is normalized, for example, after 1 month, the drops are tapered to the minimum dosing regimen required to maintain a therapeutic effect.
***
[00156] All of the references cited in this disclosure are hereby incorporated by
reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.
[00157] The foregoing description of the specific embodiments of the present
invention will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present
specification is to be interpreted by the skilled artisan in light of the teachings and guidance. The present invention is further described by the following claims.

Claims

1. An ascorbic acid-amino acid conjugate having Formula I:
(I)
Figure imgf000040_0001
wherein SCi is amino acid side chain;
wherein Ri, R2, R3, and Rs are independently selected from -H; a carbonyl group selected from -CHO and -COX, wherein X is a Ci-6 branched or unbranched alkyl; and an acetyl group selected from CH3CO- and CH3(CH2)nCO- wherein n is 1-6; and
wherein R4 and R5 are independently selected from -H, -CH3, -CH2CH3, and -CF3; with the proviso that at least one of R1-R7 is not H.
2. An ascorbic acid-amino acid conjugate having Formula II:
(Π)
Figure imgf000040_0002
wherein SCi and SC2 are the same or different amino acid side chains;
wherein Ri, R2, R3, and R5 are independently selected from -H, a carbonyl group selected from -CHO and -COX, wherein X is a Ci-6 branched or unbranched alkyl, and an acetyl group selected from CH3CO- and CH3(CH2)nCO-, wherein n is 1-6;
wherein R4 and R5 are independently selected from -H, -CH3, -CH2CH3, and -CF3; and
wherein R7 is H or CH3;
with the proviso that at least one of R1-R7 is not H.
3. The ascorbic acid-amino acid conjugate of claim 1 or claim 2, wherein at least one C-H is replaced with C-F, C-Cl, C-Br, or C-I.
4. The ascorbic acid-amino acid conjugate of claim 1, wherein SCi is selected from the group consisting of: an L-DOPA side chain, a Tyr side chain, an Arg side chain, and a Sec side chain.
5. The ascorbic acid-amino acid conjugate of claim 2, wherein SCi and SC2, respectively, are selected from the group consisting of: (i) an Asn side chain and an Arg side chain, (ii) an Arg side chain and an Asn side chain, (iii) a Cys side chain and an Asp side chain, (iv) an Asp side chain and a Cys side chain, (v) a His side chain and a Cys side chain, (vi) a Cys side chain and a His side chain, (vii) a Glu side chain and an Ala side chain, (viii) a Lys side chain and an Ala side chain, (ix) a Phe side chain and an Ala side chain, (x) a Sec side chain and an Ala side chain, (xi) a Ser side chain and an Asn side chain, (xii) a Gly side chain and a Cys side chain, (xiii) an L-DOPA side chain and a Cys side chain, (xiv) a Ser side chain and a Gin side chain, (xv) a Trp side chain and a Gin side chain, (xvi) an Ala side chain and a Glu side chain, (xvii) a Cys side chain and a Gly side chain, (xviii) a Ser side chain and a Gly side chain, (xix) a Val side chain and a Gly side chain, (xx) a Cys side chain and an L- DOPA side chain, (xxi) an Ala side chain and a Lys side chain, (xxii) a Trp side chain and a Met side chain, (xxiii) an Ala side chain and a Phe side chain, (xxiv) an Ala side chain and a Sec side chain, (xxv) an Asn side chain and a Ser side chain, (xxvi) a Gin side chain and Ser side chain, (xxvii) a Gly side chain and a Ser side chain, (xxviii) a Gin side chain and a Trp side chain, (xxix) a Met side chain and Trp side chain, and (xxx) a Gly side chain and a Val side chain.
6. An isolated trabecular meshwork cell comprising one or more ascorbic acid- amino acid conjugates of claim 1 or claim 2.
7. An isolated trabecular meshwork cell comprising at least one ascorbic acid- amino acid conjugate of claim 1 and at least one ascorbic acid-amino acid conjugate of claim 2.
8. A composition comprising one or more ascorbic acid-amino acid conjugates of claim 1 or claim 2, or an isolated trabecular meshwork cell of claim 6.
9. A composition comprising at least one ascorbic acid-amino acid conjugate of claim 1 and at least one ascorbic acid-amino acid conjugate of claim 2, or an isolated trabecular meshwork cell of claim 7.
10. The composition of claim 8, which is a pharmaceutical composition.
11. The composition of claim 9, which is a pharmaceutical composition.
12. A kit comprising one or more ascorbic acid-amino acid conjugates of claim 1 or claim 2, an isolated trabecular meshwork cell of claim 6, or a composition of claim 8.
13. A kit comprising at least one ascorbic acid-amino acid conjugate of claim 1 and at least one ascorbic acid-amino acid conjugate of claim 2, an isolated trabecular meshwork cell of claim 7, or a composition of claim 9.
14. A method of reducing intraocular pressure (IOP) in a subject in need thereof, the method comprising administering to the subject the composition of claim 10 or claim 11.
15. A method of treating high IOP in a subject, the method comprising
administering to the subject the composition of claim 10 or claim 11.
16. A method of treating glaucoma in a subject, the method comprising
administering to the subject the composition of claim 10 or claim 11.
17. The method of claim 14, wherein administration is topical, intraocular, systemic, or via a drug delivery device.
18. The method of claim 15, wherein administration is topical, intraocular, systemic, or via a drug delivery device.
19. The method of claim 16, wherein administration is topical, intraocular, systemic, or via a drug delivery device.
20. The method of any one of claims 17-19, wherein administration is via eye drops.
21. The method of any one of claims 17-19, wherein administration is oral.
22. The method of claim 21, comprising oral administration of a tablet or capsule.
23. The method of claim 16, wherein the glaucoma is open-angle glaucoma.
24. A method of increasing aqueous outflow through trabecular meshwork tissue, the method comprising exposing the trabecular meshwork tissue to one or more ascorbic acid-amino acid conjugates of claim 1 or claim 2.
25. A method of increasing aqueous outflow through trabecular meshwork tissue, the method comprising exposing the trabecular meshwork tissue to at least one ascorbic acid- amino acid conjugate of claim 1 and at least one ascorbic acid-amino acid conjugate of claim 2.
26. The method of claim 24 or claim 25, wherein the method is performed ex vivo.
27. A method of screening a candidate ascorbic acid-amino acid conjugate for its ability to modify fluid outflow through trabecular meshwork tissue, the method comprising: measuring fluid outflow through trabecular meshwork tissue before and after exposing the trabecular meshwork tissue to the candidate ascorbic acid-amino acid conjugate; wherein a change in fluid outflow after exposure to the candidate ascorbic acid-amino acid conjugate indicates that the ascorbic acid-amino acid conjugate is able to modify fluid outflow.
28. The method of claim 27, which is performed ex vivo.
29. The method of claim 27, which is performed in vivo in an animal model.
30. The method of claim 27, wherein the candidate ascorbic acid-amino acid conjugate is the ascorbic acid-amino acid conjugate of claim 1 or claim 2.
31. Use of one or more ascorbic acid-amino acid conjugates of claim 1 or claim 2 to reduce IOP in a subject in need thereof, or to treat high IOP in a subject, or to treat glaucoma in a subject.
32. Use of at least one ascorbic acid-amino acid conjugate of claim 1 and at least one ascorbic acid-amino acid conjugate of claim 2 to reduce IOP in a subject in need thereof, or to treat high IOP in a subject, or to treat glaucoma in a subject.
33. The use of claim 31 or claim 32, wherein the ascorbic acid-amino acid conjugate(s) is formulated for topical, intraocular, or systemic delivery.
34. The use of claim 31 or claim 32, wherein the ascorbic acid-amino acid conjugate(s) is formulated for delivery via a drug delivery device.
35. Use of the isolated trabecular meshwork cell of claim 6 to reduce IOP in a subject in need thereof, or to treat high IOP in a subject, or to treat glaucoma in a subject.
36. Use of the isolated trabecular meshwork cell of claim 7 to reduce IOP in a subject in need thereof, or to treat high IOP in a subject, or to treat glaucoma in a subject.
PCT/US2017/039940 2016-06-29 2017-06-29 Ascorbic acid-amino acid conjugates and their use in regulation of fluid outflow WO2018005762A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP17821235.3A EP3478259A4 (en) 2016-06-29 2017-06-29 Ascorbic acid-amino acid conjugates and their use in regulation of fluid outflow

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662356432P 2016-06-29 2016-06-29
US62/356,432 2016-06-29

Publications (2)

Publication Number Publication Date
WO2018005762A2 true WO2018005762A2 (en) 2018-01-04
WO2018005762A3 WO2018005762A3 (en) 2018-02-22

Family

ID=60785551

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/039940 WO2018005762A2 (en) 2016-06-29 2017-06-29 Ascorbic acid-amino acid conjugates and their use in regulation of fluid outflow

Country Status (2)

Country Link
EP (1) EP3478259A4 (en)
WO (1) WO2018005762A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115260170A (en) * 2021-04-30 2022-11-01 禾美生物科技(浙江)有限公司 Ascorbic acid polypeptide derivative and preparation method and application thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5674888A (en) * 1995-06-07 1997-10-07 University Of California Method for the treatment of a trabecular meshwork whose cells are subject to inhibition of cell division
CN100378087C (en) * 2001-08-24 2008-04-02 马蒂亚斯·拉特 Novel ascorbic acid compounds, methods of synthesis and application use thereof
US20030113362A1 (en) * 2001-08-24 2003-06-19 Roomi Waheed M. Unique ascorbic acid derivatives with essential amino acids, nonessential amino acid and amino acids that do not occur in protein: the process for their preparation and use in research, medicine, physiology, pharmacology, nutrition, pharmaceuticals, and for cosmetic, commercial and industrial applications
WO2016085127A2 (en) * 2014-11-27 2016-06-02 (주)셀트리온 Stable peptide-conjugated ascorbic acid derivative, method for preparing same, and cosmetic composition containing same
CN107249577A (en) * 2014-12-29 2017-10-13 奥拉西斯医药股份有限公司 Composition and method for treating glaucoma

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115260170A (en) * 2021-04-30 2022-11-01 禾美生物科技(浙江)有限公司 Ascorbic acid polypeptide derivative and preparation method and application thereof
WO2022228475A1 (en) * 2021-04-30 2022-11-03 禾美生物科技(浙江)有限公司 Ascorbic acid polypeptide derivative, preparation method therefor, and application thereof
CN115260170B (en) * 2021-04-30 2024-05-28 禾美生物科技(浙江)有限公司 Ascorbic acid polypeptide derivative and preparation method and application thereof

Also Published As

Publication number Publication date
EP3478259A2 (en) 2019-05-08
WO2018005762A3 (en) 2018-02-22
EP3478259A4 (en) 2020-02-26

Similar Documents

Publication Publication Date Title
JP2022001609A (en) Methods and compositions for preventing or treating ophthalmic conditions
US10966962B2 (en) Method for treating neurodegenerative diseases
US20180296630A1 (en) Methods and compositions for preventing or treating ophthalmic conditions
US20240024408A1 (en) Methods and compositions for preventing or treating leber's hereditary optic neuropathy
WO2006096011A1 (en) PHARMACEUTICAL COMPOSITION FOR TREATING AVELLINO CORNEA DYSTROPHY COMPRISING AN ANTIBODY AGAINST TGF-β
US20120135915A1 (en) Treatment of traumatic or degenerative neurologic, otologic, or ophthalmologic diseases with targeted protease inhibitors
US20170368024A1 (en) Compositions and Methods for Treating Glaucoma
US20220280477A1 (en) Compositions And Methods For Treating Traumatic Brain Injury
US20160038558A1 (en) Interval therapy for the treatment of loss of eyesight in humans with glaucoma and other degenerative eye diseases
WO2018005762A2 (en) Ascorbic acid-amino acid conjugates and their use in regulation of fluid outflow
US20150253308A1 (en) Compositions and methods of treating glaucoma
US20130316983A1 (en) Drug screening method, compositions and methods of treating glaucoma
WO2022046919A2 (en) Compositions and methods for the treatment of ocular neuroinflammation
EP4161498A1 (en) Treatment of cystinosis
CN118434434A (en) Methods and compositions comprising peptide mimetics for treating, preventing, inhibiting, ameliorating, or delaying the onset of an ophthalmic condition
WO2007148744A1 (en) Prophylactic or therapeutic agent for corneal/conjunctival disorder comprising fumaric acid derivative as active ingredient
WO2023150791A1 (en) Peptides and methods of use thereof in treating ocular disorders

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17821235

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

ENP Entry into the national phase in:

Ref document number: 2017821235

Country of ref document: EP

Effective date: 20190129

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17821235

Country of ref document: EP

Kind code of ref document: A2