EP1272170A1 - Croissance oculaire et antagonistes nicotiniques - Google Patents

Croissance oculaire et antagonistes nicotiniques

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Publication number
EP1272170A1
EP1272170A1 EP01903112A EP01903112A EP1272170A1 EP 1272170 A1 EP1272170 A1 EP 1272170A1 EP 01903112 A EP01903112 A EP 01903112A EP 01903112 A EP01903112 A EP 01903112A EP 1272170 A1 EP1272170 A1 EP 1272170A1
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EP
European Patent Office
Prior art keywords
nicotinic
eye
nicotinic antagonist
antagonist
growth
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Legal status (The legal status 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 status listed.)
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EP01903112A
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German (de)
English (en)
Inventor
Richard A. Stone
Jon M. Lindstrom
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University of Pennsylvania Penn
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Valley Forge Pharmaceuticals Inc
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Publication of EP1272170A1 publication Critical patent/EP1272170A1/fr
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    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • 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
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4747—Quinolines; Isoquinolines spiro-condensed
    • 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
    • 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
    • A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia

Definitions

  • the present invention relates to the control of eye growth by nicotinic receptor antagonists, more particularly to the inhibition of postnatal ocular growth and the prevention of myopia in a host animal by ocular administration of nicotinic receptor antagonists.
  • retinal amacrine cells hypothesized to influence refractive development include those containing vasoactive intestinal peptide (Pickett Seltner and Stell, 1995,
  • Ophthalmol. Vis. Sci., 35:3691-701) or lesioning the pre-ganglionic input to the ciliary ganglion at the Edinger-Westphal nucleus each fail to have a major impact on the development of experimental myopia.
  • Pharmacologic evidence also argues against a role for accommodation in myopia development.
  • An Ml -selective muscarinic antagonist with minimal cycloplegic activity is at least as effective as atropine against experimental myopia in the rhesus monkey (Tigges et al, 1999, Optometry & Vision Science, 76:397-407).
  • the chick ciliary body contains striated rather than smooth muscle, that avian accommodation is controlled by nicotinic rather than muscarinic mechanisms, and that atropine fails to paralyze accommodation in the chick (Stone et al, 1991, Exp. Eye Res., 52:755-8). That atropine blocks myopia in the chick further argues against an accommodation mechanism (Stone, 1997, Myopia Updates: Proceedings of the 6th International Conference on Myopia, pp. 241-254).
  • muscarinic antagonists might inhibit myopia development by acting at extra-retinal sites such as sclera or choroid.
  • vecuronium bromide a neuromuscular blocking agent and nicotinic antagonist
  • a neuromuscular blocking agent and nicotinic antagonist were applied to chick corneas, it paralyzed accommodation but failed to influence the ocular elongation following spectacle- induced hyperopic defocus, again arguing against an accommodative mechanism for myopia (Schwahn and Schaeffel, 1994, Invest. Ophthalmol. Vis. Sci., 35:3516-24).
  • Charged antagonists at the neuromuscular junction typically penetrate poorly into the central nervous system and bind to all nicotinic receptor subtypes with low affinity (Gotti et al, 1997, Progress in Neurobiology, 53:199-237).
  • Vecuronium bromide is also highly charged; thus, while it diffuses readily to block the neuromuscular junctions of intraocular muscles, it may not have access to receptor sites in lipophilic tissues potentially involved in eye growth control, such as the neural retina.
  • the chick eye also has well-characterized nicotinic receptor subtypes in both retina (Hamassaki-Britto et al, 1994a, Vis. Neurosci., 11:63-70; Hamassaki-Britto et al, 1994b, J Comp. Neurol, 347:161-170; Keyser et al, 1993, J Neurosci., 13:442-454; Vailate et al, 1999, Mol.
  • the invention concerns the use of nicotinic antagonists of suitable solubility to penetrate to the relevant targets that regulate postnatal eye growth and that inhibit postnatal ocular growth and the development of myopia.
  • the invention provides a method of controlling postnatal ocular growth by ocular administration of therapeutically effective amounts of a nicotinic antagonist to control postnatal ocular growth. Further provided is a method of inhibiting the abnormal postnatal axial growth of the eye of a host animal by administering therapeutically effective amounts of a nicotinic antagonist during postnatal development. The invention also provides a method of inhibiting abnormal equatorial expansion of the eye of a host animal by administering therapeutically effective amounts of a nicotinic antagonist during postnatal development. The invention further provides a method of inhibiting the abnormal vitreous cavity expansion of the eye of a host animal by administering therapeutically effective amounts of a nicotinic antagonist during postnatal development. Another aspect of the invention provides a method of preventing or inhibiting development of myopia by ocular administration of therapeutically effective amounts of a nicotinic antagonist.
  • the invention provides for the use of a nicotinic antagonist for the preparation of a medicament adapted for ocular administration for the control of postnatal ocular growth.
  • the invention further provides for the use of a nicotinic antagonist for the preparation of a medicament for uses such as inhibiting the abnormal axial growth of the eye of a host animal during postnatal development, inhibiting the abnormal equatorial expansion of the eye of a host animal during postnatal development, inhibiting the abnormal vitreous cavity expansion of the eye of a host animal during postnatal development.
  • the invention also provides for the use of a nicotinic antagonist for the preparation of a medicament adapted for ocular administration for the prevention or treatment of myopia.
  • the nicotinic antagonist may be a competitive nicotinic antagonist such as methyllcaconitine or dihydro- ⁇ -erythroidine.
  • the nicotinic antagonist may be a channel-blocking nicotinic antagonist such as chlorisondamine or mecamylamine.
  • the nicotinic antagonist may be a noncompetitive nicotinic antagonist such as sertraline, paroxetine, nefaxodone, venlafaxine, fiuoxetine, buproprion, phencyclidine, and ibogaine.
  • the nicotinic antagonist may be an antibody inhibiting nicotinic receptor function. In yet another embodiment of the invention, the nicotinic antagonist may be an agonist that acts like a nicotinic antagonist.
  • the invention provides method of detecting the ability of a nicotinic antagonist to control postnatal ocular growth of the eye of a host animal by contacting an animal eye with a therapeutically effective amount of a nicotinic antagonist, detecting the change in growth of the eye exposed to a therapeutically effective amount of a nicotinic antagonist, then applying a known control agent in a second eye, observing the results of the control agent on the change in growth of the second eye, and comparing the change in growth of the first eye exposed to a therapeutically effective amount of a nicotinic antagonist with the change in growth of the second eye exposed to a known control agent, thereby identifying the nicotinic antagonist as having the ability to control postnatal ocular growth.
  • a method of making a pharmaceutical including the steps of identifying a nicotinic antagonist as an active agent having the ability to control postnatal ocular growth and combining the active agent in admixture with a pharmaceutical excipient.
  • the invention provides a method of identifying compounds that can be used to modulate myopia including the steps of incubating a cell that expresses a nicotinic receptor in the presence and absence of a test compound, determining whether the test compound binds to at least one nicotinic receptor, selecting a test compound that binds to at least one nicotinic receptor, administering the selected test compound to a test animal, determining whether the test compound alters the development of myopia of the test animal, and selecting a compound that alters the development of myopia of a test animal.
  • Figure 2A shows drug effects on axial length measured by ultrasound.
  • Figure 2B shows drug effects on vitreous cavity length measured by ultrasound.
  • Figure 2C shows drug effects on axial length measured by digital calipers.
  • Figure 2D shows drug effects on equatorial diameter measured by calipers. Chlorisondamine (CHL), mecamylamine (MEC) and methyllycaconitine (MLA) each reduced the excessive ocular growth occurring beneath a goggle; the statistically significant effects (P ⁇ 0.05) and suggestive trends are identified by the ANOVA results directly on each data set.
  • CHL Chlorisondamine
  • MEC mecamylamine
  • MMA methyllycaconitine
  • Figure 3 shows effects of chlorisondamine on non-goggled eyes.
  • N 9-20 chicks per group. The data are illustrated as the difference between the drug treated and contralateral vehicle treated eye (mean ⁇ S.E.M.).
  • Table A presents a summary of nicotinic receptor subtypes.
  • Table 1 shows post hoc pairwise comparisons of drug effects using the Tukey test
  • Table 2 shows the longer term effects of a single-dose of chlorisondamine (200 ⁇ g) on form deprivation myopia.
  • the nervous system in large part through the retina, controls eye growth postnatally, and the development of refractive errors (the need for glasses) appears to be chiefly dependent on neural mechanisms. The most common refractive error clinically is myopia.
  • the present disclosure includes a pharmaceutical drug class, nicotinic antagonists, with activity against an experimental model of myopia, but which in addition also inhibits "normal" eye growth.
  • the inventors tested the effects on eye chick growth of several nicotinic antagonists with favorable pharmacologic properties, using the efficient delivery route of intravitreal injection.
  • nicotinic antagonists selected were chlorisondamine (CHL), mecamylamine (MEC), methyllcaconitine (MLA) and dihydro- ⁇ - erythroidine (DHBE).
  • CHL chlorisondamine
  • MEC mecamylamine
  • MMA methyllcaconitine
  • DHBE dihydro- ⁇ - erythroidine
  • Animal models using chicks offer advantages of good optics as a model for the human eye.
  • antagonists with established profiles against neuronal nicotinic receptors and having lipophilic properties compatible with diffusion into neural tissue we found evidence for an actual role, perhaps a central role, of nicotinic receptors in control of ocular growth.
  • the invention is directed to the use of nicotinic antagonists having suitable solubility properties to penetrate to the relevant target sites that regulate postnatal eye growth and that inhibit postnatal ocular growth or the development of myopia.
  • All drug classes previously identified for controlling eye growth show activity only against the form deprivation myopia model (eye remains covered by a goggle beginning at approximately 1 week of age).
  • the present invention identifies a drug class that is also active against open eyes (vision has not been deprived by goggles during maturation); and it is the first class of agents identified to show this activity. This activity against open eyes is advantageous in application to human myopia where form deprivation is not the usual circumstance.
  • Nicotinic receptor subtypes While chicks utilize nicotinic receptors in the control of pupil size and accommodation, mammalian eyes use a subclass of muscarinic receptors to control pupil size and accommodation; therefore, nicotinic antagonists are expected to be well tolerated following local application in the human eye, without inducing pupil dilation and paralysis of accommodation in children. Section 1. Nicotinic receptor subtypes
  • Nicotinic acetylcholine receptors subtypes are composed of five homologous subunits that form an acetylcholine-gated cation channel (Lindstrom, 1997, Mol. Neurobiology, 15:193-222). There are 17 known nicotinic receptor subunits ( ⁇ l-10, ⁇ l-4, ⁇ , ⁇ , and ⁇ ). Each subunit possesses four transmembrane domains.
  • the acetylcholine binding site is formed by at least three peptide loops on the ⁇ -subunit (principal component), and two on the adjacent subunit (complementary component).
  • the receptors fall into three general classes: a muscle class and two neural classes.
  • the muscle types exist in only two forms - a fetal and an adult form, each with ⁇ l subunits and other subunits specific for muscle receptors.
  • One class of neuronal receptors binds ⁇ - bungarotoxin and is composed of ⁇ 7, ⁇ 8, ⁇ 9 or ⁇ lO subunits, often as homomeric receptors.
  • the other class of neural receptors does not bind ⁇ -bungarotoxin and is formed from combining ⁇ 2, ⁇ 3, ⁇ 4 or ⁇ 6 subunits with ⁇ 2 or ⁇ 4 subunits. Rapid desensitization and limited availability of selective drugs suited for in vivo studies have impaired defining physiologic functions for these biochemically defined receptor subtypes.
  • a summary of nicotinic receptor subtypes is presented in Table A.
  • the nicotinic receptor subcommittee of NC-IUPHAR has recommended a nomemclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally-expressed nACh receptor subtypes and/or on subtypes formed by heterologous expression (Lukas et al. (1999) IUPHAR XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharm. Rev. 51, 397-401). Headings for Table A reflect abbreviations designating nACh receptor subtypes based on the predominant ⁇ subunit contained in that receptor subtype.
  • An asterisk following the indicated ⁇ subunit means that other subunits are known to or might assemble with the indicated ⁇ subunit to form the designated nACh receptor subtype(s). The absence of an asterisk indicates that the indicated subunit is known to assemble into a homomeric nACh receptor subtype. Where subunit stoichiometries are known, numbers of a particular subunit in a specific nACh receptor subtype are indicated by a subscript following the subunit in brackets. All subunits are of mammalian origin with the exception of ⁇ 8 (avian).
  • Section 1.1 Nicotinic receptor subtypes and the eye.
  • the chick retina contains several classes of cholinergic neurons (Miller et al, 1987, Neurosci., 21:725-743). Besides several subtypes of muscarinic acetylcholine receptors (Fischer et al, 1998a, J Comp. Neurol, 392:273-84), the chick retina expresses a multiplicity of nicotinic acetylcholine receptor subunits, including ⁇ 3, ⁇ 6, ⁇ 7, ⁇ 8 and ⁇ 2, ⁇ 3, ⁇ 4. The cellular patterns of neural localization of nicotinic receptors in chick retina are complex (Hamassaki-Britto et al, 1994a, Vis.
  • the chick ciliary ganglion is similarly enriched with a diversity of nicotinic receptor subtypes that include the ⁇ 3 subunits that typifies autonomic ganglia, ⁇ 5, ⁇ 7, ⁇ 2 or ⁇ 4 subunits, with both synaptic and extrasynaptic localizations (Berg et al, 1998, Neuronal Nicotinic Receptors: Pharmacology and Therapeutic Opportunities, pp. 187-196; Conroy and Berg, 1995, J. Biol. Chem., 270:4424- 4431; Horch and Sargent, 1995, J. Neurosci., 15:7778-7795; Pugh et al, 1995, Mol. Pharmacol, 47:717-725).
  • nicotinic acetycholine receptors can be antagonized by various compounds.
  • the action of these compounds against NR may be complex, may involve more than one mechanism of antagonism, and may not yet have been fully characterized in all details.
  • Specific drugs for discussion purposes are classified in terms of their currently best understood mechanism of action. Nicotinic antagonists are defined as compounds that inhibit, block, compete, prevent, or otherwise interfere with any effect of a nicotinic agonist on a target. We claim use of competitive nicotinic antagonists in this invention.
  • Competitive nicotinic antagonists are defined as compounds that appear to compete for the agonist binding sites on nicotinic receptors, where competitive antagonists appear to inhibit receptor function by preventing activation of the receptor by agonists.
  • Examples of competitive antagonists may include, but are not limited to, dihydro- ⁇ -erythroidine, bungarotoxins, tubocurarine, methyllcaconitine, the peptide conotoxins derived from snails, including MI, El, GI, SI, SIA, SII, as well as other naturally occurring peptide antagonists and synthetic peptide antagonists derived from expression libraries (Lindstrom, 1997, Mol. Neurobiology, 15:193-222). We claim use of channel-blocking nicotinic antagonists in this invention.
  • Channel- blocking nicotinic antagonists are defined as compounds that appear to block the ion channel of nicotinic receptors (NR), thereby preventing the transmembrane ion flux required for nicotinic receptor function.
  • Examples of channel-blocking nicotinic antagonists may include, but are not limited to, chlorisondamine, mecamylamine, hexamethonium, amantadine, memantine, dizocilpine [(+)-MK-801], 8 (dethylamino)octyl- 3,4,5-trimethoxybenzoate (TMB-8), and zinc.
  • Noncompetitive nicotinic antagonists are defined as compounds that antagonize the functions of nicotinic receptors (NR), but do not appear to block the ligand binding site or directly block the ion channel. Functional blockade by a noncompetitive nicotinic antagonist of ion flux through the ion channel of nicotinic receptors is insurmountable by increasing agonist concentration (Fryer and Lukas, 1999a, J. Pharmacol, and Exper. Therapeutics, 288: 88-92; Fryer and Lukas, 1999b, J. Neurochem., 72: 1117-1124).
  • Ethanol and volatile anesthetics including tetracaine and procaine are noncompetitive functional nicotinic antagonists for diverse nicotinic receptor subtypes (Bencherif et al, 1995, J. Pharmacol, and Exper. Therapeutics, 275: 1418-1426; Lindstrom, 1997, Mol. Neurobiology, 15:193-222).
  • Unexpected noncompetitive nicotinic antagonists include psychoactive compounds such as buproprion, phencyclidine, ibogaine, sertraline, paroxetine, nefaxodone, venlafaxine, and fluoxetine (Fryer and Lukas, 1999a, J. Pharmacol, and Exper.
  • Noncompetitive nicotinic antagonists may act as negative allosteric effectors, acting via allosteric sites used by known postive allosteric effectors such as ivermectin, or acting on distinct sites on the receptor (Krause et al, 1998, Mol. Pharmacol, 53: 283-294).
  • Certain voltage-dependent mechanisms can also function as noncompetitive antagonists, including voltage-sensitive Mg 2+ block of nicotinic receptor ion channels, voltage-dependent channel blockage by intracellular spermine, and nicotinic receptor inactivation triggered by membrane depolarization.
  • Antibodies can also act as nicotinic antagonists; for example, the monoclonal antibody mAb 319 blocks the function of nicotinic receptors (NR) when injected into cells (Cuevas and Berg, 1998, J Neurosci. 18: 10335-10344).
  • Antibodies include polyclonal antibodies, monoclonal antibodies, humanized or chimerized antibodies, single chain antibodies, FAb fragments F(Ab)' 2 fragments, fragments produced by a FAb expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • agonists that function as nicotinic antagonists in this invention can also function as nicotinic antagonists under certain circumstances, for example, based on their time-averaged antagonist effects. Reversible desensitization is observed following stimulation by all agonists including, but not limited to, acetylcholine, nicotine, epibatidine, cytisine, methylcarbamylcholine, and DMPP. Some compounds have a bifunctional effect, acting as an agonist for some nicotinic receptor subtypes and as an antagonist for others.
  • heterocyclic substituted pyridine derivative (+/-)-2-(-3- pyridinyl)-l-azabicyclo [2.2.2]octane also known as RJR-2429, selectively activates human muscle nicotinic receptors and a putative ⁇ 3 ⁇ 4-containing receptor, but inhibits nicotinic receptors in preparations of rat thalamus.
  • This compound is a partial agonist on nicotinic receptors mediating dopamine release from rat synaptosomal preparations. (Bencherif et al, 1998, J. Pharmacol, and Exper. Therapeutics, 284: 886-894).
  • any agonist can cause reversible and irreversible desensitization and/or inactivation of nicotinic receptors. Exposure to high agonist concentrations thus give rise to time-averaged antagonist effects on the exposed cell. By way of example, this phenomenon is observed following chronic nicotine exposure at concentrations comparable to circulating nicotine levels found in human tobacco smokers, and can lead to inactivation of certain nicotinic receptor subtypes (Lindstrom, 1997, Mol. Neurobiology, 15:193-222). Section 3. Screening assays for compounds that modulate nicotinic receptor activity
  • the following assays are designed to identify compounds that interact with nicotinic receptors (NR) that control postnatal eye growth, compounds that interfere with NR, and compounds which modulate the activity of NR genes or modulate the levels of NR. Assays may additionally be utilized which identify compounds which bind to NR and which may modulate NR levels.
  • NR nicotinic receptors
  • the compounds which may be screened in accordance with the invention include, but are not limited to peptides, antibodies and fragments thereof, and other organic compounds (e.g., peptidomimetics) that bind to NR and either mimic the activity triggered by the natural ligand (i.e., agonists) or inhibit the activity triggered by the natural ligand (i.e., antagonists), as well as peptides, antibodies or fragments thereof, and other organic compounds that mimic a domain of the NR (or a portion thereof) and bind to and "neutralize" natural ligand.
  • organic compounds e.g., peptidomimetics
  • Such compounds may include, but are not limited to, naturally occurring peptides such as conotoxins, synthetic peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries, (see, e.g., Lam et al, 1991, Nature, 354:82-84; Houghten et al, 1991, Nature, 354:84-86), and combinatorial chemistry- derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; (see, e.g., Songyang et al, 1993, Cell, 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti- idiotypic, chimeric or single chain antibodies, and FAb, F(Ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorgan
  • Other compounds which can be screened in accordance with the invention include but are not limited to small organic molecules that are able to cross the blood-retinal or blood-aqueous humor barrier, gain entry into an appropriate cell and affect the expression of the NR gene or some other gene involved in the NR signal transduction pathway (e.g., by interacting with the regulatory region or transcription factors involved in gene expression); or such compounds that affect the activity of the NR or the activity of some other intracellular factor involved in the NR signal transduction pathway.
  • Computer modelling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate NR activity. Having identified such a compound or composition, the active sites or regions are identified.
  • Such active sites might typically be ligand binding sites, such as the interaction domains of ligands with NR itself.
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found. Next, the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure.
  • solid or liquid phase NMR can be used to determine certain intra-molecular distances.
  • Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.
  • the methods of computer based numerical modelling can be used to complete the structure or improve its accuracy.
  • Any recognized modelling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. (Bencherif et al, 1998, J. Pharmacol, and Exper. Therapeutics, 284: 886-894)
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential NR modulating compounds.
  • these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand.
  • the composition of the known compound can be modified and the structural effects of modification can be determined using experimental and computer modelling methods such as those described above applied to the new composition.
  • the altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • CHARMM performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modelling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function NR, and for ameliorating myopia.
  • Assays for testing the effectiveness of compounds identified by, for example, techniques such as those described in Section 3.1 through 3.3, are discussed, below, in Section 3.4. Section 3.1. In Vitro Screening Assays for Compounds that Bind to NR
  • In vitro systems may be designed to identify compounds capable of interacting with (e.g., binding to) NR.
  • Compounds identified may be useful, for example, in modulating the activity of wild type and/or mutant NR gene products; may be useful in elaborating the biological function of NR; may be utilized in screens for identifying compounds that disrupt normal NR interactions; or may in themselves disrupt such interactions.
  • the principle of the assays used to identify compounds that bind to NR involves preparing a reaction mixture of the NR and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture.
  • the NR species used can vary depending upon the goal of the screening assay. For example, where agonists of the natural ligand are sought, the full length NR, or a truncated NR, a peptide corresponding to the extracellular domain or a fusion protein containing the NR ligand binding site fused to a protein or polypeptide that affords advantages in the assay system (e.g., labeling, isolation of the resulting complex, etc.) can be utilized. Where compounds that interact with the cytoplasmic domain (CD) are sought to be identified, peptides corresponding to the NR CD and fusion proteins containing the NR CD can be used.
  • CD cytoplasmic domain
  • the screening assays can be conducted in a variety of ways.
  • one method to conduct such an assay would involve anchoring the NR protein, polypeptide, peptide or fusion protein or the test substance onto a solid phase and detecting NR/test compound complexes anchored on the solid phase at the end of the reaction.
  • the NR reactant may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.
  • microtiter plates may conveniently be utilized as the solid phase.
  • the anchored component may be immobilized by non-covalent or covalent attachments.
  • Non- covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying.
  • an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface.
  • the surfaces may be prepared in advance and stored.
  • the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g. , using an immobilized antibody specific for NR protein, polypeptide, peptide or fusion protein or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
  • cell-based assays can be used to identify compounds that interact with
  • cell lines that express NR or cell lines that have been genetically engineered to express NR (e.g., by transfection or transduction of NR DNA) can be used.
  • Interaction of the test compound with, for example, the heterologous NR expressed by the host cell can be determined by comparison or competition with native ligands.
  • Any method suitable for detecting protein-protein interactions may be employed for identifying transmembrane proteins or intracellular proteins that interact with NR.
  • traditional methods which may be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns of cell lysates or proteins obtained from cell lysates and the NR to identify proteins in the lysate that interact, with the NR.
  • the NR component used can be a full length NR, a soluble derivative lacking the membrane-anchoring region (e.g., a truncated NR in which the transmembrane region is deleted resulting in a truncated molecule containing the extracellular domain fused to the cellular domain), a peptide corresponding to the cellular domain or a fusion protein containing the cellular domain of NR.
  • a soluble derivative lacking the membrane-anchoring region e.g., a truncated NR in which the transmembrane region is deleted resulting in a truncated molecule containing the extracellular domain fused to the cellular domain
  • a peptide corresponding to the cellular domain e.g., a truncated NR in which the transmembrane region is deleted resulting in a truncated molecule containing the extracellular domain fused to the cellular domain
  • a fusion protein containing the cellular domain of NR
  • amino acid sequence of an intracellular protein which interacts with the NR can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique. (See, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, pp.34-49).
  • the amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such intracellular proteins. Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known.
  • methods may be employed which result in the simultaneous identification of genes which encode the transmembrane or intracellular proteins interacting with NR. These methods include, for example, probing expression libraries, in a manner similar to the well known technique of antibody probing of ⁇ gtl 1 libraries, using labeled NR protein, or an NR polypeptide, peptide or fusion protein, e.g., an NR polypeptide or NR domain fused to a marker (e.g., an enzyme, fluor, luminescent protein, or dye), or an IgG- Fc domain.
  • a marker e.g., an enzyme, fluor, luminescent protein, or dye
  • plasmids are constructed that encode two hybrid proteins: one plasmid consists of nucleotides encoding the DNA-binding domain of a transcription activator protein fused to a nucleotide sequence encoding NR, an NR polypeptide, peptide or fusion protein, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an unknown protein which has been recombined into this plasmid as part of a cDNA library.
  • the DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site.
  • a reporter gene e.g., HBS or lacZ
  • Either hybrid protein alone cannot activate transcription of the reporter gene: the hybrid containing the DNA-binding domain cannot activate transcription because it does not provide activation function, and the hybrid containing the activation domain cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.
  • the two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product.
  • NR may be used as the bait gene product.
  • Total genomic or cDNA sequences are fused to the DNA encoding an activation domain.
  • This library and a plasmid encoding a hybrid of a bait NR gene product fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene.
  • a bait NR gene sequence such as the open reading frame can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.
  • a cDNA library of the cell line used to detect proteins that interact with bait NR gene product can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4.
  • This library can be co-transformed along with the bait NR gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence.
  • a cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait NR gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene.
  • Colonies which express HIS3 can be detected by growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the protein that interacts with the bait NR gene product using techniques routinely practiced in the art.
  • binding partners The macromolecules that interact with the NR are referred to, for purposes of this discussion, as "binding partners". These binding partners are likely to be involved in the NR signal transduction pathway, and therefore, in the role of NR in controlling postnatal ocular growth. Therefore, it is desirable to identify compounds that interfere with or disrupt the interaction of such binding partners with NR which may be useful in regulating the activity of NR and control of postnatal ocular growth associated with NR activity.
  • the basic principle of the assay systems used to identify compounds that interfere with the interaction between the NR and its binding partner or partners involves preparing a reaction mixture containing NR protein, polypeptide, peptide or fusion protein as described in Sections 3.1 and 3.2 above, and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex.
  • the reaction mixture is prepared in the presence and absence of the test compound.
  • the test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the NR moiety and its binding partner. Control reaction mixtures are incubated without the test compound, or with a placebo.
  • any complexes between the NR moiety and the binding partner is then detected.
  • the formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the NR and the interactive binding partner.
  • complex formation within reaction mixtures containing the test compound and normal NR protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant NR. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal NRs.
  • the assay for compounds that interfere with the interaction of the NR and binding partners can be conducted in a heterogeneous or homogeneous format.
  • Heterogeneous assays involve anchoring either the NR moiety product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction by competition can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the NR moiety and interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g.
  • NR moiety or the interactive binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
  • the anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the NR or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.
  • the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • the antibody in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody.
  • test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes.
  • test compounds which inhibit complex or which disrupt preformed complexes can be identified.
  • a homogeneous assay can be used.
  • a preformed complex of the NR moiety and the interactive binding partner is prepared in which either NR or its binding partners is labeled, but the signal generated by the label is quenched due to formation of the complex (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays).
  • the addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt the interaction between NR and an intracellular binding partner can be identified.
  • an NR fusion can be prepared for immobilization.
  • NR or a peptide fragment can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein.
  • GST glutathione-S-transferase
  • the interactive binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art.
  • This antibody can be labeled with the radioactive isotope 125 I, for example, by methods routinely practiced in the art.
  • the GST-NR fusion protein can be anchored to glutathione-agarose beads.
  • the interactive binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
  • the interaction between NR (as a gene product) and the interactive binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
  • the GST-NR fusion protein and the interactive binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads.
  • the test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the NR/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
  • these same techniques can be employed using peptide fragments that correspond to the binding domains of the NR and/or the interactive or binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins.
  • any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid surface using methods described above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin.
  • a proteolytic enzyme such as trypsin.
  • a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the intracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.
  • a GST-NR fusion protein can be prepared and anchored to a solid material as described, for example by allowing it to bind to glutathione agarose beads.
  • the interactive binding partner can be labeled with a radioactive isotope, such as 35 S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-NR fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the intracellular binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology. Section 3.4. Assays for Identification of Compounds that Ameliorate Abnormal
  • Compounds including but not limited to binding compounds identified via assay techniques such as those described, above, in Sections 3.1 through 3.3, can be tested for the ability to ameliorate abnormal postnatal ocular growth, including myopia.
  • the assays described above can identify compounds which affect NR activity (e.g., compounds that bind to the NR, inhibit binding of the natural ligand, and either activate signal transduction (agonists) or block activation (antagonists), and compounds that bind to the natural ligand of the NR and neutralize ligand activity); or compounds that affect NR gene activity (by affecting NR gene expression, including molecules, e.g., proteins or small organic molecules, that affect or interfere with splicing events so that expression of the full length or the truncated form of the NR can be modulated).
  • NR activity e.g., compounds that bind to the NR, inhibit binding of the natural ligand, and either activate signal transduction (agonists) or block activation (antagonists), and compounds that bind to
  • the assays described can also identify compounds that modulate NR signal transduction (e.g., compounds which affect downstream signalling events, such as inhibitors or enhancers of tyrosine kinase or phosphatase activities which participate in transducing the signal activated by ligand binding to the NR).
  • compounds which affect another step in the NR signal transduction pathway in which the NR gene and/or NR gene product is involved, and by affecting this same pathway may modulate the effect of NR on the development of abnormal postnatal ocular growth are within the scope of the invention.
  • Such compounds can be used as part of a therapeutic method for the treatment of myopia and other conditions resulting from abnormal postnatal ocular growth.
  • the invention encompasses cell-based and animal model-based assays for the identification of compounds exhibiting such an ability to ameliorate myopia symptoms, signs or characteristics.
  • Such cell-based assay systems can also be used as the "gold standard” to assay for purity and potency of natural ligands, including recombinantly or synthetically produced ligands.
  • Cell-based systems can be used to identify compounds which may act to ameliorate myopia symptoms, signs or characteristics.
  • Such cell systems can include, for example, recombinant or non-recombinant cells, such as cell lines, which produce NR.
  • recombinant or non-recombinant cells such as cell lines, which produce NR.
  • retinal cells or cell lines derived from retina can be used.
  • expression host cells e.g., COS cells, CHO cells, fibroblasts
  • expression host cells genetically engineered to express a functional NR and to respond to activation by the natural ligand, e.g., as measured by a chemical or phenotypic change, induction of another host cell gene, change in ion flux (e.g., Na + , K + ), tyrosine phosphorylation of host cell proteins, etc., can be used as an end point in the assay.
  • cells may be exposed to a compound suspected of exhibiting an ability to ameliorate myopia symptoms in intact eyes, at a sufficient concentration and for a time sufficient to elicit amelioration of myopia-related cellular phenotypes or cell functions in the exposed cells.
  • the cells can be assayed to measure alterations in gene expression, e.g., by assaying cell lysates for mRNA transcripts (e.g., by Northern analysis) or for NR protein expressed in the cell; compounds which regulate or modulate expression of the NR gene are good candidates as therapeutics.
  • the cells are examined to determine whether one or more myopia-related cellular phenotypes or cell functions has been altered to resemble a more normal or more wild type, non-myopic phenotype, or a phenotype more likely to produce a lower incidence or severity of myopia symptoms.
  • the expression and/or activity of components of the signal transduction pathway of which NR is a part, or the activity of the NR signal transduction pathway itself can be assayed.
  • the cell lysates can be assayed for the presence of tyrosine phosphorylation of host cell proteins, as compared to lysates derived from unexposed control cells.
  • test compound inhibits signal transduction initiated by NR activation.
  • the cell lysates can be readily assayed using a Western blot format; i.e., the host cell proteins are resolved by gel electrophoresis, transferred and probed using a anti-phosphotyrosine detection antibody (e.g., an anti- phosphotyrosine antibody labeled with a signal generating compound, such as radiolabel, fluor, enzyme, etc.) (See, e.g., Glenney et al, 1988, J Immunol Methods, 109:277-285; Frackelton et al, 1983, Mol. Cell.
  • a signal generating compound such as radiolabel, fluor, enzyme, etc.
  • an ELISA format could be used in which a particular host cell protein involved in the NR signal transduction pathway is immobilized using an anchoring antibody specific for the target host cell protein, and the presence or absence of phosphotyrosine on the immobilized host cell protein is detected using a labeled anti-phospho tyrosine antibody.
  • ion flux such as sodium or potassium ion flux
  • Membrane depolarization can also be measured as an end point for NR stimulated effects.
  • animal-based myopia models may be used to identify compounds capable of ameliorating myopia-like symptoms.
  • Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and interventions which may be effective in treating such disorders.
  • animal models may be exposed to a compound suspected of exhibiting an ability to ameliorate myopia symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of myopia symptoms in the exposed animals.
  • the response of the animals to the exposure may be monitored by assessing the reversal of characteristics, signs, or symptoms associated with myopia.
  • any treatments which reverse any aspect of myopia-like characteristics, signs or symptoms should be considered as candidates for human myopia therapeutic intervention.
  • Dosages of test agents may be determined by deriving dose-response curves. Section 4.
  • the nicotinic antagonists of this invention have been found to possess valuable pharmacological properties. Nicotinic antagonists regulate postnatal growth of the eye, with the particularly desirable effect of inhibiting postnatal ocular growth and preventing the development of myopia. This effect can be demonstrated, for example, using the methods described in the Examples below.
  • these compounds can be used to control postnatal growth of the eye, inhibit postnatal ocular growth, prevent myopia, control abnormal postnatal ocular growth, inhibit abnormal postnatal axial growth of the eye, inhibit abnormal equatorial expansion of the eye, inhibit vitreous cavity expansion, inhibit the progression of myopia, inhibit the onset of myopia, and reverse myopia. These compounds are particularly useful to inhibit the development of myopia.
  • the compounds of this invention are generally administered to animals, including but not limited to mammals including humans, as well as to birds, monotremes, reptiles, or fish.
  • the pharmacologically active compounds of this invention can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans.
  • the compounds of this invention can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (oral) or topical ocular application which do not deleteriously react with the active compounds.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, or any other suitable carrier.
  • the pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g. vitamins.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservative
  • injectable, sterile solutions perferably aqueous or oily solutions, as well as suspensions, emulsions, or implants.
  • Suitable enteral application particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules.
  • a syrup, elixir, or the like can be used wherein a sweetened vehicle is employed.
  • liquid to viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity that might be preferably greater than water.
  • suitable formulations include but are not limited to, solutions, suspensions, emulsions, creams, ointments, gels, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • sprayable aerosol preparations wherein the active ingredient, prefereably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or otherwise propelled through a vehicle capable of aerosolizing the preparation.
  • the compounds of the instant invention are useful in treating or preventing the development of myopia.
  • Therapy to inhibit axial elongation or equatorial expansion, control postnatal growth of the eye, inhibit postnatal ocular growth, prevent myopia, control abnormal postnatal ocular growth, inhibit abnormal postnatal axial growth of the eye, inhibit abnormal equatorial expansion of the eye, inhibit vitreous cavity expansion, inhibit the progression of myopia, inhibit the onset of myopia, and reverse myopia, can be administered by the use of the agent in eye drops.
  • Eye drops are typically made up at a concentration of active agent between about 0.005% and 10% in the ophthalmic medium, advantageously between about 0.01% and 5%, and preferably between about 0.1 % and 2%.
  • a pH of about 3.5 to 8.5, advantageously about 4.0 to 8.0, and preferably about 4.5 to 7.5, may be expected to be acceptable as an ophthalmic drop.
  • Phosphate buffering is also common for eye drops, but other buffers can be used.
  • a common regimen for application of eye drops is one to four times a day spaced evenly throughout waking hours. More effective agents may require fewer applications or enable the use of more dilute solutions.
  • One-day-old white leghorn chicks (Truslow Farms, Chestertown, MD) were reared in brooders on a 12 hour light-dark cycle with General Electric chroma 50 fluorescent lighting with irradiance of approximately 50 ⁇ W/cm 2 at chick eye level.
  • the chicks received Purina Chick Chow® food and water ad libitum.
  • the goggled eye received a 10 ⁇ l intravitreal injection of either drug or saline vehicle at that time.
  • Other chicks were non- goggled but similarly received intravitreal injections of either drug or vehicle to one eye.
  • drug and vehicle were administered by intraocular injections daily or every other day at approximately four hours into the light phase.
  • the experimental eye was alternated between left and right, and all contralateral eyes received injections of saline vehicle at the same time as injections to the experimental eye.
  • Chicks were anesthetized with inhalation ether for all goggle applications and drug injections.
  • the chicks were anesthetized with an intramuscular mixture of ketamine (20mg/kg) and xylazine (5mg/kg), and ocular refractometry and A-scan ultrasonography were performed as described (Stone et al, 1995, Vision Res., 35:1195-202). No intraocular injections were administered on the day of examination. While still under general anesthesia, the chicks were decapitated and the axial and equatorial dimensions of enucleated eyes were measured with digital calipers. The coronal profile of the chick eye is elliptical, and the equatorial diameter is reported as the mean of the shortest and longest equatorial dimensions of the eye.
  • Example 1 The following drugs were administered daily: dihydro- ⁇ -erythroidine hydrobromide (RBI/Sigma; Natick, MA), mecamylamine (RBI/Sigma) and methyllcaconitine citrate (RBI/Sigma). Because it is a long-acting nicotinic antagonist in mammalian brain (El-Bizri and Clarke, 1994, Br. J. Pharmacol, 113:917-925), chlorisondamine diiodide (Tocris Cookson; Ballwin, MO) was generally administered every other day by intravitreal injection in most experiments.
  • Goggled chicks As expected from previous studies, the cohorts of vehicle treated control chicks wearing a unilateral goggle developed ipsilateral myopia of about -7 to -12 diopters compared to the contralateral non-goggled eyes. The axial lengths in the goggled eyes were increased by some 0.4-0.6 mm compared to the contralateral eyes. In general, the axial length difference between goggled and open eyes was greater as measured by ultrasound which records to the inner limiting membrane than as measured by calipers which records to the outer scleral surface. Besides the greater variability of the caliper measurements, this disparity may at least partly be physiologic as both the choroid and retina of young chicks thins during goggle wear.
  • the vitreous cavity of goggled eyes was enlarged in both the axial and equatorial dimensions, with the vitreous cavity elongation largely accounting for the increase in overall axial length of the eye.
  • Goggle wearing alone induced no significant effect on anterior chamber depth in most cohorts of vehicle treated chicks.
  • post hoc pairwise comparison testing did not identify any individual differences.
  • the vehicle treated goggled eyes in the mecamylamine experiments had an anterior chamber depth slightly shallower than the contralateral non-goggled eyes (1.22 ⁇ 0.04mm in goggled eyes versus 1.34 ⁇ 0.04mm in non-goggled eyes).
  • the differences in anterior chamber depth between goggled and contralateral eyes were similar for the low mecamylamine doses; but for the 100 and 200 ⁇ g doses, the anterior chamber depths in the drug treated goggled eyes relative to the contralateral controls were no longer reduced but instead were equal (data not shown).
  • methyllycaconitine showed the greater efficacy of the two drugs and was similar to mecamylamine in that the strongest effects seemed to occur at the intermediate drug doses.
  • Dihydro- ⁇ -erythroidine exhibited only a weak effect against experimental myopia ( Figures 1, 2).
  • each of the drugs induced some pupillary dilation (change from baseline: 200 ⁇ g chlorisondamine, 0.8 ⁇ 0.1mm, P ⁇ 0.01; 50 ⁇ g mecamylamine, 0.3 ⁇ 0.1mm, not significantly changed; l ⁇ g mecamylamine, 0.8 ⁇ 0.2mm, P ⁇ 0.05; 5 ⁇ g methyllycaconitine 0.4 ⁇ 0.2mm, not significantly changed; 50 ⁇ g dihydro- ⁇ -erythroidine l.O ⁇ O.lmm, P ⁇ 0.01).
  • chlorisondamine By ultrasonography, chlorisondamine induced a 0.16 ⁇ 0.04mm (P ⁇ 0.05) reduction in axial length and a 0.20 ⁇ 0.07mm (P ⁇ 0.05) reduction in posterior chamber depth at 2 hours, each of which returned to baseline at 24 hours; chlorisondamine also reduced lens thickness by 0.12 ⁇ 0.04mm (P ⁇ 0.05) at 2 hours and by 0.16 ⁇ 0.05mm (P ⁇ 0.05) at 24 hours. None of the other drugs influenced the ultrasound measurements.
  • Example 3 Longer term effects of single-dose chlorisondamine.
  • another group of goggled chicks received a single intravitreal dose of 200 ⁇ g of chlorisondamine to the form deprived eye and saline to the contralateral eye only at the time of goggle application. They were compared to a group of unilaterally goggled chicks receiving a single saline injection to both eyes only at the time of goggle application. Neither group received subsequent intraocular injections. These chicks were evaluated by refractometry and ultrasound 4 days later, using ketamine/xylazine anesthesia.
  • Example 4 Histopathological effects.
  • the eyes were then immersion fixed in 3% glutaraldehyde/0.5% paraformaldehyde in 0.1M phosphate buffer, pH 7.4.
  • the posterior segments were either embedded in paraffin, cut at 5 ⁇ m thickness and stained with hematoxylin and eosin, or embedded in historesin, cut at 3 ⁇ m or 5 ⁇ m thickness and stained with 0.5% azure 11/0.5% methylene blue in 1% borate.
  • Acetylcholine Receptors (Nicotinic)
  • Agonists! iso, sub, ACh, cytisine 3 , cytisinea ABT418 D , epibatidine, BAC, ep ⁇ bat ⁇ d ⁇ ne d , DMPP, nicotine 0 , ep ⁇ bat ⁇ d ⁇ ne d ,

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Abstract

Cette invention concerne une méthode consistant à administrer au plan oculaire des doses efficaces au plan thérapeutique d'un antagoniste nicotinique propre à réguler la croissance oculaire postnatale et à empêcher l'apparition d'une myopie.
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FI20075498A (fi) 2007-06-29 2008-12-30 Eero Castren Menetelmä amblyopian hoitamiseksi masennuslääkkeillä
WO2018164113A1 (fr) 2017-03-06 2018-09-13 学校法人 慶應義塾 Modèle murin d'induction de la myopie et inhibiteur de stress du réticulum endoplasmique pour prévenir ou supprimer la myopie
GB2571696B (en) 2017-10-09 2020-05-27 Compass Pathways Ltd Large scale method for the preparation of Psilocybin and formulations of Psilocybin so produced
WO2020212948A1 (fr) 2019-04-17 2020-10-22 Compass Pathfinder Limited Procédés pour traiter les troubles neurocognitifs, la douleur chronique, et pour réduire l'inflammation

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RU2002120485A (ru) 2004-04-10
CA2400803A1 (fr) 2001-07-26
WO2001052832A1 (fr) 2001-07-26
CN1418095A (zh) 2003-05-14
AU775516B2 (en) 2004-08-05
JP2003520228A (ja) 2003-07-02
HK1052464A1 (zh) 2003-09-19
MXPA02007039A (es) 2003-09-25
KR20020081260A (ko) 2002-10-26

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