Patent Application
20080025973
S1P-3 (Edg-3) receptors belong to a family of G-protein coupled receptors for which either LPA or S1P are endogenous ligands. LPA is a ligand for the Edg-2, -4, and -7 receptors and S1P is a ligand for the Edg-1, -3, -5, -6, and -8 receptors. The Edg receptors have been renamed S1P receptors by the International Union of Pharmacology (Chun et al., Pharmacol Rev, Vol. 54:265-269, 2002. Therefore, as used herein, the term “Edg receptor” is synonymous with the term “S1P receptor.”
Significantly higher levels of TGFβ2 isoform has been found in aqueous humor collected from glaucomatous human eyes as compared to “normal” eyes (Tripathi et al., Exp Eye Res, Vol. 59(6):723-727, 1994; Inatani et al., Graefes Arch Clin Exp Opthalmol, Vol. 239(2):109-113, 2001; Picht et al., Graefes Arch Clin Exp Opthalmol, Vol. 239(3):199-207, 2001; Ochiai et al., Jpn J Opthalmol, Vol. 46(3):249-253, 2002). Furthermore, TGFβ2 is able to provoke substantial increases in IOP in a perfused human anterior segment model (Fleenor et al., Invest Opthalmol Vis Sci, Vol. 47(1):226-234, 2006). Therefore, TGFβ, in particular TGFβ2, appears to have a causative role in IOP related disorders such as glaucoma.
The S1P-3 receptors appear to activate Smad signaling pathways in renal mesangial cells (Xin et al., Br J Pharmacol, Vol. 147:164-174, 2006). In addition, Smad proteins are known to mediate the canonical signaling pathways activated by members of the TGF superfamily, including that of TGF-β (as shown by
Selective modulation of the TGFβ/S1P3 signaling pathway is desired since TGFβ has a positive role as well as a negative role in tissue. Positive roles include, for example, TGFβ as an anti-inflammatory agent, as an immunosuppressive agent, and as a promoter of migration and homing of T cells. Such selective modulation is provided herein.
The present inventors provide herein antagonists for ocular S1P3 receptors that result in decreased signaling through the Smad receptors, thereby decreasing downstream CTGF accumulation. Modulation of the Smad downstream pathway as provided herein results in a decrease of the negative aspects of TGFβ signaling, while leaving positive signaling effects of TGFβ substantially unaffected. Another embodiment of the invention provides a method of antagonizing S1P3 receptor binding thereby interfering with the S1P3 downstream signaling cascade, and particularly interfering with Smad signaling, for the treatment of ocular disorders in which Smad protein signaling results in inappropriate connective tissue growth factor accumulation.
Antagonists of endothelial differentiation gene subfamily 3 receptor (EDG-3, S1P-3): Antagonists of the S1P-3 receptor include agents that attenuate binding affinity or specificity between the S1P-3 receptor and its natural ligand, S1P. The antagonist may be a S1P analog. Antagonists may be a substituted thiazolidine particularly an alkyl-substituted thiazolidine or an arylalkyl-substituted thiazolidine, a substituted thiazinane particularly an alkyl-substituted thiazinane, a polysulfonated naphthylurea such as suramin (most commonly available as the hexasodium salt), or a S1P analog having structure III as cited infra; an antibody, biologically active antibody fragment thereof, peptide or a peptidomimetic having binding specificity and affinity for the S1P3 receptor; or a pharmaceutically acceptable salt of an antagonist. Antagonist agents as set forth herein may be a racemic mixture, a diastereomer or an enantiomer.
A “pharmaceutically acceptable salt of an antagonist” is a salt of an antagonist that retains the S1P3 receptor antagonistic activity and is acceptable by the human body. Salts may be acid or base salts since antagonists herein may have amino or carboxy substituents.
A substituted thiazolidine has structure I:
wherein R1 is C6-C13 alkyl, or alkyl-substituted aryl where the substitution is C5-C9 alkyl. In one embodiment of the invention, the antagonist has structure I where R1 is C10 alkyl or C11 alkyl, (2-alkylthiazolidine-4-carboxylic acid where the alkyl is C10 or C11). When R1 is C11 alkyl, the antagonist is CAY10444 available commercially from Cayman Chemical (Ann Arbor, Mich.). In another embodiment of the invention, the antagonist has structure I where R1 is alkyl-substituted phenyl and the substitution on the phenyl ring is m- or p- C7-alkyl i.e., (2-(m- or p-heptylphenyl)thiazolidine-4-carboxylic acid).
In one embodiment of the invention, the antagonist of S1P3 has structure II:
where R2 is C9-C13 alkyl.
In another embodiment of the invention, the antagonist of S1P3 has structure
where R3 is o- or m- C5-C8 alkyl; and R4 is phosphate, phosphate analog, phosphonate, or sulfate. As used herein “phosphate analog” includes the terms phosphoro-thioates, -dithioates, -selenoates, -diselenoates, -anilothioates, -anilidates, -amidates, or boron phosphates, for example.
Further compounds active in S1P3 signaling are described in U.S. Patent Application Publication No. 2005/0222422 to Lynch et al., published Oct. 6, 2005, incorporated by reference herein, and Koide et al., J Med Chem, Vol. 45:4629-4638, 2002.
An assay for identifying further antagonists of S1P3 receptor uses a competitive binding assay which may comprise combining a candidate antagonist, S1P, a S1P3 receptor and a kinase having activity for activated S1P3 receptor and measuring the amount of phosphorylated S1P3 receptor obtained. The result is compared with the amount of phosphorylated S1P3 receptor obtained from the same assay in the absence of the candidate antagonist. The candidate antagonist has antagonist activity when the level of phosphorylated S1P3 receptor is lower than when the candidate is not present. Further assays may include assays for inhibition of receptor specific antibody binding by a candidate antagonist, reduced accumulation of CTGF mRNA by a candidate antagonist, or reduced accumulation of CTGF protein by a candidate antagonist. U.S. Patent Application Publication No. 2005/0222422 to Lynch et al., published Oct. 6, 2005, previously incorporated by reference, describes a GTP binding assay for measuring S1P activity of S1P mimetics to human S1P receptors.
Substituted thiazolidines and substituted thiazinanes are synthesized using methods known in the art, for example, methods described by Koide et al. (J Med Chem, Vol. 45:4629-4638, 2002). U.S. Patent Application Publication No. 2005/0222422 to Lynch et al. published Oct. 6, 2005, previously incorporated by reference describes synthesis of S1P analog having structure III.
Antibodies having binding specificity and affinity for the S1P3 receptor are available commercially, for example, a mouse monoclonal antibody is available from GENETEX, Inc. (Catalog Number GTX12254, San Antonio, Tex.), a rabbit polyclonal antibody to sphingolipid receptor Edg3/S1P3 is available from Novus Biologics Inc. (Catalog Number NLS 1031, Littleton, Colo.), and the EDG-3 CT antibody is available from Exalpha Biologicals, Inc. (Watertown, Mass.). EDG-3 CT has binding affinity and specificity for the unique C-terminal peptide of human S1P3 receptor.
Antagonism of S1P-3 receptors and resultant inhibition of CTGF accumulation is also inferred in a human or mammal by observing an improvement in an ocular disorder. For example, in age-related macular degeneration a slowing or reversal of vision loss indicates inhibition of CTGF accumulation and, in glaucoma patients, lowered intraocular pressure and a delay or prevention of the onset of symptoms in a subject at risk for developing glaucoma indicates inhibition of CTGF accumulation.
Antagonists of the present invention may be used in combination with other agents for treating ocular disorders where CTGF accumulation or activity is inappropriate such as, for example, agents described by U.S. Published Patent Application No. 2005/0234075 to Fleenor et al., published Oct. 20, 2005, previously incorporated by reference herein.
Mode of administration: The antagonist may be delivered directly to the eye (for example: topical ocular drops or ointments; slow release devices in the cul-de-sac or implanted adjacent to the sclera (transscleral) or within the eye; periocular, conjunctival, sub-Tenons, intracameral, intravitreal, sub-retinal, retrobulbar, or intracanalicular injections) or systemically (for example: oral; intravenous, subcutaneous or intramuscular injections; parenterally, dermal delivery) using techniques well known by those skilled in the art. It is further contemplated that the antagonists of the invention may be formulated in a placement device such as a retinal pellet, intraocular insert, catheter, suppository or an implant device comprising a porous, non-porous, or gelatinous material. Intracameral injection may be through the cornea into the anterior chamber to allow the agent to reach the trabecular meshwork. Intracanalicular injection may be into the venous collector channels draining Schlemm's canal or into Schlemm's canal.
Subject: A subject in need of treatment for an ocular disorder or at risk for developing an ocular disorder is a human or other mammal having a condition or at risk of having a condition associated with Smad activation with inappropriate accumulation of CTGF. Such an ocular disorder may include, for example, hypertension, glaucoma, macular degeneration, diabetic retinopathy, choroidal neovascularization, proliferative vitreoretinopathy, ocular wound healing, and conditions with excessive scarring, with endothelial cell proliferation, or fibroproliferation. Ocular structures associated with such disorders may include the retina, choroid, lens, cornea, trabecular meshwork, rod, cone, ganglia, macula, iris, sclera, aqueous chamber, vitreous chamber, ciliary body, optic disc, papilla, or fovea, for example.
Formulations and Dosage: Pharmaceutical formulations comprise an antagonist, or salt thereof, as set forth herein up to 99% by weight mixed with a physiologically acceptable ophthalmic carrier medium such as water, buffer, saline, glycine, hyaluronic acid, mannitol, and the like. Examples of possible formulations embodied by aspects of the invention are as follows.
In a further embodiment, the ophthalmic compositions are formulated to provide for an intraocular concentration of about 0.1-100 nanomolar (nM) or, in a further embodiment, 1-10 nM of the antagonist. Topical compositions are delivered to the surface of the eye one to four times per day according to the routine discretion of a skilled clinician. The pH of the formulation should be 4-9, or 4.5 to 7.4. Systemic formulations may contain about 10 to 1000 mg of the antagonist.
An “effective amount” refers to that amount of S1P-3 receptor antagonist that is able to disrupt binding between the S1P-3 receptor and Smad. Such disruption leads to lowered Smad activity, lowered CTGF gene transcription, lowered CTGF protein accumulation and resultant lessening of symptoms in ocular disorders in a subject. Such disruption delays or prevents the onset of symptoms in a subject at risk for developing ocular disorders as set forth herein. The effective amount of a formulation may depend on factors such as the age, race, and sex of the subject, or the severity of the ocular condition, for example. In one embodiment, the antagonist is delivered topically to the eye and reaches the trabecular meshwork, retina or optic nerve head at a therapeutic dose thereby ameliorating the ocular disease process.
Acceptable carriers: An ophthalmically acceptable carrier refers to those carriers that cause at most, little to no ocular irritation, provide suitable preservation if needed, and deliver one or more S1P-3 antagonists of the present invention in a homogenous dosage. For ophthalmic delivery, a S1P-3 antagonist may be combined with opthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, or water to form an aqueous, sterile ophthalmic suspension or solution. Ophthalmic solution formulations may be prepared by dissolving the antagonist in a physiologically acceptable isotonic aqueous buffer. Further, the ophthalmic solution may include an opthalmologically acceptable surfactant to assist in dissolving the antagonist. Viscosity building agents, such as hydroxymethylcellulose, hydroxyethylcellulose, methylcellulose, polyvinylpyrrolidone, or the like, may be added to the compositions of the present invention to improve the retention of the compound.
In order to prepare a sterile ophthalmic ointment formulation, the S1P-3 antagonist is combined with a preservative in an appropriate vehicle, such as mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations may be prepared by suspending the S1P-3 antagonist in a hydrophilic base prepared from the combination of, for example, CARBOPOL®-940 (BF Goodrich, Charlotte, N.C.), or the like, according to methods known in the art for other ophthalmic formulations. VISCOAT® (Alcon Laboratories, Inc., Fort Worth, Tex.) may be used for intraocular injection, for example. Other compositions of the present invention may contain penetration enhancing agents such as cremophor and TWEEN® 80 (polyoxyethylene sorbitan monolaureate, Sigma Aldrich, St. Louis, Mo.), in the event the S1P-3 antagonists are less penetrating in the eye.
Kits: Embodiments of the present invention provide a kit that includes antagonists for attenuating S1P3 receptor signaling in a cell. The kit contains in close confinement one or more containers containing an antagonist of the present invention, a pharmaceutically acceptable carrier and, optionally, printed instructions for use.
The effect of Edg3 receptor antagonism on CTGF gene expression in cultured human trabecular meshwork cells can be determined as follows. Transformed or non-transformed human TM cell cultures (Pang et al., Curr Eye Res, Vol. 13:51-63, 1994; Steely et al., Invest Opthalmol V is Sci, Vol. 33:2242-2250, 1992; Wilson et al., Curr Eye Res, Vol. 12:783-793, 1993; Stamer et al., Curr Eye Res, Vol. 14:611-617, 1995) are treated with or without a stimulatory amount of sphingosine-1-phosphate (S1P) and with or without Edg3 receptor antagonists for a specified period of time. Separate cultures are also treated with the requisite diluent vehicle(s) used in order to serve as controls. Total RNA is then isolated from the TM cells using Qiagen RNeasy 96 system according to the manufacturer's instructions (Qiagen).
Differential expression of CTGF after cell treatment is verified by quantitative real-time RT-PCR (QRT-PCR) using an ABI Prism® 7700 Sequence Detection System (Applied Biosystems) essentially as previously described (Shepard et al., IOVS, Vol. 42:3173, 2001). Primers for CTGF amplification were designed using Primer Express software (Applied Biosystems) to anneal to adjacent exons of Genbank accession # NM 001901.1 as set forth in U.S. Published Patent Application No. 20050234075 to Fleenor et al., published Oct. 20, 2005, U.S. Ser. No. 10/510,585, filed Oct. 8, 2004, (incorporated by reference herein) to generate a 76-bp amplicon. Amplification of CTGF is normalized to 18S ribosomal RNA expression using primers designed to the 18S rRNA gene (GenBank accession #X03205) as cited by U.S. Published Patent Application No. 20050234075 to Fleenor et al. Id., to generate a 69-bp amplicon. CTGF QRT-PCR is performed in multiplex with 18S primer/probe sets in a 50 ul final volume consisting of 40 nM 18S or 900 nM CTGF primers; 100 nM 18S probe or 100 nM CTGF; 5 ul RNA; 1× Multiscribe and RNase Inhibitor Mix (ABI); and 1× TaqMan® Universal Mix (ABI). Thermal cycling conditions consist of 48° C. for 30 min and 95° C. for 10 min, followed by 40 cycles at 95° C. for 15 sec and 60° C. for 10 min. Data analysis is performed with SDS software version 1.9.1 (Applied Biosystems) and MS Excel 2002 (Microsoft). Quantification of relative RNA concentrations is done using the delta delta Ct method as described in PE Biosystems User Bulletin #2. Levels of amplified products are expressed as mean SEM of quadruplicate QRT-PCR assays. Data analysis is performed with SDS software version 1.9.1 (Applied Biosystems) and MS Excel 97 (Microsoft).
The effect of Edg3 receptor antagonism on expression of extracellular matrix-related proteins by cultured human trabecular meshwork cells is determined as follows. Human TM cell cultures are split into replicate and/or experimental and/or control groups to which are then added control solutions or experimental solutions comprising diluent vehicle(s) (as controls) and/or S1P (as stimulatory agent) and/or Edg3 receptor antagonists. Levels of extracellular matrix-related proteins, such as fibronectin, plasminogen activator inhibitor I (PAI-1), collagens, fibrillin, vitronectin, laminin, thrombospondin I, proteoglycans, or integrins, are then measured in each cell culture group via standard enzyme-linked immunoabsorbent assays (ELISA). Such assays are well-known to those skilled in the art and are sensitive immunoassays which utilize an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein. By these means, levels of various extracellular matrix-related proteins can then be compared between the groups in order to determine the effect of experimental solutions.
An example of the effect of Edg3 receptor antagonism on PAI-1 levels in supernatants from treated human TM cell cultures is shown in
The references cited herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated by reference.
Those of skill in the art, in light of the present disclosure, will appreciate that obvious modifications of the embodiments disclosed herein can be made without departing from the spirit and scope of the invention. All of the embodiments disclosed herein can be made and executed without undue experimentation in light of the present disclosure. The full scope of the invention is set out in the disclosure and equivalent embodiments thereof. The specification should not be construed to unduly narrow the full scope of protection to which the present invention is entitled.
As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more.”
This application claims priority under 35 U.S.C. §119 to U.S. Provisional Patent Application No. 60/833,080, filed Jul. 25, 2006, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60833080 | Jul 2006 | US |
